Source: http://www.google.com/patents/US5616341?dq=6978253
Timestamp: 2013-12-07 06:08:22
Document Index: 405202207

Matched Legal Cases: ['Application No. 86', 'Application No. 86', 'art 1', 'art 2', 'application No. 122', 'application No. 122', 'application No. 161', 'application No. 164', 'application No. 22', 'application No. 220', 'application No. 236', 'application No. 284', 'application No. 310', 'application No. 360', 'application No. 4', 'application No. 61', 'application No. 622', 'application No. 622', 'application No. 638', 'application No. 660', 'application No. 69', 'application No. 749', 'application No. 752', 'application No. 759', 'application No. 788', 'application No. 800', 'application No. 874']

Patent US5616341 - High drug:lipid formulations of liposomal antineoplastic agents - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Advanced Patent Search | Sign inAdvanced Patent SearchPatentsA method for encapsulation of antineoplastic agents in liposomes is provided, having preferably a high drug:lipid ratio. Liposomes may be made by a process that loads the drug by an active mechanism using a transmembrane ion gradient, preferably a transmembrane pH gradient. Using this technique, trapping...http://www.google.com/patents/US5616341?utm_source=gb-gplus-sharePatent US5616341 - High drug:lipid formulations of liposomal antineoplastic agentsPublication numberUS5616341 APublication typeGrantApplication numberUS 08/112,875Publication dateApr 1, 1997Filing dateAug 26, 1993Priority dateMar 5, 1987Fee statusPaidAlso published asUS5744158, US5795589, US6083530Publication number08112875, 112875, US 5616341 A, US 5616341A, US-A-5616341, US5616341 A, US5616341AInventorsMarcel B. Bally, Pieter R. Cullis, Richard S. Ginsberg, Lawrence D. Mayer, George N. MitilenesOriginal AssigneeThe Liposome Company, Inc.Patent Citations (42), Non-Patent Citations (109), Referenced by (23), Classifications (15), Legal Events (7) External Links: USPTO, USPTO Assignment, EspacenetHigh drug:lipid formulations of liposomal antineoplastic agentsUS 5616341 AAbstract A method for encapsulation of antineoplastic agents in liposomes is provided, having preferably a high drug:lipid ratio. Liposomes may be made by a process that loads the drug by an active mechanism using a transmembrane ion gradient, preferably a transmembrane pH gradient. Using this technique, trapping efficiencies approach 100%, and liposomes may be loaded with drug immediately prior to use, eliminating stability problems related to drug retention in the liposomes. Drug:lipid ratios employed are about 3-80 fold higher than for traditional liposome preparations, and the release rate of the drug from the liposomes is reduced. An assay method to determine free antineoplastic agents in a liposome preparation is also disclosed.
We claim: 1. A composition comprising:(i) liposomes which comprise an ionizable antineoplastic agent selected from the group consisting of doxorubicin and daunorubicin, a release-inhibiting aqueous buffer comprising citric acid and a bilayer comprising a lipid which comprises a phospholipid; and (ii) an aqueous solution external to the liposomes which is basic with respect to the release-inhibiting buffer, wherein the weight ratio of antineoplastic agent to lipid in the liposomes is from at least about 0.1:1 to about 3:1. 2. The composition of claim 1, wherein the liposome has an average diameter of from about 60 nm to about 300 nm and wherein the liposome is unilamellar.
CORRESPONDING U.S. PATENT APPLICATIONS This application is a continuation of U.S. Ser. No. 07/636,015, filed Jan. 4, 1991 and now abandoned, which is a continuation of U.S. Ser. No. 07/164,557, filed Mar. 7, 1988 and now abandoned, which-in-turn is a continuation-in-part of U.S. Ser. No. 07/022,154, filed Mar. 5, 1987 and now abandoned.
Techniques for producing large unilamellar vesicles (LUVs), such as, reverse phase evaporation, infusion procedures, and detergent dilution, can be used to produce liposomes. A review of these and other methods for producing liposomes may be found in the text Liposomes, Marc Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1, the pertinent portions of which are incorporated herein by reference. See also Szoka, Jr. et al., (1980, Ann. Rev. Biophys. Bioeng., 9:467), the pertinent portions of which are also incorporated herein by reference. A particularly preferred method for forming LUVs is described in Cullis et al., PCT Publication No. 87/00238, Jan. 16, 1986, entitled "Extrusion Technique for Producing Unilamellar Vesicles" incorporated herein by reference. Vesicles made by this technique, called LUVETS, are extruded under pressure through a membrane filter. Vesicles may also be extruded through a 200 nm filter; such vesicles are known as VET.sub.200 s. LUVETs may be exposed to at least one freeze and thaw cycle prior to the extrusion technique; this procedure is described in Mayer, et al., (Biochim. Biophys. Acta., 1985, 817:193-196), entitled "Solute Distributions and Trapping Efficiencies Observed in Freeze-Thawed Multilamellar Vesicles"; such vesicles are known as FATMLVs.
SUMMARY OF THE INVENTION The present invention discloses a liposome composition that comprises an antineoplastic agent and a lipid preferably a phospholipid, such as EPC and cholesterol, and wherein the liposomes have a transmembrane ion gradient preferably a pH gradient. The liposomes have a drug (antineoplastic agent) to lipid ratio of about greater than about 0.1:1 to about 3:1, most preferably about 0.3:1 to 3:1. The liposomes contain a release-inhibiting buffer combination such as citric acid/sodium carbonate, citric acid/sodium his phosphate, or sodium carbonate/potassium sulfate-HEPES. The antineoplastic agent can be for example, an anthracycline such as doxorubicin, daunorubicin, or epirubicin, a vinca alkaloid such as vinblastine, or vincristine, a purine or pyrimidine derivative such as 5-fluorouracil, an alkylating agent such as mitoxanthrone, mechlorethamine hydrochloride or cyclophosphamide, or an antineoplastic antibiotic such as mitomycin or bleomycin. The liposomes may comprise phospholipid such as egg phosphatidylcholine ("EPC"), hydrogenated soy phosphatidylcholine, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, or diarachidonoylphosphatidylcholine, and may additionally comprise cholesterol, for example, in about a 55:45 phospholipid:cholesterol mol ratio. The liposomes may additionally comprise alpha tocopherol. The liposomes can be about 30 nm to about 2 microns in size, preferably about 100 to about 300 nm in diameter; large unilamellar vesicles. They can contain about 50 to 200 mg/ml lipid, more preferably about 90 to about 110 mg/ml lipid. The entrapment of the antineoplastic agent in the liposomes is from about 50% to about 100%, preferably about 90% to about 100%, more preferably about 98 to about 100%. These liposomes may be large unilamellar vesicles, and may be homogeneous or unimodal with regard to size distribution. The liposomes may be administered intravenously in a patient. Pharmaceutical preparations containing the antineoplastic agents entrapped in the liposomes and pharmaceutically acceptable carriers or diluents are another embodiment of the present invention. The liposome compositions of the invention may be used to treat or stabilize a neoplastic disease, or prophylactically to prevent the onset or recurrence of a neoplastic disease. The composition of the present invention is, for example, provided as a three-component system. Where the antineoplastic agent is doxorubicin, the three component system comprises empty liposomes in an acidic solution of about pH 4.0, a basic solution, and the antineoplastic agent. The acidic solution is acetic acid buffer, oxalic acid buffer, or succinic acid buffer, preferably aqueous citric acid buffer. The basic solution is preferably sodium carbonate. The drug to lipid weight ratio is greater than about 0.1:1 to about 3:1.
Transmembrane Gradient--Uptake of Drugs As discussed above, the liposomes of the invention may be formed by any of the methods known, but preferably they are formed according to the procedures disclosed in Bally et al., PCT Application No. 86/01102, published Feb. 27, 1986. This technique allows the loading of liposomes with ionizable antineoplastic agents to achieve interior concentrations considerably greater than the drugs solubility in aqueous solution at neutral pH and/or concentrations greater than can be obtained by passive entrapment techniques. In this technique, a transmembrane ion (pH) gradient is created across the membranes of the liposomes and the antineoplastic agent is loaded into the liposomes by means of the pH gradient. The transmembrane pH gradient is generated by creating a concentration gradient for one or more charged species (e.g., Na.sup.+, Cl.sup.-, K.sup.+, Li.sup.+, .sup.- OH, and preferably H.sup.+) across the liposome membranes, and these ion gradients drive the uptake of ionizable bioactive agents (drugs) across the membranes. In the present invention, transmembrane H.sup.+ (pH) gradients are preferably employed.
Typically, a dried film of the lipid to be used is hydrated using an aqueous solution. This hydration employs a first aqueous medium, such as distilled water (e.g., USP water for injection) or aqueous buffer. When cationic drugs are to be loaded, for example, such aqueous buffer includes but is not limited to a relatively acidic buffer. Such a buffer is for example citric acid, succinic acid, acetic acid, or oxalic acid buffers. Such buffers are best used at pH about 3.5 to about 4.5. In the case of loading the drugs doxorubicin, daunorubicin, epirubicin, and vincristine, for example, it has been found most desirable to employ 300 mM citric acid at about pH 4.0 as the initial hydration medium, which makes the inside of the liposomes acidic. Citric acid has been identified as the buffering solution that best produces uptake of these drugs into the liposomes. Other buffered salines may be included in this mixture when adjusted to about pH 4 Buffered salines include phosphate buffered saline "PBS," tris-(hydroxymethyl) - aminomethane hydrochloride ("tris") buffers, N-2-Hydroxyethyl Piperazine-N'-2-Ethane sulfonic acid ("HEPES"), glycine buffers or glutamic acid, adjusted to relatively acidic pH.
Once the liposomes have been sized to the appropriate size distribution, the external medium may be replaced, by changing the original external medium to a new external medium having a different concentration of the one or more charged species (e.g., H.sup.+ ions), for example, a relatively basic or relatively acidic medium. The replacement of the external medium can be accomplished by changing the external pH, for example, in the case of doxorubicin, daunorubicin, or epirubicin, by adding a basic solution such as preferably sodium carbonate, at about pH 11.0, or a pH sufficient to result in a final pH of about 7.5-8.3, most preferably pH 7.8. In the case of vincristine, sodium bis phosphate is preferably employed, at about pH 6.8 to about pH 7.2, preferably at pH 7.0, or at a pH sufficient to result in a final pH of about 7.1. Other basic solutions that may be employed include but are not limited to sodium bicarbonate, sodium bis phosphate, sodium hydroxide, or potassium phosphate. Such a procedure creates the concentration gradient. In the case of 5-fluorouracil, the external medium is changed to a relatively acidic medium for example, with buffer such as preferably potassium sulfate/150 mM HEPES, or H.sub.2 SO.sub.4, at pH about 6.5 to about 8.5, added in sufficient amount to make the preparation relatively acidic, preferably about pH 7 Other relatively acidic solutions that may be used for FU include but are not limited to HCl, H.sub.3 PO.sub.4, to a desired pH of about 7 Other methods that may be used to change the external medium are gel filtration; (e.g. using a Sephadex column which has been equilibrated with the new medium), centrifugation, dialysis, or related techniques. This transmembrane pH gradient will load the drug into the liposomes such that the free vesicle-associated drug ratios reflect or are greater than predicted by [H.sup.+ ].sub.in /[H.sup.+ ].sub.out ratios. An ion gradient remains across liposome membranes even after the loading has been completed.
Transmembrane Gradient--Drug Release Turning now to the aspects of the invention relating to reducing the rate of release of an ionizable antineoplastic agent or other ionizable biologically-active agent from liposomes, it has been surprisingly found that the transmembrane pH gradient may also markedly reduce the rate of release across the liposome membranes. Thus, the liposomes are extremely stable regarding release of their contents. The reduced rate of drug release is created by the liposome interior buffering capacity; that is, the concentrations on the inside and outside of the liposomes of a charged species such as H.sup.+ ions (e.g., a pH gradient). For example, high interior buffering capacities, which require a larger influx of cations (such as the antineoplastic agent) to decrease the pH gradient, will lead to longer retention times. Further, once the interior buffering capacity is exhausted, the release rate of the antineoplastic agent (e.g., doxorubicins will be increased. Loading the liposomes with the drug requires adjusting the ionic concentration of the external medium of the liposomes to form a chemical potential across the liposome membrane. Where the ion is the hydrogen cation, such an adjustment may be made by changing the pH by adding a solution of relatively acidic or basic pH. As previously stated, the release rate of the bioactive agent is mediated by the buffer. Certain buffer combinations (internal aqueous medium/external aqueous medium) have been found to enhance to uptake and reduce the release of the liposome contents. For example, for the drugs doxorubicin, epirubicin, and daunorubicin, the buffer combinations found most suitable for the retention of liposomal contents are citric acid/sodium carbonate. In the case of vincristine, the buffer combination most suitable is citric acid/sodium bis phosphate. In the case of 5-FU, the preferred buffer combination is sodium carbonate/sodium hydroxide or sodium carbonate/potassium sulfate-HEPES.
Doxorubicin retention in EPC/cholesterol (55:45) vesicles exhibiting a pH gradient can be increased by employing citrate/carbonate buffer systems such that less than about 5% drug release is observed over 24 h at 37 serum components; less than 5% doxorubicin is released over 24 hours for vesicles incubated at 37 association assays, where doxorubicin was incubated with HEPES buffer at pH 7.5, and citrate buffers (sodium citrate) at pH ranging from about 4.0-7.5, citrate interacts with doxorubicin and precipitates, whereas HEPES buffer does not. Such a buffer combination, that is, citrate/carbonate, acts to reduce the rate of release of the drug from the liposomes. Other release-reducing buffer combinations can be used such as oxalic acid/potassium phosphate or succinic acid/sodium bicarbonate, with citric acid/sodium carbonate or citric acid/sodium bis phosphate preferred.
The liposomes are then incubated to facilitate encapsulation, (above 37 the length of incubation can depend on the temperature. Daunorubicin, epirubicin, and mitoxanthrone can be incubated at 25 ionizable antineoplastic agent may likewise be heated at the same temperature and the two components are admixed. The liposome-drug suspension is incubated further, and the resulting solution is of final pH about 6.9-8.3, preferably about 7.5-7.8. Such an incubation at elevated temperatures is preferred for efficient loading of doxorubicin into liposomes containing cholesterol. The solution is then diluted as needed with physiological saline, for example, and administered.
In order to determine whether an ionizable antineoplastic agent will load into liposomes in response to a transmembrane pH gradient, EPC-containing liposomes are made (about 1.0 mM EPC) with a .sub.3 H-DPPC tracer and with a relatively acidic or basic internal medium such as 300 mM citric acid at about pH 4 These liposomes are extruded about 10 times according to the LUVET procedure through 2 100 nm filters, followed by adjustment of the external pH to a relatively basic or acidic pH, for example, sodium carbonate, at about pH 11 Following the formation of the pH gradient, the agent to be loaded, spiked with a radioactive isotope of the agent, is admixed with the liposomes to about 200 uM (per 1.0 mM lipid used). The liposomes are separated from free, unentrapped agent on G50-M Sephadex minicolumns at 500 radioactivity counted in a scintillation counter. Uptake of the drug inn moles per umole of lipid is then plotted over incubation time. One hundred percent of the available doxorubicin is taken up into liposomes under these conditions.
In the case of doxorubicin, commercially available forms, such as powdered, solid, and methylparaben-containing forms (Adriamycin R. D. F., Adria Laboratories, Inc., Columbus, Ohio) may be used in the invention. When the methylparaben-containing form is employed, an aqueous solution such as saline may be added to that form, thereby dissolving it, followed by the admixing of this suspension with the liposomes which have the transmembrane pH gradient across their bilayers. Such admixing at 60 encapsulation of the doxorubicin.
Lipids which can be used in the liposome formulations of the present invention include phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI), sphingomyelin (SPM), and the like, alone or in combination. The phospholipids can be synthetic or derived from natural sources such as egg or soy. The phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) may also be used. In the preferred embodiments, egg phosphatidylcholine (EPC), and cholesterol are used in preferably a 55:45 mole ratio. In other embodiments, distearoylphosphatidyl choline (DSPC), dipalmitoylphosphatidylcholine (DPPC), or hydrogenated soy phosphatidylcholine (HSPC) may be used in a mole ratio of 55:45 with cholesterol. Dimyristoylphosphatidylcholine (DMPC) and diarachidonoyl phosphatidylcholine (DAPC) may similarly be used. Due to the elevated transition temperatures (T.sub.c) of lipids such as DSPC (T.sub.c of about 65 45 preferably heated to about their T.sub.c or temperatures slightly higher (e.g., up to about 5 these liposomes.
Doxorubicin--Drug Release Depends on Lipid Composition In vitro doxorubicin release properties demonstrate dependence on lipid composition. Preparations containing cholesterol were more resistant to drug release, and those containing cholesterol and egg phosphatidyl glycerol resulted in drug release intermediate to those containing only EPC and those containing EPC/cholesterol.
Doxorubicin--Toxicity The doxorubicin administered in the liposomes of the present invention are shown to be of lesser toxicity than doxorubicin given in free form. Toxicological evaluation of liposomal doxorubicin in mice has shown a 2.3 fold increase in acute LD.sub.50 values, with significantly less weight loss.
Apparent mouse LD.sub.50 s were dependent on lipid composition. The LD.sub.50 of liposomal doxorubicin increases as the cholesterol content of the liposomes is increased from 0 to about 45 mol %, or when the lipid formulation includes DSPC.
Variables such as liposome surface charge and size do not significantly change the acute toxicity of liposomal doxorubicin, as do changes in lipid composition. Further, the use of DSPC/cholesterol dramatically increases LD.sub.50 (&gt;200 mg/kg), which is 4- and 10- fold greater than observed for EPC/cholesterol entrapped and free drug, respectively. Such formulations also have very low drug accumulation levels in heart, lung and kidney tissues. Increasing drug to lipid ratios has a dramatic effect on amelioration of doxorubicin toxicity. Previous studies have not shown this effect, due to the limitations in doxorubicin entrapment by prior art entrapment techniques. Although such entrapment of the drug leads to its uptake by liver, acute liver damage is not observed.
Liposome Formation Several methods may be used to form the liposomes of the invention. For example, multilamellar vesicles (MLVs), stable plurilamellar vesicles (SPLVs) or reverse phase evaporation vesicle (REVs) may be used. Preferably, MLVs are extruded through filters forming LUVs of sizes dependent upon the filter pore size. Polycarbonate filters of 30, 50, 60, 100, 200, or 800 nm pore sizes are used. In this method, disclosed in Cullis, et al., PCT Publication No. WO 86/000238, Jan. 16, 1986, relevant portions of which are incorporated herein by reference, the liposome suspension may be repeatedly passed through the extrusion device resulting in a population of liposomes of homogenous size distribution. For example, the filtering may be performed through a straight-through membrane filter (a Nucleopore polycarbonate filter) or a tortuous path filter (e.g. a Nucleopore membrafil filter (mixed cellulose esters) of 0.1 um size), or by alternative size reduction techniques such as homogenization. The liposomes of the present invention may be from about 30 nm to about 2 microns in diameter; preferably about 50 nm to 300 nm, preferably about 60 nm to 300 nm and most preferably about 100 to 300 nm. This size range includes liposomes that may be MLVs, SPLVs, or LUVs. In the present invention, liposomes which are unilamellar liposomes of about 100 nm to about 300 nm are preferred; such liposomes are LUVs. The size range of SUVs is about 25-50 nm.
When lipids having a gel to liquid crystalline T.sub.c above ambient temperature are employed, an extruder having a heated barrel (thermojacket) may be employed. Such a device serves to increase the liposome suspension temperature allowing extrusion of these LUVs. Such lipids used with the thermojacketed extruder are DSPC, DPPC, DMPC and DAPC, for example. These lipids may be combined with cholesterol in a 55:45 mol ratio, for example. Liposomes containing DSPC would be extruded at about 65 85 further embodiment of this invention that LUVs employing these lipids having a T.sub.c above ambient temperatures may be formed. Previous techniques used with such lipids to form small vesicles involved sonication, which creates SUVs (size range of about 25-50 nm).
One embodiment of the present invention is a 3 component liposomal-antineoplastic agent treatment system which allows for entrapment of the agent at the clinical site. When the drug is doxorubicin or vincristine or other antineoplastic agent that will load in response to a transmembrane pH gradient where the interior of the liposomes is acidic, the first component of the system (Vial 1) is liposomes in an acidic solution, for example, in citric acid buffer (300 mmol., pH 3.8-4.2, preferably pH 4.0). The second component (Vial 2) is a base, preferably sodium carbonate or sodium bisphosphate solution at 0.5M, pH 11.5. The third component (Vial 3) is the antineoplastic agent. The above-mentioned treatment system may be provided as a 3-vial system, with a first vial containing the liposomes in acidic medium, the second vial containing the base, and a third vial containing the antineoplastic agent (e.g. doxorubicin). Where the drug is one that loads in response to a transmembrane gradient wherein the inside of the liposomes is relatively basic (such as, for example, 5-FU), the first component of the system is liposomes in relatively basic buffer (such as, for example, sodium carbonate, pH 6.8-11.0, preferably pH 9.6). The second component is a relatively acidic solution, for example, 150 mM potassium sulfate/150 mM HEPES buffer, pH 7.4. The third component comprises the antineoplastic agent. Following the formation of the pH gradient across the liposomes (by admixing the first and second vials), the liposomes may be heated prior to admixing with the drug. When loading doxorubicin, vincristine, and FU it has been found advantageous to heat the liposomes to about 60 Daunorubicin, epirubicin, mitoxanthrone, and vincristine load efficiently at 25
When the above-described vial system is used in the case of loading doxorubicin, the components may be mixed immediately prior to use according to the following method. Sodium carbonate solution from Vial 2 is added to the liposomes in Vial 1. The mixture is heated at an elevated temperature (e.g. 60 combined carbonate and liposome solutions are then added to Vial 3 containing the antineoplastic agent (doxorubicin) and lactose. This vial is vortically mixed, then heated at an elevated temperature (e.g. 60 resulting liposomal-drug suspension is then diluted with normal saline or 5% dextrose. The final solution is at pH 6.9-8.0, preferably pH 7.5.
Spectrophotometric Assay In the antineoplastic assay aspect of the invention, an assay is disclosed for determining the proportions of free and liposome-entrapped antineoplastic drug in liposomal preparations, based on a pH-dependent spectral response (e.g., infrared, ultraviolet, or visible). For example, at pH of about 7.0, doxorubicin exhibits a maximal absorbance at 489 nm, whereas at alkaline pH (about 10.0), absorbance peaks are observed at 550 and 592 nm (FIG. 15). Free doxorubicin concentrations in liposomal systems can thus be determined by monitoring the absorbance at 600 nm after alkalinizing the extravesicular media (liposomal bathing solution) with a base such as sodium hydroxide (absorbance differential). Such procedure induces the spectral shift of free doxorubicin and not liposomal entrapped doxorubicin since the lipid bilayer is able to isolate the entrapped doxorubicin from the alkaline external media. The resulting O.D..sub.600 therefore reflects the amount of unentrapped doxorubicin in the preparation. Total doxorubicin concentrations are then quantitated by repeating the measurement after solubilizing the liposomes (breaking the liposomes) by any method known in the art, for example with Triton X-100 (thereby exposing all the doxorubicin to the alkaline environment). The absorbance ratio at 600 nm is directly proportional to the percent free doxorubicin in vesicle preparations as detected by standard column chromatography techniques. The proportions of unentrapped drug are determined as the ratio of the absorbance obtained after alkalinization with NaOH divided by that observed in the presence of Triton X-100 (measuring an absorbance differential).
The spectroscopic analysis of liposomal doxorubicin preparations was compared to column chromatography methods which directly measure free and vesicle associated drug to correlate absorbance ratio values to actual free DOX/total DOX ratios over a wide range of trapping efficiencies. Since pH gradients induce the uptake of doxorubicin into liposomes such that [DOX].sub.in /[DOX].sub.out ratios reflect [H.sup.+ ].sub.in /[H.sup.+ ].sub.out ratios, EPC/cholesterol liposomes exhibiting pH gradients (acidic inside) of varying magnitude were utilized to construct liposome systems with trapping efficiencies from 10 to 99%. FIG. 5 demonstrates that the absorbance ratio at 600 nm described here accurately represents the ratio of free/total doxorubicin in the vesicle preparations over the full range of trapping efficiencies studied. The spectroscopic analysis method was also completed on EPC liposomes in which doxorubicin had been passively entrapped during vesicle formation to insure that these results were not specific to liposomal doxorubicin obtained by active entrapment. FIG. 5 (open symbol) shows that the absorbance ratio at 600 nm for this sample correlates with the free/total doxorubicin value obtained by column chromatography.
Liposomal Dehydration and Storage The liposomes formed by the procedures of the present invention may be lyophilized or dehydrated at various stages of formation. For example, the lipid film may be lyophilized after removing the solvent and prior to adding the drug. Alternatively, the lipid-drug film may be lyophilized prior to hydrating the liposomes. Such dehydration may be carried out by exposure of the lipid or liposome to reduced pressure thereby removing all suspending solvent. The liposomes may be dehydrated in the presence of a hydrophilic agent according to the procedures of Bally et al., PCT Publication No. 86/01102, published Feb. 27, 1986, entitled "Encapsulation of Antineoplastic Agents in Liposomes", and Janoff et al., PCT Publication No. 86/01103, published Feb. 27, 1986, entitled "Dehydrated Liposomes", or Schneider et al., in U.S. Pat. No. 4,229,360, issued Oct. 29, 1980. Alternatively or additionally, the hydrated liposome preparation may also be dehydrated by placing it in surrounding medium in liquid nitrogen and freezing it prior to the dehydration step. Dehydration with prior freezing may be performed in the presence of one or more protective agents, such as sugars in the preparation according, to the techniques of Bally, et al., PCT Application No. 86/01103published Feb. 27, 1986, relevant portions of which are hereby incorporated by reference. Such techniques enhance the long-term storage and stability of the preparations. For example, the liposomal-antineoplastic agent can be mixed with a sugar solution in a sugar: lipid w/w ratio of about 0.5:1 to about 50:1, and preferably about 20:1. Upon rehydration, such liposomes retain essentially all the antineoplastic agent previously loaded, for such liposomes sized through 100 and 200 nm pore size filters. In a preferred embodiment, the sugar is mannitol, or mannitol:glucose:lactose in a 2:1:1 w/w/w ratio. Following rehydration in distilled water, the preparation is preferably heated for ten minutes at an elevated temperature, for example 60 suitable methods may be used in the dehydration of the above-disclosed liposome preparations. The liposomes may also be dehydrated without prior freezing.
Once the liposomes have been dehydrated, they can be stored for extended periods of time until they are to be used. The appropriate temperature for storage will depend on the lipid formulation of the liposomes and the temperature sensitivity of encapsulated materials. For example, various antineoplastic agents are heat labile, and thus dehydrated liposomes containing such agents should be stored under refrigerated conditions e.g. at about 4 for such agents, the dehydration process is preferably carried out at reduced temperatures, rather than at room temperature.
The mode of administration of the liposomes containing antineplastic agents and the pharmaceutical formulations thereof may determine the sites and cells in the organism to which the compound will be delivered. The liposomes of the present invention can be administered alone but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. The preparations may be injected parenterally, for example, intravenously. For parenteral administration, they can be used, for example, in the form of a sterile aqueous solution which may contain other solutes, for example, enough salts or glucose to make the solution isotonic. The doxorubicin liposomes, for example, may be given, as a 60 minute intravenous infusion at a dose of at least about 20 mg/m.sup.2. They may also be employed for peritoneal lavage or intrathecal administration via injection. They may also be administered subcutaneously for example at the site of lymph node metastases. Other uses, depending on the particular properties of the preparation, may be envisioned by those skilled in the art.
EXAMPLE 1 Citric acid (1.0 ml of 150 mM, pH 4.0) was added to 200 mg of EPC/cholesterol (mole ratio of 1:1) in a test tube. The tube was vortically mixed for 5 minutes to homogeneously disperse the solution and create MLVs. The sample was transferred to a 2.0 ml capacity cryogenic vial, immersed in liquid nitrogen for 2 minutes, and then heated at 40 freeze-thaw cycle was repeated 7 times with brief vortical mixing of the sample immediately prior to the freezing step, creating FATMLVs. The sample was then extruded 7 times through 2 stacked 0.2 um polycarbonate filters according to the LUVET procedure. This sample was diluted 2-fold with unbuffered 0.85% saline. The liposome solution was preheated to 60 doxorubicin (22.2 mg dox/100 mg lipid) and powdered sodium carbonate (3.75 mg/22.2 mg dox). The sample was heated to 60 intermittently vortically mixed.
EXAMPLE 2 The procedures of Example 1 were followed, using 300 mM citric acid (pH 4.0) and a lipid concentration of 100 mg/ml. The liposomes were not diluted with saline, and sodium bicarbonate was added as a diluent, bringing the exterior pH to about pH 8.0 prior to doxorubicin addition.
EXAMPLE 3 Liposomes that actively encapsulated doxorubicin were prepared by hydrating an EPC film (dried down from CHCl.sub.3 and placed under high vacuum for 12 h) in 300 mM citric acid buffer (pH 4.0) to achieve a final lipid concentration of 100 mg/ml. These MLVs were frozen and thawed 5 times and extruded 5 times through polycarbonate filters with a pore size of 0.2 um according to the LUVET technique. The liposomes were then adjusted to pH 7.5 with 1.0M Na.sub.2 CO.sub.3, and incubated with doxorubicin at 60
Liposomes that passively entrapped doxorubicin were made using the materials as above, by suspending doxorubicin in buffer (20 mM HEPES, 150 mM NaCl, pH 7.5) to 2.0 mM doxorubicin, prior to the lipid hydration step. The liposomes were frozen and thawed and extruded as above. Active entrapment of doxorubicin was accomplished by preparing vesicles in buffer at pH 4.0, increasing the exterior pH to 7.5 with 1.0M Na.sub.2 CO.sub.3, and incubating the vesicles (20 mM lipid) with doxorubicin (10 mg lipid/ml) at 60
EXAMPLE 4 The materials and procedures of Example 3 were employed, using citric acid buffer at pH 4.2, 5.2, 5.7, 6.7, and 7.2. FIG. 5 demonstrates that the absorbance ratio (Abs. 600 NaOH/Abs..sub.600 after Triton X-100) accurately represents the ratio of free/total doxorubicin in the vesicle preparations over the full range of trapping efficiencies.
EXAMPLE 5 The materials and procedures of Example 3 were employed, but entrapment efficiency of liposome encapsulated doxorubicin was monitored by comparison of the color resulting from addition of an aliquot (0.2 ml) of the liposomes to 1.0N NaOH to a color chart.
EXAMPLE 6 EPC/cholesterol (55/45 mol/mol ratio) (200 mg) was dried to a thin film from chloroform, under reduced pressure at 37 Citric acid (1.0 ml of 150 mM at pH 4.0) was added and the film suspended. Resulting MLVs were frozen and thawed 7 times as in Example 1, and extruded 5 times through a 200 nm polycarbonate filter using the LUVET procedure. Size distributions of the resulting liposomes were determined by quasielastic light scattering (QELS) and general morphology was observed using freeze-fracture electron microscopy. Sterile saline (1.0 ml) was added to the extruded vesicle solution, yielding a total lipid concentration of 100 mg/ml. The exterior pH of the liposomes was titrated to 7.5 using 1.0N NaOH. This liposome solution (1.0 ml), and powdered doxorubicin (22 mg) (containing Na.sub.2 CO.sub.3 at a wt. ratio of 1 mg/6 mg doxorubicin) was then heated at 60 intermittent vortical mixing.
EXAMPLE 7 The materials and procedures of Example 6 were employed to determine the in vitro stability of the liposome-doxorubicin preparations. Release experiments were performed as follows: 10-fold dilute liposome samples were dialyzed for 24 hours against 1000 volumes of 20 mM HEPES, 150 mM NaCl (pH 7.5) at 37 post-preparation, a 150 ul aliquot was removed and the entrapped doxorubicin was determined.
EXAMPLE 8 The materials and procedures of Example 3 were employed except that vesicles were sized through 1.0 micron pore size filters and serum stability for the samples was determined. The diluted liposomal-doxorubicin sample was diluted with 20 volumes of fresh human serum and incubating at 37 vesicles were pelleted by centrifugation at 500 washed two times with 20 mM HEPES, 150 mM NaCl at pH 7.5 and assayed for phospholipid and doxorubicin as previously described.
EXAMPLE 9 The entrapment efficiency of doxorubicin liposomes was analyzed as follows:
After completion of the entrapment procedure according to Example 6, 20 ul of the doxorubicin-liposomes were diluted to 200 ul with 20 mM HEPES, 150 mM NaCl (pH 7.5). An aliquot of this diluted sample (20 ul) was assayed for lipid phosphate by the procedure of Bartlett, J. Biol. Chem. 1959, 234:466-468. A second 20 ul sample of the diluted preparation was removed and placed in a glass test tube, to which Triton X-100 (1.0 ml of 1% w/w) was added. The sample was heated in a water bath at 40 minutes and vortically mixed. Absorbance of the sample was read at 480 nm in a spectrophotometer. Sample readings were compared to a standard curve of doxorubicin samples containing known amounts of the agent which have been diluted with 1.0 ml of Triton X-100.
Sephadex G-50 (medium grade) columns were prepared at 1.0 ml capacity that had been pre-swollen with gel in 20 mM HEPES, 150 mM NaCl (pH 7.5). Columns were centrifuged at 500 spin, to pack columns. Doxorubicin-liposome samples were applied (150 ul of the 10 of 50 ul of buffer, and centrifuged at 3000 rpm for 5 minutes. The eluant was vortically mixed until homogenous. Aliquots (25 ul) were removed and analyzed for phosphate and doxorubicin as described above.
DBA/2 mice weighing 18-20 gms were obtained and divided into groups of 6 to 10. These mice were given i.p. injections (0.5 ml) of 1.5.times.10.sup.6 L1210 tumor cells. Treatment was initiated 24 hours after tumor injection and was given via the lateral tail vein. Animals were treated with liposomal doxorubicin based on mean body weight. Mice were weighed daily. Survival time was recorded in days and mean and median survival times were calculated.
EXAMPLE 11 LD.sub.50 studies comparing free- and liposomal-doxorubicin were carried out as follows:
EXAMPLE 12 EPC/cholesterol (2.1:1 wt. ratio) was dispersed in 150 mM citric acid (pH 4.0) to yield 200 mg total lipid/ml buffer. The resulting MLVs were frozen and thawed 7 times with vortical mixing prior to each freezing step. The resulting FATMLVs were extruded 5 times through 2 stacked 0.2 um pore size filters to make VET.sub.200 s. The liposomes were then diluted 2 fold with unbuffered saline and the pH brought to 7.5 with 1N NaOH. The equivalent of 1.0 ml of liposomes before pH adjustment were added to 133 mg of doxorubicin/lactose and 3.7 mg Na.sub.2 CO.sub.3 contained in a sealed vial (20 ml capacity). Both the liposomes and the doxorubicin-containing vial were heated to 60 admixing, the liposomes were heated at 60 vortical mixing every minute. The sample was then cooled to room temperature. An aliquot of the sample (50 ul) was removed and diluted to 0.5 ml with 20 mM HEPES, 150 mM NaCl (pH 7.5). An aliquot of this sample (150 ul) was applied to a 1.0 ml Sephadex G-50 column as described previously. Phosphate and doxorubicin were quantitated as described previously, in the eluant and the original samples.
EXAMPLE 13 VET.sub.200 samples were prepared according to Example 12 using EPC/EPG/cholesterol (0.95/0.05/1.0 mole ratio) at 200 mg total lipid in 150 mM citric acid (pH 4.0). The samples were diluted 2 times with unbuffered saline and the exterior pH of the liposomes was adjusted to 7.5 with 1.0N NaOH. After incubation of this preparation for 5 minutes at 60 containing 11.7 mg of Na.sub.2 CO.sub.3. The sample was vortexed intermittently while incubating at 60
EXAMPLE 14 A film of hydrogenated soy PC (HSPC) and cholesterol (HSPC/cholesterol 2.4:1 weight ratio, 400 mg total lipid) was hydrated with 4.0 ml of 300 mM citric acid at pH 4.0, forming MLVs. This solution was extruded 5 times through a 0.2 um pore size filter. An aliquot of sodium bicarbonate was added to the extruded liposomes to adjust the pH to 8.5+/-0.2. A vial containing 10 mg doxorubicin and the liposomes were preheated at 60 added to the doxorubicin vial, vortically mixed, and incubated for 15 minutes at 60 indicated greater than 95% trapping efficiency.
EXAMPLE 16 Release characteristics of liposomal-doxorubicin were determined as follows:
EPC/cholesterol (55/45 mole ratio) was dried from chloroform to a thin film on a 500 ml capacity round bottom flask (400 mg total lipid). The film was hydrated with 4.0 ml of 300 mM citric acid at pH 4.0, forming MLVs. These MLVs were extruded through 2 stacked 0.22 um Nucleopore membrafil filters followed by extrusion 10 times through a 0.1 um Nucleopore membrafil (tortuous path) filter. To 1.0 ml of the resulting filtrate sample as added 275 ul of 1M Na.sub.2 CO.sub.3, which raised the exterior pH to 8.3. An aliquot (0.6 ml) was heated for 3 minutes at 60 mg sample of doxorubicin. The liposome aliquot was added to the 10 mg doxorubicin and heated at 60 divided into 2 parts. Part 1 was diluted 10 times with 30 mM HEPES, 150 mM NaCl, at pH 7.5. Part 2 was diluted 10 times with 300 mM citric acid at pH 4 Both samples were placed into dialysis bags and dialyzed at 37 a 150 ul aliquot was removed and analyzed for doxorubicin and lipid phosphate as previously described, after passage down a 1.0 ml Sephadex column equilibrated in the respective buffer.
Doxorubicin was added, at 25 NaCl buffer, pH 7.5 to give a 4 mM doxorubicin solution. The sample was centrifuged to pellet any precipitate, and the supernatant assayed for doxorubicin by spectrophotometric methods as previously described.
Results are graphed in FIG. 3, a graph of a citrate-doxorubicin interaction resulting from mixing experiments at varying citrate pH values. The mM doxorubicin remaining in solution following centrifugation is plotted as a function of titrate pH: 4 mM doxorubicin, mixed at 60 cooled to 25 25 then cooled to 25 in 20 mM/HEPES, 150 mM NaCl, at 25 circle).
EXAMPLE 18 The procedures of Example 17 were followed at the following temperature conditions of mixing: 60 25 C. using 20 mM doxorubicin.
Results are graphed in FIG. 3, a graph of a citrate-doxorubicin interaction resulting from mixing experiments at varying citrate pH values. The mM doxorubicin remaining in solution following centrifugation is plotted as a function of citrate pH: 4 mM doxorubicin, mixed at 60 cooled to 25 25 then cooled to 25 in 20 mM/HEPES, 150 mM NaCl, at 25 circle).
EXAMPLE 19 EPC and cholesterol (55:45 mole ratio), total lipid 100 mg lipid per ml buffer was dried to a thin film on the walls of a reaction vessel, and hydrated with 300 mM citrate pH 4 The resulting MLVs were size reduced by passage 10 times through a 0.22 um Nucleopore membrafil filter. An aliquot of sodium carbonate (1.0 m) was added to the resulting liposomes, to adjust the external pH to 8.3. The suspension was incubated at 60 yield 29.+-.2 mg doxorubicin per 100 mg of total lipid, and the suspension incubated at 60 suspension was administered to mice according to the procedures of Example 10.
EXAMPLE 20 The procedures and materials of Example 19 were followed, with the additional steps after size reduction of passing the liposome suspension 10 times through a 0.1 um Nucleopore membrafil filter, then 10 times through 2 stacked 0.1 um Nucleopore membrafil filters. LD.sub.50 for the resulting liposomal-doxorubicin suspension were performed according to Example 10.
EXAMPLE 21 Liposomes containing DSPC were prepared by hydrating a lipid film (dried down from methylene chloride for 12 hours under high vacuum) in 300 mM citric acid pH 4.0 to achieve 100 mg total lipid per ml of citric acid solution. The resulting MLVs were frozen and thawed 7 times in liquid nitrogen, and heated for several minutes at 60 times through polycarbonate filters 0.2 um pore size using a thermojacket LUVET extrusion device. The exterior pH of these extruded liposomes was then titrated to pH 7.8 with sodium hydroxide. This liposome solution was then heated at 60 at a drug to lipid ratio of 0.25:1 and heated at 60 minutes with vortical mixing. Untrapped doxorubicin was removed from the preparation by passing 150 ul of the sample over 1 ml Sephadex G-50 column equilibrated in buffered saline. This procedure resulted in an entrapment efficiency of greater than 95%.
EXAMPLE 22 The materials and procedures of Example 21 were employed wherein the pH of the resulting liposomes was adjusted with sodium carbonate (1.0M) to pH 8.0 and maintained at 60
EXAMPLE 24 Female DBA/2 mice weighing 18-22 gms groups of 6 to 10, were inoculated via i.p. injections of 1.5.times.10.sup.6 L1210 tumor cells suspended in 0.5 ml RPMI 1640. The L1210 cell line was maintained by serial passage of ascites fluid or as a frozen (liquid N.sub.2) culture. Without treatment the mice develop a 2 to 5 gm ascitic tumor within 7 to 8 days, and had a mean survival time of 8 to 10 days. Liposomes made according to Example 22 were employed; treatment was initiated one day after tumor injection, and was given as a single i.v. dose via the lateral tail vein. The animals were treated with free or liposomal doxorubicin at 5 mg/kg doxorubicin. Control groups were treated with either sterile saline or empty liposomes at a lipid dose equivalent to that given with the highest dose of liposomal doxorubicin. Mice were weighed on the day prior to tumor injection, and weights were recorded daily until the first death within a group. Survival time was recorded in days after tumor injection. Mean and median survival times and statistical significance of the results were determined employing a two-tailed Wilcoxon's ranking test (randomized two-group design).
EXAMPLE 25 Liposomes were made according to the procedures of Example 2. Where the P388 leukemia model was employed, tumor cells (1 0.1 ml, were injected i.p into female CDF-1 mice. One day after tumor inoculation, the mice were treated with liposomal doxorubicin (5 mg/kg dose) via tail vein injection. Dosage was calculated according to the mean weight of each group, and weights were determined on day 0 (day of tumor injection) and day 5. Deaths were recorded on a daily basis.
Male shinogi mice (25-40g, 9 per group) were injected subcutaneously with 1 inoculated mice. Tumor growth was monitored by palpation and tumor measurements with a vernier caliper. Upon growth of the tumor to 0.5-2.0 g (tumor weight=[width.sup.2 were administered liposomal doxorubicin dose of 13 mg/kg i.v. at seven day intervals (3 injections of the indicated dose). Tumor growth was monitored 3 times weekly for 50 days post first treatment or until the tumor weight exceeded 9 g at which time the animal was sacrificed. Treatment doses were based on the initial animal weights prior to tumor inoculation.
EXAMPLE 27 Liposomes were prepared by hydrating a film of DSPC/cholesterol (55:45 molar ratio) in 300 mM citric acid buffer (pH 4.0) with vortex mixing. These MLVs (100 mg total lipid/ml buffer) were extruded 10 times through a 200 nm pore size polycarbonate filters in a thermojacket LUVET heated to 60 sulfate (Oncovin, available from Eli Lilly and Co., Indianapolis, Ind.) to achieve a drug to total lipid weight ratio of approximately 0.17:1. To this was added a sufficient amount of 1.0M Na.sub.2 HPO.sub.4 to bring the pH of the solution to about 7 The samples were then heated at 60 inside the liposomes at a trapping efficiency in excess of 98%.
Drug retention was measured at 21 dialysis in 20 mM HEPES, 150 mM NaCl, pH 7.5 (dialysate). Table 1 shows vincristine uptake characteristics for EPC/cholesterol, HSPC/cholesterol, and DSPC/cholesterol vesicles, employing vincristine from various sources, specifically, that from Sigma Chemical Co. (St. Louis, Mo.), and Oncovin, Eli Lilly & Co. (Indianapolis, Ind.) brand of vincristine.
The antitumor activity of free and liposomal vincristine were assessed employing an L1210 lymphocytic leukemia model. DBA/2J mice (6 mice per group) were injected i.p. with 1 the ascites fluid of a previously infected mouse. Liposomal vincristine made according to Example 27 was administered i.v. at various times after tumor innoculation and animal weights as well as mortality rates were monitored.
EXAMPLE 29 DSPC/cholesterol vesicles (55:45) were prepared by extrusion at 60 C. 10 times through 2 0.2 um Nucleopore polycarbonate straight through path filters in 300 mM Sodium carbonate pH 9.6 (adjusted with 10% H.sub.2 SO.sub.4) at a lipid concentration of 100 mg/ml. The external buffer was removed and the pH gradient established by passing the vesicles down a G-50 Sephadex column equilibrated with 150 mM K.sub.2 SO.sub.4, 20 mM HEPES, pH 7.4 (adjusted with NaOH). These vesicles were incubated with 2 mM 5-fluorouracil (FU) (Sigma Chemical Co., St. Louis, Mo.) for 60 minutes at 21 60 passage down a G-50 column equilibrated with the external buffer.
FIG. 7 demonstrates the uptake of FU as a function of temperature. Incubation of the liposomes at 60 In FIG. 7, the delta T reflects a temperature increase from 21 to 60
The above liposomes containing FU were then passed down a Sephadex G-50 column equilibrated with 150 mM NaCl at 37 according to the pH gradient (FIG. 8). FIG. 8 is a graph depicting the effect of external buffer on FU release at 37
Liposomes containing the original K.sub.2 SO.sub.4 buffer were also exchanged as above for 250 mM ammonium acetate. Complete release of FU resulted (FIG. 8).
EXAMPLE 30 Egg phosphatidylcholine (15 mg) was dispersed in 2 ml of 300 mM citric acid., .sub.p H 4.0 and the resulting MLVs frozen in liquid nitrogen and thawed in warm water (approximately 35 The lipid was then extruded 10 times through two stacked 100 nm pore size polycarbonate filters using the LUVET procedure. A proton gradient was created by passage of the vesicles over a Sephadex G-50 (fine) column (1.5 cm 7.5. An aliquot of the large unilamellar vesicles eluted from the column was diluted in 300 mM NaCl, mM HEPES, .sub.p H 7.5 to a lipid concentration of 0.75 mgml.sup.-1 in a total volume of 2 ml and then daunorubicin (113 ug) added from a stock solution (5.64 mgml.sup.-1) in distilled water. The mixture was incubated at room temperature (25 C.) and at intervals of 2, 10, 20, 30, 60 and 120 minutes, 100 ul aliquots were centrifuged through 1 ml "minicolumns" of Sephadex G-50 (fine) to remove any unencapsulated daunorubicin from the vesicles. The concentration of entrapped daunorubicin was determined from its absorbance at 500 nm in a Shimadzu UV-265 spectrophotometer following solubilization of the vesicles in 1% Triton X-100. Lipid was quantified by liquid scintillation counting using tracer levels of .sup.3 H-DPPC. In excess of 98% of the daunorubicin was encapsulated by the vesicles giving a drug to lipid molar ratio of 1:5.
EXAMPLE 31 The materials and procedures of Example 30 were employed except that epirubicin (116 ug) was addded to the vesicle suspension (2 ml) from a stock solution (5.8 mgml.sup.-1). Epirubicin uptake was quantified from its absorbance at 500 nm following solubilization of the vesicles in 1% Triton X-100. Epirubicin encapsulation by the vesicles was in excess of 98% giving a drug to lipid molar ratio of 1:5.
EXAMPLE 32 The materials and procedures of Example 30 were employed except that mitoxanthrone (103 ug) was added to the vesicle suspension (2 ml) from a stock solution (2 mgml.sup.-1). Mitoxantrone uptake was quantified from its absorbance at 670 nm following solubilization of the vesicles in 2% Triton X-100. Mitoxantrone encapsulation by the vesicles was in excess of 98% giving a drug to lipid molar ratio of 1:5.
TABLE 1______________________________________TRAPPING EFFICIENCIES OF VARIOUSLIPOSOMAL VINCRISTINE PREPARATIONS  TEM-  PERA-                        TRAPPINGSAM-   TURE     VINC      DRUG:LIPID                               EFFI-PLE    (           SOURCE    (wt:wt)   CIENCY______________________________________EPC/   60       SIGMA     0.24:1     95.0CHOLEPC/   60       ONCOVIN   0.29:1     88.0CHOLHSPC/  21       SIGMA     0.20:1     15.0CHOLHSPC/  60       SIGMA     0.20:1    100.0CHOLDSPC/  60       ONCOVIN   0.24:1    100.0CHOL______________________________________
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the effect of incubation temperature on remote loaded doxorubicin uptake into EPC/cholesterol (55:45 mol ratio) liposomes. Liposomes were prepared in 300 mM citric acid (pH 4.0) and extruded through 200 nm pore size polycarbonate filters. Prior to doxorubicin addition the external liposome solution was brought to pH 7.8 with sodium hydroxide. Doxorubicin (3.0 mg/ml) was added to the liposomes (11.0 mg lipid/ml) equilibrated at 21 (open circle), and 60
FIG. 2 is a graph of release characteristics of liposomal-doxorubicin (EPC:cholesterol, 55:45 mol:mol, 29.+-.2/100 drug/lipid wt./wt.) containing 300 mm citrate, dialyzed against buffer at 37 4.0 (open circles) and pH 7.5 (closed circles) at 37
FIG. 3 is a graph of a citrate-doxorubicin interaction resulting from mixing experiments at varying citrate pH values. The mM doxorubicin remaining in solution following centrifugation is plotted as a function of citrate pH: 4 mM doxorubicin, mixed at 60 25 (open squares); 20 mM doxorubicin mixed at 60 25 150 mM NaCl, at 25
FIG. 6 is a graph demonstrating the release of vincristine from HSPC/cholesterol (open circles), DSPC/cholesterol (closed squares), and EPC/cholesterol (closed circles) liposome systems under dialysis conditions at 37
FIG. 7 is a graph demonstrating the effect of temperature on uptake of 5-fluorouracil ("FU"). The delta T reflects a temperature increase from 21
FIG. 8 is a graph depicting the effect of external buffer on FU release at 37
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Bioeng. 9:467 509.85 *U.S. application No. 122,354, Forssen, filed Nov. 18, 1987.86 *U.S. application No. 122,613, Bally et al. filed Nov. 17, 1987 Pending.87 *U.S. application No. 161,141, Popescu et al., filed, Feb. 25, 1988 Pending.88 *U.S. application No. 164,580, Janoff et al., filed Mar. 7, 1988 Pending.89 *U.S. application No. 22,157, Janoff et al., filed Mar. 5, 1987 Abandoned.90 *U.S. application No. 220,388, Mehlhorn, filed Jul. 12, 1988.91 *U.S. application No. 236,700, Janoff, et al., filed Aug. 25, 1988.92 *U.S. application No. 284,751, Bally, et al., filed Dec. 12, 1988.93 *U.S. application No. 310,495, Cullis et al., filed Feb. 13, 1989 Pending.94 *U.S. application No. 360,964, Janoff, et al., filed Jun. 26, 1989.95 *U.S. application No. 4,762, Cullis et al., filed Jan. 7, 1987 Pending.96 *U.S. application No. 61,837, Hope et al., filed Jun. 12, 1987 Pending.97 *U.S. application No. 622,502, Cullis et al., filed Jun. 20, 1984 Abandoned.98 *U.S. application No. 622,690, Cullis et al., filed Jun. 20, 1984 Abandoned.99 *U.S. application No. 638,809, Janoff, et al., filed Aug. 8, 1984.100 *U.S. application No. 660,573, Lenk et al., filed Oct. 12, 1984 Pending.101 *U.S. application No. 69,908, Janoff et al., filed Jul. 6, 1987 Abandoned.102 *U.S. application No. 749,161, Bally, et al., filed Jun. 26, 1985.103 *U.S. application No. 752,423, Bally et al., filed Jul. 5, 1985 Abandoned.104 *U.S. application No. 759,419, Janoff, et al., filed Jul. 26, 1985.105 *U.S. application No. 788,017, Cullis et al., filed Oct. 16, 1985 Abandoned.106 *U.S. application No. 800,545, Cullis et al., filed Nov. 21, 1985 Abandoned.107 *U.S. application No. 874,575, Hope et al., filed Jun. 16, 1986 Abandoned.108Van Hoesel, et al., "Reduced cardiotoxicity and nephrotoxicity with preservation of anittumor activity of Doxorubicin entrapped in stable liposomes in the LOU/M", Chemical. 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Abstracts,. vol. 101, 1984, 163307y.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS5795589 *Feb 5, 1997Aug 18, 1998The Liposome Company, Inc.Liposomal antineoplastic agent compositionsUS6083530 *May 26, 1998Jul 4, 2000The Liposome Company, Inc.High drug:lipid formulations of liposomal-antineoplastic agentsUS6475517 *Jul 5, 2001Nov 5, 2002Mitsubishi Chemical CorporationMethod for preparing closed vesiclesUS6682758Dec 22, 1999Jan 27, 2004The United States Of America As Represented By The Department Of Health And Human ServicesWater-insoluble drug delivery systemUS6838090Jun 5, 2003Jan 4, 2005The United States Of America As Represented By The Department Of Health And Human ServicesWater-insoluble drug delivery systemUS6849616Mar 26, 1999Feb 1, 2005Pharmacia Italia S.P.A.Methods to potentiate intravenous estramustine phosphateUS7048944Sep 25, 2002May 23, 2006Mitsubishi Chemical CorporationMethod for preparing closed vesiclesUS7238367Oct 3, 2002Jul 3, 2007Celator Pharmaceuticals, Inc.Liposome loading with metal ionsUS7744921Aug 20, 2007Jun 29, 2010Celator Pharmaceuticals, Inc.Liposome loading with metal ionsUS8097276Jan 18, 2006Jan 17, 2012National University Corporation Hokkaido UniversityMethod for coating particle with lipid filmUS8241663 *Mar 25, 2005Aug 14, 2012Terumo Kabushiki KaishaLiposome preparationUS8246984Dec 1, 2006Aug 21, 2012Rigel Pharmaceuticals, Inc.Formulation of insoluble small molecule therapeutics in lipid-based carriersUS8349360Oct 6, 2005Jan 8, 2013Bc Cancer AgencyLiposomes with improved drug retention for treatment of cancerCN101229127BNov 26, 2003Oct 10, 2012吉里德科学公司Liposome preparationEP0834309A2 *Sep 18, 1997Apr 8, 1998Artur Herzog Dr. MesmerUse of a liposome solution for enhancing the activity and/or reducing the toxicity of drugsEP1233754A2 *Nov 30, 2000Aug 28, 2002The Regents of The University of CaliforniaTargeted drug delivery with a cd44 receptor ligandEP2407169A1Apr 22, 2005Jan 18, 2012Celator Pharmaceuticals, Inc.Combination formulations of anthracycline agents and cytidine analogsWO1999049716A2 *Mar 24, 1999Oct 7, 1999Ulrich MassingMethod for producing liposomal formulations of active agents by high-pressure homogenisationWO1999049869A1 *Mar 26, 1999Oct 7, 1999Asp BerylMethods to potentiate intravenous estramustine phosphateWO2004047802A2 *Nov 26, 2003Jun 10, 2004Su-Ming ChiangLiposomal formulationsWO2006052767A2Nov 4, 2005May 18, 2006Inex Pharmaceuticals CorpCompositions and methods for stabilizing liposomal camptothecin formulationsWO2007111720A2 *Dec 1, 2006Oct 4, 2007Manjeet M ParmarFormulation of insoluble small molecule therapeutics in lipid-based carriersWO2008127358A2 *Oct 10, 2007Oct 23, 2008Jina Pharmaceuticals IncAqueous systems for the preparation of lipid-based pharmaceutical compounds; compositions, methods, and uses thereof* Cited by examinerClassifications U.S. Classification424/450, 436/164, 436/829, 264/4.3, 514/908, 424/1.21International ClassificationA61K9/133, A61K9/127Cooperative ClassificationY10S436/829, Y10S436/826, Y10S514/908, A61K9/1278, A61K9/127European ClassificationA61K9/127P2, A61K9/127Legal EventsDateCodeEventDescriptionOct 6, 2008REMIMaintenance fee reminder mailedOct 1, 2008FPAYFee paymentYear of fee payment: 12Dec 17, 2007ASAssignmentOwner name: CEPHALON LIMITED, UNITED KINGDOMFree format text: CHANGE OF NAME;ASSIGNOR:ZENEUS PHARMA LIMITED;REEL/FRAME:020261/0820Effective date: 20061002Owner name: MEDEUS PHARMA LIMITED, UNITED KINGDOMFree format text: CHANGE OF NAME;ASSIGNOR:MEDEUS UK LIMITED;REEL/FRAME:020261/0793Effective date: 20040109Owner name: ZENEUS PHARMA LIMITED, UNITED KINGDOMFree format text: CHANGE OF NAME;ASSIGNOR:MEDEUS PHARMA LIMITED;REEL/FRAME:020261/0778Effective date: 20040922May 29, 2007ASAssignmentOwner name: MEDEUS UK LIMITED, ENGLANDFree format text: REDACTED LICENSE AGREEMENT;ASSIGNOR:ELAN PHARMACEUTICALS, INC.;REEL/FRAME:019353/0253Effective date: 20031223Oct 1, 2004FPAYFee paymentYear of fee payment: 8Jun 14, 2002ASAssignmentOwner name: ELAN PHARMACEUTICALS, INC., CALIFORNIAFree format text: MERGER;ASSIGNOR:LIPOSOME COMPANY, INC., THE;REEL/FRAME:012958/0601Effective date: 20011228Owner name: ELAN PHARMACEUTICALS, INC. 800 GATEWAY BOULEVARD SOwner name: ELAN PHARMACEUTICALS, INC. 800 GATEWAY BOULEVARDSOFree format text: MERGER;ASSIGNOR:LIPOSOME COMPANY, INC., THE /AR;REEL/FRAME:012958/0601Sep 21, 2000FPAYFee paymentYear of fee payment: 4RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google