CA1272686A - Anthracycline antineoplastic agents encapsulated in phospholipid micellular particles and methods for using same for tumor therapy - Google Patents

Abstract

ABSTRACT
Formulations consisting of phospholipid micellular particles encapsulating anthracycline anti-neoplastic agents and methods for using such compositions to treat neoplastic tumors are described. In a preferred embodiment, the particles are in the form of vesicles which comprise an anthracycline agent, preferably daunorubicin, distearoyl phosphatidyl choline, distearoyl phosphatidyl glycerol and cholesterol, preferably in the ratio of about 1:4:5:6 to about 1:4:20:20.
The vesicles are suspended in a low ionic solution such as 5%
dextrose at pH 7.4 and may be administered to humans to deliver the anthracycline to treat neoplastic tumors.

Description

~'7~

This invention relates to compositions consisting of phospholipid encapsula-ted anthracycline anti-neoplastic ayents.
In another aspect it rela-tes to the use of such compositions to dellver chemotherapeu-tlc agents to -tumors in a body.
Daunorublcln (also known as daunomycin), doxorubicin (also known as Adriamycln), Aclacinomycin ~ and other cationic anthracycline anti-neoplastic agents are currently of great clinical interest for the treatment of tumors, including most leukemias and solid tumors. Structurally, these compounds consist of a hydrophobic tetracycline ring system coupled to an amino sugar through a glycoside linkage. These anthracycline agents associate with phosphate containing materials, and exhib-it a high affinity for example, with cardiolipin. These com-pounds have been shown to exhibit marked activity against a wide variety of neoplasms. ~lowever, the clinical use of these drugs in humans has been limited by the chronic toxic effect of the drugs on heart -tissue. Children, for example, are highly sus-ceptible to doxorubicin-induced congestive hear-t failure. Mosij-ezuk, et al., Cancer, 44, p. 1582-1587 (1979). Long-term admin-istration of such drugs leads to an increased risk of cardiomyo-pathy. Lefrak et al., Cancer, 32, p. 302-314 (1973).
Phospholipid bilayer membrane particles in the form of unilamellar vesicles known as liposomes have received in-creasing attention as possible carriers for anthracycline drugs.
Certain formulations have been shown to increase antitumor activity, alter in vivo tissue distribution and decrease tox-lClty .
Difficulties have been encountered in producing en-capsulated anthracyclines. In part this has been due to the surfactant or detergent-like eXfect these compounds exert on the phospholipid vesicle bilayer, causing leakage and creating vesicle instability.

~;~7~

Another problem has been the aggregation of such vesicles during storage. In addition, the efficiency of entrapment of previous formulations o-f encapsulated anthracyclines has been low, and has been reported to be between 5 and 65~. Forssen and Tolces, Cancer Res. 43, ~.546-550 (1983). Gabizon et al., Canc _ Res. 43, p. 4730-3745 (1983); and Gabizon et al., Br.
J. Cancer 51, p. 681-689 (1985). Thus it has no-t been possible to achieve large scale production of stable, encapsulated anthracyclines for therapeutic purposes.
Accordingly, it is an objec~ of the present invention to provide improved formulations for encapsulating anthracycline antineoplastic agents in phospholipid bilayer membrane particles.
Another object of this invention is to provide a method for using improved formulations of encapsulated antineoplastic agents to provide decreased cardiotoxicity and increased anti- tumor efficacy in humans.
The manner in which these and other objects are realized by the present invention will be apparent from the 0 summary and detailed description set forth below.
SUMMARY OF THE INVENTION
This invention relates to a composition comprising anthracycline neoplastic agents encapsulated in phospholipid micellular particles consisting of anionic and neutral phospholipids, said particles being suspended in a low ionic strength aqueous phase.
Compositions comprising anthracycline anti-neoplastic agents encapsulated in phospholipid bilayer membrane particles consisting of anionic and neutral phospholipids and cholesterol are described. The particles are ~7~
~724-1648 suspended in a low ionic strength aqueous phase such as a 5%
dextrose solution. The anionic phospholipid may be distearoyl p'nosphatidyl glycerol. A preferred composition is daunorubicin, distearoyl phosphatidyl glycerol, distearoyl phosphatidyl choline and cholesterol. In one - 2a -7~

embodiment the ratio of these components is preferably from about 1:2:0:0 to about 1:4:20:20. Particularly preferred embodiments are ratios of 1:4:5:6 or 1:1.5:7:0 in an aqueous phase of a monosaccharide (e.q. dextrose) or a disaccharide (e.g. lactose, sucrose). The pii of the suspending solution is preferably between about 4.0 to about 8Ø Th~secompositions may be administered in multiple doses -to a human subject to treat tumors.
BRIEF DESCRIPTION OF THE DRAWINGS
.
Figure 1 illustrates 1n vivo levels of C~14 labelled daunorubicin, free and vesicle entrapped, in the blood in mice at 1, 4, 24 and 48 hours.
Figure 2 illustrates the ln vivo levels of C-14 labelled daunorubicin, free and vesicle entrapped, in solid tumors in mice at 1, 4, 24 and 48 hours.
Figure 3 illustrates the ln vivo levels of C-14 labelled daunorubicin, free and vesicle entrapped, in heart tissue in mice at 1, 4, 24 and 48 hours.
Figure 4 illustrates the ln vivo hepatic levels of C-14 labelled daunorubicin, free and vesicle entrapped, in mice at 1, 4, 24 and 48 hours.
Figure 5 depicts the rate of survival in mice bearing solid tumors treated on day 3 with a single dose of free or ves-icle-entrapped daunorubicin.
Figure 6 shows the effect on tumor volume of single doses of daunorubicin, free or vesicle entrapped, administered to mice bearing solid tumors.
Figure 7 illustrates the effect on tumor volume of multiple (20 mg/kg) doses of free or vesicle entrapped daunorub-icin in mice bearing solid tumors.
Figure 8 depicts the survival rate for mice bearing solid ~7~

60724-16a,8 tumors and receiving multiple doses (20 mg/kg) of free or vesicle entrapped daunorubicin.
The vesicles for which the results illustrated in Figures 1-8, inclusive were obtained were those of Example 1 herein below.
DETAILED DESCRIPTION OF THE INVENTION

As indicated above, according to this invention, encap-sulation and improved delivery of anthracycline agents useful in treating tumors in humans is achieved using compositions contain-ing bilayer membrane particles, preferably in the form of small, unilamellar vesicles, consisting of phospholipids, cholesterol and an anthracycline agent and suspending such micellular part-icles in a low-ionic strength aqueous phase in which a mono-saccharide, disaccharide, or other hydroxyl compound is dissolvedO
That anthracyclines exhibit a high affinity for the phospholipid cardiolipin appears to be of particular importance for mediating the biological activities of these drugs. Cardio-lipin, however, is not a desirable constituent for phospholipid vesicles, in spite of its high affinity for anthracyclines, because when interacting with an anthracycline such as daunorubi-cin, it forms micelles which destabilize the bilayer structureof encapsulating particles such as liposomes. Caridiolipin is also known to be highly antigenic in nature when incorporated in liposome membranes, and thus may cause an increased immunog-enic response when injected into a body.
As noted above, one of the difficulties associated with the entrapment of anthracycline anti-neoplastic agents in phos-pholipid bilayer membrane particles is their amphiphilic nature which can cause these drug molecules to attempt to partition 7~

nearly equally between aqueous and lipid media. This partition-ing can, in turn, cause these drugs to easily leak from lipid membranes and can dlsrupt the membranes themselves, destroying the vesicles' bilayer structure. An advantage of using an anionic phospholipid such as distearoyl phosphatidyl glycerol is that it has a negative charge which can be used to cancel the positive charge on the cationic anthracycline molecule. This permits the production of neutral vesicles, which resist dis-ruption and leakage. Furthermore, the use of a low-ionic strength aqueous phase to suspend the vesicles improves vesicle stability because it inhibits vesicle aggregation.
An additional advantage of using such negatlvely charged phospholipids is that the cancellation of the charge on the anthracycline molecule permits the formation of a water insoluble salt between the phospholipid and the anthracycline. This com-plex increases the affinity of the drug for the hydrophobic bilayer of the vesicle. While an anthracycline, such as dauno-rubicin, will bind fairly strongly to a negative phospholipid such as distearoyl phosphatidyl glycerol with a binding constant of approximately 10 M , its affinity for binding to DNA in a cell is much greater, on the order of 2 x 106M 1. Thus, the drug will be able to be released from the distearoyl phospha-tidyl glycerol and to complex with DNA present in the target tumor cells.
The micellular particles of this invention are prefer-ably in the form of small (45-55 nanometers in diameter) unilam-ellar phospholipid vesicles prepared by sonication as described by M.R. Mauk and R.C. Gamble, Anal. Bioc., 94, p. 302-307 (1979~, or by microemulsification using the procedures described in a co-pending Canadian application by R. Gamble filed January 30, ~;~7~

1986, Application No. 500,652, and assigned to the same assiynee as this application. Vesicles prepared in this fashion having the types and amounts of components taught by the invention exhibit a high efficiency of entrapment (greater than 90%) of the anthracycline anti-neoplastic agent, and a good storage life (about 90% particles intact after two weeks), adequate targett-ing of the drug to tumor tissue and little or no tendency to aggregate. One advantage of the higher entrapment efficiency is that the step of separating free drug from entrapped drug after loading procedures may be eliminated, thus simpliEying manuf-acture.
Adjustment of pH is an additional factor for maximum drug entrapment with an optimal range of from about pH 4.0 to 8Ø A suitable buffer for maintaining pH is TRIS (Tromethamine or 2-amino-2-hydroxymethyl-1, 3~propanediol) since it can read-ily be buffered over a pH range of 7 to 9. Other buffering agents may include sodium acetate and sodium benzoate.
`~ It has been found in the present invention that by ~ //Sfearo~ /
using anionic phospholipids such as ~h~Y~t~ phosphatidyl gly-cJls7~e~ro yl cerol (DSPG) with neutral phospholipids such as ~ r~1 phos-phatidyl choline (hereafter DSPC), the partitioning of an anth-racycline agent such as daunorubicin (hereaf-ter DAU) into the lipid phase may be increased leading to increased entrapment of the agent in the micellular particle and more stable particles.
It has also been found that the incorporation of cholesterol (CHOL) leads to improved stability of the particles encapsulat-ing the anthracycline. The stability of these compositions is further enhanced by suspending the particles in a low-ionic strength solution such a 5~ dextrose solution.

3~j 6072~-1648 A preferred formulation is DAU:DSPG:DSPC:CHOL in a molar ratio of from about 1:4:5:6 to about 1:4:20:20. Other embodiments inclu~e ratios of l:l.5:7:0, 1:4:5:4, L:2:6:0, 1:2:20:0 and l:2:6:1. Preferably, the DSPG is present in at least a fifty percent (molar) excess relative to DAU. It appears however, that there is no upper limit to the amount of DSPG (or other anionic phospholipid) which may be incorporated.
The preferred amount of cholesterol which may be incorporated is approximately equal to the amount of DSPG prPsent and from 0 to 20 times the amount of DAU present. Other anionic phosp~olipids, for example phosphatidyl serine and phosphatidic acid, may be used. Because neutral vesicles appear to be more e-ffective as delivery vehicles to tumors (see, Maulc and Gamble) it may be desirable to select a ratio of phospholipid components which minimizes the net negative charge of the vesicles whlle maintaining the physical integrity of the vesicles' structure by preserving the stability of t'ne anthracycline in the bilayer. Thus, for certain applications a ratio of DA~:DSPG of l:l.S may be preferred.
To prepare vesicles, the lipids and anthracycline agent, for example, daunorubicin, to be used for vesicle preparation are weighed out in the desired ratios and are either dissolved in an organic solvent such as methanol or chloroform or kept until use ~ 7~ PATENT

as dry powders. If a solvent i5 used, it must be removed prior to the addition of the aqueous phase by evaporation, for example under argon, nitrogen or by application of a vacuum.
The aqueous phases preferred for formulation of anthracycline vesicle~ with high entrapment and maximum stability are low-ionic media, such as sugar solutions or de-ionized distilled water. A 5~ dextrose in water solution at pH 7.4 is preferred. Other solutions such as a 9~ lactose or 9~ sucrose solution in water may also be used. Such solutions minimize drug leakage f~om vesicles and decrease vesicle aggregation, and are well suited for parenteral use, for example human intravenous injection.
The example which follows describes the preparation, characterization and ln vivo chemotherapeutic application in an animal model for a vesicle formulation of this invention.
The example is presented solely for purposes or illustration and is not intended to limit the present invention in any way.

7~ 3 PATENT

EXAMPL~

DAUNORUBICIN VESICLES
.

~ aration of Vesicles_Enca~sulating Daunorubicin J~s7~ea ro y I
Phospholipid vesicles were prepared using ~JH~HY~
phosphatidyl glycerol (DSPG),- ~ phosphatidyl choline (DSPC), cholesterol (CHOL), and daunorubicin (DAU) in a molar ratio of DAU:DSPG:DSPC:CHOL of 1:4:5:6.
The lipids were ob~ained erom Avanti Polar Lipids, ~irmingham, Alabama) and the daunorubicin was obtained from Sigma Chemical Co., (St. Louis, Missouri). These compounds were weighed out in the desired ratios and were dissolved in the organic solvent chloroform. The solvent was removed prior to addition of the aqueous phase by evaporation under nitrogen gas ~ollowed by vacuum. The non-ionic aqueous phase consisting of 5 dextrose solution in water, pH adjusted to 7.4 with NaOH was added to the lipid mixture and the solution was heated in a water bath at 60 to 70 C for 1 to 3 minutes then vigorously agitated to form a suspension of the drug-lipld mix. This step was repeated until all the material had been suspended in the aqueous phase. This mixture was then sonicated using a needle probe sonicator (Sonics and Materials, Danbury, Conn.~, at an output control setting of 1-2 (on a scale of 10). The sample was sonicated until clear, about 2-5 minutes for a 5 ml sample.
During sonication the mixture was heated at 10 to 80 C in a water bath. Following sonication, the sample was centrifuged to remove all particulate matter.

Characterization of Vesicles Encapsulating Daunorubicin The vesicles containing daunorubicin, prepared as described above, were characterized for size (diameter) and entrapment efficiency following preparations. Vesicle sizing was performed usin~ a Laser Particle Sizer Model 200 (~icornp Instruments, Santa Barbara, California) and was determined to be in the range of 45 to 55 nanometers in diameter.
The efficiency of association of daunorubicin within the vesicles was estimated using Sephadex G 50 gel-filtration to separate free from entrapped daunorubicin. Using the above formulations, 90-100% of the daunorubicin was found to be associated with the vesicles. Due to this high association, additional separation steps were unnecessary to remove free drug.
The daunorubicin vesicles prepared as described above were examined usiny IIPLC and were found to be stable as indicated by the lack of chemical decomposition comparing freshly sonicated vesicles with those left at room temperature for two weeks. In addition, when a 2 ml sample of these vesicles were frozen in dry ice and later thawed at 65C, the vesicles main-tained their original size as determined by light scattering using the Laser Particle Sizer, and also retained all of the previously entrapped daunorubicin as determined by Sephadex*
gel filtration. Finally, incubation of Indium-III loaded daunorubicin vesicles in serum at 37C for 24 hours following the procedures described by Mauk and Gamble, Anal. Bioc., 9~, p.
302-307 (1979), for loading In-III in phospholipid vesicles, demonstrated no loss in entrapped In-III had occurred as deter-mined by x-ray perturbed angular correlation ("PAC") (no decline in G22)-* Trademark , . ~

7~ PATENT

Biodistribution of C-14 Labeled Daunorubicin Vesicles ~ iodistribution studies of C-14 labeled daunorubicin, both free and vesicle entrapped, were conducted using a daunorubicin dose o~ 5 mg/kg in CD2Fl mice bearing intradermal P-1798 lymphosarcoma solid tumoc. Time points were taken at 1, 4, 24 and 48 hours. The results are presented in FIG. 1 through 4 showing that daunorubicin vesicles remain in the blood for longer periods of time than free drug and that in tumor tissue the level of vesicle encapsulated daunorubicin was significantly higher than free daunorubicin.

Toxicity It appears that daunorubicin vesicles are not more toxic and are most likely less toxic than unencapsulated drug in animals bearing tumors as determined by survival in a small sample of mice using doses of 10, 20 and 30 mg/kg. In this limited study, toxicity induced deaths occurred only for the high dose ùnencapsulated daunorubicin (30 mg/kg), at a 100~ rate. In contrast, no deaths occurred in mice receiving an equal dose of vesicle encapsulated daunorubicin.

Chemotherapeutic Efficacy of Daunorubicin Vesicles CD2~1 mice implanted with intradermaI p-1798 solid lymphosarcoma received free and vesicle-encapsulated daunorubicin in a single dose injection of 20 mg/kg, and in multiple dosages oE 5, 10 and 20 mg/kg.
In the first investigation, groups of 10 mice received Eree daunorubicin or daunorubicin-vesicles in 20 mg/kg single doses at three or four days following tumor implantation. Tumors were measured using calipers and the survival over time of PATENT

~ ~ 7 ~ ~ 8~

treated and control mice was recorded. Controls consisted of injections of a 5% dextrose in water solution at 2 ml/20 gm doses.
Representative results of these investigations are shown in FIG. 5 for treatment commencing on day 3 after tumor implantation. Typically, wlth tumor metatastasis, mice with tumors die within 14-17 days. Survival times or daunorubicin-vesicle treated mice increased in comparison to free drug. The median life span of mice injected with daunorubicin-vesicles was 21 days. All mice receiving free or vesicle entrapped daunorubicin demonstrated significant inhibition of tumor growth compared with untreated controls. Mice receiving vesicle entrapped daunorubicin had less tumor growth than those receiving free doses, as depicted in FIG. 6.
In a second study of chemotherapeutic effects of injections of daunorubicin-vesicles, groups of ten mice received multiple doses of 5, 10 and mg/kg. Treatments were ini~iated on day 4 and followed at weekly intervals (day 11, day 18) for a total of three doses. Body weight and tumor size were monitored during the study. As shown in FIG. 7, at 20 mg/kg significant inhibition of tumor growth occurred in daunorubicin-vesicle treated mice compared with those treated with free drug.
Survival times were investigated in a group of 19 mice and as indicated in FIG. 8 following tumor implantation were significantly increased for daunorubicin-vesicle treated mice relative to free drug a~ doses of 20 mg/kg.
These results clearly demonstrate the usefulness and efficacy of the vesicle formulations of the present invention as improved vehicles Eor delivering an~hracycline to tumors in a body.

~ PATENT

Although this invention has been described with reference to particular applications, the principles involved are susceptible of other applications which will be apparent to those skilled in the art. The invention is, therefore, to be limited only as indicated by the scope of the claims appended hereto.

~_~7~P~;

SUPPLEMENTARY DISCLOSURE

Formula-tions consisting of phospholipid bilayer mem-brane particles made from mixtures of anionic and neutral phos-pholipids encapsulating anthracycline anti-neoplastic agents, suspended in a low ionic strength aqueous phase, are described.
In a preferred embodiment, the particles are in the form of vesicles which comprise daunorubicin, distearoyl phosphatidy-lglycerol and distearoyl phosphatidylcholine, the mol ratio of daunorubicin to distearoyl phosphatidylglycerol is at least about 1:1.25, and the suspending medium is an aqueous lactose solution containing a small amount of base.
Compositions comprising anthracycline anti-neoplastic agents encapsulated in phospholipid bilayer membrane particles consisting, in one embodiment, of anionic phospholipids such as distearoyl phosphatidylglycerol admixed with neutral phospholip-ids such as distearoyl phosphatidylcholine are described. In another embodiment of this invention the composition can also contain cholesterol or like-acting substances, but this is not essential to the practice of the invention. The particles are suspended in a low ionic strength aqueous phase such as an aqueous solution oE a physiologically acceptable nonionic hydroxyl-containing compound, e.g., a monosaccharide such as dextrose or a polysaccharide such as lactose. Thi~ low ionic strength aqueous phase will be one having as low a content of extraneous anions, e.g., chloride ions from an anthracycline anti-neoplastic agent such as daunorubicin hydrochloride, as can practicably be achieved, e.g., an anion concentration of about 5 mMolar (millimolar) or less, and a pH preferably be-tween about 6.0 and about 8Ø

~;~7~

A particularly preferred composition comprises dauno-rubicin, distearoyl phosphatidylglycerol and distearyol phosph-atidylcholine in a molar ratio of these components of 1:1.5:7, respec-tively, suspended in an aqueous phase comprising a dis-accharide such as lactose, preferably a 9-11% lactose solution containing 5 mM TRIS base (Tromethamine or 2-amino-2-hydroxy-methyl-1,3-propanediol) at a pH of abou-t 6.0 to 8Ø
These compositions may be administered in multiple doses to a human subject to treat tumors.
FIG~ 9 illustrates the uptake of tritiated daunorubicin by whole blood, tumor tissue (P-1798 lymphosarcoma) and three other tissues in mice as determined for the daunorubicin-cont-aining vesicles of Examples II, III and IV hereinbelow, for free daunorubicin and for daunorubicin simply admixed with dis-tearoyl phosphatidylglycerol in a 1:1 mol ratio.
FIG~ 10 indicates the therapeutic effects of the daunorubicin-containing vesicle formulations of Examples II and IV hereinbelow, compared to each other and to free daunorubicin, for a solid tumor in mice.
FIG~ 11 illustrates the results of a study on the effect on tumor sizes of the daunorubicin-containing vesicle formulations of Examples II and IV hereinbelow.
FIGo 12 illustrates the in vlvo levels (biodistribution) of tritiated daunorubicin, free and vesicle entrapped (Example III hereinbelow), in the blood, in solid tumors, in heart tissue and in the livers of mice over a 48 hour periodO
DETAILED DESCRIP rION
As indicated above, according to this invention encap-sulation and improved delivery of anthracycline anti-neoplastic .~ , ' ayents useful in treatiny tumors in humans is achieved using compositions containing bilayer membrane particles, preferably in the form of small, unilamellar vesicles consisting of a mixtuxe of anionic and neutral phospholipids, and a cationic anthracycline anti-neoplastic agent, suspended in a low-ionic strength aqueous phase in which a physiologically acceptable nonionic hydroxyl-containing compound is dissolved and which contains as low a content of extraneous anions as can practic-ably be achieved.
Although I do not wish to be bound by any particular theory or mechanism advanced to explain the operation of this invention, I believe that the drug becomes entrapped in the vesicle membrane itself rather than simply being present within the vesicle's interior aqueous space.
The micellular particles of this invention are pref-erably in the form of small [less than about 60 nm tnanometers), and preferably about 45-55 nm in diameter] unilamellar phosph-olipid vesicles prepared by sonication as described by M.R. Mauk and R.C. Gamble, op. cit. 307 (1979) or by microemulsification using the procedures described by R. Gamble op. cit.
Adjustment of pH is an additional factor for maximum drug entrapment when practicing this invention, with the optimal pH range being from about 6.0 to 8Ø A suitable substance for adjusting pH is TRIS base (Tromethamine or 2-amino-2-hydroxy-methyl-1,3-propanediol) since it can readily be buffered over a p~I range of 7 to 9. Other bases such as sodium hydroxide or potassium hydroxide, amine bases such as N-methylglucamine, and the like, which will not contribute unwanted anions, can also be used.

~ 16 -It has been found in the present invention that by using anionic phospholipids such as distearoyl phosphatidylgly-cerol (sometimes referred to hereinafter as DSPG) with neutral phospholipids such as distearoyl phosphatidylcholine (sometimes referred to hereinafter as DSPC), the partitioniny of an anth-racycline anti-neoplastic agent such as daunorubicin (sometimes referred to hereinafter as DAU) into the lipid phase may be increased leading to inereased entrapment of the anthracyeline anti-neoplastic agent in the micellular particle and more stable particles. The incorporation of cholesterol (sometimes referred to hereinafter as CHOL) can further improve the stability of the particles encapsulating the anthracycline anti-neoplastic agent, and in all eases the stability of these eompositions is further enhanced by suspending the partieles in a low-ionie strength aqueous phase in whieh a physiologieally aeceptable anionie hydroxyl-containing compound is dissolved and which contains as low a content of extraneous anions as can practic-ably be achieved.
Among the anionic phospholipids which can be employed 20 in praeticing this invention are phosphatidylglycerols, phosph-atidylserines, phosphatidylinositols and phosphatidic acids, sueh as distearoyl phosphatidylglycerol, dipalmitoyl phosphat-idylglycerol, distearoyl phosphatidylserine, dioleoyl phosphat-idylinositol, and the like. Neutral phospholipids which can be used together with an anionic phospholipid include phosphat-idyleholines and phosphatidylethanolamines, such as distearoyl phosphatidylcholine, l-palmitoyl-2-oleoyl phosphatidylcholine, dilinoleoyl phosphatidylethanolamine, and the like.

~L~ 7;~

The mol ratio of anthracycline anti-neoplastic agent to total phospholipid [anionic plus neutral phospholipid(s)] in the compositions of this invention should preferably be no more than about 1:20, with mol ratios of about 1:10 or less being particularly preferred, although there is no upper limit, other than one imposed by the practical considerations one faces when working with injectable substances, on the total amount of phospholipids which can be used. The mol ratio of anthracycline anti-neoplastic agent to the anionic phospholipid(s) alone will be at least about 1:1.25, and preferably at least about 1:1.5.
From about 1 to about 50 percent, and preferably from about 10 to about 20 percent, by weight, of the total weight of phosph-olipids present will preferably be anionic phospholipid(s), the balance being neutral phospholipid(s), but here too these amounts are not critical.
Compositions prepared in accordance with this invention having the aforementioned drug to anionic phospholipid mol ratios, particularly when prepared using an aqueous 9-11% lact-ose solution containing a small amount of base - typically 5 mM
TRIS base - have been found to provide adequate targeting of the drug to tumor tissue (targeting efficiencies of about 90% or more have been observed) while, at the same time, limiting or eliminating the tendency of the phospholipid vesicles to aggre-gate. And, since neutral vesicles appear to be more effective for delivering anthracycline anti-neoplastic agents to tumors (see Mauk and Gamble, loc. cit.), the foregoing ratios of anthracycline anti-neoplastic agent to phospholipid components, which minimize the net negative charge of the vesicles while maintaining the physical integrity of the vesicles' structure by preserving the stability of the drug in the bilayer, are generally pre~erred when practicing this invention for this reason as well.
Cholesterol and like-acting substances, e.g., other sterols, when used, can be present in the compositions of this invention in mol ratios of cholesterol or the like to total phospholipid(s) ranginy from about 1:1 to about 0:1, respect-ive]y, and in mol ratios of cholesterol or the like to anthracy-cline anti-neoplastic agent ranging from about 0:1 to about 20:1, respectively.
The aqueous phases preferred for formulation of anthracycline vesicles with high entrapment and maximum stabil-ity are low-ionic strength media whlch contain one or more physiologically acceptable nonionic hydroxyl-containing com-pounds and which also contain a low o~ minimal amount of extra-neous anions. Extraneous anions include, for example, chloride ions from an anthracycline anti-neoplastic agen-t such as dauno-rubicin hydrochloride, and will be present in amounts as low as can practicably be achieved, e.g., an anion concentration of about 5 mM or less, such as can be achieved in sugar solutions in deionized distilled water. Sugars which can be used include monosaccharides such as dextrose, fructose and galactose and disaccharides such as lactose, sucrose, maltose and trehalose.
An aqueous 9-11% lactose solution containing a small amount of base, e.g., S m~l TRIS base, is particularly preferred. Such solutions minimize drug leakage from vesicles and decrease ves-icle aggregation, and are well suited lor parenteral use, for example human intravenous injection.
E~AMPLES II-IV

The procedure of Example IA above was repeated in every essential detail except for the materials used to prepare the anthracycline anti-neoplastic agent-containing phospholipid vesicles, i.e.:

-in Example II radioactive labelled (tritiated) daunorubicin was used to prepare vesicles having the same D~U:DSPG:DSPC:CHOL molar ratio (1:4:5:6, respectively) as in Example IA (cholesterol was used);
-in Example III the vesicles were prepared using tritiated daunorubicin with distearoyl phosphatidylglycerol and distearoyl phosphatidylcholine in a molar ratio of DAU:DSPG:
DSPC=1:1.5:7, respectively (no cholesterol was used);
-in Example IV the vesicles were prepared in a DAU(tri-tiated):DSPG:DSPC:CHOL molar ratio of 1:1.5:7:2, respectively (cholesterol was used);
-a 9% lactose solution in deionized distilled water containing 5 mM TRIS base was used in each instance as the low ionic strength aqueous phase.
The uptake of tritiated daunorubicin by whole blood, tumor tissue (P-1798 lymphosarcoma) and three other tissues in mice was then determined for the daunorubicin-containing vesicles of Examples II, III and IV, for free daunorubicin and for daunorubicin simply admixed with distearoyl phosphatidylglycerol in a 1:1 mol ratio. In all cases the adminstered dose of daunorubicin was 20 mg/kg (normalized to the hydrochloride form;
equivalent to about 35.5 ~M/kg3. The results of these determin-ations are shown graphically in FIG. 9, in which the numbers below the legends "Dau:Ves.." indicate the mol ratios of each component in the particular formulation [the first formulation listed under the legend "Dau:Ves." (1:1.5:7:0) is that of Example III, the second (1:1.5:7:2) is that of Example IV, the third is that of Example II~.

As indicated in FIG. 9, merely combining daunorubicin with distearoyl phosphatidylglycerol produced no increase in tumor uptake. A vesicle formulation with a relatively high proportion of distearoyl phosphatidylglycerol (1:4:5:6; Example II) did increase tumor drug levels over those for free dauno-rubicin. However, two other formulations, either with(1:1.5:7:2;
Example IV) or without cholesterol (1:1.5:7:0), which had lower mol proportions of distearoyl phosphatidylcholine produced even greater tumor drug levels.
The therapeutic effects of the daunorubicin-containing vesicle formulations of Examples II and IV, indicated in FIG. 10 as "Fmln-A" and "Fmln-B", respectively, were compared to each other and to free daunorubicin for a solid tumor in mice. The results shown in FIG. 10 indicate that the formulation with the lower targeting abil:ity (Fmln-A) did little to improve median survival times relative to free daunorubicin at doses of 25 mg/kg or below. Only when tested at dose levels of 30 mg/kg and above did Fmln-A demonstrate improved efficacy. However, the formula-tion with improved targeting characteristics (Fmln-B) demonstrat-ed improved therapeutic efficacy at all tested dose levels.
Tumor sizes were determined in a repeated study of Fmln-A and Fmln-B. The results of this study, shown in FIG. 11, demonstrated that the formulation which does target more effect-ively to tumor tissue (Fmln-B) has the direct effect of enhanc-ing tumor growth suppression.

EXAMPLE V_ Biodistribution studies of tritiated daunorubicin, both free and vesicle entrapped (DAU.DSPG:DSPC mol ratio = 1:1.5:7;
Example III hereinabove) were conducted over a 48 hour period ~7~

using a daunorubicin dose of 20 mg/kg in CD2Fl mice bearing in-tradermal P-1798 lymphosarcoma solid tumor. The results of these studies are illustrated in FIG. 12, in which the error bars are for the standard error, n=5 for each data point.
Although this lnvention has been described with refer-ence to particular applications, the principles involved are susceptible of other applications which will be apparent to t~iose skilled in the art. The invention is, therefore, to be limited only as indicated by the scope of the claims appended hereto.

Claims (64)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A composition comprising anthracycline neoplastic agents encapsulated in phospholipid micellular particles consisting of anionic and neutral phospholipids, said particles being suspended in a low ionic strength aqueous phase.

2. The composition according to Claim 1 wherein the low ionic strength aqueous phase contains a hydroxyl-containing compound.

3. The composition according to Claim 2 wherein the hydroxyl containing compound is a sugar.

4. The composition according to Claim 3 wherein the hydroxyl containing compound is dextrose.

5. The composition according to Claim 1 wherein the aqueous phase is a sugar solution containing 5% dextrose.

6. The composition according to Claim 1 wherein the phospholipids are selected from the group consisting of a phosphatidylglycerol, a phosphatidyl choline, a phosphatidyl serine, a phosphatidic acid, and a phosphatidyl inositol.

7. The composition according to Claim 6 wherein the phospholipids are distearoyl phosphatidyl glycerol and distearoyl phosphatidyl choline.

8. The composition according to Claim 7 containing an anthracycline agent,distearoyl phosphatidyl glycerol, distearoyl phosphatidyl choline and from 0 to 20 times the amount of anthracycline agent present of cholesterol.

9. The composition according to Claim 8 wherein the anthracycline agent, distearoyl phosphatidyl glycerol, distearoyl phosphatidyl choline and cholesterol are in the molar ratio of 1:4:5:6.

10. The composition according to Claim 8 wherein the anthracycline agent, distearoyl phosphatidyl glycerol, distearoyl phosphatidyl choline and cholesterol are in the molar ratio of 1:1.5:7:0.

11. The composition according to Claim 9 wherein the pH
of the suspending solution is in the range of from about pH 4.0 to 8Ø

12. The composition according to Claim 11 wherein the pH
is 7.4.

13. The composition according to Claim 1 wherein the micellular particles are in the form of unilamellar vesicles about 45 to about 55 nanometers in diameter.

14. The composition according to claim 1, 6, 8, 9 or 11 wherein the anthracycline agent is selected from the group consisting of daunorubicin, doxorubicin, N-trifluoroacetyl-doxorubicin-14- valerate and aclacinomycin A.

15. The composition of Claim 1, 6, 8, 9 or 11 wherein the anthracycline agent is daunorubicin.

16. A process for preparing a composition as defined in Claim 1 which process comprises (a) sonicating an unclear mixture of the anthracycline neoplastic agent and phospholipid with a low ionic strength aqueous phase until the mixture is clear, or, (b) homogenizing a mixture of the anthracycline neoplastic agent and phospholipid with a low ionic strength aqueous phase in a homogenizing apparatus at high pressure and a selected temperature for a selected time, thereby subjecting the mixture to very high shearing forces, to generate a microemulsion containing a composition as defined in Claim 1, and separating said composition from unencapsulated materials.

17. A process according to Claim 16 wherein said low ionic strength aqueous phase contains a hydroxyl-containing compound.

18. A process according to Claim 16 wherein the hydroxyl-containing compound is a sugar.

19. A process according to claim 16 wherein the hydroxyl-containing compound is glucose.

20. A process according to Claim 16 wherein the aqueous phase is a sugar solution containing 5% dextrose.

21. A process according to Claim 16 wherein the phos-pholipids are selected from the group consisting of phosphatidyl glycerol, phosphatidyl choline, phosphatidyl serine, phosphatidic acid, phosphatidyl inositol.

22. A process according to Claim 21 wherein the phospholipids are distearoyl phosphatidyl glycerol and distearoyl phosphatidyl choline.

23. A process according to Claim 22 wherein the anthr-acycline agent, distearoyl phosphatidyl glycerol, distearoyl phosphatidyl choline and cholesterol are in a molar ratio ranging from about 1:2:0:0 to about 1:4:20:20.

24. A process according to Claim 23 wherein the anth-racycline agent, distearoyl phosphatidyl glycerol, distearoyl phosphatidyl choline and cholesterol are in the molar ratio of 1:4:5:6.

25. A process according to Claim 23 wherein the anthracycline agent, distearoyl phosphatidyl glycerol, distearoyl phosphatidyl choline and cholesterol are in the molar ratio of 1:1.5:7:0.

26. A process according to Claim 25 wherein the pH of the suspending solution is in the range of from about pH 4.0 to 8Ø

27. A process according to Claim 26 wherein the pH is 7.4.

28. A process according to Claim 16 wherein the micellular particles are in the form of unilamellar vesicles about 45 to about 55 nanometers in diameter.

29. A process according to Claim 16, 21, 23, 24 or 26 wherein the anthracycline agent is selected from the group consisting of daunorubicin, doxorubicin, N-trifluoroacetyl-doxorubicin-14- valerate and aclacinomycin A.

30. A process according to claim 16, 21, 23, 24 or 26 wherein the anthracycline agent is daunorubicin.

31. Use of a composition according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 to treat a neoplastic tumor in a mammal.

32. Use of a parenterally-administrable multiple doses of a composition according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 to treat a neoplastic tumor in a mammal.

33. Use of intravenously injectible multiple doses of a composition according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 to treat a neoplastic tumor in a mammal.

34. Use of a composition according to claim 14 to treat a neoplastic tumor in a mammal.

35. Use of parenterally-administrable multiple doses of a composition according to claim 14 to treat a neoplastic tumor in a mammal.

36. Use of intravenously injectible multiple doses of a composition according to claim 14 to treat a neoplastic tumor in a mammal.

37. Use of a composition according to claim 15 to treat a neoplastic tumor in a mammal.

38. Use of parenterally-administrable multiple doses of a composition according to claim 15 to treat a neoplastic tumor in a mammal.

39. Use of intravenously injectible multiple doses of a composition according to claim 15 to treat a neoplastic tumor in a mammal.

CLAIMS SUPPORTED BY THE SUPPLEMENTARY DISCLOSURE

40. A composition comprising an anthracycline anti-neoplastic agent encapsulated in phospholipid bilayer membrane particles consisting of anionic and neutral phospholipids, said particles being suspended in a low ionic strength aqueous phase, the mol ratio of anthracycline anti-neoplastic agent to anionic phospholipid being at least about 1:1.25.

41. A composition according to claim 40 wherein the low ionic strength aqueous phase contains a physiologically accept-able nonionic hydroxyl-containing compound.

42. A composition according to claim 41 wherein the hydroxyl-containing compound is a sugar.

43. A composition according to claim 42 wherein the sugar is a monosaccharide.

44. A composition according to claim 43 wherein the mono-saccharide is dextrose.

45. A composition according to claim 42 wherein the sugar is a disaccharide.

46. A composition according to claim 45 wherein the dis-accharide is lactose.

47. A composition according to claim 46 wherein the lactose is present as a 9-11% solution and the pH is about 6.0 to 8Ø

48. A composition according to claim 40 wherein the anionic phospholipid is a phosphatidylglycerol, phosphatidylserine, phosphatidylinositol or phosphatidic acid and the neutral phospholipid is a phosphatidylcholine or phosphatidylethanolamine.

49. A composition according to claim 48 wherein cholesterol is also present.

50. A composition according to claim 48 wherein the anionic phospholipid is distearoyl phosphatidylglycerol and the neutral phospholipid is distearoyl phosphatidylcholine.

51. A composition according to claim 50 wherein the anthracycline anti-neoplastic agent, distearoyl phosphatidylglycerol and distearoyl phosphatidyl choline are in the molar ratio of 1 1.5:7.

52. A composition according to claim 49 wherein the anthracycline anti-neoplastic agent, distearoyl phosphatidylglycerol, distearoyl phosphatidyl choline and cholesterol are in the molar ratio of 1:1.5:7:2.

53. A composition according to claim 49 wherein the anthracycline anti-neoplastic agent, distearoyl phosphatidylglycerol, distearoyl phosphatidylcholine and cholesterol are in the molar ratio of 1:4:5:6.

54. A composition according to claim 40, 48 or 51 wherein the anthracycline anti-neoplastic agent is selected from the group consisting of daunorubicin, doxorubicin and aclacinomycin A.

55. A composition according to claim 40, 48 or 51 wherein the anthracycline anti-neoplastic agent is daunorubicin.

56. Use of a composition according to claim 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 to treat a neoplastic tumor in a mammal.

57. Use of parenterally-administrable multiple doses of a composition according to claim 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 to treat a neoplastic tumor in a mammal.

58. Use of intravenously-injectible multiple doses of a composition according to claim 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 -to treat a neoplastic tumor in a mammal.

59. Use of a composition according to claim 54 to treat a neoplastic tumor in a mammal.

60. Use of parenterally-administrable multiple doses of a composition according to claim 54 to treat a neoplastic tumor in a mammal.

61. Use of intravenously injectible multiple doses of a composition according to claim 54 to treat a neoplastic tumor in a mammal.

62. Use of a composition according to claim 55 to treat a neoplastic tumor in a mammal.

63. Use of parenterally-administrable multiple doses of a composition according to claim 55 to treat a neoplastic tumor in a mammal.

64. Use of intravenously injectible multiple doses of a composition according to claim 55 to treat a neoplastic tumor in a mammal.