Note: Descriptions are shown in the official language in which they were submitted.
~'7~
60724-1648
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.
-- 1 --
~;~7~
60724-1648
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~
60724-1648
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~
60724-1648
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~
60724-1648
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
171/279
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
171/279
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.
-10- 60724-1648
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
171/279
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
171/279
~ ~ 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.
-12-
~ 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.
-13-
~_~7~P~;
60724-1648
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
- 14 -
~;~7~
60724-1648
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
- 15 -
.~ , '
60724-1648
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 -
60724-1648
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;~
60724-1648
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
- 18 -
60724-1648
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.:
-- 19 --
60724-1648
-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~.
- 20 -
60724-1648
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
- 21 -
~7~
60724-1648
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.
- 22 -
