Note: Descriptions are shown in the official language in which they were submitted.
W092/02208 PCT/US91/05519
,
208~75
STABLE DOXORUBICIN/LIPOSOME COMPOSITION
1. Field of the Invention
The present invention relates to a stable, }yophilized
; doxorubicin/liposome composition.
2. References
Anchordogu~, T., et al., Biochim Biophys Acta,
946(2):299 (1988).
Anchordoguy, T., et al., Cryobiology, 24(4):324
~ (1987). Aubel-Sadron, G., et al., Biochemie, 66:333
; ~1984).
Bearer, E.L., et al., Biochim Biophys Acta, 693(1):93
(1982)-
Crowe, L.M., et al., et al., Biochim Biophys Acta,
769:141 (1983).
Forssen, E.A.,, et al., Proc Nat Acad Sci, USA,
20 78(3):1873 (1981).
Gabizon, A., et al., Cancer Research 42:4734 (1982).
~ Gabizon, A., et al., Cancer Research 43:4730 (1983).
< Gabizon, A., et al., Brit J Cancer, 51:681 (1985).
Higgins, J., et al., J Pharm Pharmacol, 39(8):577
25 (1987).
Higgins, J., et al., J Pharm Pharmacol, 38(4):259
(1986)-
-:, ,
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~ W092/02208 PCT/US91/~551~
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Juliano, R.L., et al., Biochem Pharmacol, 27:21
(1978) .
Madden, T.D., Biochim Biophys Acta, 817: 67 (1~85) .
Strauss, G., et al., Biochim Biophys Acta, 858 (1): 169
5(1986).
Szoka, ~., Jr., et al., Proc Nat Acad Sci (USA)
75:4194 (1978).
Szoka, F., Jr., et al., Ann Rev Biophys Bioeng 9:467
(1980) -
- 10Young, R.C., et al., N Eng ~ Med, 305: 139 (1981) .
3. Backqround of the Invention
Doxorubicin is a potent chemotherapeutic agent effec-
; tive against a broad spectrum of neoplasms ~Aubel~Sadron,
Young). However, the use of the drug is limited by serio~s
side effects. Its acute toxicity includes malaise, nausea,
vomiting, myelosuppression, and severe alopecia. In addi-
tion, cumulative and irreversible cardiac damage occurs
with repeated administration, which seriously limits the
use of the drug in protracted treatment (Young).
When administered in liposomal form, doxorubicin re-
tains its therapeutic effectiveness against animal tumors,
but is significantly less toxic (Forssen, Gabizon, 1985).
The drug-protectiVe effect of liposomes is due, at least in
part, to a marked alteration in plasma pharmacokinetics and
tissue disposition of the injected drug (Gabizon, 1982,
1983; Juliano).
Recently, it has been recognized that liposomal doxo-
rubicin preparations are relatively unstable on storage in
liquid form, as evidenced by rapid breakdown of both doxo-
- rubicin and liposomal lipids. The instability of doxorubi-
cin, when combined in liposomal form, appears to involve
-
` free radical and related oxidative mechanisms, since the
rate and extent of lipid and drug damage is substantially
reduced by free-radical quenchers, such as alpha-tocopher-
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ol, and b~ an iron specific chela~or, sucn as desferriox-
amine (~ s ?a~en, ~,~9~,73~)
~ he rate and ex ent of drug and lipid damage in a
doxorubicin/liPcsome formulation can also be reduced in a
lyophilized storage form However, experiments conducted in
support of the present invention show tha; relatively high
rates of doxorubicin breakdown can oc_ur in a lyophilized
formula.ion under conditions OI accelerated s.orage, even in
tne presence o~ free-~adicâl ~uenchers or i~on-specific
10 chela~ors
4 Summar~- of _he ~nven.ion
~ = is one general object of _ne invention _o
p-o~-ide a lyophilized doxorubicin/liposome composi.ion which
is stable agains- doxorubicin breakdown on long-term s.orage
1~ In one aspect .he invention includes a lyophilized
doxorubicin/liposome (L-D0~) composi.ion which is
charac;erized by less .han 1~%, and prefe-ably less than
about '0%, d~xorubicin breakdown after ac-elerated storage in
lyophi1ized form at 40C fo- ~ weeks The composition is
20 prepared by lyophilizing an aqueous liposome suspension
having a pH be-ween 3 0 and 4 ~, and preferably between 3 5
and 4 0, and con~aining (i) liposomes whose do~inan. lipid
componen~s are neutral phosphclipids, chcles~erol, and a
negatively charged lipid, (ii) doxorubicin,'a~ a drug lipid
2~ ratio of between ~-10 mole percen~, and a doxorubicin
concentration of less than 10 mg/ml, preferably between about
4-6 mg/ml, and (iii) a bulking agent
These and other objects and features of the
invention will become more fully apparen~ when the following
30 detailed description of the invention is read in conjunc-tion
with the accompanying drawings
Brief Descri~tion of the Drawinqs
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SVE~STITUTE SHEET
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W092/02208 PCT/US91/05~19
Figure l is an HPLc chromatogram showing doxorubicin
and breakdown products in a lyophili~ed L-DOX composition
of the invention after 2 weeks at 50C;
Figure 2 is an HPLC chromatogram showing doxorubicin
and breakdown products in the same L-DOX composition, but
after storage in liquid suspension form for 2 weeks at 40C;
and
- Figure 3 is an HPLC chromatogram showing doxorubicin
and breakdown products in a lyophilized L-DOX composition
at pH 4.3 in the presence of succinate after 2 weeks at
50C.
Detailed DescriPtion of the Invention
A. Preparation of Lyophilized L-DOX Composition
15Liposomes produced by the method of the present inven-
tion are formed from standard vesicle-forming lipids, typi-
cally including neutral phospholipids, such as phosphati-
dylcholine (PC), negatively charged lipids, such as
~ phosphatidylglycerol (PG), phosphatidylserine (PS),
: .20 phosphatidylinositol (PI), phosphatidic acid (PA) or
negatively charged sterol lipids, such as cholesterol
sulfate and cholesterol hemisuccinate, and cholesterol or
.:neutral cholesterol analogs. One preferred formulation
includes 40-60 mole percent PC, 10-30 mole percent of a
.25 negatively charged lipid, such as PG or cholesterol
~sulfate, and remainder cholesterol.
.,The phospholipid components may contain either satura-
ted acyl chains, such as dipalmitoyl acyl chains, or
;.unsaturated acyl chains, such as the mixture of unsaturated
::30 chains present in egg PC or egg PG. The effect of acyl
~chain composition, lipid purity, and negatively charged
~-lipid on storage properties, under accelerated storage
conditions, is discussed in Section B below.
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The liposomal lipids may also include a lipophilic
- free-radical quencher, such as alpha-~ocopherol (~-T), or an
acià or sal ,hereof, e.g. alpha-toc~pherol s~ccinc_e (~-TS),
or bu.yl2~ed hyd~o~:~toluene (3HT), at a preferred
concentration of abou- 0-~-2 mole percent of to,al lipids.
One preferred lipid composition, described in
Example 1, contains 47.1 mg/ml egg PC (EPC), 19.9 mg1ml egg
PG (E?G), 13.4 mg/ml cholesterol, and 0.91 mg/ml alphz-
tocopherol acid succinate (~TS), in prelyophilized liquid-
10 suspension form- .~nother preferred composi.ion is given in
~ ~xample 2.
- ~he L-DOX compcsi~ion can be prepared by a varie.v
of liposome-forming methods, such as have been re~iewec
; (Szok2, 19~0). 'n one lipid-hydration method suitable for
; 15 large-scale liposome production, the lipid components are
initially dissolved in a volatile non-polar solvent or
i solvent sys-em, such as chloroform, or a chlorofl~orocar30n
solvent. Freon ~ 11 or a solven~ Sys~em con~aining FreonT~
~; and 2-5 ~/v percen_ ethanol are well suited for dissolving
20 lipid components for use in the present invention. The low
1 bolling point of this solven- permits rapid, and
;I substantially complete solvent removal under the solvent-
removal conditions of the inven~ion~ The solven. poses
minir,al health and safety-hazard risks and can be read~ly
25 reclaimed by condensation.
The lipid solution is dried, under vacuum, in a
suitable drying vessel. For efficient large-scale liposome
production, drying and rehydration steps are prefera~ly
: carried out in a planetary mixer par~ially filled with
30 chemical-inert, preferably hydrophobic, spherical particles
which become coated with the lipid during dehydration, as has
been detailed in co-owned U.S. patent application for "Large-
; Scale Liposome ~roduction," filed March 30, 1990, Serial No.
502,222. One suitable particle is a ~/16 inch
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W092/02208 ~ ~ PCT/~S91/0~19
Teflon bead, preferably having a roughened surface, such as
obtained from Clifton Plastics ~Clifton Heights, PA). The
quantity of particles added to the vessels is preferably
~; such as to produce an area of the particles of between
5about 0.02 and 0.04, and preferably about Q.025 and 0.03
cm2/~mol total lipid added to the vessel.
Planetary mixers having mixer volumes between about 1-
10 liters and greater are commercially available. One pre-
ferred mixer is a 2- or 4- gallon mixer supplied by Charles
Ross and Sons (Hauppauge, NY). Suitable mixing speeds are
given in Example 1. Drying with mixing is typically car-
~- ried out for 3-4 hours for large lipid-salution volumes.
After complete solvent removal, the particles in the mixer
are coated with an irregular film of lipid, providing a
high surface area o~ dried lipids.
After solvent removal, the lipids are hydrated with an
aqueous volume to a ~inal concentration of lipids of
between about 100-300 ~mol/ml. The aqueous medium contains
~ doxorubicin, at a concentration of between about 10-20
;~ 20 mg/ml, and preferably about 15-16 mg/ml doxorubicin in a
pyrogen-free aqueous medium. Typically, the mole concen-
tration ratio of drug to phospholipid is between 1:10 and
1:50, depending on the drug being e~capsulated. For
:~ example, common doxorubicin/phospholipid concentration
ratios are on the order of 1:15.
Other components in the hydration medium may include
desferioxamine, at a concentration of about 50 ~M to l mM
and preferably about 0.2 mM, and a physiological salt, such
as NaCl, at a f inal concentration which gives a substan-
tially isoosmolar solution. The hydration medium may
. include a bulking agent ~Section B), at a weight concentra-
... .
tion between about 1-10%, and preferably about 5%.
Alternatively, the bulking agent may be added to the re-
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W092/02208 P~T/US91~05519
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hydrated liposome suspension just prior to lyophilization,
as described in Section B.
According to an important feature of the invention,
the pH of the aqueous medium is between about 3.0 and 4.4,
and preferably between about 3.5-4Ø As will be seen in
-Section C below, a pH of 4.4 or below significantly
enhances the stability of doxorubicin against breakdown
under accelerated storage conditions in lyophilized L-DOX
form. At the same time, maintaining the pH above 3.0
reduces the extent of phospholipid hydrolysis, which is
promoted by low pH.
One exemplary aqueous medium, used for producing doxo-
rubicin liposomes, is prepared by dissolving desferal in
pyrogen-free water, then adding doxorubic~- with stirring
until the drug is dissolved. 'rO ~his solution is added the
bulking agent and NaCl solution, to a final concentration
of components of 200 ~M desferal, 5 mg/ml doxorubicin, 0.45
NaCl, and 5% (w/v) bulking agent. The solution is
adjusted to pH 3.8 by addition of HCl (Example l).
20Hydration of the lipid-coated particles preferably
occurs under mixing conditions similar to those used in
preparing the lipid-coated particles. In particular, for
large-scale preparation, the hydration procedure is prefer-
ably carried out in a planetary mixer, under the mixing
condition described above. The thorough mixing action pro-
~ vided by the bi-axial motion of the mixing blades breaks up
`~ particle-particle aggregates, and thus exposes more lipid-
coated surface area for hydration. The greater degree of
lip:d surface exposure to the aqueous medium enhances the
rate and final yield of liposome formation.
The liposome suspension may be sized to achieve a
selected size distribution of vesicles in a slze range less
than about l micron and preferably between about 0.05 to
0.5 microns, and most preferably between about 0.05 and 0.2
. :
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W092/0220~ ~ PCT/US91/055l9
,. ,:
microns. The sizing serves to eliminate larger liposomes
and to produce a defined size range having optimal pharma-
cokinetic properties.
~everal techniques are available for reducing the
sizes and size heterogeneity of liposomes. Extrusion of
liposomes through a small-pore polycarbonate membrane is an
; effective method for reducing liposome sizes down to a
well-defined size distribution whose average is in the
range between about 0.08 and l micron, depending on the
pore size of the membrane. Typically, the suspension is
cycled through the membrane several times until the desired
liposome size distribution is achieved. The liposomes may
be extruded through successively smaller-pore membranes, to
~9 achieve a gradual reduction in liposome size.
}5 ~ree doxorubicin, i.e., doxorubicin present in the
bulk a~ueous phase of the medium, is preferably removed to
increase the ratio of liposome-entrapped to free drug. The
drug removal is designed to reduce the final concentration
of free doxorubicin to less than about 20% and preferably,
less than about 10% of the total drug present in the
composition.
Several methods are available for removing free druq
from a liposome suspension. A sized liposome suspension
can be pelleted by high-speed centrifugation, leaving free
drug and very small liposomes in the supernatant. Another
method involves concentrating the suspension by ultrafil-
- tration, then resuspending the concentrated liposomes in a
drug-free replacement medium. Alternatively, gel filtra-
tion can be used to separate larger liposome particles from
solute (free drug) molecules.
One preferred procedure for removing free doxorubicin,
or analog thereof, utilizes an ion-exchange resin capable
of bindinq drug in free, but not in liposome-entrapped,
form. The preferred resin is a cation-exchanger, since the
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W092/02208 PCT/US91/0~5]9
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drug is positively charged at neutral pH. One preferred
ion exchange resin is Dowex SOW-X4 50-lO0 mesh resin.
After free drug removal, the liposomes may be diluted
by addition of a physiological buffer, or concentrated,
- 5 e.g., by ultrafiltration, to a desired drug concentration.
Bulking agent, if not already present, is added to the
suspension at this stage. The liposomes are then lyophi-
lized for storage, as will now be described.
: ;
B. LYoPhilization and Storaqe
; In general, storage of drug/liposomes in liquid
suspension form over prolonged periods can lead to loss of
entrapped drug from the liposomes, liposome size changes,
and degradative changes in the lipid and/or drug molecules
in the composition. In general, degradative changes in a
liposome or cell suspension can be arrested, for long-term
storage, by freezing or lyophilization, i.e., freezing
followed by water removal by sublimation under vacuum.
As is known, the steps of freezing, dehydrating,
and/or rehydrating dried liposomes can themselves cause
undesired changes in drug/liposome properties. Studies
conducted in support of the present invention, as well as
studies report@d by others (e.g., Anchord~guy, 1988, 1987;
Higgins, 1987, 1986; Strauss), suggest that liposomes may
-; 25 undergo two types of disruptive damage during freezing,
prior to lyophilization. One type of damage is membrane
;;~ rupture caused by ice crystal formation inside and outside
the vesicle spaces during freezing. This type of damage
can lead to substantial loss of an encapsulated, water-
~- 30 soluble drug molecule. Such drug solute loss due to
`membrane rupture can be reduced by cryoprotectants such as
ylycerol, DMSO, polyethylene glycol, polypropylene glycol,
1,3,-butanediol, 2,3, butanediol, 1,3-propanediol, a
variety of mono- and disaccharides, such as lactose,
sucrose, and trehalose, and polysaccharides, such as
~ , .
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W092/02208 ~ PCT/US9]/05~19
"' 10
dextran and hydroxyethyl starch (HES) which appear to
interrupt or minimize ice crystal formation, when present
: both inside and outside the liposomes.
In addition to membrane rupture, as evidenced by a
- 5 loss of encapsulated solute, liposomes tend to show a pro-
gressive size growth with freezetthaw cycles. The liposome
size growth may be due to a solute-exclusion effect in
. which the formation of ice crystals concentrates solutes
and liposomes into microenvironments of very high lipid and
salt concentrations, in effect, forcing liposome together.
- This effect can be reduced by including in the bulk ~extra-
liposomal) phase of a liposome suspension, a bulking agent
which is effective to interrupt formation of large ice
crystals.
15The bulking agent should also have the property of
forming a solid, somewhat porous, non-crystalline matrix on
drying, to allow escape of water by sublimation from the
frozen sample, and to reduce additional solvent exclusion
effects due to crystal formation in the bulking agent.
Note that the requirement for a solid bulking agent
-~` excludes a variety of small viscous-liquid cryoprotectants
such as DMSO, glycerol, ethylene glycol, and propylene
glycol as the bulking agent in the present-rinvention.
; one general class of compounds which are suitable as
; 25 bulking agents are non-crystalline carbohydrates, such as
j trehalose and lactose, and polysaccharides, such as
dextrans and hydxoxyethylcellulose. The use of these
compounds as cryoprotectants or bulking agents for lipo-
somes, microsomal membranes or blood cells has been
reported (e.g., Crowe; Anchordoguy, 1988; Strauss; Madden).
Another general class of bulking agents incIudes amino
acids, and amino acid analogs, such as 2-aminobutyric acid,
4-hydroxyproline, sarcosine, glycine betaine, and basic
amino acids, such as lysine and histidine, which may inter-
: i
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W O 92/02208 PC~r/US91/05519
2B~89 75
.. . 11
act with the negatively charged head groups of the lipo-
-~ somes (Anchordoguy, 1988).
A third general class of bulking agents includes a
variety of non-sugar glycolytic pathway compounds, such as
:i5 sodium or potassium salts of tartrate, oxaloacetate, fuma-
rate, malate, ketoglutarate, and pyruvate. The bulking
agent is preferably a mixture of two or more of these com-
pounds, to minimiæe crystal formation effects on freezing
and dehydration. As will be seen in Section C below, suc-
cinate is not a suitable bulking agent since it enhances
doxorubicin breakdown on storage in lyophilized form, nor
are di- or tri-dicarboxylic acid compounds, such as ci-
trate, since these may precipitate with doxorubicin, which
is positively charged. A ~ourth class of bulking agents
includes non-saccharide polymeric compounds, such as higher
molecular weight polyethylene glycol (PEG), which are (a)
solids at room temperature, (b) readily soluble in water,
and ~b) pharmaceutically acceptable for parenteral adminis-
tration. In particular, PEG polymers with molecular
weights above about 1,500-2,000 daltons are contemplated.
In one embodiment, the polymer is included in the bulk
phase of the suspension, at a weight concentration between
about 1-10 percent.
In another embodiment, the liposomes themselves are
;25 derivatized with a PEG or other polyalkyl oxide, for pur-
poses of enhanced lifetime in the bloodstream when admini-
stered intravenously, as described in co-owned U.S. patent
application for "Liposomes with Enhanced Circulation
Times," filed October 10, 1989, Serial No. 425,224. Here
the surface-derivatized molecules act to prevent ice cry-
stal growth in the region of the liposomes. The liposome
-suspension is preferably concentrated to insure a relative-
ly high polymer concentration in the bulk phase. Ihe
,iposome suspension may also contain a solution-phase
. ~
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W092/0220~ PC~/~S91/05519
12
bulking agent selected from one of the classes above, in
addition to the liposome-bound agent.
The bulking agents may be included in the aqueous
hydration medium used in forming the liposomes, yielding a
suspension with an equal concentration of agent in the bulk
and encapsulated liposomal aqueous phases. Alternatively,
the agent can be added to the final sized liposome suspen-
sion, wherein the agent is present only in the bulk phase
of the suspension. As indicated above, the final concen-
tration of the bulking agent in the suspension is between
about l-lO weight percent and preferably about 4-6 weight
percent.
In still another embodiment, liposome fusion on
~- freezing and dehydration is minimized by forming the
initial liposome dispersion in a low-ionic-strength medium
in which the doxorubicin/liposomes have a partially ordered
gel structure, by virtue of the surface charge repulsion
effects. This type of liposome gel has been described in
co-owned U.S. application ~or "Liposome Gel Composition and
20 Method," filed May 22, 1989, Serial No.356,262. The gel
formulation re~uires an ionic strength comparable to that
of NaCl, at a concentration of less than about 20 mM NaCl.
The medium may contain non-ionic species, such as mono or
disaccharide protective agents.
In forming the lyophilized freeze-dried suspension,
the liposome dispersion is frozen and lyophilized as
described below. The lyophilized material can be recon-
stituted, for parenteral injection, by addition of a
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W092/02208 PC~`/US9~/OS519
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rehydration medium containing physiological salts or other
ionic species.
In a preferred lyophilization method, the suspension
is first cooled to 4-5~C, then frozen to -45C and main-
tained at this temperature for 12 hours in a conventionallyophiliæer. The lyophilizer chamber is pumped down to
- about 200 ~ pressure, after which the chamber temperature
is raised, at about 10C/hr, to -20C. The chamber is
maintained at this temperature until the lowest reading
product thermocouple is within 3C of shelf temperature.
The chamber temperature is now allowed to rise, again
at about 10C/hour, to room temperature, and the material is
held under vacuum at this temperature for an additional 15
hours. The chamber is backfilled with nitrogen to a
pressure of about 2 inches Hg, and the sample vessels
stoppered ~or storage. One suitable lyophilizer is a
; Edwards Lyoflex 08 lyophilizer supplied commercially from
Edwards High Vacuum, lnc. (New York).
The stability of the lyophilized preparation can be
examined by accelerated studies conducted at elevated
temperatures, according to known principles. The storage
conditions which were used in the studies described below
were 40C for 4 weeks, and 50C for two ~eeks. In each
storage study, the lyophilized sample in a stoppered vessel
was placed in an incubator at the selected temperature and
analyæed after the two- or four-week incubation period for
doxorubicin breakdown, with the results discussed in
Section C.
C. Doxorubicin Stability on Storaqe
: 30 Table I below shows composition and pH variables in
. several lyophilized L-DOX preparations which were studied
under accelerated storage conditions. Each of the 20 pre-
parations were prepared according to the methods outlined
;~ above, and detailed for Preparation No. 11 in Example 1.
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$ W092/02208 PCT/VS~1/0~519
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TABLE l
, Anionic Anti-
-~ 5 NO. Lipid Oxidant Desferal Succlnate PH
1 EPG oTS .2 ~ 4.8
2~ EPG oTS .2 + 4.8
3 EPG oTS .2 - 4.8
4 EPG - .2 ~ 4.8
EPG BHT .2 ~ 4.8
6b DPPG ~TS .2 ~ 4.8
7 EPG uTS - + 4.8
8 EPG oTS l.O + 4.8
; gc EPG oTS .2 + 4.8
CS oTS .2 ~ 4.8
11 EPG oTS .2 ~ 3.8
12 EPG oTS .2 - 3.o
, 13 EPG oTS .2 - 3.8
14 EPG ~TS .2 ~ 3.8
EPG B~T .2 - 4.0
16 EPG BHT .2 - 4 2
17 EPG oT .2 - 3 8
18 - _ _
19 - - .2 + 4.8
- - .2 - 5.3
99~ pure EPC and EPG from Avanti Polar Lipids, Inc.
(Birmingham, AL) were used.
bA 99% pure dipalmitoylphosphatidylcholine (DPPC) and di-
palmitoylphosphatidylglycerol (DPPG) (Avanti Polar
- Lipids) were used to assess the effect of the degree of
fatty acid saturation on the stability profile.
C99% pure EPG from Genzyme Corp. tBoston, MA) was uti-
lized.
, dEPG was completely removed from the formulation; sodium
; cholesterol sulfate was incorporated in its place to
, 40 provide the negative charge for the optimal incorporation of doxorubicin into liposomes.
, elOmM lactic acid substituted for l0 mM succinic acid in
; the aqueous phase.
:~ 45
After storage under the above accelerated storage condi-
tions, each preparation was reconstituted by addition of dis-
tilled water to a final doxorubicin concentration of about 5
mg/ml. An aliquot of the material was diluted with mobile
phase to a final dilution of 1:50 at a final liquid concentra-
,
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~ W092/02208 PCT/US91/0~519
~ 2088975
.
tion of ab~ut 2.5 ~mol/ml (1.75 mg/ml). At this concentra-
tion, the lipid and drug components of the L-DOX suspension
; are in solution. A typical example of this material, chroma-
tographed by HPLC is detailed in Example 3.
Figure 1 shows an HPLC profile of doxorubicin and doxoru-
bicin breakdown products observed in a lyophilized preparation
corresponding to composition No. 11 in Table 1, after acceler-
ated storage for 2 weeks at 50C. The peaks in the chromato-
gram represent absorbance at the doxorubicin absorbance peak
of at 480 nm. Three distinct peaks, with RT (retention time)
values of 9.92, 12.75, and 14.4 ~doxorubicin) were observed,
with the measured relative peak areas shown. The vertical
markers on either side of each peak indicate the portion of
the curve that was integraked.
TABLE 2
Peak# Area% RT
1 0.695 9.92
- 20 2 1.227 12.75
3 98.077 14.4
, :
The concentration of doxorubicin in the reconstituted
sample, both before and after storage at 50C for two weeks,
was determined from a standard curve of known concentrations
of doxorubicin as a function of peak areas on X~LC. The
values calculated were 4.96~0.03 and 4.51~0.05 mg/ml doxorubi-
cin before and a~`ter storage (Table 5). Thus, about 9% of the
total doxorubicin present in the pre-stored sample was lost to
breakdown products on storage.
The doxorubicin peak in the Figure 1 HPLC profile consti-
tutes all but about 2% of the detected products, leaving
approximately 7% of the breakdown products unaccounted for.
One possible source of this discrepancy is that one or more of
the breakdown products have lower absorbance coefficients at
- 480 nm, so that these product are either not seen or underes-
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W092/02208 PCT/US91/0~5]9
9 ~ ;i , S
16
timated in amount. Another possible source of discrepancy is
that the breakdown products do not have well-defined peaks,
and therefore are not included in the total peak area calcula-
tions. Finally, it is possible that some of the breakdown
products are not eluted from the column under the chromato-
graphy conditions employed.
Figure 2 shows an HPLC profile of the same L-DOX formula-
tion as in Figure 1, but after accelerated storage at 40~C for
two weeks in liquid suspension form. The relative peak areas
of the four peaks are given in Table 3 below. The total
-- amount of doxorubicin in the main peak in the figure repre-
sents about 67% of the doxorubicin present prior to storage.
- Thus, storage in liquid suspension form substantially increa-
ses the breakdown of doxorubicin in the a low-pH L-DOX formu-
lation.
. . .
TABLE 3
Peak# Area~ RT
` 1 5.193 10.16
2 87.212 14.88
3 4.808 20.06
, . .
4 2.407 21.15
~- 25 Figure 3 shows an HPLC profile of a lyophilized L-DOX
formulation corresponding to composition No. 2 in Table 1,
` after accelerated storage at 50C for 2 weeks. As seen in the
;' Figure, and in Table 4 below, at least 16 different peaks were
identified, with the doxorubicin peak accounting for about 85%
of the total peak area. The total amount of doxorubicin in
the main peak in the figure represents about 74% of the doxo-
rubicin present prior to storage.
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. TABLE 4
Peak~ Area~ RT
. 1 0.069 3.34
. 2 0.104 3.6
3 0.129 4.03
: 4 0.216 7.35
0.427 9.25
~ 6 0.729 10.38
~ 7 0.077 11.11
~ .:
- 8 0.705 13.63
: 9 85.509 15.31
0.344 20.75
11 0.623 21.S8
~ 12 0.631 22.31
. 13 0.124 25.45
. 14 0.302 30.48
0.756 31.55
16 1.255 69.61
. , ,
. Each of the 20 formulations in Table 1 were tested as
. above, for loss of doxorubicin after storage at 40C or 50C
for 4 or 2 weeks, respectively. The results are given in
Table 5 below, along with the pH for the suspension, prior to
lyophilization.
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TABLE 5
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::~ Formulation Formulation ~DOX~3 mgJmL (~ of Original)
No. Description p~
Original 2 weeks 4 weeks
at 50c b at 40Cb
.
1 5.2 4.92+0.072.44+0.043.g9+0.03
(50%) (81%)
2 hiyh purity 5.2 4.90+0.042.13+0.093.58+0.07
EPC & EPG (443) (73%)
3 No Succinate 6.0 4.86+0.043.41+0~07 4.08+0.01
15 4 No aTs 5.3 4.90+0.022.64+0.093.94_0.10
BHT, no aTs 5,3 5.05+0.232.48+0.253.94+0.08
(49%) (7~3%)
6 DPPC/DPPG' 5.0 4.32 1.69 3.72
(39~) (86~)
7 No Desferal 5.0 4.76+0.063.21+0.074.05+0.06
;-' (67%) (85~)
8 1 mM Desferal 5.1 4.68+0.162.32~0.88 4.1010.02
25 9 high purity 5.1 4.69+0.06~50~) (88%)
Chol. Sulphate, 5.2 4.92+0.113.51~0.02 4.28+0,oo
11 No Succinate, 3.8 4.96~0.03 t71%) (87~)
, ~ 30 pH 3.8 (91%) (92%)
12 No Succinate, 3.2 4.75+0.034.50+0.03 4.51+0.02
pH 3.0 (95%) (95%)
,~- 13 Lactate, 3.8 4.73+0.023.88+0.124.31+0.03
, No Succinate (82%) (91%)
' 3514 Succinate, 3.9 5.08+0.003.61+0.094.26+0.02
pH 3.8 (71%) (84
No Succinate, 3.9 5.03i0.054.41+0.04 * d
pH 4.0 (88~)
' 16 No succinate, 4.1 5.11~0.074.38+0.04 ~ d
pH 4.2 (86~)
, 17 oT, No ~Ts, 3.7 5.34+0.034.81+0.07 ~ d
No Succinate, pH 3.8 (90%)
18 DOX --- 5.07 5.06+0.034.74+0.06
19 DOX in Suc- 4.8 5.25~0.22 3.64_0.og ~94~)
cinate buffer (69%) (91~)
DOX in Buffer, 5.1 5.49+0.055.15+0.04 5.28+0.14
No Succinate (94%) ~96%)
' ~DOX] was measured using HP~C. Triplicate vials were analyzed at
each time point, with the exception that 4 weeks, 40C samples 2-5,
19 and 20 were done in duplicate.
. b 4.5 mL of pre-lyophilization bulk suspension ~nominal [DXN] = 5.0
mg/mL) was filled into 20cc clear type I glass vials for lyophiliza-
tion. Reconstitution was accomplished by adding 4.0 mL of water to
provide a target ~DXN] of 5.0 mg/mL. All vials were stored upright
during the incubation period.
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WO9~/02208 PCT/US9l/05519
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19
The most important factors effecting doxorubicin stabi-
- lity were reduced pH (between pH 3-4) and the absence of suc-
: cinate. Removal of succinic acid from the L-DOX formulation
significantly increased drug stability (sample ~3 vs. sample
#1). Less doxorubicin degradation was also seen when the pH
of the formulation was lowered to 3.8 (sample #14 vs. sample
#1). Maximal stability was obtained with the simultaneous
elimination of succinic acid and lowering of pH. For example,
only 5% degradation was observed in a pH 3.0 formulation that
did not contain succinic acid (sample #12) after two weeks
incubation at 50c., whereas a 9-10% decrease in drug potency
was exhibited in similar formations at pH 3.8 under the same
: condition (sample #11 and sample ~17). Increasing the pH
further to 4.0 and 4.2 (sample #15 and sample #16) resulted in
slightly more DOX degradation. Adding lactate resl~lted in
significantly more DOX degradation (sample ~13 ~. sample
#11), although lactate had a less detrimental effect than
succinate in this regard (sample #13 vs. sample #14). A less
dramatic effect was observed in samples incubated at 40OC than
those incubated at 50OC. No lysophosphatidylcholine (lyso-PC)
was detected in any of these samples after incubation at 40OC
or 50C (data not shown).
No systematic trend relating the stability profile to the
purity, degree of saturation of phospholipid used, or type of
negatively charged lipid used was seen, based on results from
, samples incubated at 50 C. The use of EPG from Genzyme
Corporation and sodium cholesterol sulfate
' DPPC - Dipal~itoylphosphatidy}choline
DPPG - dipalmitoylphosphatidylglycerol
50 mL batch size was prepared for this formulation; only
- one vial was analayzed for each time point.
~ Data are not yet available at this time.
pH of the prelyophilization bulk suspension except for samples 6-10
~pH of reconstituted samples).
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~ W092/0~208 P~T/US9l/05519
1.9~ ;
appears to result in some improvement in DOX potency preser-
vation compared to the original formulation (sample #l) at the
end of the incubation period. No dramatic effect was observed
in samples incubated at 40C.
To assess the effect of the antioxidant on the stability
profile, a-Ts was removed (sample #4) and replaced with BHT
(sample #5). No significant difference in DOX stability was
observed in these samples at either 50C or 40.
Doxorubicin stability was compared in formations con-
taining no Desferal (sample #7), 200 ~M (sample #l) or 1 mM
Desferal (sample #8). The samples without Desferal, incubated
at 50C, showed a slight improvement in DOX stability.
The present invention is useful as an improved storagable
form of doxorubicin/liposomes, for use in the treatment of a
variety of tumor types. Reduced side effects have been
observed in Phase I and Phase II clinical trials with doxoru-
bicin liposome formulations prepared by reconstituting
lyophilized preparations. The present ~-DOX invention
provides the additional advantage of long-term stability on
storage, without significant loss of active drug or generation
of undesired breakdown products. The preparation is easily
prepared by a method that is suitable for large-scale manufac-
ture.
It will be appreciated that the advantages and features
of the present invention will apply to a variety of related
anthracene glycoside anti-neoplastic drugs, such as daunomy-
cin, carcinomycin, N-acetyladriamycin, N-acetydaunomycin,
rubidazone, 5-imidodaunomycin, and epirubicin.
The following example illustrates the method o~ the in-
vention for preparing doxorubicin liposomes. The example il-
lustrates, but in no way is intended to limit the scope o~ the
invention.
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Example 1
Preparation of LYophilized L-DOX Composition
(Pre~aration~ rom Table 1)
:A 300 ml chloroform solution containing 20.1 g EPC
(Lipoid), 8.5 g EPG (Asahi), 5.7 g CH (Croda~, and 0.4 g ~-
tocopherol succinate (Henkel~) was added to a 2-liter round-
bottomed flask containing 180 g of 3 mm diameter glass beads
and the whole dried in vacuo using a rotary evaporator. The
; lipid film was subsequently exposed to a vacuum of 50 mTorr
overnight to complete drying.
The lipids were hydrated to a final total lipid concen-
tration of about 240 ~mole/ml by addition of a doxorubicin-
containing solution and mechanically agitating the mixture for
2 hours at room temperature. This solution contained about 15
mg/ml DOX, 5% (w/v) lact.ose monohydrate, 0.4% (w/v) sodium
chloride, 200 ~M desferal in water and was prepared by dissol-
ving the doxorubicin in water and adding the other excipients
when complete dissolution was achieved. The pH of the mixture
was adjusted to 3.8 with HCl. The resultant liposome suspen-
sion was sized by passage five times through 0.4 ~m and fivetimes through 0.2 ~m pore-size polycarbonate membranes.
Unincorporated DOX was removed by passing the sized liposome
suspension over a Dowex 50W-X4 cation exchange resin.
Finally, the suspension was diluted to a DOX concentration of
about 5 mg/ml prior to lyophiliæation, using a 5% lactose,
0.4% sodium chloride, 200 ~M desferal solution, pH 3.8. The
characteristics o~ this pre-lyophilization solution are given
in Table 6.
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W092/022~8 PCT/US91/0~519
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TABLE 6
Theoretical.
. Com~osition .m~tml
DOX HCl 5 0
-:- EPC 47.1
- EPG 19.9
CH 13.3
- ..................... a -TS 0. 91
.~ 10 Desferoxamine mesylate 0.132
... Lactose monohydrate 52.6
NaCl 4.0
Water for injection qs to 1 ml
HCl qs to pH 3.80
Assay values for pre-lyophilization solution:
Doxorubicin HCl (mg/ml) 4.91
% encapsulation 96
Liposome diameter ~nm) 290
. Example 2
Large Scale L-DOX Production
:' 25
A lipid solution containing 1500 ml Freon llTM, 151.1 g
EPC (Lipoid), 63.9 g EPG (Asahi), 42.9 g cholesterol (Croda),
and 0.86 g o~ butylated hydroxytoluene (Penta) was added to a
2-gallon double planetary mixer (Ross mode~ 130 LD~) which
. 30 contained about 4,100 PTFE Teflon beads (Clifton Plastics)
having a mean diameter o~ 1/4 inch. The beads had been washed
in an aqueous solution containing a detergent (Alconox) to
: remove any oily residues from the sur~aces o~ the beads,
rinsed with ethanol, and dried, before placement into the
mixer.
The mixer was sealed and the pressure inside the mixer
was reduced while the solution and beads were mixed at an
orbital stirring rate of 20 rpm and an axial stirring rate of
: 26 rpm for about two hours, to trans~orm the lipids to a solid
state attached to the beads. A vacuum was maintained for an
additional 3 hours without mixing to achieve additional sol-
.~ vent removal.
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W092/02208 PCT/US91/05519
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After solvent removal, an aqueous solution of doxorubicin
HCl to a final lipid concentration of about 262 ~mol/ml was
added to the lipid coated particles in the planetary mixer.
The drug solution was prepared to contain 0.132 mg/ml deferox-
amine mesylate in pyrogen-free distilled water. Doxorubicin
hydrochloride (Farmitalia Carlo Erba) was then added with
stirring to a final concentration of about 12.5 mg/ml. After
complete dissolution of the drug, lactose was added to a final
concentration of 5 weight percent and NaCl 0.4 weight percent.
The pH of the solution was then adjusted to 3.7 with HCl
solutlon .
Hydration of the lipid was carried out at a temperature
of 25 C. for 2 hours, with mixer stirring at abou, 25 rpm.
The resulting liposome suspension was sized by passes through
0.4 and 0.2 micron polycarbonate filters. Free doxorubicln
was reduced by treating the sized-liposome suspension with a
Biorad AG 50w-X4 50-lOO ion exchange resin. The preparation
was then diluted with a solu~ion containing deferoxamine
mesylate, lactose, sodium chloride and hydrochloric acid in
pyrogen-free distilled water.
The approximate final composition of the liposome suspen
sion was:
4.3 mg/ml doxorubicin hydrochloride
40.0 mg/ml EPC
16.9 mg/ml EPG
11.3 mglml cholesterol
0.23 mg.ml butylated hydroxytoluene
0.132 mg.ml deferoxamine mesylate
50.0 mg/ml lactose
4.0 mg/ml sodium chloride
The preparation had the following properties: pH of approxi-
mately 3.8, mean particle size of approximately 232 nm;
approximately 96~ of the total drug was liposome associated.
Example 3
HPLC Chr~mato~ra~hV
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~ W092/02208 PCT/US91/05519
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; A. HPLC Chromatography
- Lyophilized L-DOX was reconstituted with water to a final
doxorubicin concentration of about 5 mg/ml. A 0.25 ml aliquot
of the rehydrated suspension was diluted to 5 ml with mobile
phase.
The separation of the doxorubicin and its degradation
products was carried out on a Waters HPLC using a Whatman
Partisil ODS-3 column, 250 x 4.6 mm held at ambient tempera-
ture. The mobile phase was 43~ aqueous buffer in methanol.
The aqueous buffer was 95 mM ammonium phosphate/ 5 mM tri-
ethylamine, pH 4Ø 15 ~l of sample was injected in the
system at a 1 ml/min flow rate. Effluent was monitored at 480
nm. An external standard, prepared fresh each day, consisted
of doxorubicin powder dissolved in mobile phase to a final
concentration of 0.35 mg/ml.
B. Analysis after Storage
After an i~cubation period of two weeks at 50C, the L-
- DOX was analysed by HPLC, as above. Doxorubicin potency was
reduced 9~. The level of doxorubicin breakdown appeared to
; plateau at this level: a further two-week incubation at 40C
did not result in further reduction of doxorubicin concentra-
tion.
Although preferred methods and formulations have been
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fications may be made without departing from the invention.
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