Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
_ / ~ TL~-130A
1 ~378~8
HIGH INTEGRITY LIPOSOMES
AND METHOD OF PREPARATION AND USE
Field of Invention
This invention relates to a high integrity liposome comprising
at least one stabile lipid and at least one peptide-like therapeutic
agent associated with said liposome, adapted for parenteral
administration to an animal, including a human, and methGd of
manufacture and use. Such liposome is particularly useful in the
extended release and of peptide therapeutic agents as well as
serving to protect said agents from degradation in the physiological
environment.
Back~round of the Invention
Peptide therapeutic agents are well known and of increasing use
in the pharmaceutical arts. Hormones, i r -dulators, and a host
of newly discovered peptide and peptide-like compounds are presently
- being administered to ~ni~ls, including humans, in therapeutic
regimens.
Consistent drawbacks to the parenteral administration of such
peptide compounds have been the rapidity of breakdown or
denaturation (loss of "native state configuration") of such
compounds in the physiological environment and the difficulty of
obt~ning extended therapeutically effective dosage levels of such
agents. Infusion pumps have been employed for chronic
1 337898
-- 2 --
- administration of therapeutic agents as well as wax or oil implants
in an effort to both prolong the presence of peptide-like
therapeutic agents and preserve the integrity of such agents.
Furthermore, in the particular case in which the peptide-like
therapeutic agent (which will be understood to include a protein or
haptene) is to function as an immunogen, the peptide-like agent
should (with particular reference to each epitope of the
peptide-like agent), ideally maintain native state configuration for
an extended period of time and additionally be presented a fashion
suitable for triggering an immunogenic responce in the challenged
animal.
SummarY of the Invention
This invention includes a high integrity liposome comprising at
least one stabile lipid and at least one peptide-like therapeutic
agent associated with the liposome, adapted for parenteral
administration to an animal. In some embodiment~ the stabile lipid
is hydrogenated phosphatidylcholine or distearoyl
phosphatidylcholine. In particular embodiments the therapeutic
agent comprises an antigen. This invention further comprise-~ the
liposomes wherein the liposome further comprises a lipid diluent,
such a~ cholesterol. In some embodiments containing cholesterol the
cholesterol i9 present in a lipid:cholesterol molar ratio of from
about 4:1 to about 1:1 (molar weight basis). In one embodiment
distearoyl phosphatidylcholine and cholesterol are used in about a
7:3 mole% ratio of phospholipid to cholesterol.
- The peptide-like therapeutic agent in the liposomes of this
invention include a hormone, an i o~~dulator, glycosylated
carrier protein, or galactosylated carrier protein, with particular
reference to the peptide-like therapeutic agent being
galactose-albumin, or the hormone being a somatotropin or calcitonin
1 337~98
_ including analogues or deravatives thereof. In embodiments wherein
the therapeutic agent is an immunomodulator, a particular example is
an interleukin, such as IL2.
This invention also includes a method of treating an animaL by
administering to the animal a therapeutically effective dose of
peptide-like therapeutic agent encapsulated in high integrity
liposomes. In embodiments of this method the administration is
intramuscular, parenteral, intra-arterial, subcutaneous,
intravenous, intraperitoneal which include the peptide-like
therapeutic agent being a hormone or ~ I odulator.
The method of treating animals also includes the peptide-like
therapeutic agent being a hormone, an immunomodulator or
glycosylated carrier protein, or galactosylated carrier protein,
such as wherein the peptide-like therapeutic agent are the hormones
bovine somatotropin or calcitonin or wherein the peptide-like
therapeutic agent is the ~ ~ odulator IL2 (including analogues
and deravatives of the foregoing).
In some embodiments of the method of treating An1 ~1s included is
the protein-like therapeutic agent being a growth promotant as well
as the animal being a pig, a chicken, a salmon a cow or a human.
In a particular aspect, this invention provides a method of
increasing milk production in dairy An~ o1~ by administering to the
animals a therapeutically effective amount of somatotropin in high
integrity liposomes. In one aspect the dairy animal is a cow and
the somatotropin is bovine somatotropin or analogues or deravatives
thereof.
In yet another embodiment this invention includes a high integrity
liposome comprising at least one stabile lipid and at least one
peptide-like therapeutic agent associated with said liposome,
adapted for topical administration to an animal.
i 33-~9&
Brief Description of the Drawin~s
Figure 1 compares in situ retention of high integrity liposomes and liposomes ofunhydrogenated lipid.
Figure 2 depicts weight gain by hypophysectomized rats administered BSTH in highintegrity liposomes.
Figure 3. discloses in situ retention of dosage at injection site.
Detailed Description of the Invention
Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous
volume. Liposomes may be unilamellar vesicles (possessing a single bilayer membrane ) or
multilamellar vesicles (onion-like structures characterized by multiple membrane bilayers,
each separated from the next by an aqueous layer). The bilayer is composed of two lipid
monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. The
structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid
monolayers orient toward the center of the bilayer while the hydrophilic "head" orient
towards the aqueous phase.
The original liposome preparation of Bangham, et al. (J. Mol. Biol., 1965, 13:238-252)
involves suspending phospholipids in an organic solvent which is then evaporated to
dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of
aqueous phase is added, the mixture is allowed to "swell," and the resulting liposomes
which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means. This
technique provides the basis for the development of the small sonicated unilamellar vesicles
described by Papahadjopoulos et al. (Biochim. Biophvs. Acta., 1963, 135:624-638), and
large unilamellar vesicles.
Unilamellar vesicles may be produced using an extrusion apparatus by a method described
in Cullis et al., PCT Application No. W0
1 - 3-l~`q~
86100238, published January 16, 1986, entitled "Extrusion Technique for Producing
Unilamellar Vesicles" incorporated herein by reference. Vesicles made by this technique,
called LUVETS, are extruded under pressure through a membrane filter.
Another class of liposomes are those characterized as having substantially equal lamellar
solute distribution. This class of liposomes is denominated as stable plurilamellar vesicles
(SPLV) as defined in U.S. Patent No. 4,522,803 to Lenk, et al., monophasic vesicles as
described in U.S. Patent No. 4,588,578 to Fountain, et al. and frozen and thawedmultilamellar vesicles ~FATMLV) wherein the vesicles are exposed to at least one freeze and
thaw cycle; this procedure is described in Bally et al., PCT Publication No. 87/00043,
January 15, 1987, entitled "Multilamellar Liposomes Having Improved Trapping
Efficiencies" and each of which are incorporated herein by reference. Honda et al.
Japanese Patent Pub. No. 60-155109 describes hydrogenated liposomes requiring 5% fatty
acid. The liposomes of this invention may be prepared with substantially less than 5%
fatty acid or without the presence of fatty acid at all.
For use in the present invention, a variety of sterols and their water soluble derivatives may
be used to form liposomes; see specifically Janoff et al., PCT Publication No. WO
85/04578, published October 24, 1985, entitled "Steroidal Liposomes." Mayhew et. al.,
PCT Publication No. WO 85/00968, published March 14, 1985, described a method for
reducing the toxicity of drugs by encapsulating them in liposomes comprising alpha-
tocopherol and certain derivatives thereof, each incorporated herein by reference. Also, a
variety of tocopherols and their water soluble derivatives have been used to form
liposomes, see Janoff et al., PCT Publication No. WO 87/02219, published April 23, 1987,
entitled "Alpha Tocopherol-Based Vesicles".
The term lipid as used herein shall mean any suitable material resulting in bilayer such that a
hydrophobic portion of the lipid
1 337898 6 -
_ material orients toward the interior of the bilayer while a
hydrophilic portion orients toward the aqueous phase. Lipids
further include highly hydrophobic compounds such as triglycerides,
sterols such as cholesterol which can be incorporated into the
bilayer. The term lipid does not include fatty acids. Specific
lipids are phospholipids such as phosphatidylcholine (PC),
phosphatidylethanolamine (PE), destearoyl phosphatidylcholine
(DSPC), phosphatidylserine (PS), phosphatidylglycerol (PG),
phosphatidic acid (PA), phosphatidylinositol (PI), sphingomyelin
(SPM), and the like, alone or in combination. The phospholipids can
be synthetic or derived from natural sources such as egg or soy.
Some synthetic phospholipids are dymyristoylphosphatidylcholine
(DMPC) and dimyristoylphosphatidylglycerol (DMPG). Liposomes can
also contain other steroid components such as polyethylene glycol
derivatives of cholesterol (PEG-cholesterols), coprostanol,
cholestanol, or cholestane, and combinations of PC and cholesterol.
Liposomes may also contain glycolipids.
"Stabile lipid" shall be understood to be lipids which are resistant
to oxidative catabolism initiated by changes in pH, temperature,
oxygen free radicals (e.g., such aq those produced by infiltrating
immune cells during inflamatory reaction) or other stress of the
physiological environment.
It iq to be understood that stabile is a property in the nature of a
continuum whereby normal lipid rigidity is modified by a stabilizing
- process such as hydrogenation. When used in an environment of low
pH-such as ~he intragastric environment, excluded from the grouping
of stabile lipids are lipids such as cholesterol hemisuccinate or
tocopherol hemisuccinate which are deconstructed at the physiologic
pH ranges found in animal gastrointestinal systemq. In such low pH
applicationq theqe are excluded without regard to rigidity. Thus a
stabile lipid is first a lipid resistant to oxidative catabolism
initiated by changes in pH, as well as resistant to temperature,
oxygen free radicals or other stress of the physiological
environment and second not deconstructed at common physiologic pH
13~ /&
ranges presented in the in vivo environment of use. Stabile lipids, when organized into
liposomes, will maintain structural integrity for an extended period of time, particularly as
compared to other liposomes.
The term peptide-like will be understood to mean short chain peptides as well as well as
proteins, but will also, for convenience, include such nonprotinaceous labile molecules as
vitamins, small steroids, azidothymidine, or free primaquine. A preferred class of peptides
are immunomodulators such as interleukins, colony stimulating factors and interferons. An
additionally preferred class of proteins are antigens such as are used in vaccines.
The term native state configuration will be understood to mean that organization of a
moiety having at least one epitope, such as a peptide, as it is configured when present in
vivo, to be distinguished from non-native state configuration Idenatured) wherein the
moiety is altered as to bioactivity or immuno-reactivity from that of the in vivo organization.
The term epitope will be understood to mean the smallest part of an antigen moiety
recognizable by the combining site of an immunoglobulin.
When using a protein merely as a proteinic macromolecule carrier of a therapeutic agent,
maintenance of the native state of such protein is of lesser importance, so long as the
carrying function is not substantially comprised. For example, albumin has been reported
as used as a "carrier protein" for therapeutic agents, particularly as linked to galactose
("galactose-albumin") or glucose ("glycosylated-albumin"), thus facilitating hepatic uptake
when presented in liposomal form (e.g., U.S. Patent No. 4,376,765). As used herein
"galactose-albumin" (and similarly glycose-albumin) refers to the moiety of galactose ~or
glucose) joined to albumin. Such moiety -- glucose or galactose/carrier protein -- is useful in
133789~ 8 -
- targeting a therapeutic agent at the liver. The moiety i9
preferentially taken up by the liver, and by covalently joining a
therapeutic agent to the carrier protein the therapeutic agent is
similarly taken up by the liver. By way of example, the therapeutic
agents doxorubicin, daunarubicin, or primaquine may be joined to the
carrier protein to then be concentrated in the liver, thus
localizing the therapeutic action of the agent (here, anticancer and
antiparisitic).
In some embodiments, liposomes of this invention, in association
with peptides, function as ad~uvants.
"Labile" therapeutic agents as used herein refers to the propensity
for destruction or denaturation of the therapeutic agent in an
animal by reactions other than the intended therapeutic reactions.
Preferred liposomes of this invention were prepared from fully
hydrogenated soy phosphatidylcholine and cholesterol in ratios of
from about 4:1 to about l:l (phosphatidylcholine:cholesterol mole
ratio). These liposomes were administered to An~ ~19 and the
An1 ~19 then tested for retention of administered material for
periods up to about 2 weeks post administration. Administered
material was found to be present at the site of administration for
up to two weeks at a level of about 20~ when administered via
intramuscular or subcutaneous administration. The extended
retention at in~ection site has facilitated extended release of
peptides over this period, while maintaining much of the integrity
of the administered material, particularly peptides. Other
preferred liposomes are of DSPC.
The administration profile that is therapeutically indicated will be
- influenced by many considerations including the increasing ease of
handling the stabile lipids, the therapeutic agent to be
incorporated into the liposome, the site of administration of the
liposome preparation, and the nature and condition of the animal
being treated.
1 3378~8 9
_ The term extended elaboration as used herein is understood to mean
the release of therapeutic agents from liposomal encapsulation over
a period in excess of about 24 hours and in some embodiments as long
as about 2 to 3 weeks.
;
Structural integrity of liposomes as used herein shall mean the
substantial maintenance of the pharmaceutical activity of the
encapsulated substance during a period of extended elaboration.
1 This structural integrity is presumed to arise from the persistence
of the bilayer arrangement of the lipid material comprising the
liposomes and the concomitant substantial maintenance of an
entrapped aqueous phase for the period of extended elaboration.
Structural integrity may be imparted by forming liposomes from
combinations of lipids comprising sufficient stabile lipid to
maintain the required structure when challenged by the physiological
conditions present in the sub~ect animal. In particular embodiments
the high integrity liposome of this invention structural integrity
will be maintained even if stabile lipid is ~ Yed with a diluent
lipid or secondary lipid which is not stabile. In the practice with
this invention liposomes of sufficient of structural integrity for
the intended use may be designed by varying the rigidity of lipid
membrane constituents or by varying the proportion in which stabile
lipid is a~ ~xed with a diluent or secondary lipid.
In the instant invention modifications of these above noted
procedures for making liposomes are required due to the high
rigidity of stabile lipids. In the most stabile lipids -- such as
fuLly hydrogenated phosphatidylcholine -- if a lipid film in the
preparation process it may be solubilized in organic solvent at an
elevated temperature often about 50-60C. This increases the
flexibility of the lipid and permits formulation of liposomes.
Stabile lipids of greater flexibility may be prepared by a variety
of methods. Hydrogenation of lipid to less than full hydrogenation
produces a lipid of increased but less than ~Y~ rigidity.
Additionally, fully or partially hydrogenated lipids or other
1 337898 lo
- stabile lipids may be admixed with unsaturated flexible lipids.
Cholesterol as an a~- ~xlng material is unique in that it tends to
make stabile lipids more flexible while conversely having the
property of rendering flexible lipids more ridged. Cholesterol is a
preferred ~1 lxlng lipid to increase the flexibility of the stabile
lipid component of the liposomes of this invention. Additionally,
alpha-tocopherol may function as an a~ l~lng lipid.
High integrity liposomes of this invention may be incorporated with
therapeutic agents by the methods well known in the art such as
those of U.S. Patent Nos. 4,522,803 and 4,588,57.8 and the Mayer
article, su~ra. Particular embodiments in the practice of this
invention are the incorporation of peptide therapeutic agents such
as growth hormone or growth hormone releasing factor.
Particular benefits of the liposomes of this invention are the
enhancement of drug entrapment upon formation of the liposomes, and
the enhancement of amount of drug per amount of lipid (loading).
The entrapment of up to about 70~ of available peptide-like
therapeutic agents are obtainable by the instant method. It is
important to note that peptide entrapment levels are dependent on
the specific peptide-like therapeutic agents being entrapped.
Liposomes entrap an aqueous medium which is enclosed by the lipid
bilayers. The aqueous medium can be for example, water or water
containing a dissolved salt or buffer. Examples of such salts or
buffers can be sodium chloride and phosphate buffered saline (PBS).
- 30 Other buffers include but are not limited to borate, citrate,
Tris-HCl(Tris-(hydroxymethyl)-~rln- ?thane hydrochloride), and HEPES
(N-2-hydroxyethyl piperazine-Nl-2-ethane sulfonic acid). Buffers
- may be in the pH range of between about 2.0 and about 14Ø In
particular embodiments, the preparations are hydrated with HEPES
buffer (150 mM NaCl, 20mM HEPES), pH 7.0, borate buffer (100 mM
Na2HC03, 50 mM H3B03), pH 8.5, or citrate buffer (150 MM
Na-citrate), pH 8.5, or O.OlM sodium carbonate buffer (pH 9-11).
1 ~3~B98 11
_ The loading of liposomes (i.e.,lipid:peptide) is also enhanced by
the use of stabile lipids in the practice of this invention. For
example, when liposomes entrapping BSTH were formed in the presence
of lipid:BSTH at 2:1 the liposomes thus formed were 6.1:1
lipid:peptide as opposed to 16.2:1 for similar liposomes formed from
unhydrogenated lipids. In another instance liposomes prepared from
lipid:galactose-albumin (1.8:1 feed) yielded a
lipid:galactose-albumin liposome of 4.3:1 as opposed to 7.1:1 for
unhydrogenated liposomes at a similar lipid:galactose-albumin feed
ratio.
In another example, DSPC liposomes, optionally mixed with
cholesterol (preferably 7:3 mole% ratio of phospholipid to
cholesterol), were formed entrapping calcitonin. Calcitonin is
available in many forms and analogues and derivatives of calcitonin
are being developed or are now available and all are understood to
be included in the term calcitonin. DSPC is obtainable from Avanti
Polar Lipids (Bil ~nghr , Ala.). The high integrity liposomes of
this invention extended the presence of detectable calcitonin from
about 1 hour for free calcitonin to about 3 to 7 days.
In a liposome-drug delivery system, the therapeutic agent i9
encapsulated in the liposome (either in the lipid or aqueous phase),
and then administered to the sub~ect being treated. For example,
see Rahman et al., U.S. Patent No. 3,993,754; Sears, U.S. Patent No.
4,145,410; Papahad~opoulos et al., U.S. Patent No. 4,235,871;
Schneider, U.S. Patent No. 4,224,179, Lenk, et al., U.S. Patent No.
4~522,803, and Fountain et al., U.S. Patent No. 4,588,578.
The liposomes may be dehydrated, thereby enabling storage for
extended periods of time until use. Standard freeze-drying
equipment or equivalent apparatus may be used to dehydrate the
liposomes. Liposomes may also be dehydrated simply by placing them
under reduced pressure. Alternatively, the liposomes and their
surrounding medium can be frozen in liquid nitrogen prior to
dehydration. Dehydration with prior freezing may be performed in
133789~ 12 -
the presence of one or more protective sugars in the preparation,
according to the process of Janoff et al., Canadian Patent No.
1,270,197,issued June 12, 1990, entitled "Dehydrated Liposomes".
Examples of protective sugars that may be used include, but are
not limited to, trehalose, maltose, sucrose, glucose, lactose and
dextran. When the dehydrated liposomes are to be used, rehydration
is accomplished by methods which include simply adding an aqueous
solution, e.g., distilled water, to the liposomes and allowing them
L0 to rehydrate.
The therapeutic agents of this invention are administered associated
with liposomes, in admixture with a pharmaceutically-acceptable
carrier selected with regard to the intended route of administration
and standard pharmaceutical practice.
Therapeutic agents are pharmacoactive agents, d~ag~c~tic a8ents,
ad~uvants, contrast agents, radioactive agents, drug targeting
carrier agents such as galactose-albumin, and the like. Preferred
agents are peptide-like therapeutic agents which include the
peptide-like agents such as galactose-albumin carriers,
i3munomodulators (e.g., interleukins) ard hor~ones
(e.g., somatotropins). Particular therapeutic agents referred to
are understood to include aralogues and deravitives of such agents
also including biologically ac~ive f.age~ents unless specifically
indicated to not so include.
Pharmaceutical dosage forms of the present inventions are comprised
of liposomes and any suitable phar aceutical carrier. A preferred
class of carrier i9 aqueous including both distiiled water and
isotoric salire. Administrat~on of high integrity liposomes may ~e
accomplished by any usual route with particular reference to
subcutaneous and intramuscular admiristration. Parenteral
administration as used herein refers to intra muscular, intravenous,
intra-articular, and intra-ocular administration. However, dosages
adapted to parenteral administration may be used in a variety of
administration methods.
1 337898 13 -
- Dosages for therapeutic agents associated with liposomes will often
be about that of the therapeutic agent alone; dosages will be set by
the prescribing medical professional considering many factors
including the age, weight and condition of the patient. The ratio
of therapeutic agent to carrier will naturally depend on the
chemical nature, solubility, trapping efficiency, and stability of
the therapeutic agent, as well as the dosage contemplated. For
parenteral administration or in~ection via such routes as
intravenous, intraperitoneal, intramuscular, subcutaneous, or
intra sry route, sterile solutions of the liposome composition
are prepared. For intravenous use, the total c~ncentration of
solutes should be controlled to render the preparation isotonic.
In the application of this invention to animal husbandry those
skilled in the art will understand the use of high integrity
liposome~ in the administration of a number of therapeutic agents
including growth promotant~ (e.g., growth hormome and growth
releasing factor) as well as lactation promotlng agents such as
BSTH. A~- ~n~ ~tration of such agents in therapeutically effective
amounts can increase productivity,
Dosage and administration of therapeutic agents, in this invention,
presumes therapeutically effective amountQ. Therapeutically
effective amounts of therapeutic agents as used herein will mean
that amount of therapeutic agent that produces therapeutic action.
This amount will be understood to vary with the particular agent or
analog or derivative thereof, the condition being treated, the site,
manner and duration of administration and other considerations known
to those skilled in the art.
In the application of this invention to the vaccine art, the
- necessary dosage will be understood to be an immunogenic dosage. It
will be understood that an immunogenic amount of of an antigen
protein such as GM2 is that amount which will stimulate the response
cells of a sub~ect animal (if in the GM2 example a human is presumed
to be the sub~ect animal then a response cell is a B-cell) to
1 3 3 7 8 q 8 - 14 _
- produce immunoglobulins against the antigen (here, GM2). This
amount will vary with the potency of adjuvant, with the mode of
administration and with the type and condition of animal but is
easily determined by any of the well known tests for immunoglobulins
with an increase in immunoglobulin representing immunogenic response.
In another example of their use, high integrity liposomes may be
incorporated into a broad range of topical dosage forms including
but not limited to gels, oils, emulsions and the like. For
instance, the suspension containing the high integrity liposomes may
be added to the aqueous phase as an ingredient in the liposome
preparation. Such preparations may be administered as topical
creams, pastes, ointments, gels, lotions and the like for direct
application.
ExamDle
PreDaration of Hi~h Inte~ritY LiDosomes
A solution of 14g of bovine somatotrophic hormone (BSTH) in 140ml
carbonate buffer, pH 10.9 was prepared. Then 21.1g of hydrogenated
soy phosphatidylcholine in powdered form and 6.9g of cholesterol in
powdered form were dissolved in 50ml chloroform and dried to powder
by rotoevaporation. The lipid film thus formed was resuspended in
140ml diethyl ether and placed in a round bottom flask, and
sonicated in a water bath at 47C during addition of the BSTH in
buffer. Sonication was continued until substantially all ether was
evaporated. A stream of 20C nitrogen was then applied to the
resulting material until residual ether was removed.
The resulting material was then resuspended in 800ml of buffer at 47
~ - 50C and the liposomes therein washed two times by repeated
centrifugation at approximately 20,000 time~ gravity for 30
minutes. The washed liposomes were then resuspended to a final
volume of 164ml.
1 337898 - 1S
_ The resulting high integrity liposome suspension contained 27.5mg
BSTH/ml, 128.0mg HSPC/ml and 42.0mg cholesterol/ml.
Example 2
;
Increasin~ Milk Production
The liposome preparation of Example 1 was administered to COW9 at
two 350mg doses per cow with the second dose two weeks after the
original dose. This regimen produced a 14.9% increase in milk
production as compared to untreated COW9. Furthermore, as compared
to cows treated with daily in~ection of 12.5mg free BSTH, two
injections of BSTH in hydrogenated soy phosphatidylcholine liposomes
was 28.8X as effective. Liposomes of unhydrogenated soy
phosphatidylcholine, delivered in 3 weekly doses of 175mg BSTH over
three weeks, were only 15.7X as effective as daily doses.
ExamDle 3
Extended Elaboration/Retention of Hi~h Inte~ritY LiDosomes In Situ
Preparation:
6.0g of BSTH was dissolved on 60.0ml of carbonate buffer pH 9.4.
Powdered HSPC, 3.52g and powdered cholesterol, 1.73g were dissolved
in 20ml chloroform in a 500ml round bottom flask. To this solution
was added 1.2 x 106dpm of 3H-dipalmitoyl phosphatidylcholine.
The lipids were then dried by rotoevaporation, and resuspended in
75ml diethyl ether. The flask and contents were then placed in a
- 45C water bath sonicator. The BSTH solution was then added to the
diethyl solution while sonicating as the ether evaporated. After 15
minutes residual ether was removed by applying a stream of nitrogen
to the contents of the flask. The contents of the flask was then
resuspended in 150ml carbonate buffer and the resulting liposomes
1 337898 - 16 -
_ washed two times by centrifugation and brought up to a final volume
~ of 34.Oml.
Elaboration/Retention:
;
A 0.320ml dose of the liposome suspension was in~ected
intramuscularly (leg) into each of 30 Swiss-Wistar mice. This
corresponded to 9.77 x 105dpm per animal. Three mice were
sacrificed at each time point over a period of 27 days. The
percentage of radioactivity r; ~n~ng at the site of injection was
compared to similar in~ection of unhydrogenated liposomes (egg
phosphatidylcholine with egg phosphatidylethanolamine).
Radioactivity in the mice receiving high integrity liposomes was
still present after 27 days while the radioactivity of the
unhydrogenated liposomes had almost entirely dissipated as is shown
in Fig. 1.
Example 4
Extended Elaboration/Retention of Hi2h InteRritY LiPosomes In Situ
Preparation:
0.75g of BSTH were dissolved in 15ml carbonate buffer, pH 10.9.
- 1.13g of HSPC and 0.37g of cholesterol are dissolved in 5ml
chloroform. The mixture was dried under rotoevaporation as in
Example 3, and the lipid resuspended in 20ml of diethyl ether, and
placed in a round bottom flask.
The flask and contents were placed in a 47C water bath sonicator,
the sonicator turned on, and then the BSTH in the aqueous phase was
added. Sonication was continued until the weight of the mixture was
equal to the additive weights of the dried lipid film and the
aqueous phase. A stream of nitrogen gas WaQ then applied to remove
1 337898
- 17 -
_ any residual ether. The contents of the flask was then resuspended
in 40ml of carbonate buffer at 47-50C and the liposomes therein
were washed two times by repeated centrifugation at approximately
20,000 times gravity for 30 minutes. The liposome suspension was
then brought to a final volume of ll.lml.
Fig 2 depicts the effect of these liposomes showing substant weight
gain over 28 days by hypophysectomized rats
(75-85gm/rat)administered BSTH in the high integrity liposomes as
compared to hypophysectomized rats administered single identical
amounts of 80ug/day (2,400ug in total per animal~ in~ected i.m. in
the hind leg of BSTH in unhydrogenated liposomes.
Example 5
IL-2 Retention and Release
67.0mg HSPC and 33.0mg cholesterol were dried from chloroform by
rotoevaporation in a round bottom flask. Then 5ml of anhydrous
diethyl ether was added to the flask. To this was added lmg of IL-2
in lml of 5mM ammonium acetate (pH5), 0.9X sodium chloride along
with 6.0 x 105 DPM of 3H-IL-2.
The resulting dispersion of aqueous solution in ether was sonicated
in a water bath at 45-50C and simultaneously dried with a stream
of nitrogen gas until no trace of ether was detectable by smelling.
To the resulting dried liposome paste was added to lOml of phosphate
buffered saline. The mixture was vortexed vigorously to remove any
material that had adhered to the flask wall. Aliquots of this
mixture were taken for tritium counting.
The re ~in~ng mixture, a liposome suspension, was centrifuged for 20
minutes in a J-20 rotor (Beckman Instruments, Mountainview, CA.) at
lO,OOOrpm. The supernatant was removed and an additional lOml of
phosphate buffered saline was used to resuspend the liposomes of the
pellet.
1 337898 - 18 -
- Table 1 shows the substantial period (14 days) over which IL-2remains at the in~ection site in mice receiving 2.7mg /kg in lOmg of
high integrity liposomes.
;
Table 2 Shows that the retention was accompanied by persistence of
available bioactive interleukin. Such persistence is seen to be
quite pronounced as compared to free interleukin or to liposomal
interleukin with other than high integrity liposomes.
Exam~le 6
Galactose-Albumin-Primaauine
HSPC:Cholesterol liposomes were prepared according to the method of
Example 1. Briefly, lipid in chloroform was dried as a thin film on
the bottom of a round bottom flask by rotoevaporation. 5ml of
diethyl ether were added to the flask and the lipid dislodged from
the flask walls by swirling. In this example the material to be
entrapped was human serum albumin conjugated to tritiated galactose
("galactose-albumin") and primaquine
("galactose-albumin-primaquine"). The galactose-albumin-primaquine
in 0.3ml of aqueous solution was added to the 5ml of ether-lipid
mixture.
Con~ugate formation was effected by simultaneous sonication and
drying of the mixture using a gentle stream of nitrogen. Sonication
and drying were discontinued when no odor of ether could be
detected. The con~ugate material was in the form of a paste which
wa-~ washed with 10-20ml of phosphate buffered saline (P8S). This
was accomplished by adding PBS to the flask and vortexing vigorously
to remove all residues from flask walls.
The resulting liposome suspension was then centrifuged at 4C for
10-20 minutes at lO,OOOrpm (J-20 Centrifuge, JA-20 rotor, Beckman).
The resulting supernatant was poured off and the pellet resuspended
in fresh PBS, vortexed and centrifuged again under the same
1 337898 - 19 -
- conditions two times to remove non-entrapped material.
193 mg of the resulting encapsulated contA~n~ng 107disentegrations
; per minute material was in;ected intramuscularly into the hind leg
of mice.
The retention of high integrity liposomes containing
galactose-albumin can be seen from Table 3 and Table 4 to be at
least 14 days. Table 3 discloses the retention at site of in~ection
of radioactivity for up to 14 days when the radioactive material was
encapsulated in high integrity liposomes, while, in contrast, free
radioactive material was eliminated in a single day. Table 4
discloses that in~ected encapsulated material r o~n~ng at the site
of in~ection retain~ bioactivity for en extended period as compared
to the activity retention of 1mencapsulated material.
Exam~le 7
Calcitonin-DSPC
8.27mg DSPC (1:1 initial L:D ratio) and 1.73mg cholesterol (7:3
mole% ratio DSPC: cholesterol), both in chloroform, were transferred
to a 50 ml round bottom flask. The lipids were dried to a film
under vacuum using rotoevaporation. The lipids were then
solubilized in 0.500 ml methanol with heating for 1-2 mins. in a 60
water bath.
Ten milligrams of calcitonin (Mitsubishi Chemical Co., Japan
MCI-536) was solubilized in 0.100 ml sodium acetate buffer. This
solution was added to the solvent mixture.
The solvent was removed under vacuum, using rotoevaporation in a 60
water bath. Once the solvent was removed, the lipid/drug film was
resuspended in 0.5 ml 60 sodium acetate buffer. The preparation
was washed by addition of 0.5 ml of 60 sodium acetate buffer
follwed by centrifugation at 12,100 xg for 10 minutes. The
1 337898 - 20 -
~ supernatant was decanted and the liposomal pellet resuspended with 1
ml of 60 sodium acetate buffer. The suspension was again
centrifuged at 12,100 xg for 10 minutes and resuspended. The
resuspended DSPC-cholesterol liposomes contA~nlng calcitonin were
administered s.c. to mice to compare retention of the liposomal
preparation to that of free calcitonin. Free calcitonin was
undetectable after one hour in mice. The calcitonin of liposomes of
the instant invention was at least about 70X present after one day
from administration and persisted for about 3 to 7 days disclosing a
substantial increase in retention time over free calcitonin.
1 337898
TABLE 1
I.M. SLOW RELEASE OF 3H LABELED ~ ES
FROM HSPC/CHOL LIPOSOMES IN MICE
% of Dose Remaining
Time Afterat In~ection Site
In~ectionInterleukin 2
1 Hr 84.4
1 Day 78.1
4 days 64.9
7 Days 69.4
10 Days 58.9
14 Days 28.1
TABLE 2
RECOVERY OF "BIOACTIVE" PEPTIDE AT THE
INJECTION SITE (IL2)
% of Initial Bioactive IL2 ~em~ining at In~ection Site
Time Free IL2 IL2-EPCIL2-HSPC/CHOL
SPLV SPLV
1 Hr 2.1 53.5 52
1 Day ND 15 44
4 Days ND 4.8 20
7 Days ND 1.3 11
10 Days ND ND 18
14 Days ND ND 14
- 22-
1 3 3 7 8 q 8 TABLE 3
I M SLOW RELEASE OF 3H r~R~?~ PEPTIDES
FROM HSPC/C LIPOSOMES IN MICE
X OF DOSE REMAI~ING AT INJECTION SITE
Time After HSPC Free
In~ectionGalactose-Albumln Galactose-Albumin
1 Hr 109 55 7
7 Hr- 29 7S
1 Day 107 25 5
1 7S Day- 11 23
2 7S Day~ 5 8
3 Day- 97
S DayJ 7S
5 75 Day~ 1 8
7 Day- 59
10 Day~ 47
14 D-y- 23
- 23 -
I 3~ /8 9~
TABLE 4
CHANGE IN FRAGMENTATION OF PEPTIDE
FOLLOWING LIPOSOME ENCAPSULATION
% OF PEPTIDE AT INJECTION SITE THAT IS FRAGMENTED
Fre~ HSPC/C ENCAPSULATED
Tim~Galactos~-Albumln-Primaquine Galactos~-Albumin-Primaquine
1 Hr 16
7 Hr~ 87
2 Day~ 100
3 Day~ 100 7
5 Day~ 100 15
7 Day~ 100 21
10 Day~ 100 17
14 DayJ 100 16
