U.S. patent application number 13/123112 was filed with the patent office on 2011-10-13 for liposomal systems comprising sphingomyelin.
This patent application is currently assigned to Yissum Research Development Company of the Hebrew University of Jerusalem, Ltd.. Invention is credited to Yechezkel Barenholz, Rivka Cohen.
Application Number | 20110250266 13/123112 |
Document ID | / |
Family ID | 41625213 |
Filed Date | 2011-10-13 |
United States Patent
Application |
20110250266 |
Kind Code |
A1 |
Barenholz; Yechezkel ; et
al. |
October 13, 2011 |
LIPOSOMAL SYSTEMS COMPRISING SPHINGOMYELIN
Abstract
The present disclosure provides a liposomal system comprising an
aqueous medium having dispersed therein liposomes encapsulating in
their intraliposomal aqueous compartment at least one active agent,
the aqueous medium being in iso-osmotic equilibrium with said
intraliposomal aqueous compartment, the liposomes having a membrane
comprising a liposome forming lipids, at least one of which being
sphingomyelin (SPM), the liposomal system having increased
stability as compared to the same liposomes free of SPM, and in one
embodiment being stable during long-term storage, said stability
being characterized in that no more than 30% of the at least one
active agent is present in the aqueous medium after said storage.
Further provided by the present disclosure are a method for storage
of liposomes making use of the liposomal system; use of the
liposomal system for the treatment of a medical condition or for
the diagnostic of a medical condition; a pharmaceutical or
diagnostic composition comprising the liposomal system, and a
method of treating or diagnosing of a medical condition comprising
administering to a subject an amount of the liposomal system.
Inventors: |
Barenholz; Yechezkel;
(Jerusalem, IL) ; Cohen; Rivka; (Jerusalem,
IL) |
Assignee: |
Yissum Research Development Company
of the Hebrew University of Jerusalem, Ltd.
|
Family ID: |
41625213 |
Appl. No.: |
13/123112 |
Filed: |
October 11, 2009 |
PCT Filed: |
October 11, 2009 |
PCT NO: |
PCT/IL2009/000966 |
371 Date: |
June 22, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61103440 |
Oct 7, 2008 |
|
|
|
Current U.S.
Class: |
424/450 ;
514/179; 514/330 |
Current CPC
Class: |
A61P 29/00 20180101;
A61K 31/573 20130101; A61K 9/127 20130101; A61K 31/445 20130101;
A61P 23/02 20180101 |
Class at
Publication: |
424/450 ;
514/330; 514/179 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61P 29/00 20060101 A61P029/00; A61K 31/573 20060101
A61K031/573; A61K 31/445 20060101 A61K031/445 |
Claims
1. A liposomal system comprising an aqueous medium having dispersed
therein liposomes encapsulating in their intraliposomal aqueous
compartment at least one active agent, the aqueous medium being in
iso-osmotic equilibrium with said intraliposomal aqueous
compartment, the liposomes having a membrane comprising liposome
forming lipids, at least one of which being sphingomyelin (SPM),
the liposomal system having increased stability as compared to the
same liposomes free of SPM.
2. The liposomal system as claimed in claim 1, being stable during
long-term storage, said stability being characterized in that no
more than 30% of the at least one active agent is present in the
aqueous medium after said storage.
3. The liposomal system as claimed in claim 2, wherein no more than
10% of the at least one active agent is present in the aqueous
medium after said storage.
4. The liposomal system as claimed in claim 1, wherein said SPM is
a C12-C24 SPM, and is selected from a synthetic or semi-synthetic
SPM.
5-6. (canceled)
7. The liposomal system as claimed claim 1, wherein said membrane
comprises SPM in an amount between 25 to 75 mole % of the total
phospholipids in said membrane.
8. The liposomal system as claimed in claim 1, comprising a mole
ratio between the liposome forming lipids other than SPM and said
SPM in the range of 1:1 to 2:1.
9. The liposomal system as claimed in claim 1, wherein said
liposome forming lipids have together a solid ordered to liquid
disordered phase transition temperature (T.sub.m) above 37.degree.
C.
10. The liposomal system as claimed in claim 1, wherein said
membrane comprises a sterol.
11. (canceled)
12. The liposomal system as claimed in claim 1, wherein said
liposomes are multilamellar vesicles (MLVs) or multivesicular
vesicles (MVVs).
13. (canceled)
14. The liposomal system as claimed in claim 1, wherein said
aqueous medium and said intraliposomal aqueous compartment have an
osmolarity between 50 to 600 mOsm/kg.
15. (canceled)
16. The liposomal system as claimed in claim 1, wherein said
aqueous medium and said intraliposomal aqueous compartment have an
osmolarity difference of no more than 50 mOsmole.
17-22. (canceled)
23. The liposomal system as claimed in claim 1, wherein the mole
ratio between said active agent and said liposome forming lipids
being above 0.5 mole/mole.
24. (canceled)
25. A method for storage of liposomes encapsulating in their
intraliposomal aqueous compartment at least one active agent, the
liposomes having a membrane comprising liposome forming lipids, at
least one liposome forming lipid being sphingomyelin (SPM), the
method comprising forming a liposomal system where said liposomes
are dispersed in an aqueous medium being in an iso-osmotic
equilibrium with the intraliposomal aqueous compartment of said
liposomes and storing said liposomal system, said liposomal system
having increased stability as compared to the same liposomes free
of SPM.
26. The method as claimed in claim 25, where no more than 30% of
the at least one active agent is present in the aqueous medium
after said storage.
27-33. (canceled)
34. The method as claimed in claim 25, wherein said membrane
comprises SPM in an amount between 25 to 75 mole % of the total
lipids in said membrane.
35. The method as claimed in claim 25, comprising a mole ratio
between the liposome forming lipids other than SPM and said SPM in
the range of 1:1 to 2:1.
36. The method as claimed in claim 25, wherein said liposome
forming lipids have together a solid ordered to liquid disordered
phase transition temperature (T.sub.m) above 37.degree. C.
37-42. (canceled)
43. The method as claimed in claim 25, wherein said aqueous medium
and said intraliposomal aqueous compartment have an osmolarity
difference of no more than 50 mOsmole.
44-53. (canceled)
54. A method of treating or diagnosing of a medical condition
comprising administering to a subject an amount of the liposomal
system as claimed in claim 1.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the field of liposome
technology.
BACKGROUND OF THE INVENTION
[0002] Among other applications, liposomes are used as carriers of
drugs for delivery via a plurality of mechanisms. To this end,
various types of liposomes are used, from small unilamellar
vesicles (SUV), large unilamellar vesicles (LUV), multilamellar
vesicles (MLV), multivesicular vesicles (MVV), large multivesicular
vesicles (LMVV, also referred to, at times, by the term giant
multivesicular vesicles, "GMV"), oligolamellar vesicles (OLV), and
others. It is appreciated by those versed in the art that LMVV are
somewhat different from unilamellar vesicles of various sizes and
of the "onion like" MLV structure. In LMVV the amount of aqueous
medium forming the aqueous phase per the amount of lipid is greater
than that in MLV, this potentially allowing higher amount of drug
to be loaded into the aqueous phase, namely, higher drug to lipid
mole ratio in the LMVV when compared to MLV system of similar size
distribution. This difference was exemplified by Grant et al. 2004
[Anesthesiology 101(0:133-7, 2004] and in U.S. Pat. No. 6,162,462.
It has been found that the difference in structure between MLV an
LMVV not only allows higher loading of the drug into the liposomes
but also a prolonged release of the drug from the LMVV system.
[0003] Specifically, U.S. Pat. No. 6,162,462 discloses liposomal
bupivacaine compositions in which the bupivacaine is loaded by a
transmembrane ammonium sulfate gradient, the liposomes being giant
multivesicular vesicles (GMV, a synonym for LMVV) having a molar
ratio of encapsulated drug to lipid in said liposomal composition
of at least 1.0. A specific drug encapsulated in the liposomes of
U.S. Pat. No. 6,162,462 is the amphipathic analgesic drug
bupivacaine (BUP). These bupivacaine loaded LMVV have shown to be
provide superior analgesia in mice and humans [Grant et al. 2004
and U.S. Pat. No. 6,162,462, ibid.]. However a phenomenon that
still remains unresolved with these LMVV relates to leakage of
bupivacaine from the LMVV during storage at 4.degree. C. or room
temperature. Thus, after time, free drug is contained in the system
(the amount being above drug MTD) and the administration of the
liposomal system containing such free drug may result in toxicity
and unwanted side effects (from exposure high amounts of free
drug), unfavorable pharmacokinetics and shorter duration of the
therapeutic effect. Thus, there is a need in the art to provide a
system where leakage of drug from liposomes encapsulating same
during storage is reduced or prevented.
SUMMARY OF THE INVENTION
[0004] The present disclosure is based on the finding that large
multivesicular vesicles (LMVV) loaded with high amount of an
amphipathic drug (bupivacaine, BUP) can be stabilized, in terms of
reduced BUP leakage, if the liposomes' membranes comprise
sphingomyelin and the LMVV are within an aqueous medium being in an
iso-osmotic equilibrium with the intraliposomal aqueous medium.
[0005] Thus, the present disclosure provides, in accordance with a
first of its aspects a liposomal system comprising an aqueous
medium having dispersed therein liposomes encapsulating in their
intraliposomal aqueous compartment at least one active agent, the
aqueous medium being in iso-osmotic equilibrium with said
intraliposomal aqueous compartment, the liposomes having a membrane
comprising a liposome forming lipids, at least one of which being
sphingomyelin (SPM), the liposomal system having increased
stability as compared to the same liposomes free of SPM (namely,
where there is no SPM in the liposome forming membrane). In one
embodiment, the liposomal system is stable during long-term
storage, said stability being characterized in that no more than
30% of the at least one active agent is present in the aqueous
medium after said storage.
[0006] The present disclosure also provides, in accordance with a
second of its aspects, a method for storage of liposomes
encapsulating in their intraliposomal aqueous compartment at least
one active agent, the liposomes having a membrane comprising
liposome forming lipids, at least one liposome forming lipid being
sphingomyelin (SPM), the method comprising forming a liposomal
system where said liposomes are dispersed in an aqueous medium
being in an iso-osmotic equilibrium with the intraliposomal aqueous
compartment of said liposomes and storing said liposomal system,
the liposomal system having increased stability as compared to the
same liposomes free of SPM.
[0007] Also provided by some aspects of the present disclosure is
the use of a liposomal system as defined herein, for the
preparation of a pharmaceutical or diagnostic composition; as well
as the liposomal system as defined for use in the treatment of a
medical condition or for the diagnostic of a medical condition.
[0008] Further, an aspect of the present disclosure provides a
pharmaceutical or diagnostic composition comprising the liposomal
system as defined herein and at least one physiologically
acceptable carrier.
[0009] Yet further, the present disclosure provides a method of
treating or diagnosing of a medical condition comprising
administering to a subject an amount of the liposomal system as
defined herein.
[0010] In one preferred embodiment, the active agent is an
amphipathic compound, being loaded into the liposomes by remote
loading technique; the SPM is synthetic or semi-synthetic C16 or
C18 SPM and the liposomes are large multivesicular vesicles
(LMVV).
[0011] A particular liposomal system in accordance with the present
disclosure comprises LMVV formed from a combination of at least
hydrogenated soy phosphatidylcholine (HSPC), C16SPM, cholesterol
and encapsulating BUP.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] In order to understand the invention and to see how it may
be carried out in practice, embodiments will now be described, by
way of non-limiting example only, with reference to the
accompanying drawings, in which:
[0013] FIGS. 1A-1B are graphs showing the release of Bupivacaine
(BUP), during storage at 4.degree. C. (FIG. 1A) or at 37.degree. C.
(FIG. 1B), from large multivesicular vesicles (LMVV) of different
lipid compositions (BUP to phospholipid mole ratio of each is
given) which have been loaded with BUP using remote loading driven
by trans-membrane ammonium sulphate (AS) gradient.
[0014] FIGS. 2A-2B are graphs showing the release of Bupivacaine
(BUP), during storage at 4.degree. C. (FIG. 2A) or at 37.degree. C.
(FIG. 2B), from large multivesicular vesicles (LMVV) of different
lipid compositions (BUP to phospholipid mole ratio of each is
given) which have been loaded with BUP using remote loading driven
by trans-membrane calcium acetate (CA) gradient.
[0015] FIGS. 3A-3B are graphs showing the release of Bupivacaine
(BUP), during storage at 4.degree. C. (FIG. 3A) or at 37.degree. C.
(FIG. 3B), from LMVV of different lipid compositions (HSPC/CHOL 6/4
mole ratio; HSPC/C16SPM/CHOL 3/3/4 mole ratio; and HSPC100/CHOL 6/4
mole ratio, BUP to phospholipid mole ratio of each composition is
given) which have been loaded with BUP using the passive loading
approach.
[0016] FIGS. 4A-4C are graphs showing the duration of analgesia in
mice using various liposomal systems identified in Table 8 as
formulations 1 to 8 (identified in the Figures with in the
corresponding formulation number "x" as "lip x"), FIG. 4A showing
the effect of injected volume of liposomal BUP or in free form, the
amount of BUP being constant 6 mg/mouse; FIG. 4B showing the effect
of 5 different LMVV formulations, the amount of BUP being constant
3 mg; and FIG. 4C which describes a comparison of the eight
different LMVV formulations (Table 8) at a dose of 3 mg/mouse.
[0017] FIGS. 5A-5F are graphs comparing analgesia duration of two
different doses of BUP (3 mg/mouse and 6 mg/mouse) for the five
different LMVV formulations identified in Table 8 ("lip x" in FIGS.
5A-5E) and 2 different amounts (0.375 and 0.75 mg/mouse) of
non-encapsulated (free) BUP (in FIG. 5F); FIG. 5A comparing the
effect of lip 2 (3 and 6 mg BUP/mouse), FIG. 5B comparing the
effect of lip 3 (3 and 6 mg BUP/mouse), FIG. 5C comparing the
effect of lip 4 (3 and 4.5 mg BUP/mouse), FIG. 5D comparing the
effect of lip 5 (3 and 6 mg BUP), FIG. 5E comparing the effect of
lip 8 (3 and 6 mg BUP/mouse), and FIG. 5F comparing the effect of
free (non liposomal) BUP at 0.375 mg/mouse using two volumes (150
and 300 .mu.l) and 0.75 mg/mouse at a volume of 150 .mu.l.
[0018] FIG. 6 is a graph showing in vivo analgesia after 20 hours
of LMVV comprising HSPC:C16SPM:cholesterol [3/3/4] 3 mg BUP and
LMVV as described by Grant et al. 2004 and free BUP 0.75 mg/mouse
(the maximal tolerated dose, MTD).
[0019] FIG. 7 is a graph showing in vivo analgesia after 40 hours
of LMVV comprising HSPC:C16SPM:cholesterol [3/3/4] 3 mg BUP LMVV
and free BUP 0.75 mg/mouse (the maximal tolerated dose, MTD).
[0020] FIGS. 8A-8E are graphs comparing the change in level of free
bupivacaine (as % in storage medium) during the indicated storage
period, at 4.degree. C. of HSPC100/C16SPM/CHOL (3/3/4 mole ratio)
LMVV loaded with bupivacaine via the AS trans-membrane as is when
stored in various storage media (Saline, 0.5% BUP or 2.0% BUP).
DETAILED DESCRIPTION OF SOME NON-LIMITING EMBODIMENTS
[0021] The present invention is based on the understanding that
existing bupivacaine liposomal formulations such as those described
in U.S. Pat. No. 6,162,462, and Grant et al. (Grant et al. 2004,
ibid.) have a tendency to leak during long term storage at low
temperatures which may impose a risk of toxicity when administered
to subjects in need of the drug. These bupivacaine liposomal
formulations contained high drug to phospholipid ratio (>0.5
mole/mole) in large multivesicular vesicle (LMVV, referred to in
U.S. Pat. No. 6,162,462 as giant multivesicular vesicles, GMV),
albeit, following storage, a substantial amount of the a priori
encapsulated drug was found to be present in the external medium.
Thus, a novel liposomal system was designed where the amount of
free bupivacaine in the medium external to the liposomes was
significantly reduced after long term storage at 4.degree. C., as
compared to the hitherto existing bupivacaine liposomal
formulations. It was further found that while the liposomal system
was stable during storage at 4.degree. C., at physiological
conditions, namely, at 37.degree. C., bupivacaine was released from
the liposomes at a controlled and prolonged rate sufficient to get
long term (prolonged) analgesia.
[0022] Specifically, it has been found that liposomes comprising in
the liposome's bilayer sphingomyelin at the amount of up to 75% of
the total phospholipids (or 50% of total lipids (which include
.about.33 mole % cholesterol) forming the liposome's bilayer
decreased the amount of leakage without compromising the rate of
bupivacaine release from the liposomes at 37.degree. C. and without
compromising the high loading of the drug into the liposomes.
[0023] Thus, in accordance with a first of its aspects, the present
disclosure provides a liposomal system comprising an aqueous medium
having dispersed therein liposomes encapsulating in their
intraliposomal aqueous compartment at least one active agent, the
aqueous medium being in iso-osmotic equilibrium with said
intraliposomal aqueous compartment, the liposomes having a membrane
comprising liposome forming lipids, at least one of which being a
sphingomyelin (SPM), the liposomal system being stable.
[0024] It has been found that the stability of the SPM containing
liposomes is significantly greater than that of liposomes which do
not contain SPM in their lipid membrane. The stability of the
liposomal system is also determined in terms of long-term storage,
the stability being characterized in that no more than 30%, at
times, not more than 20% and even not more than 10% of the at least
one active agent of the system is present in the aqueous medium
after said storage.
[0025] As used herein, the term "liposomal system" denotes a system
comprising an organized collection of lipids forming at least one
type of liposomes, and enclosing at least one intraliposomal
aqueous compartment. In addition to the liposomes, the system
comprises an aqueous medium in which the liposomes are dispersed or
suspended.
[0026] The aqueous medium is any water based buffer solution having
a desired osmolarity and ion concentration and is to be understood
as encompassing a variety of physiologically acceptable buffers.
The buffer system is generally a mixture of a weak acid and a
soluble salt thereof, e. g., sodium citrate/citric acid; or the
monocation or dication salt of a dibasic acid, e. g., potassium
hydrogen tartrate; sodium hydrogen tartrate, phosphoric
acid/potassium dihydrogen phosphate, and phosphoric acid/disodium
hydrogen phosphate. A weak acid buffer is a buffer solution with
constant pH values of between 4 and 7 and a weak base buffer is a
buffer solution with constant pH values between 7 and 10. Some
non-limiting examples of buffers that may be used for producing the
aqueous medium in accordance with the present disclosure include
physiological saline (0.9% NaCl), phosphate buffered saline (PBS),
sucrose buffer, histidine buffer etc., set at a pH of between about
4 to 8, or between 5.5 to 7 (as typically used in liposomal drug
delivery system).
[0027] In one embodiment, the aqueous medium comprises an amount of
free active agent, the presence of said free active agent in the
aqueous medium allows or participates in the formation of said
iso-osmotic equilibrium. The amount of free active agent is
determined such to form said iso-osmotic equilibrium. As shown in
the examples herein, the presence of the free agent in the aqueous
medium, also reduced the leakage of eth agent from the liposomes
(this being comparable the same formulation without free drug in
the aqueous medium).
[0028] In the aqueous medium are dispersed liposomes. The term
"dispersed" is used to denote the distribution or suspension of the
liposomes in the aqueous medium.
[0029] As appreciated, liposomes are comprises of a lipid bilayer
comprising liposome forming lipids, discussed hereinbelow, and an
aqueous intraliposomal core. According to the present disclosure
the aqueous medium external to the liposomes and the intraliposomal
aqueous compartment are in iso-osmotic equilibrium. The iso-osmotic
equilibrium should be understood as meaning that the aqueous medium
and the medium of the intraliposomal aqueous compartment have
similar osmolarities, the similarity being defined by a difference
in osmolarity of not more than 50 mOsmole. In accordance with one
embodiment, the osmolarity of the aqueous medium and of the
liposomal aqueous phase are in the range of about 50 to about 600
mOsm/kg, or even between about 250 to about 550 mOsm/kg. The
iso-osmotic equilibrium may be obtained by washing the liposomes
encapsulating the active agent with the buffer solution having an
osmolarity similar to that of the intraliposomal aqueous
compartment. Specifically, once the active agent is loaded into the
liposomes, the non-encapsulated agent may be washed out by the
selected buffer solution.
[0030] The liposomes' membrane is a bilayer membrane and may be
prepared to include a variety of physiologically acceptable
liposome forming lipids. As used herein, the term "liposome forming
lipids" is used to denote primarily glycerophospholipids and
sphingomyelins. The glycerophospholipids have a glycerol backbone
wherein at least one, preferably two, of the hydroxyl groups at the
head group is substituted by one or two of an acyl, alkyl or
alkenyl chain, a phosphate group, or combination of any of the
above, and/or derivatives of same and may contain a chemically
reactive group (such as an amine, acid, ester, aldehyde or alcohol)
at the head group, thereby providing the lipid with a polar head
group. The sphingomyelins consists of a ceramide unit with a
phosphorylcholine moiety attached to position 1 and thus in fact is
an N-acyl sphingosine The phosphocholine moiety in sphingomyelin
contributes the polar head group of the sphingomyelin.
[0031] In the liposome forming lipids the acyl chain(s) are
typically between 14 to about 24 carbon atoms in length, and have
varying degrees of saturation being fully, partially or
non-hydrogenated lipids. Further, the lipid matrix may be of
natural source, semi-synthetic or fully synthetic lipid, and
neutral, negatively or positively charged.
[0032] Examples of liposome forming glycerophospholipids include,
without being limited thereto, glycerophospholipid.
phosphatidylglycerols (PG) including dimyristoyl
phosphatidylglycerol (DMPG); phosphatidylcholine (PC), including
egg yolk phosphatidylcholine, dimyristoyl phosphatidylcholine
(DMPC), 1-palmitoyl-2-oleoylphosphatidyl choline (POPC),
hydrogenated soy phosphatidylcholine (HSPC),
distearoylphosphatidylcholine (DSPC); phosphatidic acid (PA),
phosphatidylinositol (PI), phosphatidylserine (PS).
[0033] As appreciated, the liposome forming lipids may also include
cationic lipids (monocationic or polycationic lipids). Cationic
lipids typically consist of a lipophilic moiety, such as a sterol
or the same glycerol backbone to which two acyl or two alkyl, or
one acyl and one alkyl chain contribute the hydrophobic region of
the amphipathic molecule, to form a lipid having an overall net
positive charge. Preferably, the headgroup of the lipid carries the
positive charge.
[0034] Monocationic lipids may include, for example,
1,2-dimyristoyl-3-trimethylammonium propane (DMTAP)
1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP);
N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium
bromide (DMRIE);
N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethyl-ammonium
bromide (DORIE); N-[1-(2,3-dioleyloxy)
propyl]-N,N,N-trimethylammonium chloride (DOTMA);
3.beta.[N-(N',N'-dimethylaminoethane) carbamoly] cholesterol
(DC-Chol); and dimethyl-dioctadecylammonium (DDAB).
[0035] Polycationic lipids may include a similar lipophilic moiety
as with the mono cationic lipids, to which spermine or spermidine
is attached. These include, without being limited thereto,
N-[2-[[2,5-bis[3-aminopropyl)amino]-1-oxopentyl]amino]ethyl]-N,N-dimethyl-
-2,3-bis[(1-oxo-9-octadecenyl)oxy]-1-propanaminium (DOSPA), and
ceramide carbamoyl spermine (CCS). The cationic lipids may form
part of a derivatized phospholipids such as the neutral lipid
dioleoylphosphatidyl ethanolamine (DOPE) derivatized with
polylysine to form a cationic lipopolymer.
[0036] According to the present disclosure, the liposome forming
lipid comprises at least a sphingomyelin. The term "sphingomyelin"
or "SPM" as used herein denotes any N-acetyl sphingosine conjugated
to a phosphocholine group, the later forming the polar head group
of the sphingomyelin (N-acetylsphingosylphosphorylcholines). The
acyl chain bound to the primary amino group of the sphingosine may
be saturated or unsaturated, branched or unbranded. In one
embodiment, the acyl chain comprises between 12 to 24 carbon atoms
(C12-C24), at times between 14 to 20 carbon atoms. In some
preferred embodiments, the SPM is a C16:0 or C18:0 sphingomyelin,
namely, saturated C16 or C18 SPM. The SPM is preferably a synthetic
or semi-synthetic SPM, i.e. a derivative of a naturally occurring
SPM and may include the natural D-erythro (2S, 3R) isomer and the
non naturally occurring L-threo (2S, 3S) isomer, although the
former, i.e. the naturally occurring isomer is preferable.
[0037] In addition, in the context of the present disclosure, the
sphingomyelin is also used to denote the corresponding dihydro
species, namely, any dihydrosphingomyelins (DHSM) corresponding to
the SPM defined herein above.
[0038] In one embodiment, the liposomal system comprises SPM
content in the liposomes membrane in an amount between 25 to 75
mole % of the total phospholipids (liposome forming lipid) in said
membrane, or about 50 mole % of the total lipids when including
cholesterol.
[0039] In yet a further embodiment, the mole ratio between the
liposome forming lipids other than SPM and said SPM is typically in
the range of 1:1 to 2:1, irrespective of the SPM used in accordance
with the present disclosure.
[0040] Typically, the liposome forming lipids have when assembled
into the liposome membranes have a solid ordered (SO) to liquid
disordered (LD) phase transition temperature having a
characteristic temperature defined as T.sub.m>37.degree. C.
T.sub.m is the temperature within the range of the SO to LD phase
transition temperatures in which the maximal change in the heat
capacity of the phase transition occurs. Interestingly, it has been
found and also shown hereinbelow that the combination HSPC having a
solid ordered to liquid disordered with a T.sub.m at
.about.53.degree. C. with C16SPM having its T.sub.m at
.about.41.4.degree. C. surprisingly led to the formation of a
stable liposomal system, i.e. reduced drug leakage during 4.degree.
C. storage, as compared to a liposomal system lacking C16SPM which
was less stable, namely, showing higher rate of drug leakage during
4.degree. C. storage (i.e. same storing conditions).
[0041] The term "stablility" in the context of the present
disclosure is used to denote that the resulting liposomes were more
stable (less agent being leaked from the liposomes during or
following storage, the difference in leakage being statistically
significant) as compared to the same liposomes, albeit free of SPM,
namely, the liposome's membrane does not comprise SPM as part of
the liposome forming lipids. The stability may also be defined that
the drug loaded liposomes are chemically and physically unaltered
when stored at 4.degree. C. and for a period of at least 3 months.
The stability is determined, for example, by measuring the amount
of free active agent that present or was released (leaked) to the
extra-liposome aqueous medium, i.e. non-encapsulated active agent,
the amount indicative of stability being less than 30%, 20% and at
times even less than 10% from the total amount of active agent in
the liposomal system (the total amount including encapsulated and
non-encapsulated agent). Surprisingly, the results presented herein
show that when comparing a liposome formulation e.g. comprising
HSPC and Cholesterol with the amount of leakage of an encapsulated
agent from the same formulation, albeit with SPM in the lipid
membrane, leakage of the agent was reduced.
[0042] The liposomes may also comprise other lipids typically used
in the formation of liposomes, e.g. for stabilization, for
affecting surface charge, membrane fluidity and/or assist in the
loading of the active agents into the liposomes. Examples of such
lipids, may include sterols such as cholesterol, cholesteryl
hemisuccinate, cholesteryl sulfate, or any other derivatives of
cholesterol.
[0043] The liposomes may further comprise lipopolymers. The term
"lipopolymer" is used herein to denote a lipid substance modified
at its polar headgroup with a hydrophilic polymer. The polymer
headgroup of a lipopolymer is typically water-soluble and may be
covalently or non-covalently attached to a hydrophobic lipid
region. Typically, the hydrophilic polymer has a molecular weight
equal or above 750 Da and may be polar or apolar. Lipopolymers such
as those that may be employed according to the present disclosure
are known to be effective for forming long-circulating liposomes.
There are numerous polymers which may be attached to lipids to form
such lipopolymers, such as, without being limited thereto,
polyethylene glycol (PEG), polysialic acid, polylactic (also termed
polylactide), polyglycolic acid (also termed polyglycolide),
apolylactic-polyglycolic acid, polyvinyl alcohol,
polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline,
polyhydroxyethyloxazoline, polyhydroxypropyloxazoline,
polyaspartamide, polyhydroxypropyl methacrylamide,
polymethacrylamide, polydimethylacrylamide, polyvinylmethylether,
polyhydroxyethyl acrylate, derivatized celluloses such as
hydroxymethylcellulose or hydroxyethylcellulose. The polymers may
be employed as homopolymers or as block or random copolymers. The
lipids derivatized into lipopolymers may be neutral, negatively
charged, as well as positively charged. The most commonly used and
commercially available lipids derivatized into lipopolymers are
those based on phosphatidyl ethanolamine (PE), usually,
distearylphosphatidylethanolamine (DSPE).
[0044] One particular family of lipopolymers that may be employed
according to the present disclosure are the monomethylated PEG
attached to DSPE (with different lengths of PEG chains, in which
the PEG polymer is linked to the lipid via a carbamate linkage
resulting in a negatively charged lipopolymer, or the neutral
methyl polyethyleneglycol distearoylglycerol (mPEG-DSG) and the
neutral methyl poly ethyleneglycoloxy
carbonyl-3-amino-1,2-propanediol distearoylester (mPEG-DS)
[Garbuzenko O. et al., Langmuir. 21:2560-2568 (2005)]. Another
lipopolymer is the phosphatidic acid PEG (PA-PEG).
[0045] The PEG moiety has a molecular weight of the head group is
from about 750 Da to about 20,000 Da, at times, from about 750 Da
to about 12,000 Da and typically between about 1,000 Da to about
5,000 Da. One specific PEG-DSPE commonly employed in liposomes is
that wherein PEG has a molecular weight of 2000 Da, designated
herein .sup.2000PEG-DSPE or .sup.2kPEG-DSPE.
[0046] The liposomes of the liposomal system encapsulate at least
one active agent. Encapsulation includes the entrapment/enclosure,
in the intraliposomal phase, of at least one active agent. The
entrapment is a non-covalent entrapment, namely in the liposomal
aqueous phase the active agent is freely dispersed and may, under
appropriate conditions, be released from the liposomes in a
controlled manner.
[0047] The active agent may be a small molecular weight compound as
well as a polymer (e.g. peptide, protein, nucleic acid sequence
etc.). The term "active agent" is used to denote that the
encapsulated agent, once administered has a beneficial effect, e.g.
as a therapeutic, as a contrasting agent (e.g. radionuclei dyes or
dye-conjugates to carrier, chromophor or fluorophor producing agent
etc.), as a nutraceutical compound etc. The active agent may be a
water soluble, hydrophilic compound as well as an amphipathic
compound.
[0048] In one embodiment, the active agent is an amphipathic
compound. The term "amphipathic compound" is used to denote a
active agent possessing both hydrophilic and lipophilic properties.
There are various biologically active amphipathic compounds known
in the art. One example includes the anti cancer compound
doxorubicin. The loading of doxorubicin (e.g., DOXIL.TM.) into
preformed liposomes is driven by transmembrane ammonium sulfate
gradient (U.S. Pat. No. 5,192,549, U.S. Pat. No. 5,316,771 and
Haran et al., [Haran G, et al. (1993) Transmembrane ammonium
sulfate gradients in liposomes produce efficient and stable
entrapment of amphipathic weak bases. Biochim Biophys Acta.
1151(2):201-15].
[0049] In one other embodiment, the amphipathic active agent is an
analgesic drug. The analgesic drug would typically be for local
analgesic. A non-limiting group of analgesic drugs are selected
from the group consisting of benzocaine, chloroprocaine, cocaine,
cyclomethycaine, dimethocaine, propoxycaine, procaine,
proparacaine, tetracaine, articaine, bupivacaine, carticaine,
cinchocaine, etidocaine, levobupivacaine, lidocaine, mepivacaine,
piperocaine, prilocaine, ropivacaine, trimecaine, saxitoxin and
tetrodotoxin. A preferred group of analgesic drugs include, without
being limited thereof, bupivacaine, lidocaine, ropivacaine,
levobupivacaine, procaine, chloroprocaine, benzocaine, etidocaine,
mepivacaine, prilocaine, ciprocaine, tetracaine, dibucaine,
heptacaine, mesocaine, propanocaine, carbisocaine, and butacaine. A
specific analgesic drug according to the present disclosure is
bupivacaine (hereinafter referred to, at times, as "BUP").
[0050] In another embodiment, the active agent is a water soluble
molecule such as a peptide, protein or nucleic acid sequences,
including, for example, cytokines, antibodies, immunostimulating
oligonucleotides (ISS-ODN), siRNA etc.
[0051] As appreciated, liposomes in general may have various shapes
and sizes. The liposomes may be multilamellar liposomes (MLV) or
multivesiclular vesicles (MVV). MVV liposomes are known to have the
form of numerous non-concentric, closely packed internal aqueous
chambers separated by a network of lipid membranes and enclosed in
a lipid membrane. In the context of the present invention, the MVV
are referred to as large multivesicular vesicles (LMVV), also known
in the art by the term giant multivesicular vesicles (GMV). In
accordance with one embodiment, the liposomes typically have a
diameter of at least 200 nm, typically in the range of about 200 nm
and 25 .mu.m, at times between about 250 nm and 25 .mu.m.
[0052] When the liposomes are MVV or LMVV, it is to be understood
that the loading of the agent into the LMVV includes containment of
the agent in more than one aqueous compartment formed by the lipid
membranes, and typically also in the aqueous environment
surrounding the non-concentric lipid membrane. At times, the agent
may be entrapped (embedded) in the lipid membrane, e.g. when the
active agent is lipophilic compound.
[0053] The liposomal system disclosed herein is characterized by a
high active agent to lipid ratio, namely, high level of active
agent per liposome. Although not exclusively, the high loading
would typically depend on the type of liposomes used, their size,
the loading conditions etc. In one embodiment, a high loading is
achieved by active loading (see below) of the active agent into
LMVV under condition of high initial active agent concentration. In
the context of the present disclosure, high loading is used to
denote a loading with a active agent to lipid ratio in the
resulting liposomal system of at least about 0.5 mole drug per mole
liposome forming phospholipid ratio (mole/mole) (this being
characteristic of the LMVV according to the present
disclosure).
[0054] Loading of the active agent into the liposomes may be by any
technique known in the art. Such techniques typically include
passive loading or active ("remote loading") loading of agents into
the liposomes.
[0055] Passive loading techniques of encapsulating agents into
liposomes typically involve loading of the agent during preparation
of the liposomes, e.g. by hydrating dry liposome forming lipids
with a solution of the active agent. By passive loading the agent
may be associated to the liposomal membrane or encapsulated within
the aqueous core. One method for passive loading was described by
Bangham, et al., [Bangham A D, Standish M M, Watkins J C (1965)
Diffusion of univalent ions across the lamellae of swollen
phospholipids. J MoI Biol. 13(1):238-52], where an aqueous phase
containing the agent of interest is put into contact with a film of
dried liposomes-forming lipids deposited on the walls of a reaction
vessel. Upon agitation by mechanical means, swelling of the lipids
occurs and multilamellar vesicles (MLV) are thus formed. A further
method for passive loading is the Reverse Phase Evaporation (REV)
method described by Szoka and Papahadjopoulos, [Szoka F. C.
Jr.sub.5 Papahadjopoulos D. (1978) Procedure for preparation of
liposomes with large internal aqueous space and high capture by
reverse-phase evaporation. Proc Natl Acad Sci USA. 75(9):4194-8.],
according to which a solution of lipids in a water insoluble
organic solvent is emulsified in an aqueous carrier phase and the
organic solvent is subsequently removed under reduced pressure.
Other methods of passive loading include subjecting liposomes to
successive dehydration and rehydration treatment, or freezing and
thawing. Dehydration is carried out by evaporation or freeze-drying
[Kirby C and Gregoriadis G (1984) Dehydration-Rehydration Vesicles:
A Simple Method for High Yield Drug Entrapment in Liposomes. Nat.
Biotechnol. 2, 979-984], or mixing liposomes prepared by sonication
in aqueous solution with the solute to be encapsulated, and the
mixture is dried under nitrogen in a rotating flask. Upon
rehydration, large liposomes are produced in which a significant
fraction of the solute has been encapsulated [Shew R L, Deamer D W.
(1985) A novel method for encapsulation of macromolecules in
liposomes. Biochim Biophys Acta. 816(1):1-8]. Loading may be
improved co-lyophilizing the active agent with the dried liposome
forming lipids [International Patent Application Publication No.
WO03000227]
[0056] Active loading techniques are also used. For example,
liposomes may be loaded using an ion gradient or pH gradient as the
pre-formed liposome loading driving force. Loading using a pH
gradient may be carried out according to methods described in U.S.
Pat. Nos. 5,616,341, 5,736,155 and 5,785,987, U.S. Pat. No.
5,192,549, U.S. Pat. No. 5,316,771 and Haran et al., [Haran G, et
al. (1993) Transmembrane ammonium sulfate gradients in liposomes
produce efficient and stable entrapment of amphipathic weak bases.
Biochim Biophys Acta. 1151(2):201-15], incorporated herein by
reference. The pH gradient may be calcium citrate-based or ammonium
sulphate-based gradient.
[0057] According to one embodiment, the liposomes have the form of
multilamellar vesicles (MLV) or multivesicular vesicles (MVV),
preferably, large multivesicular vesicles (LMVV).
[0058] The present disclosure also provides a method for storage of
liposomes as defined above, i.e. encapsulating in their
intraliposomal aqueous compartment at least one active agent, the
liposomes having a membrane comprising liposome forming lipids, at
least one liposome forming lipid being sphingomyelin (SPM), the
method comprising forming a liposomal system where said liposomes
are dispersed in an aqueous medium being in an iso-osmotic
equilibrium with the intraliposomal aqueous compartment of said
liposomes and storing said liposomal system, whereby no more than
30%, at times no more than 20% and even no more than 10% of the at
least one active agent is present in the aqueous medium after said
storage.
[0059] The method allows long term stable storage (at low
temperatures, e.g. 4.degree. C.) of the liposomes. While at minimum
stable storage is for a period of 3 months, as will be shown in the
following non-limiting examples, stable storage was also obtained
for a period of four months (120 days), 4.5 months and even up to 6
months storing at 4.degree. C. However, as indicated above, the
stability would be retained at any other temperature that is lower
than the physiological temperature of the body, namely, below
37.degree. C. When referring to lower temperatures it is to be
understood that the reasonable storage temperature should be at
least 15.degree. C. below body core temperature, i.e. below
22.degree. C. According to one embodiment, storing is at a
temperature between about 2.degree. C. to 8.degree. C.
[0060] Due to the low leakage of the active agent during storage of
SPM-containing LMVV there it has been found that there is no need
to wash the liposomal system prior to administration to a subject
in need thereof. The liposomal system may be administered to the
subject in need thereof as is or may be combined with a
physiologically acceptable additive.
[0061] Thus, the present invention also provides the use of the
liposomal system as defined hereinabove for the preparation of a
pharmaceutical or diagnostic composition, for, respectively,
treatment of a medical condition or for diagnostic purposes. The
composition typically comprises, in addition to said liposomal
system, at least one physiologically acceptable additive.
[0062] Further, the present invention provides a method for the
treatment or diagnostic of a medical condition, the method
comprising administering to a subject in need of said treatment or
diagnostic an amount of the liposomal system as defined hereinabove
or physiologically acceptable composition comprising the same.
[0063] The liposomal system alone or in combination with
physiologically acceptable additives may be administered by any
route acceptable in the art. According to one embodiment, the
administration of the liposomal system is by parenteral injection
or infusion. This would include, without being limited thereto,
intravenous, intraarterial, intramuscular, intracerebral,
intracerebroventricular, intracardiac, subcutaneous, intraosseous
(into the bone marrow), intradermal, intratheacal, intraperitoneal,
intravesical, and intracavernosal and epiduaral (peridural)
injection or infusion. Pareneral administration may also include
transdermal, e.g. by transdermal patches, transmucosal (e.g. by
diffusion or injection into the peritoneum), inhalation and
intravitreal (through the eye).
[0064] When the active agent is an analgesic drug, a preferred mode
of administration is local administration by any acceptable route,
as can be determined by a medical doctor or any other appropriate
physician.
[0065] The amount of liposomal system administered, and thereby the
amount of active agent encapsulated therein should be effective to
achieve the desired effect by the active agent, at the target site.
For example, if the active agent is a drug, the amount of the
liposomal systems should be determined so that at the target site
the amount of the drug encapsulated therein is sufficient to
achieve the desired therapeutic effect. Such desired therapeutic
effect may include, without being limited thereto, amelioration of
symptoms associated with a medical condition, prevention of the
manifestation of symptoms associated with a medical condition, slow
down of a progression state of a medical condition, enhance of
onset of a remission period, prevent or slow down irreversible
damage caused by the medical condition, lessen the severity of the
medical condition, cure the medical condition or prevent it from
developing, etc. The medical condition to be treated by the
liposomal system may be any such condition treatable by the active
agent encapsulated in the liposomes according to the present
disclosure.
[0066] Further, if the active agent may be a diagnostic agent. To
this end, the amount of the liposomal system should be such that it
would be possible to image the marker at the target site.
[0067] The amount of the liposomal systems will be determined by
such considerations as may be known in the art, typically using
appropriately designed clinical trials (dose range studies
etc.).
[0068] As used herein, the forms "a", "an" and "the" include
singular as well as plural references unless the context clearly
dictates otherwise. For example, the term "a liposome forming
lipid" includes one or more lipids capable of forming a
liposome.
[0069] Further, as used herein, the term "comprising" is intended
to mean that the liposomal system include the recited constituents,
i.e. the liposome forming lipid, SPM and the active agent, but not
excluding other elements, such as physiologically acceptable
carriers and excipients as well as other active agents. The term
"consisting essentially of" is used to define liposomal systems
which include the recited elements but exclude other elements that
may have an essential significance on the effect to be achieved by
the liposomal system. "Consisting of" shall thus mean excluding
more than trace amounts of other elements. Embodiments defined by
each of these transition terms are within the scope of this
invention.
[0070] Further, all numerical values, e.g. when referring the
amounts or ranges of the elements constituting the liposomal system
comprising the elements recited, are approximations which are
varied (+) or (-) by up to 20%, at times by up to 10% of from the
stated values. It is to be understood, even if not always
explicitly stated that all numerical designations are preceded by
the term "about".
[0071] The invention will now be exemplified in the following
description of experiments that were carried out in accordance with
the invention. It is to be understood that these examples are
intended to be in the nature of illustration rather than of
limitation. Obviously, many modifications and variations of these
examples are possible in light of the above teaching. It is
therefore, to be understood that within the scope of the appended
claims, the invention may be practiced otherwise, in a myriad of
possible ways, than as specifically described hereinbelow.
DESCRIPTION OF SOME NON-LIMITING EXAMPLE
Materials
[0072] Drugs:
[0073] Bupivacaine hydrochloride (B UP) USP XXIII (Orgamol, SA,
Evionnaz, Switzerland).
[0074] Methylprednisolone sodium succinate (MPS) (PHARMACIA NV/SA
Puurs-Belgium).
[0075] Lipids:
[0076] Cholesterol (CHOL) (NF; Solvay Pharmaceuticals (Veenedaal,
Netherlands).
[0077] Fully hydrogenated soy phosphatidylcholine (HSPC-100),
Phospholipon.RTM. 100H batch no 50190 (Phospholipids GmbH
Nattermannallee 1*D 50829 Koln, Germany). HSPC100 is 99.5 pure,
i.e. comprising lysoPC and fatty acid in an amount less than the
detectable limit.
[0078] Fully hydrogenated soy phosphatidylcholine (HSPC) (Lipoid
Gmbh, Ludwigshafen, Germany). 98.0 pure, i.e. comprising less than
1.2% lysoPC and about 1% fatty acid.
[0079] Fully synthetic
N-Palmitoyl-D-erythro-sphingosine-1-phosphocholine, N-palmitoyl
sphingomyelin, (C16-SPM) >98% pure, Lot no. 546701 (Biolab Ltd.,
POB 34038 Jerusalem 91340).
[0080] Buffer:
[0081] Ammonium sulfate (AS, MERCK);
[0082] Calcium acetate monohydrate (CA, Aldrich);
[0083] Calcium chloride-dihydrate (MERCK);
Methods
Preparation of Drug Loaded LMVV
Preparation of Large Multi Vesicular Vesicles (LMVV)
[0084] Powder mixtures of lipids at the desired mole ratio (see
Table 1 for details regarding constituents and mole ratios) were
dissolved in ethanol at 60-65.degree. C. and added to an aqueous
solution (ammonium sulfate (AS), calcium acetate (CA) or another
buffer, as indicated below) to reach a final phospholipid (PL)
concentration of 60 mM and final ethanol concentration of 10%.
[0085] The resulting solutions were mixed for 30 min at 65.degree.
C. to obtain multilamellar vesicles (MLV). Alternative methods to
prepare MLV can also be used (see for example: Barenholz &
Crommelin, 1994, In: Encyclopedia of Pharmaceutical Technology.
(Swarbrick, J. and Boylan, J. C., Eds.), Vol. 9, Marcel Dekker, NY
pp. 1-39).
[0086] LMVV were prepared from the MLV with the desired aqueous
phase (for example: ammonium sulfate 250 mM or 127 mM, calcium
acetate 250 mM, or 200 mM; or a desired buffer) from the MLV by
exposing the MLV to 10 cycles of freezing in liquid nitrogen and
thawing in a water bath at 60.degree. C. thereby forming the LMVV.
At each cycle, each 1 ml of dispersed LMVV solution was kept at the
liquid nitrogen for 1 minute. For example, a dispersion of 3 ml was
kept in liquid nitrogen for 3 minutes.
Gradient Creation
[0087] Transmembrane AS or CA gradient were created by removal of
AS or CA (respectively) from the extra liposome aqueous phase and
replacing it with NaCl.
[0088] Three methods were used for creating the pH gradient:
[0089] (i) Centrifugation (Grant et al 2004, ibid.) for both AS and
CA gradients at 1000 g, for 5 min and temperature of 4.degree. C.
Supernatant was removed and pellet was washed with saline at
4.degree. C. The washing process was repeated 7 times.
[0090] (ii) Dialysis using MWCO 12-14000 Dalton dialysis tubing
[0091] (iii) Diafiltrating using Midjet benchtop system with hollow
fiber cartridge 500000 NMWC (GE Healthcare Bio-Sciences Corp.
Westborough, Mass. 01581 USA).
Loading of Bupivacaine
[0092] LMVV were loaded with Bupivacaine (B UP) using two
alternative approaches:
[0093] Remote loading of preformed liposomes having a
trans-membrane ammonium sulfate (AS) gradient (Haran et al.,
(1993), BBA, 1151 201-215), modified to fit the LMVV (Grant et al
2004, ibid.); or into preformed LMVV having a trans-membrane
calcium acetate (CA) gradient (Clerc & Barenholz. (1995), BBA,
1240, 65-257, Avnir et al (2008) Arthritis & Rheumatism, 58,
119-129). This method makes use of the fact that BUP, like
doxorubicin, is an amphipathic weak base.
[0094] (ii) Passive loading was performed by lipid hydration using
aqueous solutions of BUP to form the BUP loaded MLV from which BUP
loaded LMVV were prepared as described above (LMVV
preparation).
[0095] In both approaches loading was performed at 60-65.degree.
C., which is above the HSPC and C16SPM solid-ordered (SO) to
liquid-disordered (LD) phase transition temperature range
(T.sub.m). It is noted that HSPC and C16SPM are the
liposome-forming lipids of the LMVV described here.
[0096] For remote loading, loading was performed for 30 min. at
60-65.degree. C. using 4.5%, 5.5%, or 5.7% BUP, which is equivalent
to osmolarity of (saline=0.9% weight per volume), or 6% BUP in
distilled water as the liposome external aqueous phase. An amount
0.5 ml of a wet LMVV pellet and 2 ml of BUP solution were used for
the remote loading. The mixture was then cooled to 4.degree. C.
overnight.
[0097] Passive loading of BUP was performed by hydrating the
ethanol lipid solution with aqueous solution of distilled water
containing 4.5% (231 mOsm/kg), or 5.5% (285 mOsm/kg), or 6% (301
mOsm/kg) or 7% (346 mOsm/kg), or 8% (373 mOsm/kg) or 10% (454
mOsm/kg) BUP (W/V) at 65.degree. C. for 30 min. For this process
0.5 ml ethanolic lipids solution containing 225 mg phospholipids
and 77 mg CHOL were used. This solution was mixed with 5 ml of one
of the above indicated BUP aqueous solutions. The suspension was
processed by 10 freezing and thawing cycles (as described above)
and than kept overnight in a cold room (4-6.degree. C.).
Free Drug Removal
[0098] Non-encapsulated BUP was removed from LMVV by washing with
saline (1 ml liposomes/4 ml saline) and centrifuging the dispersion
at 1000 g for 5 min at 4-5.degree. C. The washing process was
repeated 7 times. The final medium (referred to herein as the
"aqueous medium") used to replace extra-liposome from CA gradient
loaded liposomes was PBS. The use of PBS was preferred over saline.
AS and the medium used for passive loading of liposomes was
replaced and LMVV were washed with un-buffered saline.
[0099] The LMVV was concentrated to a final solution of 2% BUP for
the passive loading and AS gradient loading. For CA gradient
loading LMVV with 1% BUP final concentration was used, due to the
large volume of these LMVV. These concentrations were close to the
highest concentrations used for injection of BUP.
[0100] The stability of LMVV thus formed was measured with respect
to the release rate of BUP from liposomes during storage at
4.degree. C.
Bupivacaine Loading Under Iso-Osmotic Conditions
[0101] When referring to iso-osmotic conditions, it should be
understood to mean that the osmolarity of the intraliposomal
aqueous core an the external medium inside and outside the
liposomes are essentially identical or close, all as defined
hereinabove.
[0102] Three osmomolar concentrations were tested:
[0103] (i) 280 mOsm/kg isoosmotic to physiological saline (0.9%
NaCl) condition: the AS and CA gradient LMVV were prepared with
.about.20 mg/ml AS or CA solution adjusted by AS or CA solutions to
280 mOsm/kg. BUP loading concentration was 5.7% BUP in water or
4.5% BUP in NaCl solution to reach 280 mOsm/kg.
[0104] (ii) 550 mOsm/kg, isoosmotic to 250 mM AS: the washing
solution for creating the AS gradient and the solution for removal
of the free drug after loading was NaCl solution. adjusted to 550
mOsm/kg. The drug loading conc. was 4.5% BUP in NaCl solution, or
4.5% BUP in sucrose sol. to make 550 mOsm/kg.
[0105] (iii) 650 mOs, iso-osmotic to 250 mM CA.
Bupivacaine to Lipids Ratio
[0106] BUP was loaded into AS-LMVV using three types of BUP to
lipid v/v ratios:
[0107] (i) wet LMVV pellet: 5.7% BUP:lipid, 1:4 vol/vol.
[0108] (ii) wet LMVV pellet: 5.7% BUP:lipid, 1:2 vol/vol.
[0109] (iii) wet LMVV pellet: 5.7% BUP:lipid 1:1 vol/vol.
[0110] The characteristics of the resulting LMVV are provided in
Table 1:
TABLE-US-00001 TABLE 1 BUP loaded LMVV Lipid/Chol ratio Loading
method Mean size (.mu.m) SPM/CHOL 6/4 CA gradient 8.33 .+-. 4.71
SPM/CHOL 6/4 AS gradient 5.7 .+-. 2.6 HSPC/CHOL 6/4 passive 6.0
.+-. 3.2
[0111] Further, FIGS. 1A and 1B compare the loading stabilities of
BUP-LMVV (prepared by similar procedure, albeit with H100), as
measured with respect to release rate at 4.degree. C. (FIG. 1A) and
37.degree. C. (FIG. 1B). The comparison relates to different lipid
compositions of LMVV as follows: [0112] (i) Previous formulation of
HSPC (of Lipoid GmbH) and CHOL as described in U.S. Pat. No.
6,162,46, the content of which is incorporated herein by reference;
[0113] (ii) HSPC-100 (Phospholipids GmbH, Germany) and CHOL; [0114]
(iii) HSPC/C16SPM and CHOL; [0115] (iv) HSPC 100/ C16SPM and
CHOL.
[0116] The data presented in FIGS. 1A and 1B show that the release
rates of BUP during 60 days storage at 4.degree. C. of the
HSPC/CHOL liposomes was the highest, followed by the release rate
from HSPC100/CHOL liposomes. The lowest release rate was achieved
for HSPC100/C16SPM/CHOL liposomes. In 24 hours, the release at
37.degree. C. reaches the level of 60% to 70% of the BUP from the
liposome--this being without reaching a plateau. It was thus
concluded that although a slight lower loading of BUP (lower BUP/PL
ratio) reached with the LMVV composed of HSPC100/C16SPM/CHOL, the
low release rate of BUP from this particular formulation at
4.degree. C. rendered this combination a preferred formulation. It
was thus further concluded that the presence of SPM reduced leakage
as compared to the same formulation without SPM.
[0117] The release rate from liposomes comprising
HSPC100/C16SPM/CHOL 3/3/4 (either SUV or LMVV as indicated)
employing the different loading techniques, different active agents
(BUP or MPS, the "Drug") and different aqueous medium (washing
buffer) were examined. The results are presented in Table 2.
TABLE-US-00002 TABLE 2 Drug to lipid ratio and stability loading
(at 4.degree. C.) of liposomes formed from HSPC100/C16SPM/CHOL
3/3/4 Aqueous Drug/PL % Drug release at 4.degree. C. Liposome type
Loading technique medium mole ratio 17 d 21 d 35 d 40 d 76 d 90 d
120 d 4.5 month 6 month LMVV-BUP Passive by 4.5% BUP Saline2% BUP
1.5 17.8 LMVV-BUP Passive by 5.5% BUP Saline2% BUP 1.7 20.9 36.3
LMVV-BUP Passive by 6% BUP Saline2% BUP 1.7 23.5 36 LMVV-BUP
Passive by 7% BUP Saline2% BUP 1.9 25.8 40.6 LMVV-BUP 250 mM CA
gradient PBS, 1% BUP 0.8 11 19.9 44 LMVV-BUP 107 mm CA gradient
Saline0.6% BUP 1.2 9 36.2 LMVV-BUP 107 mm CA gradient Saline0.7%
BUP 1.1 7.5 43 LMVV-BUP 250 mm AS gradient Saline 2% BUP 1.6 8 11.1
21 LMVV-BUP 250 mm AS gradient 1.75% NaCl 1.4 2.5 9.9 22 LMVV-BUP
250 MM AS gradient 1.75% NaCl 2 8 9 LMVV-BUP 127 mm AS gradient
Saline0.9% BUP 2.3 3 9.6 13 LMVV-BUP 127 mm AS gradient Saline0.7%
BUP 1.5 2.8 13.5 LMVV-BUP 127 mm AS gradient saline 1.5 3.3 20
LMVV-MPS 107 mm CA gradient saline 0.6 1.4 SUV-MPS 250 mm CA
gradient saline 0.3 20 ml LMVV- 127 mm AS gradient saline 1.35 5 9
11.1 BUP 20 ml LMVV- 127 mm AS gradient saline 1.56 3.3 BUP
dialysis tube 10 ml LMVV- 127 mm AS gradient saline 1.17 BUP
diafiltration
[0118] FIGS. 2A and 2B demonstrate the release rate at 4.degree. C.
(FIG. 2A) and 37.degree. C. (FIG. 2B) of BUP from LMVV having the
same lipid compositions as used in FIGS. 1A-1B, wherein BUP was
remotely loaded using Ca acetate gradient. The SPM used was C16
SPM, and comparison with HSPC/SPM/CHOL and HSPC100/SPM/CHOL was
also made at 4.degree. C.
[0119] The ratio BUP/PL for the CA gradient loading was lower than
that obtained for the AS gradient loading. Stability was assessed
from the release at 4.degree. C. This ratio was also lower (i.e.
higher release rate) than that obtained for LMVV remote loaded by
AS gradient at 37.degree. C. The release rates are similar to those
of the LMVV loaded BUP by AS gradient, except that rate of release
is faster at the first 10 hours followed by an almost plateau. It
is apparent from FIG. 2A that the HSPC100 LMVV has better stability
(i.e. lower leakage at 4.degree. C.) than HSPC based LMVV, and that
C16 SPM effect on improving stability is much greater than the
difference between the two HSPC preparations. C 16 SPM also reduces
leakage rate for both HSPC compositions by a similar extent.
[0120] FIGS. 3A and 3B demonstrate the release rate at 4.degree. C.
(FIG. 3A) and 37.degree. C. (FIG. 3B) of BUP loaded LMVV of the
same lipid compositions used in FIGS. 1A and 1B, wherein LMVV were
passively loaded with BUP. The SPM used is C16 SPM, and a
comparison of HSPC/SPM/CHOL and HSPC100/SPM/CHOL was also made at
4.degree. C.
[0121] In general, release rates at 4.degree. C., for passively
loaded LMVV of the 3 lipid compositions used, were higher than for
the remote loading via CA gradient and even higher when compared
with AS remote loading LMVV.
[0122] However the effect of LMVV lipid composition on release
rates at 4.degree. C. and 37.degree. C. were similar (but larger in
magnitude) to that observed for the remote loading driven by AS and
CA gradient, thus indicating that the ion gradient driven remote
loading increases loading stability at 4.degree. C.
LMVV Optimization
[0123] Various formulations with different mole ratio of
HSPC100:C16SPM were prepared in order to determined the optimized
ratio between these two constituents. The different formulations
are provided in Tables 3A and 3B.
TABLE-US-00003 TABLE 3A Effect of HSPC100:C16SPM mole ratio in
HSPC100/C16SPM/CHOL LMVV formed by active loading with AS gradient
% SPM/ BUP HSPC100 BUP/PL % BUP release at 4.degree. C. load- mole
mole 2 3.5 ing ratio ratio 8 d 22 d 30 d 38 d month month 4.5 0/1
2.2 2.5 8.2 18.9 4.5 1/0 1.8 4 9.5 15.5 4.5 1/1 1.68 8 5.7 1/1 1.96
7.5 8.7 5.7 5/4 2.03 5.2 7 5.7 2/1 1.5 5.8 7.8 5.7 7/2 1.6 5.3 7.5
5.7 0/1 1.8 4.3 5.7 1/1 1.55 2.6 5.7 2/1 1.44 2.4
TABLE-US-00004 TABLE 3B Effect of HSPC100:C16SPM mole ratio in
HSPC100/C16SPM/CHOL LMVV formed by active loading with CA gradient.
% SPM/ BUP HSPC100 BUP/PL % BUP release at 4.degree. C. load- mole
mole 2 3.5 ing ratio ratio 8 d 22 d 30 d 38 d month month 4.5 0/1
1.7 2 19.2 41.2 4.5 1/0 1.45 7.4 8.8 20.8 4.5 1/1 1.77 15 4.5 0/1*
1.16 2 25.8 4.5 1/1* 1.5 3 12.6 34 4.5 1/3* 1.5 3.7 16 41 *HSPC and
not HSPC100
[0124] Further, pre-formed LMVV were centrifuged for 5 min at
4.degree. C. at 2000 g to give packed LMVV. For remote loading the
packed LMVV were suspended in various volumes of 5.7% BUP. The
volume ratio of BUP to LMVV or PL is given in Table 4.
TABLE-US-00005 TABLE 4 Optimization of passive loading to the
volume ratio of 5.7% BUP to packed LMW (during loading). BUP/LMVV %
free BUP volume ratio* BUP/PL mole ratio t = 0 4 1.17 0.4 2 1.23
0.6 1 1.13 2.8
In Vivo Experiments
Bupivacaine Loaded LMVV Preparations:
[0125] Eight formulations were prepared (as specified below) under
sterile conditions and were tested for sterility in the Clinical
Microbiology Department, Hadassah Hospital, Jerusalem, Israel. The
liposomes were shipped from Jerusalem Israel to Dr G. J. Grant,
Department of Anesthesiology, NYU, School of Medicine, NYC, USA at
controlled temperature of 2.degree. C.-8.degree. C. HPLC analysis
(not shown) before shipment and after arrival to destination
indicated that no leakage during shipment took place.
TABLE-US-00006 TABLE 7 Liposome Composition Characterization Ratio
Pi Bupivicaine/ Pellet Total % of Bupivicaine .mu.mol/ml = Pi mM
date of sample Liposomes volume volume free (total) mmol/1 =
Bupivicane/ Sample preparation number Gradient sort type ml bupiv.
bupiv. mM mM mM Pi H100/SPM.sub.c16/ 15 Jul. 2007 1 AS (in saline)
MLV 3.5 15 5.08 17.11 28.12 0.61 CHOL 3/3/4 1 ml lipos (instead 0.5
ml) + 2 ml 4.5% bup. 09 Jul. 2007 2 CaAc MLV 4 15 3.09 17.86 19.53
0.91 (in PBS) 10 Jul. 2007 3 AS LMVV 5 15 2.81 27.36 13.71 2.00 (in
saline) 11 Jul. 2007 & 4 CaAc LMVV 15 30 3.56 17.28 21.06 0.82
15 Jul. 2007 (in PBS) H100/CHOL 16 Jul. 2007 5 AS LMVV 7 15 3.27
32.23 15.91 2.03 6/4 (in saline) HS 16 Jul. 2007 6 AS LMVV 6 15
6.81 33.32 14.89 2.24 (in saline) H100/CHOL 17 Jul. 2007 7 CaAc
LMVV 7 15 6.11 14.67 19.82 0.74 6/4 (in PBS) H100/SPM.sub.c16/ 18
Jul. 2007 8 6% passive LMVV 4 15 1.20 23.00 20.13 1.14 CHOL
3/3/4
[0126] All liposomal formulations were analyzed for free BUP and
total BUP before the in vivo experiment and concentrated to reach
the level of 2% (w/w) BUP (liposomes formulations #1, 2, 3, 5, 6,
8) or 1% (w/w) BUP (liposomes formulations #4, 7). BUP was loaded
into the liposomes either by active loading (CA or AS gradient) or
by passive loading.
TABLE-US-00007 TABLE 8 Liposome composition analysis prior to in
vivo experimentation Liposome # Lipids* Loading technique type %
free BUP 1 H100/SPM/CHOL AS gradient MLV 3.88 2 H100/SPM/CHOL CA
gradient MLV 3.95 3 H100/SPM/CHOL AS gradient LMVV 3.69 4
H100/SPM/CHOL CA gradient LMVV 4.52 5 H100/CHOL AS gradient LMVV
3.68 6 HSPC/CHOL AS gradient LMVV 7.80 7 H100/CHOL CA gradient LMVV
7.66 8 H100/SPM/CHOL 6% BUP passive LMVV 1.90 loading *with SPM the
ratio is 3/3/4 and without SPM the ratio is 6/4
Analgesic Efficacy in Mouse Model:
[0127] Testing for analgesia was done by electrical stimulation of
the skin directly overlying the abdomen at the site of injection
using a current generator (model S48, Grass Instruments).
[0128] Mice (male Swiss-Webster, 26.+-.3 gr) were tested prior to
injection to determine the vocalization threshold than were
injected with encapsulation BUP liposomes than determine analgesic
duration (G. J. Grant et al, pharmaceutical research, vol 18, no.
3, 336-343, 2001).
[0129] The duration of the main in vivo screening study was 2 days
and started after a preliminary study using two different injection
volumes of formulation #4 (referred to as the PILOT in Table 9A)
was performed.
[0130] In order to evaluate the effect of altering the volume and
BUP concentration of the injection, in each group, three mice
received 150 .mu.L of the 2% formulation and 3 mice received 300
.mu.L of a 1:1 diluted 2% formulation.
[0131] It has been previously determined (Grant et al. 2004, ibid.,
Bolotin et al. 2000, ibid. and U.S. Pat. No. 6,162,462) LMVV (GMV)
encapsulated BUP provide an analgesic effect for approximately 75
minutes post injection.
[0132] The analgesic efficacy of the various formulations 1 to 8,
at different BIP concentration, different injection volume etc. is
presented in Tables 9A to 9C. In these Tables, an numeric score of
"1" denotes full analgesia, a numeric score of "0" was given when
there was no analgesic effect, and a numeric value of "10" when
there was partial analgesia. In the following tables the numeric
value "10" is replaced by "0.5".
[0133] In Table 9A results of mice injected with LMVV formulation
#4, two mice with 300 .mu.l and two mice with 150 .mu.l are
presented as "PILOT 1-4" Testing was done at 4, 17, and 21 hours
following injection.
[0134] FIGS. 4A-4C, 5A-5F, 6 and 7 show the duration of analgesia.
The difference in these figures is in the formulations used, FIGS.
4 and 5 making use of the various formulations identified in Table
8, and FIGS. 6 and 7 making use of HSPC100/C16SPM/CHOL (3/3/4). The
in vivo results show that SPM containing liposomes have a
significantly greater analgesic effect as compared to free BUP.
These results specifically show that the inclusion of SPM into the
liposomes did not reduce the analgesic effect to the system, as
compared to prior art formulations [Grant et al. 2004, ibid.].
TABLE-US-00008 TABLE 9A Duration of analgesia at different BUP
concentrations (administered as liposomal-BUP) and different
injected volumes Aug. 9, 2007 1 indicates mice under analgesia, 0
indicates mice lacks analgesia; 10 indicates mice is under partial
analgesia Note: On Aug. 8, 2007, we injected four animals with LMW
formulation #4 (2 animals with 300 ul and 2 mice with 150 ul);
testing was done at 4, 17, and 21 hours. These are labeled "PILOT"
in the spreadsheet below animal # lipo # bup conc volume (ul) mg
Bup 4 hr 8 hr 12 hr 15 hr 18 hr 21 hr 1 1 2% 150 3 1 1 10 0 0 0 2 1
2% 150 3 1 1 1 1 0 0 3 1 2% 150 3 1 1 1 1 0 0 4 1 1% 300 3 1 1 1 0
0 0 5 1 1% 300 3 1 1 1 0 0 0 6 1 1% 300 3 1 1 1 0 0 0 7 2 2% 150 3
1 1 1 1 0 0 8 2 2% 150 3 1 1 0 0 0 0 9 2 2% 150 3 1 1 1 0 0 0 10 2
1% 300 3 1 1 1 1 0 0 11 2 1% 300 3 1 1 0 0 0 0 12 2 1% 300 3 1 1 0
0 0 0 13 3 2% 150 3 1 1 1 1 0 0 14 3 2% 150 3 1 1 1 0 0 0 15 3 2%
160 3 1 1 1 1 0 0 16 3 1% 300 3 1 1 1 0 0 0 17 3 1% 300 3 1 1 1 1 1
0 18 3 1% 300 3 1 1 1 1 0 0 19 4 1% 300 3 1 1 1 1 1 0 20 4 1% 300 3
animal eliminated from study 21 4 1% 300 3 1 1 1 10 10 0 22 4 1%
300 3 1 1 1 1 10 10 23 4 1% 300 3 1 1 1 1 10 0 24 4 1% 300 3 1 1 1
1 0 0 17 hr PILOT 1 4 1% 300 3 1 1 0 PILOT 2 4 1% 300 3 1 1 0 PILOT
3 4 1% 150 1.5 1 0 PILOT 4 4 1% 150 1.5 1 0 25 5 2% 150 3 1 1 1 0 0
0 26 5 2% 150 3 1 1 1 1 1 0 27 5 2% 150 3 1 1 1 0 0 0 28 5 1% 300 3
1 1 1 1 0 0 29 5 1% 300 3 1 1 1 0 0 0 30 5 1% 300 3 1 1 10 0 0 0 31
6 2% 150 3 1 1 0 1 0 0 32 6 2% 150 3 1 1 1 1 0 0 33 6 2% 150 3 1 0
0 0 0 0 34 6 1% 300 3 1 1 0 0 0 0 35 6 1% 300 3 1 1 1 10 10 0 36 6
1% 300 3 1 1 1 0 0 0 37 7 1% 300 3 1 1 1 10 10 0 38 7 1% 300 3 1 1
1 1 0 0 39 7 1% 300 3 1 0 0 0 0 0 40 7 1% 300 3 1 1 0 0 0 0 41 7 1%
300 3 1 1 1 1 0 0 42 7 1% 300 3 1 1 1 1 0 0 43 8 2% 150 3 1 1 1 1 0
0 44 8 2% 150 3 1 1 1 0 0 0 45 8 2% 150 3 1 1 1 0 0 0 46 8 1% 300 3
1 1 1 1 0 0 47 8 1% 300 3 1 1 1 1 0 0 48 8 1% 300 3 1 1 1 0 0 0
TABLE-US-00009 TABLE 9B Analgesic effect at different PBU
concentrations and at different injected volumes Aug. 13, 2007
Standard Bupivacaine (Control) 1 = analgesia; 0 = no analgesia; 10
= partial analgesia Mouse # Bup Conc Volume(ul) mg Bup 15 min 30
min 45 min 60 min 75 min 90 min 105 min 120 min 135 min 1 0.25% 150
0.375 1 1 1 0 0 0 2 0.25% 150 0.375 1 1 1 0 0 0 3 0.25% 150 0.375 1
1 1 0 0 0 4 0.25% 150 0.375 1 1 1 1 0 0 5 0.25% 150 0.375 1 1 1 1
10 0 6 0.25% 150 0.375 1 1 1 0 0 0 7 0.25% 150 0.375 1 1 1 10 0 0 8
0.25% 150 0.375 1 1 1 0 0 0 1 0.25% 300 0.75 1 1 1 1 0 0 0 0 0 2
0.25% 300 0.75 1 1 1 1 1 1 0 0 0 3 0.25% 300 0.75 1 1 1 1 1 10 10 0
0 4 0.25% 300 0.75 1 1 1 1 1 10 0 0 0 5 0.25% 300 0.75 1 1 1 1 1 1
1 10 0 6 0.25% 300 0.75 1 1 1 1 1 1 0 0 0 7 0.25% 300 0.75 1 1 1 1
1 1 0 0 0 8 0.25% 300 0.75 1 1 1 1 1 1 1 0 0 1 0.50% 150 0.75 1 1 1
1 1 0 0 2 0.50% 150 0.75 1 1 1 1 1 0 0 3 0.50% 150 0.75 1 1 1 1 1
10 0 4 0.50% 150 0.75 1 1 1 1 0 0 0 5 0.50% 150 0.75 1 1 1 1 1 1 0
6 0.50% 150 0.75 1 1 1 1 1 10 0 7 0.50% 150 0.75 1 1 1 1 1 1 0 8
0.50% 150 0.75 1 1 1 10 10 0 0 Liposomal (LMVV) Bupivacaine Pilot
Study Mouse # LipoForm# Conc. Volume mg Bup 15 hr 18 hr 21 hr 1 3
2% 300 6 1 1 10 2 3 2% 300 6 1 1 0 1 4 1% 450 4.5 1 1 0 2 4 1% 450
4.5 1 1 0 1 5 2% 300 6 1 1 0 2 5 2% 300 6 0 1 0
TABLE-US-00010 TABLE 9C Analgesic effect at different PBU
concentrations and different injected volumes Aug. 13, 2007
Standard Bupivacaine (Control) 1 = analgesia; 0 = no analgesia; 10
= partial analgesia Mouse Bup Volume 15 30 45 60 75 90 105 120 135
# Conc (ul) mg Bup min min min min min min min min min 1 0.25% 150
0.375 1 1 1 0 0 0 2 0.25% 150 0.375 1 1 1 0 0 0 3 0.25% 150 0.375 1
1 1 0 0 0 4 0.25% 150 0.375 1 1 1 1 0 0 5 0.25% 150 0.375 1 1 1 1
10 0 6 0.25% 150 0.375 1 1 1 0 0 0 7 0.25% 150 0.375 1 1 1 10 0 0 8
0.25% 150 0.375 1 1 1 0 0 0 1 0.25% 300 0.75 1 1 1 1 0 0 0 0 0 2
0.25% 300 0.75 1 1 1 1 1 1 0 0 0 3 0.25% 300 0.75 1 1 1 1 1 10 10 0
0 4 0.25% 300 0.75 1 1 1 1 1 10 0 0 0 5 0.25% 300 0.75 1 1 1 1 1 1
1 10 0 6 0.25% 300 0.75 1 1 1 1 1 1 0 0 0 7 0.25% 300 0.75 1 1 1 1
1 1 0 0 0 8 0.25% 300 0.75 1 1 1 1 1 1 1 0 0 1 0.50% 150 0.75 1 1 1
1 1 0 0 2 0.50% 150 0.75 1 1 1 1 1 0 0 3 0.50% 150 0.75 1 1 1 1 1
10 0 4 0.50% 150 0.75 1 1 1 1 0 0 0 5 0.50% 150 0.75 1 1 1 1 1 1 0
6 0.50% 150 0.75 1 1 1 1 1 10 0 7 0.50% 150 0.75 1 1 1 1 1 1 0 8
0.50% 150 0.75 1 1 1 10 10 0 0 Liposomal (LMVV) Bupivacaine Pilot
Study Mouse # LipoForm# Conc. Volume mg Bup 15 hr 18 hr 21 hr 1 3
2% 300 6 1 1 10 2 3 2% 300 6 1 1 0 1 4 1% 450 4.5 1 1 0 2 4 1% 450
4.5 1 1 0 1 5 2% 300 6 1 1 0 2 5 2% 300 6 0 1 0
[0135] As indicated above, the numerical score to the spreadsheet
was introduced for the evaluation of the analgesic effect of
various liposome preparations performance in vivo: For each time
period (e.g. 4 hrs, 8 hrs etc) a numeric value of 1 was given if
the anesthesia was complete; 10 or 0.5 was given when analgesia was
partial (incomplete) and 0 for no anesthesia. The mean for each
subgroup was calculated separately (i.e. 1% 300 .mu.l, 2% 150
.mu.g).
[0136] The results show that formulation 4, where BUP was actively
loaded into LMVV with CA gradient and the iso-osmotic aqueous
medium was saline provided the best analgesic effect, although the
differences between the various formulations was not significant,
when compared to the 10 fold increase in analgesia when compared to
BUP formulations as the reference liposomal GMV formulation [Grant
et al. 2004, ibid., Bolotin et al. 2000, ibid. and U.S. Pat. No.
6,162,462].
[0137] In a separate experiment the effect of repeated injection of
bupivacaine loaded LMVV In mice was evaluated. The results showed
(data not shown) that the analgesia obtained after the second
(repeated) injection was identical to the one achieved at the first
injection without any observed side effect. The conclusion was that
analgesia can be prolonged by repeated injections and the time
period of analgesia after the second injection was at least of the
same duration as that obtained after the first injection.
* * * * *