U.S. patent application number 14/520542 was filed with the patent office on 2015-02-05 for methods for joint lubrication and cartilage wear prevention making use of glycerophospholipids.
This patent application is currently assigned to TECHNION RESEARCH AND DEVELOPMENT FOUNDATION LTD.. The applicant listed for this patent is HADASIT MEDICAL RESEARCH SERVICES & DEVELOPMENT LIMITED, TECHNION RESEARCH AND DEVELOPMENT FOUNDATION LTD., YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM. Invention is credited to Yechezkel BARENHOLZ, Izhak ETSION, Grigory HALPERIN, Dorit NITZAN, Avi SCHROEDER, Sarit SIVAN.
Application Number | 20150037404 14/520542 |
Document ID | / |
Family ID | 39144533 |
Filed Date | 2015-02-05 |
United States Patent
Application |
20150037404 |
Kind Code |
A1 |
BARENHOLZ; Yechezkel ; et
al. |
February 5, 2015 |
METHODS FOR JOINT LUBRICATION AND CARTILAGE WEAR PREVENTION MAKING
USE OF GLYCEROPHOSPHOLIPIDS
Abstract
A method for lubricating a joint of a mammal by administering
into a cavity of the joint a composition of liposomes that are
multilamellar vesicles (MLV) dispersed in a fluid medium, the
liposomes having a mean diameter of between about 0.8 .mu.m to
about 10 .mu.m and including membranes of at least one
glycerophospholipid (GPL) having two C.sub.12-C.sub.16 hydrocarbon
chains which are the same or different. These membranes have a
phase transition temperature in which solid ordered (SO) to liquid
disordered (LD) phase transition occurs, the phase transition
temperature being at a temperature of about 20.degree. C. to about
39.degree. C. and being lower than the temperature of the
joint.
Inventors: |
BARENHOLZ; Yechezkel;
(Jerusalem, IL) ; NITZAN; Dorit; (Bar Giora,
IL) ; ETSION; Izhak; (Haifa, IL) ; SCHROEDER;
Avi; (Moshav Massuot Yitzhak, IL) ; HALPERIN;
Grigory; (Or-Akiva, IL) ; SIVAN; Sarit;
(Zichron Yaakov, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TECHNION RESEARCH AND DEVELOPMENT FOUNDATION LTD.
HADASIT MEDICAL RESEARCH SERVICES & DEVELOPMENT LIMITED
YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF
JERUSALEM |
Haifa
Jerusalem
Jerusalem |
|
IL
IL
IL |
|
|
Assignee: |
TECHNION RESEARCH AND DEVELOPMENT
FOUNDATION LTD.
Haifa
IL
HADASIT MEDICAL RESEARCH SERVICES & DEVELOPMENT
LIMITED
Jerusalem
IL
YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF
JERUSALEM
Jerusalem
IL
|
Family ID: |
39144533 |
Appl. No.: |
14/520542 |
Filed: |
October 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12411855 |
Mar 26, 2009 |
8895054 |
|
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14520542 |
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PCT/IL2007/001215 |
Oct 7, 2007 |
|
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12411855 |
|
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60847651 |
Sep 28, 2006 |
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Current U.S.
Class: |
424/450 ;
514/121 |
Current CPC
Class: |
A61P 19/02 20180101;
A61K 9/0019 20130101; A61P 17/06 20180101; A61P 29/00 20180101;
A61P 41/00 20180101; A61K 9/127 20130101; A61L 27/50 20130101; A61L
2430/24 20130101; A61K 31/685 20130101; A61K 9/1271 20130101 |
Class at
Publication: |
424/450 ;
514/121 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 9/00 20060101 A61K009/00 |
Claims
1. A method for lubricating a joint of a mammal, which comprises
administering into a cavity of the joint having a first temperature
a composition consisting essentially of liposomes that are
multilamellar vesicles (MLV) dispersed in a fluid medium, the
liposomes having a mean diameter of between about 0.8 .mu.m to
about 10 .mu.m and consisting essentially of membranes of at least
one glycerophospholipid (GPL) having two C.sub.12-C.sub.16
hydrocarbon chains which are the same or different, the membranes
having a phase transition temperature in which solid ordered (SO)
to liquid disordered (LD) phase transition occurs, the phase
transition temperature being at a temperature of about 20.degree.
C. to about 39.degree. C.; the phase transition temperature being
lower than the first temperature.
2. The method of claim 1, wherein said at least one GPL comprises
two C.sub.14-C.sub.16 acyl chains.
3. The method of claim 1, wherein the two hydrocarbon chains are
saturated.
4. The method of claim 1, wherein said at least one GPL is a
phosphatidylcholine (PC).
5. The method of claim 4, wherein said PC is
dimyristoylphosphatidylcholine (DMPC).
6. The method of claim 4, wherein said PC is
1,2-dipalmitoyl-sn-glycero-3-phosphocoline (DPPC).
7. The method of claim 4, wherein said PC is a combination of DMPC
and DPPC.
8. The method of claim 4, wherein said PC comprises
1,2-dipentadecanoyl-sn-glycero-3-phosphocholine.
9. The method of claim 1, wherein said fluid medium comprises a
buffer.
10. The method of claim 1, for the treatment of or prevention of an
articular disorder or symptoms arising therefrom or for the
treatment, management or prevention of deterioration of locked
joints, sports injury or traumatic injury towards osteoarthritis
(OA) or disorders secondary to rheumatoid arthritis, and psoriatic
arthritis.
11. The method of claim 10, wherein said articular disorder is
selected from arthritis, osteoarthritis, osteoarthritis in
rheumatoid arthritis patients, traumatic joint injury, locked
joint, sports injury, status post arthrocentesis, arthroscopic
surgery, open joint surgery, and joint replacement.
12. A method for reducing or preventing a mammal's cartilage wear,
which method comprises: administering into a cavity of a mammal's
joint having a first temperature a composition consisting
essentially of liposomes, wherein said liposomes are multilamellar
vesicles (MLV) dispersed in a fluid medium, the liposomes having a
mean diameter of between about 0.8 .mu.m to about 10 .mu.m and
consisting essentially of membranes of at least one
glycerophospholipid (GPL) having two C.sub.12-C.sub.16 hydrocarbon
chains which are the same or different, the membranes having a
phase transition temperature in which solid ordered (SO) to liquid
disordered (LD) phase transition occurs, the phase transition
temperature being at a temperature of about 20.degree. C. to about
39.degree. C.; the phase transition temperature being lower than
the first temperature.
13. The method of claim 12, wherein said at least one GPL comprises
two C.sub.14-C.sub.16 acyl chains.
14. The method of claim 12, wherein the two hydrocarbon chains are
saturated.
15. The method of claim 12, wherein said at least one GPL is a
phosphatidylcholine (PC).
16. The method of claim 15, wherein said PC is
dimyristoylphosphatidylcholine (DMPC).
17. The method of claim 15, wherein said PC is
1,2-dipalmitoyl-sn-glycero-3-phosphocoline (DPPC).
18. The method of claim 15, wherein said PC is a combination of
DMPC and DPPC.
19. The method of claim 15, wherein said PC comprises
1,2-dipentadecanoyl-sn-glycero-3-phosphocholine.
20. The method of claim 12, wherein said fluid medium comprises a
buffer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/411,855 filed Mar. 26, 2009, which is a
continuation-in-part of International application no.
PCT/IL2007/001215 filed Oct. 7, 2007, which claims priority to U.S.
provisional application No. 60/847,651 filed Sep. 28, 2006, the
entire content of each of which are incorporated herein by
reference thereto.
FIELD OF THE INVENTION
[0002] This invention generally concerns liposomes and methods for
their therapeutic use.
LIST OF PRIOR ART
[0003] The following is a list of prior art, which is considered to
be pertinent for describing the state of the art in the field of
the invention. [0004] Hills, B. A. Phospholipid and propylene
glycol based lubricant. U.S. Pat. No. 6,133,249, 1998. [0005]
Hills, B. A. Lubricant Composition for Rheumatism. U.S. Pat. No.
5,403,592, 1990. Hills, B. A.; Monds, M. K., Enzymatic
identification of the load-bearing boundary lubricant in the joint.
Br. J. Rheumatol. 1998, 37, (2), 137-142. [0006] Oloyede, A.,
Gudimetla, P., Crawford, R., Hills, B. A., Biomechanical responses
of normal and delipidized articular cartilage subjected to varying
rates of loading. Connective Tissue Research 2004, 45, (2), 86-93.
[0007] Ethell, M. T.; Hodgson, D. R.; Hills, B, A., The synovial
response to exogenous phospholipid (synovial surfactant) injected
into the equine radiocarpal joint compared with that to prilocaine,
hyaluronan and propylene glycol. New Zealand Veterinary Journal
1999, 47, (4), 128-132. [0008] Pickard, J. E.; Fisher, J.; Ingham,
E.; Egan, J., Investigation into the effects of proteins and lipids
on the frictional properties of articular cartilage, Biomaterials
1998, 19, (19), 1807-1812. [0009] Kawano, T.; Miura, H,; Mawatari,
T,; Moro-Oka, T.; Nakanishi, Y.; Higaki, H.; Iwamoto, Y.,
Mechanical effects of the intraarticular administration of high
molecular weight hyaluronic acid plus phospholipid on synovial
joint lubrication and prevention of articular cartilage
degeneration in experimental osteoarthritis. Arthritis Rheum. 2003,
48, (7), 1923-1929. [0010] Forsey, R. W.; Fisher, J.; Thompson, J.;
Stone, M. H.; Bell, C.; Ingham, E., The effect of hyaluronic acid
and phospholipid based lubricants on friction within a human
cartilage damage model. Biomaterials 2006, 27, (26), 4581-4590.
[0011] Klein, J., Molecular mechanisms of synovial joint
lubrication. J. Proc. Inst. Mech Eng., Part J: J. Eng. Tribology
2006, 220, (8), 691-710. [0012] Burdick et al., Biological
lubricant composition and method of applying lubricant composition.
U.S. Pat. No. 6,800,298. [0013] International patent application
publication No. WO2003/000190; [0014] International patent
application publication No. WO2004/047792; [0015] International
patent application publication No. WO2002/078445.
[0016] A complete list of prior art, which is referred to
occasionally in the text below, appears at the end of the
description before the claims. Reference to the publications will
be made by indicating their number from the complete list of
references.
BACKGROUND OF THE INVENTION
[0017] Joint dysfunctions affect a very large portion of the
population. Sufficient biolubrication is a prerequisite for proper
joint mobility, which is crucial for prevention and amelioration of
degradative changes of the joint.sup.1.
[0018] A common joint dysfunction is osteoarthritis, with
prevalence exceeding 20 million in the United States alone.sup.2.
The etiology of osteoarthritis is multifactorial, including
inflammatory, metabolic and mechanical causes.sup.3-5. Among the
list of risk factors involved are age, gender, obesity, occupation,
trauma, atheromatous vascular disease and immobilization.sup.1,
3-7. osteoarthritis may arise as a result of articular cartilage
breakdown; or conversely, subchondral bone sclerosis may actually
precede cartilage degeneration and loss.sup.8, 9. Once articular
cartilage is injured, damage progresses.sup.10.
[0019] Current treatment focuses on reducing overloading of joints,
physical therapy, and alleviation of pain and inflammation, usually
by systemic or intra-articular administration of drugs.sup.11.
[0020] Articular cartilage forms a smooth, tough, elastic and
flexible surface that facilitates bone movement. The synovial space
is filled with the highly viscous synovial fluid (SF), containing
hyaluronic acid (HA) and the glycoprotein lubricin.sup.12-14. HA is
a polymer of D-glucuronic acid and D-N-acetylglucosamine, which is
highly unstable and degrades under the inflammatory conditions of
osteoarthritis.sup.15, 16. Lubricin is composed of .about.44%
proteins, .about.45% carbohydrates and .about.11% phospholipids
(PL).sup.12-14, of which .about.41% are phosphatidylcholines (PCs),
.about.0.27% phosphatidylethanolamines (PE) and .about.32%
sphingomyelins.sup.17-19. These PL are referred to as
"surface-active phospholipids" (SAPL). The PE and PC of SAPL
contain two hydrocarbon chains, one of which is the monounsaturated
oleic acid (18:1).
[0021] Many studies are found in the literature on the effect of
SAPL on joint friction, but only a few of them deal with wear. Due
to problems of acquiring suitable human specimens and the
complicated nature of the experiments, most of the studies
addressing this issue used animal cartilage.
[0022] Boundary lubrication, in which layers of lubricant molecules
separate opposing surfaces, occurs under loading of articular
joints.sup.17, 18, 20. Several different substances have been
proposed as the native boundary lubricants in articular cartilage.
In the past, HA was thought to be the major lubricant.sup.21,
however, a recent tribiological study states that HA "by itself . .
. is not responsible for the nearly frictionless boundary
biolubrication found in articular cartilage", but may contribute to
load bearing and wear protection.sup.22. Many reports have shown
lubricin to play the major role in the lubricating properties of
synovial fluid.sup.12, 14, 19, 20, 23, 24. Pickard et al..sup.25
and Schwartz and Hills.sup.19 demonstrated that phospholipids
defined as surface active phospholipids (SAPL) of lubricin
facilitate joint lubrication in articular cartilage.
[0023] Special wear experiments.sup.58 were conducted on intact
sheep knee joints of which some were injected with lipid solvent
prior to the wear tests. The wear progression of the `naturally
worn` joints was compared with that of the `artificially worn`
dissolved lipid ones. It was found that severe depletion of the
SAPL layer, which is strongly related to osteoarthritis, resulted
in accelerated wear of the articular cartilage. It was concluded
that the lipid layer acts as a boundary lubricant and is critically
important to the proper functioning of synovial joints. In another
wear test.sup.59, artificially worn lipid-depleted sheep knee
joints were injected with two concentrations of the phospholipid
dipalmitoyl phosphatidylcholine (DPPC) and worn further. The
results indicated that a solution of DPPC may decrease cartilage
wear in synovial joints.
[0024] Cartilage surfaces of human osteoarthritis hip and knee
joints, which were replaced by artificial ones, showed deficiency
of the outermost lubricating layer of SAPL.sup.60.
[0025] Hills et al..sup.17 demonstrated that osteoarthritis joints
have a SAPL deficiency, and that injection of the surface-active
phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)
into joints of osteoarthritis patients resulted in mobility
improvement lasting up to 14 weeks.sup.26 without major side
effects.sup.27. In another study, utilizing a unique cryogenic
cartilage preservation technique, Watanabe et al. observed lipidic
globular vesicles on the surface of healthy cartilage, which are
assumed to play a major role in lubrication.sup.28. Kawano et
al..sup.29 and Forsey et al..sup.30, using animal models, have
shown that use of high molecular weight HA (.about.2000 kDa)
combined with DPPC improved lubricating ability. DPPC in the form
of multilamellar vesicles (MLV) has a phase transition temperature
in which solid ordered (SO) to liquid disordered (LD) phase
transition occurs of 41.4.degree. C.
[0026] U.S. Pat. No. 6,800,298 discloses dextran-based hydrogel
compositions containing lipids, particularly phospholipids, for
lubrication of mammalian joints.
[0027] International patent applications publications Nos.
WO2003/000190.sup.63, WO2004/047792.sup.64 and WO2002/078445.sup.65
describe liposomal formulations for intraarticular delivery of
active ingredients, such as steroids so as to treat an inflammatory
condition.
[0028] Recently, Klein et al..sup.31 summarized various issues of
joint lubrication at the molecular level. They point to the
potential role of highly-hydrated brush-like charged macromolecules
at the surface of cartilage as major contributors to cartilage
lubrication.sup.31-33.
SUMMARY OF THE INVENTION
[0029] The present invention is based on the discovery of a
liposomal platform for joint lubrication and on studies of the
effect of different phospholipids (PL) compositions, liposomal
sizes and lamellarity on joint friction and/or on cartilage wear,
using a cartilage-on-cartilage apparatus that mimics articular
joints.
[0030] Thus, in accordance with the invention, a novel formulation
based on a liposome system comprising PL is proposed, for
introduction into synovial joints in order to improve or restore
joint mobility. It was been found that the novel liposomal platform
is effective as a lubricant as well as for reducing cartilage
wear.
[0031] Thus, in accordance with a first of its aspects, there is
provided the use of liposomes comprising one or more membranes with
at least one phospholipid (PL) of the group consisting of a
glycerophospholipid (GPL) having two, being the same or different,
C.sub.12-C.sub.16 hydrocarbon chain and a sphingolipid (SPL) having
a C.sub.12-C.sub.18 hydrocarbon chain, the one or more membranes
having a phase transition temperature in which solid ordered (SO)
to liquid disordered (LD) phase transition occurs, the phase
transition temperature being within a temperature of about
20.degree. C. to about 39.degree. C., the use being for lubrication
and/or reducing wear rate of joints (cartilage) having a joint
temperature which is above the phase transition temperature of the
membrane.
[0032] In accordance with another aspect there is provided a method
for lubricating a joint of a mammal, comprising: administering into
a cavity of the joint having a joint temperature a therapeutically
effective amount of liposomes comprising one or more membranes with
at least one phospholipid (PL) of the group consisting of
glycerophospholipid (GPL) having two, being the same or different
C.sub.12-C.sub.16 hydrocarbon chain and a sphingolipid (SPL) having
a C.sub.12-C.sub.18 hydrocarbon chain, the one or more membranes
having a phase transition temperature in which solid ordered (SO)
to liquid disordered (LD) phase transition occurs, the phase
transition temperature being at a temperature of about 20.degree.
C. to about 39.degree. C.; the phase transition temperature being
lower than the joint temperature.
[0033] In accordance with yet another aspect there is provided a
method for preventing or reducing a mammal's cartilage wear,
comprising: administering into a cavity of the joint having a joint
temperature a therapeutically effective amount of liposomes
comprising one or more membranes with at least one phospholipid
(PL) of the group consisting of glycerophospholipid (GPL) having
two, being the same or different C.sub.12-C.sub.16 hydrocarbon
chain and a sphingolipid (SPL) having a C.sub.12-C.sub.18
hydrocarbon chain, the one or more membranes having a phase
transition temperature (Tm, the temperature in which the maximum
change in the heat capacity during the phase transition from solid
ordered (SO) to liquid disordered (LD) occurs), the phase
transition temperature being at a temperature of about 20.degree.
C. to about 39.degree. C.; the phase transition temperature being
lower than the joint temperature.
[0034] It is of particular importance to note that the criteria
above are cumulative criteria, namely, (a) that the liposomes
comprise one or more membranes with at least one phospholipid (PL)
of the group consisting of glycerophospholipid (GPL) having two
(being the same or different) C.sub.12-C.sub.16 hydrocarbon chain
and a sphingolipid (SPL) having a C.sub.12-C.sub.18 hydrocarbon
chain, and (b) that this combination of lipids form a membrane with
a phase transition temperature in which solid ordered (SO) to
liquid disordered (LD) phase transition occurs at a temperature of
about 20.degree. C. to about 39.degree. C. (the phase transition
temperature being lower than the joint temperature).
[0035] Thus, for example, while
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) is encompassed
within criteria (a) above, when used as a sole liposome forming PL,
the membrane thus formed has phase transition temperature above
39.degree. C. and thus DPPC was found to be ineffective in joint
lubrication and cartilage wear prevention. Thus, DPPC alone is
excluded from the scope of the present invention. The lack of
effective lubricating/wear reducing effect of liposomal membrane
composed of DPPC alone is exemplified hereinbelow.
[0036] By a still further aspect of the invention there is provided
a pharmaceutical composition for joint lubrication and/or reducing
wear rate of joints having a joint temperature and comprising a
physiologically acceptable carrier and liposomes; the liposomes
comprising one or more membranes with at least one phospholipid
(PL) of the group consisting of glycerophospholipid (GPL) having
two, being the same or different, C.sub.12-C.sub.16 hydrocarbon
chains and a sphingolipid (SPL) having a C.sub.12-C.sub.18
hydrocarbon chain; the one or more membranes having a phase
transition temperature in which solid ordered (SO) to liquid
disordered (LD) phase transition occurs, the phase transition
temperature being within a temperature of about 20.degree. C. to
about 39.degree. C. and being below said joint temperature.
[0037] The GPL, SPL or their combination form liposomes, preferably
liposomes with a mean diameter greater than about 0.3 .mu.m,
typically greater than about 0.5 .mu.m and at times greater than
about 0.8 .mu.m. The mean diameter of the liposomes is usually less
than about 10 .mu.m, typically less than about 8, 7, 6 or 5 .mu.m
and at times less than 3.5 .mu.m. The liposomes may be a
single-membrane liposome or may be, according to one embodiment,
multilamellar vesicles (MLV) liposomes. According to other
embodiments the liposomes may also be large multivesicular vesicles
(LMVV) or dehydrated rehydrated vesicles (DRV) liposomes.
[0038] In one embodiment said C.sub.12-C.sub.16 or
C.sub.12-C.sub.18 hydrophobic chains are saturated.
[0039] The liposomal compositions of the invention may be
administered to an afflicted joint through intra-articular
injection, orthoscopic administration, surgical administration and
in general any form of administration that can be used to instill
such a formulation into the joint synovium or onto the joint
cartilage. Afflicted joints treatable according to the invention
may be associated with a variety of conditions, such as arthritis,
rheumatoid arthritis, osteoarthritis (as well as osteoarthritis in
rheumatoid arthritis patients), traumatic joint injury, sports
injury, locked joint (such as in temporomandibular joint (TMJ)),
status post surgical intervention such as arthrocentesis,
arthroscopic surgery, arthroplasty, knee and hip replacement. A
preferred condition to be treated or prevented by the invention is
primary or secondary osteoarthritis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] 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:
[0041] FIG. 1 is a bar graph showing the friction coefficients
(static and dynamic) obtained for various lubricating media,
including inflamed synovial fluid (ISF); histidine buffer (HB, 5
mM), dispersions comprising multilamellar vesicles (MLV, carried in
5 mM HB, the lipids being at a concentration range of between
35-140 mM) with the phospholipid being DMPC, MLV comprising DMPC,
or DMPC-cholesterol, or mixture of DMPC and.sup.2000PEG-DSPE DMPC
or a mixture of DMPC and DPPC, or small unilamellar vesicles (SUV)
comprising DMPC. All measurements were performed at 37.degree. C.
under contact pressure of 2.4 MPa (30N load) and sliding velocity
of 1 mm/s. Saline was used as a control.
[0042] FIG. 2 is a graph showing cartilage wear as a function of
sliding distance in the presence of two different potential carrier
media HB and saline, the cartilage wear being measured by analyzing
the glycosamineglycan (GAG) content weight in the debris present in
the aqueous medium of the cartilage as a result of wearing.
[0043] FIG. 3 is a graph showing cartilage wear as a function of
sliding distance in the presence of DOPC, HSPC, DPPC and DMPC, all
dispersed in HB.
[0044] FIG. 4 is a graph showing cartilage wear as a function of
sliding distance in the presence of DPPC, DMPC and a mixture of
DMPC/DPPC, all dispersed in HB.
[0045] FIG. 5 is a graph showing cartilage wear as a function of
sliding distance in the presence of ISF, ISF+HA and
ISF+DMPC/DPPC.
[0046] FIG. 6 is a bar graph showing the constant wear rates
obtained with different lubricating fluids (Saline, HB or HA) and
phospholipids (DOPC, HSPC, DPPC, DMPC/DPPC or DMPC), used as
liposome additives, following a run-in period.
[0047] FIG. 7 is a graph showing the effect of the various
lubricants and media on total phospholipid concentration, in
cartilage specimens from healthy individuals after being subjected
to similar friction tests in the presence of the different
lubricants. The controls were not subjected to friction tests.
[0048] FIG. 8 is a graph showing PC concentration as a function of
vertical depth into cartilage where cartilage specimens were
subjected to similar friction tests in the presence of: DMPC-MLV
(0.8-3.5 .mu.m in diameter) 141 mM in 5 mM HB (.box-solid.);
DMPC-SUV (.about.100 nm in diameter) 141 mM in 5 mM HB
(.tangle-solidup.); or HB alone 5 mM (x); sliced into discs and
tested for their DMPC concentration as a function of cartilage
depth.
[0049] FIGS. 9A-9F are scanning electron microscope (SEM)
micrographs of cartilage specimens in the presence and absence of
lubricating media and friction tests. SEM micrographs of control
specimens, in the absence of friction test: FIG. 9A is a micrograph
of healthy cartilage, showing its naturally occurring lipidic
vesicle structures on the surface (.times.3000); FIG. 9B is a
micrograph of arthritic cartilage (.times.3000); and healthy
cartilage subjected to friction tests in the presence of the
following lubricants: saline (.times.6000, FIG. 9C); ISF
(.times.800 FIG. 9D); DMPC-SUV (.times.800, FIG. 9E); and DMPC-MLV
(.times.6000 FIG. 9F).
DETAILED DESCRIPTION OF SOME NON-LIMITING EMBODIMENTS
[0050] The present invention is based on results, inter alia,
making use of a human cartilage-on-cartilage setup (Merkher, Y. et
al..sup.40) where the following were determined (i) friction
coefficient measurements, (ii) wear of human articular cartilage
(iii) cartilage morphological studies based on SEM, (iv) cartilage
quantitative phospholipid and phosphatidylcholine (PC)
determinations, and (v) physicochemical characteristics of
different PC-based liposomes, which demonstrated the potential of
large (diameter greater than 0.3 .mu.m) multilamellar vesicles,
such as DMPC-MLV and of DMPC/DPPC-MLV (0.6/1.0 mole ratio),
dispersed in low ionic-strength histidine buffer (HB), as effective
cartilage lubricants and wear reducers at temperature slightly
above (e.g. about 1.degree. C., 2.degree. C., 3.degree. C.,
5.degree. C., 8.degree. C., 11.degree. C. and at times up to about
15.degree. C.) the Solid Order to Liquid Disorder (SO-to-LD) phase
transition temperature.
[0051] Initially, the lubricating efficacy of multilamellar
liposomes composed of various PCs, with two hydrocarbon chains from
14 to 22 carbons, fully saturated or with varying degrees of
un-saturation, was compared. C.sub.12-C.sub.16 hydrocarbon chains
where shown to be of preferred length.
[0052] Then, using the most effective single-component lubricant,
DMPC, the effects of liposome size, lamellarity, and of
incorporating either cholesterol, mPEG-DSPE or an additional PL
into the lipidic bilayer of DMPC liposomes was investigated. These
studies showed that MLV, such as DMPC-MLV or DMPC/DPPC-MLV (0.8-3.5
.mu.m in diameter), when used as lubricants at temperature slightly
above the SO-to-LD phase transition temperature, were most
effective. This was confirmed by the performance of DMPC/DPPC-MLV
at 37.degree. C., which is slightly above the range of its SO-to-LD
phase transition temperature, i.e., T.sub.m=.about.34.degree. C.,
in comparison to its performance at 24.degree. C. (SO phase).
[0053] The results presented herein below further show the
following: [0054] DMPC, which was identified as one preferred
component of the liposomal biolubricant composition (when used
alone or in combination with DPPC) has saturated, medium-length
acyl chains (14 carbons), having a T.sub.m slightly lower than the
physiological temperature (T.sub.m=23.2.degree. C. for DMPC-MLV and
T.sub.m=.about.34.degree. C. for DMPC/DPPC [0.6/1.0 mole/mole]
used), thus both PC compositions providing liposomes which are in
the LD phase at 37.degree. C. (see Table in Materials and Methods
below). When in the LD phase, PC polar headgroup is highly hydrated
(.about.9.7 water molecules per DMPC or DPPC headgroup, in
comparison to <4.3 water molecules per headgroup when below the
T.sub.m in the SO phase).sup.53; [0055] The adiabatic
compressibility data presented herein below demonstrate the
differences between PC in the solid-ordered (SO) phase (low K
values) and the LD phase (higher K values) and the superiority of
the LD phase. Partial adiabatic lipid bilayer compressibility (K),
which correlates well with the thermotropic behavior.sup.54 and was
found to reflect the level of hydration, physical state and the
volume of cavities (free volume) in the lipid bilayer.sup.45. Bound
water molecules, which interact with the PC headgroup, are
suggested to affect the total volume of cavities in the bilayer,
thus affecting intermolecular interactions, as well as the
adiabatic compressibility. Specifically, both DOPC and DMPC are in
the LD phase (above their T.sub.m Table 1 below) at (24.degree. C.
as well as at 37.degree. C. However, the lubrication ability of
DMPC liposomes was substantially superior to that of DOPC. Without
being bound by theory, it is believed that the difference in
behavior between DMPC and DOPC resides in the fact that under
physiological conditions, i.e. at a temperature of between
36.degree. C. and 43.degree. C. DMPC is only slightly above the
T.sub.m. Moreover, the temperature in synovial joints of the hand
can be as low as .about.28 C. Under such conditions DMPC is also
slightly above the T.sub.m. In addition, DMPC is the PC with the
shortest acyl chains capable of forming stable liposomes, thus
composing the mechanically "softest" bilayer of all other
single-component PC bilayers exemplified herein.sup.44. [0056] The
lubrication ability of MLV composed of DMPC/DPPC (0.6:1.0
mole/mole) mixture having good miscibility and nearly ideal mixing
properties, and a combined SO-to-LD phase transition temperature of
.about.34.degree. C. The DMPC/DPPC-MLV showed high lubricating
efficacy at 37.degree. C. (static and dynamic friction coefficients
of 0.017 and 0.0083, respectively) but not at 24.degree. C. (0.042
and 0.021, respectively), compared with DPPC-MLV alone (T.sub.m of
41.4.degree. C.) which were inferior at 37.degree. C. (0.029 and
0.022, for the static and dynamic friction coefficients,
respectively); [0057] The more efficient lubrication achieved by
using liposomes with T.sub.m only slightly below physiological
temperature. [0058] The protection of cartilage from wear using MLV
composed of DMPC/DPPC, where a mixture of DMPC/DPPC added to ISF,
substantially reduced the wear in comparison to ISF alone and ISF
with an addition of HA; this protection effect being significantly
higher than the other exemplified membrane, in particular that
composed of DPPC alone. [0059] The "softness" and hydration level
of DMPC-MLV and the impact of changes in these features on
cartilage lubrication. The first modification in formulation
included introduction of .about.33 mole % cholesterol into liposome
membranes. As shown below, this resulted in a physical transition
from the LD phase to the liquid-ordered (LO) phase.sup.34. Such a
change is known to "dry" the lipid bilayer.sup.56, and is also
reflected in a reduction in the adiabatic compressibility and
therefore in bilayer softness. Therefore, lubricating cartilage
with DMPC/cholesterol-MLV was substantially inferior to lubricating
of cartilage with DMPC-MLV (FIG. 1). In another modification 5 mole
% of the lipopolymer mPEG-DSPE into the lipid bilayer of DMPC-MLV
was introduced. The PEG moieties, extending 4-10 nm from the
liposome surface (depending on the polymer chain state, being
either in a mushroom or brush configuration.sup.39), and are highly
flexible and highly hydrated (3 to 4 water molecules per ethylene
oxide group).sup.45. However, addition of mPEG-DSPE to DMPC
liposomes did not improve lubrication (FIG. 1), which seemed to be
contradictory to the role of hydration in lubrication. This may be
explained by the fact that the PEG moiety although highly polar is
nonionic and therefore its hydration differs from that of the
hydration of ionic the PC headgroup.sup.45. It must be noted, that
these grafted PEG moieties may still be beneficial in vivo as they
can protect the liposomes from interacting with macromolecules of
interstitial fluid.sup.34, similarly to the cartilage-protecting
behavior of HA.sup.22; [0060] Friction coefficients obtained by
different media (saline, ISF, and low ionic strength HB)
demonstrated that HB was superior to saline and to ISF (FIGS. 1, 2,
5-7). Furthermore, the total PL concentration of cartilage
specimens lubricated with HB was nearly twice that of cartilage
lubricated with ISF and substantially higher than that of cartilage
lubricated with saline (FIG. 7). Suggesting that HB may better
retain naturally-occurring cartilage SAPLs, thereby improving
lubrication. The superiority of HB over saline (FIG. 1) can also be
explained by its lower ionic strength, which induces a less compact
PL packing in the lipid bilayer, thus enabling rapid bilayer
recovery after frictional events.sup.34, 57. This further supports
the importance of bilayer softness as a major contributor to
effective lubrication. From the above, it became apparent that HB
is an effective and supportive medium for liposomes as lubricants;
[0061] Large multilamellar DMPC-MLV were found to be superior to
small unilamellar liposomes (<100 nm). Without being limited by
theory as it is not required for the establishment of the
invention, it is believed that this superiority stems from the way
the former are retained near the cartilage surface, as demonstrated
by the PC distribution along cartilage depth (FIG. 8), due to the
large size of MLV (0.8-3.5 .mu.m in diameter). Maroudas et al.
reported the presence of 100-nm gaps between collagen fibers in
cartilage.sup.50. Stockwell and Barnett.sup.51 and Barnett and
Palfrey.sup.52 state that these fibers act as barriers against
penetration of large particles into the cartilage, reporting that
small silver proteinate particles penetrated deeper than large
particles into cartilage. The results presented herein show that
smaller DMPC-SUV penetrated deeply into cartilage, while DMPC-MLV
remained near the surface (FIG. 8). This is in agreement with the
similarity of friction levels obtained from cartilage lubricated
with DMPC-SUV in HB and of cartilage lubricated with HB alone (FIG.
1), as DMPC-SUV penetrate deeply into the cartilage the effect of
lubrication is primarily of the media (i.e. HB). [0062] SEM
morphological studies, in which naturally-occurring globular
structures, in the size range of DMPC-MLV, seemed to be present on
the surface of healthy non-lubricated cartilage prior to conducting
friction tests (FIG. 9A), and absent after friction tests of
healthy cartilage lubricated with saline or ISF (FIGS. 9C and 9D,
respectively). Cartilage specimens lubricated with DMPC-MLV seemed
to have globular lipidic structures on their surface, after
conducting friction tests (FIG. 9F).
[0063] In light of these results, it has been envisaged that
phospholipids (PL) selected from glycerophospholipids (GPL) and
sphingolipids (SPL), are potential substituents for the
naturally-occurring lipidic globular structures, being capable of
reducing friction and protecting against cartilage wear.
[0064] Further, it has been envisaged that when present near
cartilage surface liposomes comprising GPL, SPL or their
combination as the liposome forming phospholipids act as a
reservoir for replenishing a protective lipid bilayer coating the
cartilage surface, thus assisting in preservation of
naturally-occurring PL, as indicated by the higher total PL level
in cartilage lubricated with DMPC-MLV in comparison to cartilage
lubricated with other lubricants and media (FIG. 8 2).
[0065] In accordance with some embodiments of the invention, the
GPL is carrying a phosphocholine headgroup (phosphatidylcholine,
PC-based lipid) or a phosphoglycerol headgroup
(phosphatidylglyccrol, PG-based lipid), and the SPL is a ceramide
(N-acyl sphingosine carrying a phosphocholine headgroup, also
referred to as N-acyl sphigosyl-phsphocholine (SM-based lipid).
[0066] As appreciated by those versed in lipid based technologies,
PCs and SMs are zwitterionic phospholipids with the cationic
choline and anionic diester phosphate moieties (constituting the
phopshocholine head group) remain fully ionized over a broad pH
range with no net charge (zeta potential=0 mV).sup.34. The PG is
negatively charged over broad pH range as evident from it negative
zeta potential. The hydrophobic part of the PC and PG includes 2
hydrocarbon (e.g. acyls and alkyls) chains. The SM also has two
hydrophobic hydrocarbon chains of which one is the chain of the
sphingoid base itself and the other is N-acyl chain. PC, SM and PG
in which the hydrocarbon chains is above 12 carbon atoms are all
cylinder like in shape as their packing parameter is in the range
of 0.74-1.0. They form lipid bilayers which above the SO to LD
phase transition become highly hydrated and vesiculate to form
lipid vesicles (liposomes).sup.34, 35. The PC and PG liposome
bilayer can be either in a solid ordered (SO) phase (previously
referred to as gel or solid phase), or in a liquid disordered (LD)
phase (previously referred to as liquid crystalline or fluid
phase).sup.34. The transformation between the SO to LD phases
involves an endothermic, first order phase transition referred to
as the main phase transition. T.sub.m is the temperature in which
the maximum change in the heat capacity change during the SO to LD
phase transition occurs. T.sub.m and the temperature range of the
SO to ID phase transition of PCs depend, inter alia, on PC
hydrocarbon chain composition. In the LD phase (but not in the SO
phase), the charged phopshocholine and phosphoglycerol head group
are highly hydrated.
[0067] It is further noted that PGs and SM have T.sub.m that are
similar to that of the corresponding PC (the same length of
substituting hydrocarbon chain(s)). For instance, the T.sub.m of
DMPG is identical to that of DMPC, namely, 23.degree. C., and that
of DPPG or N-palmitoyl SM is identical to that of DPPC, namely,
41.4.degree. C. Thus, while the following examples make use of
PC-based lipids, the PL in accordance with the invention may also
be a PG- or SM-based lipid.
[0068] In accordance with the invention, a mixture of two or more
PLs (e.g. two different PCs, a PC with PG, two different PGs, two
SM, a PC or PG with SM, etc) may be used, as long as the mixture
formed is in a LD state and the lipid headgroups are highly
hydrated, when in situ (either at the articular region of a healthy
or a dysfunctioning joint).
[0069] Having considered the above, the inventors have developed
liposomal systems for joint lubrication, which are chemically
stable, oxidative-damage-resistant and free of HA.
[0070] Thus, in accordance with an aspect of the invention, the use
of a liposome comprising at least one PL selected from
glycerophospholipid (GPL) or sphingolipid (SPL), for joint
lubrication is provided.
[0071] By another aspect of the invention, there is provided the
use of a liposome comprising at least one PL selected from
glycerophospholipid (GPL) or sphingolipid (SPL), for the
preparation of a pharmaceutical composition for joint
lubrication.
[0072] The liposomes in accordance with both aspects being
characterized in that they comprise one or more membranes with at
least one phospholipid (PL) of the group consisting of a
glycerophospholipid (GPL) having two, being the same or different,
C.sub.12-C.sub.16 hydrocarbon chains and a sphingolipid (SPL)
having a C.sub.12-C.sub.18 hydrocarbon chain. The phase transition
temperature in which solid ordered (SO) to liquid disordered (LD)
phase transition occurs is within a temperature range of about
20.degree. C. to about 39.degree. C. The liposomes are used to
lubricate joints that have a joint temperature that is somewhat
higher than the phase transition temperature. Accordingly the
liposomes are in an LD phase within the joint. The fact that the
joint temperature is typically only slightly (e.g. within the range
of about 1.degree. C. to about 15.degree. C., as detailed above)
above the phase transition temperature seems to be of importance
for efficient lubrication.
[0073] In one preferred embodiment said C.sub.12-C.sub.15 or
C.sub.12-C.sub.18 hydrophobic chains are saturated.
[0074] It is noted that the above conditions are cumulative,
namely, the selection of PL (either a single PL or a combination of
PL with additional PLs) contained in the liposome is so that the
liposome will have SO-LD phase transition temperature between about
20.degree. C. to about 39.degree. C.
[0075] In accordance with additional embodiments of the invention,
the liposomal systems making use the said GPL or SPL further
encompass one or more of the following, all of which require to
exhibit a liposomal system having a phase transition temperature as
defined herein:
[0076] The GPL or SPL have alkyl, alkenyl or acyl C.sub.12 to
C.sub.16 hydrocarbon chain. In the case of GPL, the two chains may
be the same or different.
[0077] One particular embodiment concerns the use of liposomes
having GPL or SPL with at least one C.sub.14 acyl chain.
[0078] Another particular embodiment concerns the use of a GPL
having C.sub.14 and C.sub.16 acyl chains.
[0079] Another particular embodiment concerns the use of liposomes
having SPL with a C.sub.16 acyl chain.
[0080] Another particular embodiment concerns the use of a
combination of any of the above liposomes.
[0081] Some GPL or SPL have a ionic headgroup and, according to
embodiments of the invention, this headgroup is highly ionized at a
wide range of pH. A wide range may be defined by a pH between 3 and
14.
[0082] The GPL as well as the SPL are highly hydrated, namely, the
number of water molecules per lipid headgroup is at least about 6;
7 or at times at least 8 water molecules that are complexed to the
ionized head group of the GPL or SPL.
[0083] The GPL or SPL are capable of forming MLV (as well as the
other type of liposomes mentioned above), preferably MLV having a
mean diameter above 0.3 .mu.m.
[0084] According to one embodiment, the MLV are defined by a mean
diameter in the range of between 0.3 .mu.m and 5 .mu.m. According
to another embodiment, the MLV are defined by a mean diameter in
the range of between 0.8 .mu.m and 3.5 .mu.m.
[0085] As cholesterol was found to reduce lubrication properties of
the MLV being formed from GPL, SPL or their combinations, as
defined herein, the MLV or the other types of liposomes that may be
used in accordance with the invention should not include in their
bilayers a membrane active sterol, such as cholesterol. A membrane
active sterol is defined as affecting short- and long-range lipid
order within membranes, minimizing volume, and decreasing membrane
permeability. Specifically, the sterol should possess 1), a flat,
fused ring system, 2), a hydroxyl or other small polar group at
position 3, 3), a "cholesterol-like" tail, and 4), a small area per
molecule (<40 .ANG..sup.2 when assembled at the air/water
interface at a surface pressure of 12 mN/m).
[0086] It is to be noted that the compositions of the invention
preferably do not contain propylene glycol.
[0087] It should further be noted that the compositions of the
invention preferably do not contain dextran.
[0088] A particular group of GPLs encompassed by one or more of the
above embodiments comprise a GPI, carrying a phosphocholine
headgroup (PC or SM-based lipids). One preferred PC in accordance
with the invention is dimyristoylphosphatidylcholine (DMPC).
[0089] Non-limiting examples of PC-based lipids which may be used
in accordance with the invention comprise
1,2-dipalmitoyl-sn-glycero-3-phosphocoline (DPPC, T.sub.m
41.4.degree. C.); 1,2-dipentadecanoyl-sn-glycero-3-phosphocoline
(C15, T.sub.m 33.0.degree. C.), albeit, these two being suitable
only when combined with one or more other lipids so as to form a
liposomal system having a phase transition temperature as defined
herein. SPL which may be in accordance with the invention comprise
a sphingomyelin (SM) carrying a phosphocholine headgroup, and
non-limiting examples include N-palmitoyl SM T.sub.m 41.0.degree.
C. and 1,2-dimyristoyl-sn-glycero-3-PC. T.sub.m values of various
PC-based lipids may be found in "Thermotropic Phase Transitions of
Pure Lipids in Model Membranes and Their Modifications by Membrane
Proteins", John R. Silvius, Lipid-Protein Interactions, John Wiley
& Sons, Inc., New York, 1982, and also in the Lipid
Thermotropic Phase Transition Data Base--LIPIDAT, and in Marsh
(1990).sup.36.
[0090] It is noted that in accordance with the invention the MLV
liposomes (or the other liposomes useful according to the
invention) have an offset temperature (upper limit) of the SO to LD
phase transition which is not higher than 15.degree. C. from the
temperature in situ, i.e. in the joint, within the range of about
20.degree. C. to about 39.degree. C. In accordance with the
invention the MLV liposomes are formed from GPL, SPL or their
combination, and the SO to ED phase transition temperature
described above thus concerns MLV liposomes which are formed from
GPL, SPL and combinations thereof, thus providing a liposome in
which the PLs or their mixture are in LD phase.
[0091] A particular embodiment in accordance with the invention
concerns the use of DMPC-MLV or DMPC/DPPC-MLV for the preparation
of a replacement of naturally-occurring cartilage PL, namely as a
cartilage lubricant and wear reducer. These MLV have major
practical advantages as well. They can be prepared simply and at
low cost. DMPC and DPPC are both resistant to oxidative damage and
stable for long periods of time. Furthermore, these PCs are already
approved for human use. According to one embodiment, when using a
mixture of DMPC and DPPC, the mole ratio between DMPC and DPPC
depends on the temperature of the joint to be treated and is
designed such that the T.sub.m of the combination provides MLV in
ED phase. One example of a suitable ratio is about 0.6/1.0 which
provides MLV in LD phase at a joint temperature between 35.degree.
C. to 39.degree. C.
[0092] In accordance with an additional aspect of the invention
there is provided a method for lubricating a joint of a mammal, the
method comprises administering into a cavity of said joint
containing synovial fluid an amount of liposomes effective to yield
a lubricating effect.
[0093] It is noted that the temperature of joints in patients
afflicted with reduced joint lubrication or with joint wear, such
as osteoarthritis varies as the disease proceeds [Hollander, J. L.;
Moore, R., Studies in osteoarthritis using Intra-Articular
Temperature Response to Injection of Hydrocortisone. Ann. Rheum.
Dis. 1956, 15, (4), 320-326]. In fact, this temperature change was
used as a clinical tool for assessing osteoarthritis inflammation
[Thomas, D.; Ansell, B. M.; Smith, D. S.; Isaacs, R. J., Knee Joint
Temperature Measurement using a Differential Thermistor
Thermometer. Rheumatology 1980, 19, (1), 8-13]. In hand joints of
osteoarthritis patients temperature was shown to vary from
.about.28 to .about.33.degree. C. [Varju, G.; Pieper, C. F.;
Renner, J. B.; Kraus, V. B., Assessment of hand osteoarthritis:
correlation between thermographic and radiographic methods.
Rheumatology 2004, 43, 915-919], while the temperature of healthy
Temporomandibular joint (TMJ) varies from .about.35 to 37.degree.
C. [Akerman, S.; Kopp, S., Intra-articular and skin surface
temperature of human temporomandibular joint. Scand. J. Dent. Res.
1987, 95, (6), 493-498].
[0094] Thus, in accordance with the invention it is essential and
in fact a pre-requisite that the GPL or the mixture thereof with
additional PLs, be in a LD phase, in situ, at the joint region to
be lubricated therewith.
[0095] The method of the invention may be used to treat, alleviate,
retard, prevent, manage or cure any articular disorder or symptoms
arising there from which is associated with joint dysfunction. For
the purposes of this disclosure the term "articular disorder" shall
be held to mean any affliction (congenital, autoimmune or
otherwise), injury or disease of the articular region which causes
degeneration, pain, reduction in mobility, inflammation or
physiological disruption and dysfunction of joints. The disorder
may be associated with reduced joint secretion and lubrication as
well as from complications of knee and hip replacement.
[0096] The joint in accordance with the invention may be any one of
the knee, hip, ankle, shoulder, elbow, tarsal, carpal,
interphalangeal and intervertebral.
[0097] Specific articular disorders include, but are not limited
to, deficiencies of joint secretion and/or lubrication arising from
arthritis, including conditions of joint erosion in rheumatoid
arthritis, osteoarthritis, osteoarthritis in rheumatoid arthritis
patients, traumatic joint injury (including sports injury), locked
joint (such as in temporomandibular joint (TMJ)), status post
arthrocentesis, arthroscopic surgery, open joint surgery, joint
(e.g. knee or hip replacement) in mammals, preferably humans. A
preferred disorder to be treated or prevented by the method of the
invention is osteoarthritis.
[0098] The method of the present invention could be used as a
prophylactic measure to prevent future damage or degeneration. For
example, the PL based MLV liposomes could be administered
intra-articularly to athletes intermittently throughout their
career to minimize the risk of stress related injury or cartilage
degeneration.
[0099] The method of the present invention may be used exclusive
of, or as an adjunct to, anti-inflammatory agents, analgesic
agents, muscle relaxants, anti-depressants, or agents that promote
joint lubrication commonly used to treat disorders associated with
joint stiffness, such as arthritis. A combined therapeutic approach
is beneficial in reducing side effects associated with agents, such
as non-steroidal, anti-inflammatory drugs (NSAIDs), commonly used
to prevent, manage, or treat disorders such as osteoarthritis
associated with reduced joint lubrication. In addition to enhancing
safety, a combined therapeutic approach may also be advantageous in
increasing efficacy of treatment.
[0100] The administration of the liposomes into an articular cavity
of a patient may be by a method chosen from the group consisting of
intra-articular injection, arthroscopic administration or surgical
administration.
[0101] The invention also provides, in accordance with yet another
aspect of the invention, a pharmaceutical composition for joint
lubrication comprising a physiologically acceptable carrier and
liposomes comprising at least one PL selected from GPL or SPL as
defined herein.
[0102] In accordance with one embodiment, the physiologically
acceptable carrier is hyaluronic acid (HA) or histidine buffer
(HB). The composition may also include polymers such as those
described by Klein et. al..sup.31.
[0103] The composition according to the invention is preferably in
a form suitable for administration by a route selected from
intra-articular injection, arthroscopic administration or surgical
administration.
[0104] The amount of liposomes in the composition will vary
depending on the liposome's PL composition, the disease, its
severity and treatment regimen, as well a on the age, weight, etc.,
of the mammal to be treated. The amount for purposes herein is
determined by such considerations as may be known in the art. The
amount must be effective to achieve an improvement in the
lubrication of the treated joint, namely, to reduce friction
between the cartilages forming the joint, the improvement may be
exhibited by clinical tests as well as by an improvement in the
well-being of the subject undergoing said treatment (e.g. reduced
pain in the afflicted joint, improvement in mobility). The
effective amount is typically determined in appropriately designed
clinical trials (dose range studies) and the person versed in the
art will know how to properly conduct such trials in order to
determine the effective amount.
[0105] Throughout the description and claims of this specification,
the singular forms "a" "an" and "the" include plural references
unless the context clearly dictates otherwise. Thus, for example, a
reference to "a PL" is a reference to one or more PLs and "a
liposome" refers to one or more liposomes. Throughout the
description and claims of this specification, the plural forms of
words include singular references as well, unless the context
clearly dictates otherwise.
[0106] Yet, throughout the description and claims of this
specification, the words "comprise" and "contain" and variations of
the words, for example "comprising" and "comprises", mean
"including but not limited to", and are not intended to (and do
not) exclude other moieties, additives, components, integers or
steps.
[0107] The invention will now be described by way of non-limiting
examples.
DESCRIPTION OF NON-LIMITING EXAMPLES
Example 1
Materials and Methods
Lipids:
[0108] Lipids used in this study and their sources are described in
Table 1; all are >98% pure. Table 1 also presents the
solid-ordered (SO) to liquid-disordered (LD) phase transition
temperatures, T.sub.m, of phospholipid bilayers,.sup.34-36 as well
as the bilayer state at 37.degree. C.
TABLE-US-00001 TABLE 1 phase transition temperatures Phase
transition Phase at temperature Lipid Chemical name (source)
37.degree. C. (T.sub.m), .degree. C. HSPC hydrogenated soybean SO
52.5 phosphatidylcholine (Lipoid, Ludwigshafen, Germany) DPPC
1,2-dipalmitoyl-sn-glycero-3- SO 41.4 phosphocholine (Avanti,
Alabaster, AL, USA) DMPC 1,2-dimyristoyl-sn-glycero-3- LD 23.2
phosphocholine (Lipoid or Avanti) DOPC 1,2-dioleoyl-sn-glycero-3-
LD -21 phosphocholine) (Lipoid or Avanti) Mixture of LD 34
DMPC/DPPC (0.6/0.1)
Water:
[0109] Water was purified using a WaterPro PS HPLC/Ultrafilter
Hybrid system (Labconco, Kansas City, Mo.), providing pyrogen-free
water with low levels of total carbons and inorganic ions (18.2
M.OMEGA.).
Reagents:
[0110] All other reagents used are of analytical grade or
better.
Liposomes:
[0111] Multilamellar liposomes (MLV) were prepared by dissolving
the desired lipids in tert-butanol, followed by lyophilization to
form a dry "cake". This was hydrated in low ionic strength (5 mM)
histidine buffer (HB) pH 6.7, at a temperature at least 5.degree.
C. above the T.sub.m.sup.34. When desired, MLV were downsized to
form small unilamellar vesicles (<100 nm, SUV) by stepwise
extrusion through polycarbonate membranes (GE-Osmonics, Minnetonka,
Minn.), starting with a 400-nm and ending with a 50-nm-pore-size
membrane, using a 10-mL extrusion system (Northern Lipids,
Vancouver, Canada) heated at least 5.degree. C. above the
T.sub.m.sup.37.
[0112] Initial screening of cartilage lubricants was performed with
MLV of different PC compositions--DMPC, DPPC, HSPC, DBPC, DOPC and
POPC (for abbreviations see Table 1). In this screening it was
found that DMPC liposomes acted as the best friction reducers
(FIGS. 1 and 3). Therefore, DMPC-based liposomes were further
investigated comparing liposomes composed of either DMPC alone, of
different sizes and lamellarities, or of a DMPC/DPPC mixture
(0.6:1.0 mole ratio), or of DMPC combined with cholesterol (2:1
mole ratio), or of DMPC combined with the lipopolymer mPEG-DSPE
(95:5 mole ratio). The mPEG-DSPE used consists of a 2000 Dalton
polyethylene glycol attached to the primary amino group of
distearoyl phosphatidylethanolamine.
Liposome Characterization:
[0113] Liposomes were characterized for: [0114] (i) phospholipid
(PL) concentration, using the modified Bartlett assay.sup.37, 38;
[0115] (ii) size distribution, for liposomes under 1 .mu.m by
dynamic light scattering using an ALV-NIBS High Performance
Particle Sizer (Langen, Germany) at a scattering angle of
173.degree.; and for liposomes above 400 inn by light diffraction
using a Beckman Coulter LS Particle Size Analyzer 13-320
(Fullerton, Calif.), equipped with polarization intensity
differential scattering (PIDS) to provide a dynamic detection range
from 40 nm to 2000 .mu.m; [0116] (iii) partial specific adiabatic
compressibility, by calculation from the density of the liposome
dispersion (using a DMA 5000 density meter, Anton Paar, Graz,
Austria) and the velocity of an 5 MHz ultrasonic wave traveling
through it (using a UCC-12 ultrasonic velocimeter, NDT Instruments,
Jerusalem, Israel), as described by Garbuzenko et al..sup.39; and
[0117] (iv) structure, using scanning electron microscopy
(SEM).
Cartilage:
[0118] Articular cartilage from healthy or osteoarthritis humans
(aged 65 to 86 years) was obtained from femoral head fracture
operations or total hip replacements. Tissue was classified as
normal or pathological according to the visual diagnosis. For wear
rate determination, only femoral heads with normal tissue were
selected. Specimens were frozen at -20.degree. C. until sample
preparation in order to keep the mechanical properties close to
those of live tissue. Pairs of cylindrical plugs, having 4 mm and 8
mm in diameter, each pair from the same region of the joint, were
prepared. These cylindrical plugs, consisting of about 2 mm thick
cartilage on top of about 8 mm long bone, were removed from the
femoral head using a cork borer. The plugs were glued to holders
through the bone part, using cyanoacrylate-based adhesive glue,
leaving the cartilage projecting out of the holders. Thereafter,
these plugs were refrozen at -20.degree. C. until tested. Cartilage
with completely intact and smooth surface was used.
Friction and Wear Testing:
[0119] Liposomes covering a wide range of sizes and concentrations,
dispersed in HB, were screened as potential lubricants to reduce
friction and wear between two discs of human cartilage at
24.degree. C. and 37.degree. C. Friction measurements were carried
out with a cartilage-on-cartilage setup (Merkher, Y.; Sivan, S.;
Etsion, I.; Maroudas, A.; Halperin, G.; Yosef, A., A rational human
joint friction test using a human cartilage-on-cartilage
arrangement. Tribol. Lett. 2006, 22, 29-36, the content of which is
incorporated herein by reference in its entirety), using two discs
of cartilage immersed in a liposomal dispersion in HB, or as
controls, in HB alone, or in physiological saline (0.9% w/v; pH
5.0; Teva Medical, Israel), or in inflamed synovial fluid (ISF)
obtained from osteoarthritis patients. These discs were subjected
to relative sliding over a wide range of loads (1 to 30 N),
equivalent to physiological pressures in joints (0.08 to 2.4 MPa).
Various sliding velocities (0.5 to 2 mm/s) and dwell times (5 to
300 s) were used to simulate, together with various loads, a range
of physiological movements.
[0120] For the a qualitative evaluation of wear, the effect of
friction tests on the concentration of total PL in cartilage, and
on the structure of the cartilage surface was determined.
PL Extraction and Quantification:
[0121] Total PL were extracted from cartilage specimens before and
after lubrication tests, using the Bligh and Dyer extraction
procedure.sup.41, 42. For this, cartilage specimens were incubated
in a chloroform-methanol solution (1:1 v/v) for 1 h at 37.degree.
C. Water was added to a final chloroform-water-methanol ratio of
1:1:1, the solution was Vortexed for 1 min and then centrifuged,
using a desk centrifuge, to form two phases. The chloroform-rich
lower phase, containing the PL, was collected, dried under vacuum
(Concentrator 5301, Eppendorf), and the residual (containing
lipids) was re-dissolved in a small volume of chloroform-methanol
solution (2:1 v/v) and then loaded onto low-phosphorus silica gel
TLC glass plates (Uniplate--Silica Gel G, Analtech, Newark, Del.).
A chloroform-methanol-water (65:25:4 v/v/v) solvent system was used
for TLC.sup.41. Commercial markers of sphingomyelin, PC and PE were
also loaded on the plates for spot identification. Lipid spots were
detected after spraying the dried TLC plates with a UV-detectable
primulin (Sigma) solution (1 mL of 0.1% w/v primulin in water,
added to 100 mL acetone-water, 4:1 v/v). Each PL spot was scraped
from the TLC plate, and its PL content was quantified by the
modified Bartlett procedure..sup.37, 38.
[0122] PL concentration was also quantified as a function of
cartilage depth. For this, cartilage specimens were sectioned by
microtome into slices 20 or 50 .mu.m thick, from the cartilage
surface inwards, parallel to the face of the cartilage. PL
concentration of each slice was quantified, after PL were extracted
as mentioned above, by the modified Bartlett procedure.sup.37,
38.
Wear Determination Test:
[0123] For the wear test, human inflamed synovial fluid (ISF) was
retrieved from several inflamed joints. It was pooled in order to
obtain a large quantity of uniform ISF for different tests with and
without liposome additives. Commercially available HA (10 mg/mL, pH
6.7, of rooster comb MW 1-4.times.10.sup.6, catalog no. H5388
Sigma, USA) which is used for intra-articular administration into
osteoarthritis joints, was tested as another additive to ISF in
order to investigate its effect on articular cartilage wear in
comparison to liposome additives.
[0124] The reciprocating sliding amplitude was set to 1 mm,
assuring full contact between the two cartilage samples during the
wear test. Each test was carried out for 7.5 hours with an average
reciprocating velocity of 4 m/min. This resulted in a maximum of
450,000 reciprocating cycles for each test, which amounts to a
total sliding distance of 1800 m. Normal load of 60 N corresponding
to contact pressure of 4.8 MPa, which is well in the range of
physiological pressures in joints, was used. In order to compensate
for any possible loss of cartilage components (which can occur, for
example, from loose ends at the circumferential cylindrical cut
surface of the plugs during agitation in the lubricating fluid),
experiments with identical test conditions but with no load and no
contact between the cartilage specimens were also conducted. The
actual wear was calculated by subtracting the results at no load
from these obtained when load was applied.
[0125] A pair of frozen specimens was thawed, and the upper and
lower specimens were fixed to a wear test loading mechanism and a
bath, respectively. A volume of 1.5 ml of the lubricating fluid was
placed in the bath and the reciprocating sliding motion started
after applying a normal load (compensation at 0 N, and wear test at
60 N).
[0126] As more than 95% of the glycosaminoglycans (GAGs) in
articular cartilage is sulphated (mainly chondroitin sulfate and
keratan sulfate).sup.61, analyzing for GAG content.sup.62 in the
aqueous cartilage medium containing the wear particles (particles
of the wear debris released from both cartilage surfaces into the
lubricating fluid) was used to assess the wear of the two cartilage
specimens,
Cartilage Structure:
[0127] Cartilage structure was examined by SEM. Specimens were
preserved by rapid cooling in liquid nitrogen and kept under vacuum
(.about.15 mbar) for 48 h to remove excess water. Next, specimens
were mounted on stubs and sputter-coated with gold in a Polaron
E5100 Sputter Coater (Watford, England). The specimens were
examined using an FEI Quanta 200 scanning electron microscopy
system (Polaron) using an accelerating voltage of 30 kV.
Results
Liposome Carrier:
[0128] it was found that the lubrication efficiency of HB is better
than that of saline, or of ISF (FIG. 1). Furthermore, liposomes
dispersed in HB were better lubricants than liposomes dispersed in
saline (FIG. 2). Thus, in the following, liposome additives to be
screened for their cartilage-lubricating abilities were all
dispersed in HB.
[0129] The surface-active phospholipids (SAPL) tested were
phosphatidylcholines (PCs), which are also naturally present in
cartilage and synovial fluid.
[0130] Screening liposomes for cartilage lubrication and wear
reduction, involved comparison of a cartilage wear as well as
static and dynamic friction coefficients obtained with MLV composed
of various single-component PCs. The exemplified PCs differ in
their acyl chains, which determine the basic characteristics of the
liposomes, especially the T.sub.m and physical state.
[0131] Screening MLV (0.8 to 3.5 .mu.m in diameter) composed of
different PCs (DMPC, DPPC, HSPC, DBPC, DOPC and POPC) revealed that
both at 24.degree. C. and 37.degree. C., DMPC was the
best-performing cartilage lubricant. This was confirmed in FIG. 3
which show the amount of cartilage wear found in the presence of
DOPC, HSPC, DPPC and DMPC, all dispersed in HB as a function of
sliding distance. Similar behavior of wear to that in the presence
of media alone (FIG. 2), but with lower wear values, was shown for
all liposome types. Comparing cartilage wear in the presence of
different liposome additive showed that DMPC (Tm=23.2.degree. C.)
is superior lubricant additive, both in lower wear values during
the run-in period and in lower constant wear rate thereafter.
Similar results were found for the friction tests described in FIG.
1.
[0132] In addition, FIG. 4 shows the amount of cartilage wear found
in the presence of DPPC, DMPC and a mixture of DMPC/DPPC, all
dispersed in HB, as a function of sliding distance. Cartilage wear
in the presence of the DMPC/DPPC mixture (0.6/1.0 mole/mole
T.sub.m=34.degree. C.) and DMPC (T.sub.m=23.2) were similar and
both were significantly lower than that exhibited in the presence
of DPPC MLV (T.sub.m 41.4.degree. C.) liposomes alone.
[0133] Using liposomes composed of a mixture of close to ideally
miscible PC's (no phase separation) may enable fitting their
T.sub.m to suit a wide range of temperatures. For example, the
ratio of DMPC/DPPC can be adjusted, so that phase transition would
take place at all physiological temperatures occurring in different
conditions of osteoarthritis.
ISF, ISF+DMPC/DPPC, ISF+HA:
[0134] In order to determine whether an addition of liposomes in
ISF can reduce wear, comparative tests were performed in the
presence of ISF alone and ISF+DMPC/DPPC. Of all the liposomes
tested earlier, the mixture of DMPC/DPPC was chosen as it was found
to be the most efficient lubricant additive (see FIGS. 3 and 4).
ISF+DMPC/DPPC (150 mM) was prepared by adding a mixture of
DMPC/DPPC in HB with a concentration of 300 mM to ISF with a ratio
of 1:1.
[0135] One of the treatments commonly used in osteoarthritis
patients is intra-articular administration of HA into inflamed
joints. The effectiveness of this treatment is still a
controversial issue and therefore it is of interest to study the
effect of ISF+HA on cartilage wear reduction in comparison with
that of ISF alone and ISF+DMPC/DPPC. A common symptom in
osteoarthritis patients is excessive synovial fluid production.
Therefore, aspiration of the joint is usually performed before the
HA injection. The excessive fluid production, which continues after
the HA injection, constantly changes the ratio of HA and ISF in the
injected inflamed joint. Since the actual ratio of HA and ISF as a
function of time is not known, a fixed typical ratio of 1:1 was
selected for the current tests in accordance with the same ratio of
ISF and DMPC/DPPC. This ratio is probably higher than the one in
actual injected osteoarthritis joints and therefore its effect on
wear reduction may be considered as an upper limit.
[0136] FIG. 5 shows the amount of cartilage wear found in the
presence of ISF, ISF+HA and ISF+DMPC/DPPC as a function of sliding
distance. Similar behavior of wear to that in the presence of media
alone and other liposome based lubricants was shown, where a
relatively short run-in period was followed by a long constant wear
rate period. Comparing cartilage wear in the presence of the
different lubricants shows that although the addition of HA to ISF
induces less wear than ISF alone, the addition of DMPC/DPPC mixture
to ISF is much more effective in terms of reducing cartilage
wear.
[0137] A summary of the constant wear rates following the run-in
period, that were obtained with all the different lubricating
fluids and additives is presented in FIG. 6. As can be seen, the
constant wear rate of cartilage in the presence of HB is lower in
comparison with saline and therefore HB was selected as the
preferred carrying media for all the tested liposome types.
Screening the different liposomes shows that the lowest constant
wear rate is achieved in the presence of the mixture of DMPC/DPPC.
Moreover, adding DMPC/DPPC to ISF reduces the constant wear rate by
.about.40% compared with ISF alone, while adding HA to ISF reduces
the constant wear rate only by .about.10%. Based on these results
it was suggested that intra-articular injections of DMPC/DPPC may
be used to improve cartilage lubrication in osteoarthritis
patients.
Friction and Wear in Cartilage Lubricated with Several DMPC-Based
Liposomes:
[0138] Investigating the effect of liposome size and lamellarity,
the lubricating efficacy of multilamellar DMPC liposomes (DMPC-MLV)
was compared to that of <100-nm unilamellar DMPC liposomes
(DMPC-SUV). In addition, the efficacy as cartilage lubricants of
DMPC-MLV enriched with lipids which are non-liposome-forming,
although are common liposome components, such as cholesterol or
mPEG-DSPE, was studied. Cholesterol, having a packing parameter of
.about.1.2.sup.39, was added at .about.33 mole % to form
DMPC/cholesterol-MLV, thus causing the transformation of the lipid
bilayer from the solid-ordered (SO, if PL are below the T.sub.m) or
liquid-disordered (LD, if PL are above T.sub.m) phase to a new
physical phase termed liquid-ordered (LO).sup.43, 44. Thereby, it
was possible to compare the effect of liposomes at the three
different bilayer phases LD, SO and LO on lubrication. Another
component added to DMPC-MLV was the lipopolymer mPEG-DSPE, having a
relatively low packing parameter of .about.0.5.sup.39, which
introduces a highly-hydrated extended steric bather that surrounds
the liposome.sup.39, 45. mPEG-DSPE was added at 5 mole % to form
DMPC/mPEG-DSPE-MLV.
[0139] The static and dynamic friction coefficients of DMPC-MLV in
HB (0.020 and 0.011, respectively) were lower than those obtained
with DMPC/cholesterol-MLV in HB (0.040 and 0.036, respectively) or
DMPC/mPEG-DSPE-MLV in HB (0.022 and 0.023, respectively), as shown
in FIG. 1, and were similar to the low friction coefficients which
exist in healthy synovial joints.sup.46. Furthermore, the static
and dynamic friction coefficients of cartilage lubricated with
DMPC-MLV were lower than those of cartilage lubricated with
DMPC-SUV (0.045 and 0.036, respectively) which were only slightly
lower than those of HB alone (0.053 and 0.037, respectively), FIG.
1.
[0140] Statistical evaluation, by Student's t test, indicated the
superiority of DMPC-MLV over the other liposome formulations tested
at this assay and media (p<0.008).
Compressibility of the Lipid Bilayer:
[0141] The partial specific adiabatic compressibility, K, is a
measure of both the physical phase of the lipid bilayer (SO, LD or
LO) and its hydration state, which is postulated herein to have an
important contribution to the liposomes' efficacy as friction and
wear reducers.sup.45 Values of K for DMPC, DPPC and hydrogenated
soy phosphatidylcholine (HSPC) determined at 37.degree. C. were
50.7, 31.2 and 33.3.times.10.sup.-6 mL/(g-atm), respectively. A
similar profile, with somewhat lower values of K, 46.4, 28.0 and
30.3.times.10.sup.-6 mL/(g-atm), was found at 24.degree. C. for
DMPC, DPPC and HSPC, respectively. These K values reflect the
higher phase transition temperatures, T.sub.m, of DPPC and HSPC
(41.4.degree. C., 52.5.degree. C.) than that of DMPC (23.2.degree.
C.) and thus the superiority of liposomes having a membrane having
a phase transition in the range defined in the present invention
(T.sub.m between 20.degree. C. to 39.degree. C. inclusive). In
DMPC/cholesterol liposomes (2:1 mole ratio) K is reduced to 42.2
and 45.5.times.10.sup.-6 mL/(g-atm) at 24.degree. C. and 37.degree.
C., respectively. Introducing 5 mole % mPEG-DSPE into HSPC
liposomes (T.sub.m 53.degree. C.).sup.39 raised compressibility to
32.8 and 35.5.times.10.sup.-6 mL/(g-atm) at 24.degree. C. and
37.degree. C., respectively. While in HSPC/cholesterol liposomes
(2:1 mole ratio) K is reduced to 30.0 and 33.6.times.10.sup.-6
mL/(g-atm) at 24.degree. C. and 37.degree. C.
[0142] Without being bound by theory, the above results suggest
that the physical phase of the MLV bilayers are important for
cartilage biolubrication, and that the optimal conditions for
lubrication are being at the LD phase, not to far above the
SO-to-LD phase transition temperature (T.sub.m). To further test
this hypothesis the inventors tested MLV composed of 0.6/1.0
(mole/mole) DMPC/DPPC. This composition was selected so as to
enable the formation of a liposome having a T.sub.m of
.about.34.degree. C..sup.47 (being possible due to the nearly ideal
mixing of these two PCs). These MLV were studied at 24.degree. C.
and 37.degree. C. The results clearly support the above hypothesis,
as they show (FIG. 1) that DMPC/DPPC-MLV are the most effective
lubricants at 37.degree. C. (static and dynamic friction
coefficient of 0.017 and 0.0083, respectively) but not at
24.degree. C. (static and dynamic friction coefficient of 0.042 and
0.021, respectively). Furthermore DMPC/DPPC-MLV were superior to
DPPC-MLV (T.sub.m=41.3) alone, which are inferior at 37.degree. C.
(static and dynamic friction coefficient of 0.029 and 0.022,
respectively).
PL Levels in Lubricated Cartilage Specimens:
[0143] The total PL (which includes naturally-occurring SAPLs and
PLs from liposomes) levels of healthy cartilage specimens
(thickness .about.4200 .mu.m), before and after being subjected to
friction tests, in the presence of different lubricants and media,
was measured. It can be seen (FIG. 8) that the total PL
concentration in cartilage lubricated with DMPC-MLV is the highest
among all specimens tested. The PL concentration of cartilage
obtained from healthy subjects and lubricated with HB is higher
than that of similar cartilage lubricated with saline or ISF, the
latter (ISF), has similar PL levels to that of cartilage obtained
from osteoarthritis patients.
Effect of Liposome Size and Lamellarity on their Penetration into
Cartilage:
[0144] PC concentration, as a function of cartilage depth (0-800
.mu.m, in 20-50-.mu.m increments), was measured after friction
tests for specimens lubricated with DMPC-MLV and DMPC-SUV, both
dispersed in HB, and for specimens lubricated with HB alone
(control). Among these specimens, cartilage lubricated with
DMPC-MLV had the highest PC concentration near the cartilage
surface (FIG. 8). PC concentration reached a maximum at a depth of
.about.100 .mu.m, below which, it decreased. On the other hand, in
cartilage lubricated with DMPC-SUV the highest PC concentration
occurred deep (.about.600 .mu.m) inside the cartilage, while at the
surface PC concentration was similar to that of the control
(cartilage lubricated with HB).
Cartilage Morphology:
[0145] SEM was used to study cartilage surface morphology and
wear.sup.28. In FIG. 9 we present SEM images of cartilage specimens
subjected to different treatments. The two control specimens (FIGS.
9A and 9B) were not subjected to friction tests, whereas all other
specimens (FIGS. 9C-9F) of cartilage were obtained from healthy
people and subjected to identical friction tests in the presence of
different lubricants, FIG. 9A shows healthy cartilage, where
naturally-occurring globular lipidic structures are dispersed on
its porous surface, as previously shown on the surface of rat
cartilage by Ohno et al..sup.28,48 On the other hand, the surface
of osteoarthritic cartilage lacks these structures (FIG. 9B), as
does friction-tested healthy cartilage lubricated with saline (FIG.
9C) or ISF (FIG. 9D), indicating poor protection against wear by
these lubricants. On the surface of cartilage lubricated with
DMPC-SUV (FIG. 9E), very few lipidic structures can be noticed
after friction testing. With DMPC-MLV (FIG. 9F), large lipidic
structures, resembling those on healthy cartilage, are present
after friction testing.
Example 2
Toxicity study
[0146] The present example was conducted to assess local reactions
(histopathology of fermorotibial joint) at different times post
intraarticular injection of DMPC-based MLV composed of either DMPC
alone or of a DMPC/DPPC mixture (0.6:1.0 mole ratio) in Sprague
Dawley (SD) rats.
Materials & Methods
Animals:
[0147] 46 male SD rats aged 9 weeks old (purchased from Harlan
Laboratories Ltd. Israel) were randomly assigned to 5 groups, 9
animals per group. The rats were maintained at Assaf Harofe Medical
Center animal facility. The rats treated by intraarticular
injection according to an initial assignment (Table 2, Group
composition, treatments and Identification of animals). Rat in
general good condition were included. All rats were treated on Day
1, and one group was treated again on Day 14. Both knees were
treated; each knee was injected with 100 .mu.L of one of the test
liposomes or control substance. Cages were marked with a clear
label and permanent marker. The rats were marked on their tail.
TABLE-US-00002 TABLE 2 Group composition, treatments and
Identification of animals Animal No. Cage No. Tail No. Left knee
Right Knee 1, 10, 19, 28, 1, 4, 7, 10, 1 HB DMPC 37 13 2, 11, 20,
29, 2 HB DMPC 38 3, 12, 21, 30, DMPC 39 4, 13, 22, 31, 2, 5, 8, 11,
1 Physiological DMPC + DPPC 40 14 saline 5, 14, 23, 32, 2
Physiological DMPC + DPPC 41 saline 6, 15, 24, 33, HB DMPC + DPPC
42 7, 16, 25, 34, 3, 6, 9, 12 1 DMPC DMPC 43 15 8, 17, 26, 35, 2
DMPC + DPPC DMPC 44 9, 18, 27, 36, DMPC + DPPC DMPC 45 46 15 4 DMPC
+ DPPC DMPC *Physiological saline: 0.9% NaCl
Test Compositions:
[0148] Liposomal DMPC and DMPC+DPPC were prepared as described
above (the liposomes being dispersed in HB) and stored in 4.degree.
C. till one hour before the application. The last hour before the
application they were left in room temperature. The concentrations
of the liposomes were for DPPC 100.6 mM and for DMPC+DPPC 92.0
mM.
[0149] The physiological solution that was used is 0/9% w/v sodium
chloride inj. (purchased from B. Braun batch No. 7281C12 exp.
08.2010).
Treatment Procedure:
[0150] Before the toxicity test the rats were anesthetized by
inhalation of Isoflurane (Abbot Laboratories). The rats knees were
shaved by an electric machine, skin sprayed with 70% alcohol. A 1
mL syringes were used with a replaceable 27 gauge needles. The
needles were chanced after each injection. 100 .mu.L was injected
each time. At the end of the study, rats were euthanized by
exposure to carbon dioxide.
[0151] Table 3 summarizes the weights and treatment of each
rat.
TABLE-US-00003 TABLE 3 Table of weights and treatments Rat Cage
Tail Initial 2.sup.nd Final 2.sup.nd No. No. No. Left knee Right
Knee Weight weight Weight treatment Eutanization 1 1 1 HB DMPC 298
303 No day (19/12) 2 1 2 HB DMPC 310 316 3 1 3 Physiological saline
DMPC 300 295 4 2 1 Physiological saline DMPC + DPPC 310 318 5 2 2
Physiological saline DMPC + DPPC 298 285 6 2 HB DMPC + DPPC 290 292
7 3 1 DMPC + DPPC DMPC 285 277 8 3 2 DMPC + DPPC DMPC 290 295 9 3
DMPC + DPPC DMPC 290 286 10 4 1 HB DMPC 302 307 No 4 days (22/12)
11 4 2 HB DMPC 303 324 12 4 Physiological saline DMPC 298 317 13 5
1 Physiological saline DMPC + DPPC 315 330 14 5 2 Physiological
saline DMPC + DPPC 314 330 15 5 HB DMPC + DPPC 282 292 16 6 1 DMPC
+ DPPC DMPC 280 280 17 6 2 DMPC + DPPC DMPC 305 305 18 6 DMPC +
DPPC DMPC 298 298 19 7 1 HB DMPC 312 312 No fortnight (1/1) 20 7 2
HB DMPC 299 299 21 7 Physiological saline DMPC 318 318 22 8 1
Physiological saline DMPC + DPPC 303 303 23 8 2 Physiological
saline DMPC + DPPC 300 300 24 8 HB DMPC + DPPC 300 300 25 9 1 DMPC
+ DPPC DMPC 300 300 26 9 2 DMPC + DPPC DMPC 321 321 27 9 DMPC +
DPPC DMPC 300 300 28 10 1 HB DMPC 313 405 No 4 weeks (15/1) 29 10 2
HB DMPC 310 409 30 10 Physiological saline DMPC 298 318 31 11 1
Physiological saline DMPC + DPPC 289 356 32 11 2 Physiological
saline DMPC + DPPC 298 364 33 11 HB DMPC + DPPC 308 404 34 12 1
DMPC + DPPC DMPC 290 355 35 12 2 DMPC + DPPC DMPC 312 420 36 12
DMPC + DPPC DMPC 315 389 37 13 1 HB DMPC 306 361 400 fortnight 1/1
4 weeks (15/1) 38 13 2 HB DMPC 298 342 374 39 13 Physiological
saline DMPC 300 338 359 40 14 1 Physiological saline DMPC + DPPC
301 351 383 41 14 2 Physiological saline DMPC + DPPC 298 341 355 42
14 HB DMPC + DPPC 300 338 368 43 15 1 DMPC + DPPC DMPC 306 348 384
44 15 2 DMPC + DPPC DMPC 291 336 337 45 15 DMPC + DPPC DMPC 298 338
384 46 15 4 DMPC + DPPC DMPC 298 351 352
Clinical Evaluation:
[0152] The animal technician held physical observations on the rats
throughout the study for monitoring prominent change in their
conditions such as drastic weight loss, wounds or deaths. All rats
were weighted before the applications and after euthanization.
Necropsy:
[0153] All rats were subjected to a full detailed necropsy and
gross pathological examination at the end of the study period
following euthanasia. At necropsy, all rats were thoroughly
examined for any abnormality or gross pathological changes in
tissues and/or organs. Samples of all knees were sent to slides
preparation at Patho-Lab Ltd. Ness Ziona. Tissue fixation was
obtained in formaldehyde (Bio-Lab Ltd.).
Results
[0154] No pathological changes were observed in any of the rats
during necropsy procedure.
[0155] Histopathological data indicated that the general reaction
to the test compositions, following one and four days from the
administration consisted of:
Synovial cytoplasmic vacuolation--ranged from minimal to mild;
Subsynovial vacuolated histiocytic cell accumulation--ranged from
minimal to mild; Articular cavity--exudate (fibrillar amorphous
material, histiocyes and polymorphonuclear cells)--ranged from
minimal to mild;
[0156] The nature and severity of changes were relatively
comparable in the DMPC and DMPC+DPPC treated rats.
[0157] No lesions were noted in the rats examined 2 and 4 weeks
after administration, suggesting complete recovery.
[0158] Thus, that the pathology results show that both liposomal
preparations (DMPC and DMPC:DPPC (6:1)) could be considered as a
safe intra-articular treatment.
REFERENCES
[0159] 1. Alexander, C. J. Idiopathic osteoarthritis: time to
change paradigms? Skeletal Radiol. 33, 321-324 (2004). [0160] 2.
Corti, M. C. & Rigon, C. Epidemiology of osteoarthritis:
prevalence, risk factors and functional impact. Aging Clin. Exp.
Res. 15, 359-363 (2003). [0161] 3. Bullough, P. G. & Vigorita,
V. J. Orthopaedic Pathology, Edn. 3rd. (Mosby-Wolfe, Baltimore;
1997). [0162] 4. Koopman, W. J. & Moreland, L. W. Arthritis and
Allied Conditions: A Textbook of Rheumatology, Vol. 1-2, Edn. 15th.
(Lippincott Williams & Wilkins, Philadelphia; 2005). [0163] 5.
Sokoloff, L. The biology of degenerative joint disease. Acta
Rhumatol Belg. 1, 155-156 (1977). [0164] 6. Conaghan, P. G.,
Vanharanta, H. & Dieppe, P. A. Is progressive osteoarthritis an
atheromatous vascular disease? Ann. Rheum. Dis. 64, 1539-1541
(2005). [0165] 7. Neame, R. & Doherty, M. Osteoarthritis
update. Clin. Med. 5, 207-210 (2005). [0166] 8. Imhof, H. et al.
Subchondral bone and cartilage disease: a rediscovered functional
unit. Invest. Radiol. 35, 581-588 (2000). [0167] 9. Lajeunesse, D.
& Reboul, P. Subchondral bone in osteoarthritis: a biologic
link with articular cartilage leading to abnormal remodeling. Curr.
Opin. Rheumatol. 15, 628-633 (2003). [0168] 10. Radin, E. L. Who
gets osteoarthritis and why? J. Rheumatol. Suppl. 70, 10-15 (2004).
[0169] 11, Grainger, R. & Cicuttini, F. M. Medical management
of osteoarthritis of the knee and hip joints. Med. J. Aust. 180,
232-236 (2004). [0170] 12. Swarm, D. A., Hendren, R. B., Radin, E.
L., Sotman, S. L. & Duda, B. A. The lubricating activity of
synovial fluid glycoproteins. Arthritis Rheum. 24, 22-30 (1981).
[0171] 13. Swarm, D. A. & Mintz, G. The isolation and
properties of a second glycoprotein (LGP-II) from the articular
lubricating fraction from bovine synovial fluid. Biochem. J. 179,
465-471 (1979). [0172] 14. Swarm, D. A., Slayter, H. S. &
Silver, F. H. The molecular structure of lubricating
glycoprotein-1, the boundary lubricant for articular cartilage. J
Biol. Chem. 256, 5921-5925 (1981). [0173] 15. Nitzan, D. W.,
Kreiner, B. & Zeltser, R. TMJ lubrication system: its effect on
the joint function, dysfunction, and treatment approach. Compend.
Contin. Educ. Dent. 25, 437-444 (2004). [0174] 16. Yui, N., Okano,
T. & Sakurai, Y. Inflammation responsive degradation of
crosslinked hyaluronic acid gels. J. Control. Release 22, 105-116
(1992). [0175] 17. Hills, B. A. & Butler, B. D. Surfactants
identified in synovial fluid and their ability to act as boundary
lubricants. Ann. Rheum. Dis. 43, 641-648 (1984). [0176] 18. Sarma,
A. V., Powell, G. L. & LaBerge, M. Phospholipid composition of
articular cartilage boundary lubricant. J. Orthop. Res. 19, 671-676
(2001). [0177] 19. Schwarz, I. M. & Hills, B. A. Surface-active
phospholipid as the lubricating component of lubricin. Br. J.
Rheumatol. 37, 21-26 (1998). [0178] 20. Hills, B. A. & Mends,
M. K. Enzymatic identification of the load-bearing boundary
lubricant in the joint. Br. J. Rheumatol. 37, 137-142 (1998).
[0179] 21. Ogston, A. G. & Stanier, J. E. Physiological
function of hyaluronic acid in synovial fluid; viscous, elastic,
and lubricant properties. J. Physiol. (Cambridge) 119, 244-252
(1953). [0180] 22. Benz, M., Chen, N. & Israelachvili, J.
Lubrication and wear properties of grafted polyelectrolytes,
hyaluronan and hylan, measured in the surface forces apparatus. J.
Biomed. Mater. Res. A. 71, 6-15 (2004). [0181] 23. Rhee, D. K. et
al. The secreted glycoprotein lubricin protects cartilage surfaces
and inhibits synovial cell overgrowth. J. Clin. Invest. 115,
622-631 (2005). [0182] 24. Swann, D. A., Bloch, K. J., Swindell, D.
& Shore, E. The lubricating activity of human synovial fluids.
Arthritis Rheum. 27, 552-556 (1984). [0183] 25. Pickard, J. E.,
Fisher, J., Ingham, E. & Egan, J. Investigation into the
effects of proteins and lipids on the frictional properties of
articular cartilage. Biomaterials 19, 1807-1812 (1998). [0184] 26.
Vecchio, P., Thomas, R. & Hills, B. A. Surfactant treatment for
osteoarthritis. Rheumatology (Oxford) 38, 1020-1021 (1999). [0185]
27. Gudimelta, O. A., Crawford, R. & Hills, B. A. Consilidation
responses of delipidized cartilage. Clin. Biomech. 19, 534-542
(2004). [0186] 28. Watanabe, M. et al. Ultrastructural study of
upper surface layer in rat articular cartilage by "in vivo
cryotechniquc" combined with various treatments. Med. Elect.
Microsc. 33, 16-24 (2000). [0187] 29. Kawano, T. et al. Mechanical
effects of the intraarticular administration of high molecular
weight hyaluronic acid plus phospholipid on synovial joint
lubrication and prevention of articular cartilage degeneration in
experimental osteoarthritis. Arthritis Rheum. 48, 1923-1929 (2003).
[0188] 30. Forsey, R. W. et al. The effect of hyaluronic acid and
phospholipid based lubricants on friction within a human cartilage
damage model. Biomaterials 27, 4581-4590 (2006). [0189] 31. Klein,
J. Molecular mechanisms of synovial joint lubrication. J. Proc.
Inst. Mech Eng., Part J: J. Eng. Tribology 220, 691-710 (2006).
[0190] 32. Briscoe, W. H. et al. Boundary lubrication under water.
Nature 444, 191-194 (2006), [0191] 33. Raviv, U. et al. Lubrication
by charged polymers. Nature 425, 163-165 (2003). [0192] 34.
Barenholz, Y. & Ceve, G. Structure and properties of membranes
in Physical Chemistry of Biological Surfaces. (Marcel Dekker, New
York; 2000). [0193] 35. Israelachvili, J., Intermolecular and
surface Forces, 2.sup.nd edition. Academic Pres, London (1992)
[0194] 36. Marsh, D. CRC Handbook of Lipid Bilayers. (CRC Press,
Boca Raton, Fla.; 1990). [0195] 37. Barenholz, Y. & Amselem, S.
Quality control assays in the development and clinical use of
liposome-based formulations in Liposome Technology, Edn. 2nd, (CRC,
Boca Raton, Fla.; 1993). [0196] 38. Shmeeda, H., Even-Chen, S.
& Barenholz, Y. Enzymatic assays for quality control and
pharmacokinetics of liposome formulations: Comparison with
nonenzymatic conventional methodologies. Methods Enzymol. 367,
272-292 (2003). [0197] 39. Garbuzenko, O., Barenholz, Y. &
Priev, A. Effect of grafted PEG on liposome size and on
compressibility and packing of lipid bilayer. Chem. Phys. Lipids
135, 117-129 (2005). [0198] 40. Merkher, Y. et al. A rational human
joint friction test using a human cartilage-on-cartilage
arrangement. Tribol. Lett. 22, 29-36 (2006). [0199] 41. Barenholz,
Y. Relevancy of drug loading to liposomal formulation therapeutic
efficacy. J. Liposome Res. 13, 1 (1993). [0200] 42. Bligh, E. G.
& Dyer, W. J. A rapid method of total lipid extraction and
purification. Can. J. Biochem. Physiol. 37, 911-917 (1959). [0201]
43. Biltonen, R. L. & Lichtenberg, D. The use of differential
scanning calorimetry as a tool to characterize liposome
preparations. Chem. Phys. Lipids 64, 129-142 (1993). [0202] 44.
Mouritsen, O. G. Life As a Matter of Fat. The Emerging Science of
Lipidomics. (Springer-Verlag, Berlin; 2005). [0203] 45. Tirosh, O.,
Barenholz, Y., Katzhendler, J. & Priev, A. Hydration of
polyethylene glycol-grafted liposomes. Biophys. J. 74, 1371-1379
(1998). [0204] 46. Hills, B. A. Boundary lubrication in vivo. J.
Eng. Med. 214, 83-94 (2000). [0205] 47. Mabrey, S. &
Sturtevant, J. M. Investigation of phase transitions of lipids and
lipid mixtures by high sensitivity differential scanning
calorimetry. PNAS 73, 3862-3866 (1976). [0206] 48. Yoshida, M.,
Zea-Aragon, Z., Ohtsuki, K., Ohnishi, M. & Ohno, S.
Ultrastructural study of upper surface layer in rat mandibular
condylar cartilage by quick-freezing method. Histol. Histopathol.
19, 1033-1041 (2004). [0207] 49. Klein, J. Mechanism of friction
across molecularly confined films of simple liquids. Tribology
Series 36, 59-64 (1999). [0208] 50. Maroudas, A. Distribution and
diffusion of solutes in articular cartilage. Biophys. J. 10,
365-379 (1970). [0209] 51. Stockwell, R. A. & Bartlett, C. H.
Changes in permeability of articular cartilage with age. Nature
201, 835-836 (1964). [0210] 52. Barnett, C. H. & Palfrey, A. J.
Absorption into the rabbit articular cartilage. J. Anat. 99,
365-375 (1965). [0211] 53. Faure, C., Bonakdar, L. & Dufourc,
E. J. Determination of DMPC hydration in the L(alpha) and L(beta')
phases by 2H solid state NMR of D2O. FEBS Lett. 405, 263-266
(1997). [0212] 54. Schrader, W. et al. Compressibility of lipid
mixtures studied by calorimetry and ultrasonic velocity
measurements. J. Phys. Chem. B 106, 6581-6586 (2002). [0213] 55.
Schwarz, U. S., Komura, S. & Safran, S. A. Deformation and
tribology of multi-walled hollow nanoparticles. Europhys. Lett. 50,
762-768 (2000). [0214] 56. Parasassi, T., Di Stefano, M., Loiero,
M., Ravagnan, G. & Gratton, E. Cholesterol modifies water
concentration and dynamics in phospholipid bilayers: a fluorescence
study using Laurdan probe. Biophys J. 66, 763-768 (1994). [0215]
57. Oncins, G., Garcia-Manyes, S. & Sanz, F. Study of
frictional properties of a phospholipid bilayer in a liquid
environment with lateral force microscopy as a function of NaCl
concentration. Langmuir 21, 7373-7349 (2005). [0216] 58. Ballantine
G. C., Stachowiak G. W., The effects of lipid depletion on
osteoarthritic wear, Wear 253, 385-393 (2002). [0217] 59 Jones C.
F., Stoffel K., Ozturk H. E., Stachowiak G. W., The effect of
surface active phospholipids on the lubrication of osteoarthritic
sheep knee joints: wear, Tribol. Lett. 16(4), 291-296 (2004).
[0218] 60 Hills B. A., Monds M. K., Deficiency of lubricating
surfactant lining the articular surfaces of replaced hips and
knees, Br. J. Rheumatol. 37, 143-147 (1998). [0219] 61 Freeman M.
A. R. (Ed.), Adult Articular Cartilage, 2.sup.nd ed., Chapter 3
Pitman Medical, London (1979). [0220] 62 Farndale R. W., Buttle D.
J., Barrett A. J., Improved quantification and discrimination of
sulfated glycosaminoglycans by use of dimethylmethylene, Biochim
Biophys Acta 883(2), 173-177 (1986). [0221] 63. International
patent application publication No. WO2003/000190; [0222] 64.
International patent application publication No. WO2004/047792;
[0223] 65. International patent application publication No.
WO2002/078445
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