U.S. patent application number 14/560045 was filed with the patent office on 2015-04-02 for liposomal compositions of glucocorticoid and glucocorticoid derivatives.
This patent application is currently assigned to YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERSUSALEM. The applicant listed for this patent is YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM. Invention is credited to Yuval AVNIR, Yechezkel BARENHOLZ, Alberto A. GABIZON.
Application Number | 20150093434 14/560045 |
Document ID | / |
Family ID | 35432741 |
Filed Date | 2015-04-02 |
United States Patent
Application |
20150093434 |
Kind Code |
A1 |
BARENHOLZ; Yechezkel ; et
al. |
April 2, 2015 |
LIPOSOMAL COMPOSITIONS OF GLUCOCORTICOID AND GLUCOCORTICOID
DERIVATIVES
Abstract
Provided are pharmaceutical compositions including a
glucocorticoid or glucocorticoid derivative stably encapsulated in
a liposome. The glucocorticoid or glucocorticoid derivative is
selected from an amphipathic weak base glucocorticoid or
glucocorticoid derivative having a pKa equal or below 11 and a logD
at pH 7 in the range between -2.5 and 1.5; or an amphipathic weak
acid GC or GC derivative having a pKa above 3.5 and a logD at pH 7
in the range between -2.5 and 1.5. The therapeutic effect of the
pharmaceutical composition of the invention was exhibited in vivo
with appropriate models of multiple sclerosis and cancer.
Inventors: |
BARENHOLZ; Yechezkel;
(Jerusalem, IL) ; GABIZON; Alberto A.; (Jerusalem,
IL) ; AVNIR; Yuval; (Jerusalem, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF
JERUSALEM |
Jerusalem |
|
IL |
|
|
Assignee: |
YISSUM RESEARCH DEVELOPMENT COMPANY
OF THE HEBREW UNIVERSITY OF JERSUSALEM
Jerusalem
IL
|
Family ID: |
35432741 |
Appl. No.: |
14/560045 |
Filed: |
December 4, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11662172 |
Oct 5, 2007 |
8932627 |
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PCT/IL05/00963 |
Sep 11, 2005 |
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14560045 |
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60608140 |
Sep 9, 2004 |
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Current U.S.
Class: |
424/450 ;
514/179 |
Current CPC
Class: |
A61K 9/127 20130101;
A61P 17/06 20180101; A61P 43/00 20180101; A61K 31/573 20130101;
A61P 7/06 20180101; A61K 9/1271 20130101; A61P 27/02 20180101; A61P
25/00 20180101; A61P 5/44 20180101; A61P 19/02 20180101; A61P 17/00
20180101; A61P 37/02 20180101; A61P 7/10 20180101; A61P 11/06
20180101; A61P 5/18 20180101; A61P 5/38 20180101; A61P 11/00
20180101; A61P 37/08 20180101; A61P 25/28 20180101; A61P 35/00
20180101; A61P 11/02 20180101; A61P 35/02 20180101; A61P 27/14
20180101; A61P 29/00 20180101; A61P 7/00 20180101 |
Class at
Publication: |
424/450 ;
514/179 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/573 20060101 A61K031/573 |
Claims
1-36. (canceled)
37. A method for delivery of a glucocorticoid (GC) to a target site
within a body comprising: providing a glucocorticoid (GC)
derivative that is an amphipathic weak base GC derivative having a
pKa equal to or below 11 and a logD at pH 7 in a range of from -1.5
to 1.0 or an amphipathic weak acid GC derivative having a pKa above
3.5 and a logD at pH 7 in a range of from -1.5 to 1.0 and loading
said GC into a liposome.
38. The method of claim 37, wherein said GC is a water immiscible
GC.
39. The method of claim 37, wherein the GC is selected from the
group consisting of prednisolone hemisuccinate, methylprednisolone
hemisuccinate, hydrocortisone hemisuccinate, dexamethasone
hemisuccinate, allopregnanolone hemisuccinate, beclomethasone
21-hemisuccinate, betamethasone 21-hemisuccinate, boldenone
hemisuccinate, prednisolone 21-hemisuccinate, nandrolone
hemisuccinate, 19-nortestosterone hemisuccinate,
deoxycorticosterone 21-hemisuccinate, corticosterone hemisuccinate,
and cortexolone hemisuccinate.
40. The method of claim 39, wherein said GC is selected from the
group consisting of methylprednisolone sodium hemisuccinate (MPS),
hydrocortisone sodium hemisuccinate (HYD), dexamethasone
hemisuccinate and prednisolone hemisuccinate.
41. The method of claim 37, wherein said liposome comprises a
counter ion to said GC.
42. The method of claim 37, wherein said liposomes comprises a
combination of a phospholipid, a lipopolymer and cholesterol.
43. The method of claim 42, wherein said liposome comprises at
least one of HSPC and DSPC, and a mole ratio between the GC
derivative and HSPC or DSPC of between 0.01 and 2.0.
44. The method of claim 43, wherein the mole ratio is between 0.04
and 0.25.
45. A method for treatment of a disease or disorder comprising
administration to a subject in need of said treatment a
pharmaceutical composition comprising a liposome comprising a
glucocorticoid (GC) derivative that is an amphipathic weak base GC
derivative having a pKa equal to or below 11 and a logD at pH 7 in
a range of from -1.5 to 1.0 or an amphipathic weak acid GC
derivative having a pKa above 3.5 and a logD at pH 7 in a range of
from -1.5 to 1.0, the GC derivative being selected from the group
consisting of prednisolone hemisuccinate, methylprednisolone
hemisuccinate, hydrocortisone hemisuccinate, dexamethasone
hemisuccinate, allopregnanolone hemisuccinate, beclomethasone
21-hemisuccinate, betamethasone 21-hemisuccinate, boldenone
hemisuccinate, prednisolone 21-hemisuccinate, nandrolone
hemisuccinate, 19-nortestosterone hemisuccinate,
deoxycorticosterone 21-hemisuccinate, corticosterone hemisuccinate,
and cortexolone hemisuccinate; and a counter-ion to the GC
derivative wherein the GC derivative is retained in the liposome
for at least six months.
46. The pharmaceutical composition of claim 45, wherein the GC
derivative is a pro-drug which is converted to an active GC upon
release thereof from the liposome into a body fluid.
47. The pharmaceutical composition of claim 45, wherein the GC
corresponds to an acidic GC selected from the group consisting of
methylprednisolone sodium hemisuccinate (MPS), hydrocortisone
sodium hemisuccinate (HYD), dexamethasone hemisuccinate and
prednisolone hemisuccinate.
48. The method of claim 45, wherein said liposome comprises a
combination of a phospholipid, a lipopolymer and cholesterol.
49. The method of claim 48, wherein said liposome comprises a
lipopolymer being PEG-DSPE.
50. The method of claim 48, wherein said phospholipid comprises one
or both of HSPC or DSPC, and the liposome comprises a mole ratio
between the GC derivative and HSPC or DSPC of between 0.01 and
2.0.
51. The method of claim 50, wherein the mole ratio is between 0.04
and 0.25.
52. The method of claim 45, for the treatment of a
neurodegenerative disorder.
53. The method of claim 52, for the treatment of multiple
sclerosis.
54. The method of claim 45, for the treatment of cancer.
55. A pharmaceutical composition, comprising: a liposome comprising
a glucocorticoid (GC) derivative that is an amphipathic weak base
GC derivative having a pKa equal to or below 11 and a logD at pH 7
in a range of from -1.5 to 1.0 or an amphipathic weak acid GC
derivative having a pKa above 3.5 and a logD at pH 7 in a range of
from -1.5 to 1.0, the GC derivative being selected from the group
consisting of prednisolone hemisuccinate, methylprednisolone
hemisuccinate, hydrocortisone hemisuccinate, dexamethasone
hemisuccinate, allopregnanolone hemisuccinate, beclomethasone
21-hemisuccinate, betamethasone 21-hemisuccinate, boldenone
hemisuccinate, prednisolone 21-hemisuccinate, nandrolone
hemisuccinate, 19-nortestosterone hemisuccinate,
deoxycorticosterone 21-hemisuccinate, corticosterone hemisuccinate,
and cortexolone hemisuccinate; and a counter-ion to the GC
derivative wherein the GC derivative is retained in the liposome
for at least six months.
Description
FIELD OF THE INVENTION
[0001] This invention relates in general to liposome technology,
and specifically, to the use of this technology for the delivery
within the body of glucocorticoids.
LIST OF PRIOR ART
[0002] 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. [0003] Gonzalez-Rothi, Ricardo J et al.
Pharmaceutical Research 13(11):1699-1703 (1996); [0004] Schmidt J
et al. Brain 126(8):1895-1904 (2003); [0005] Fildes F J et al. J
Pharm. Pharmacol. 30(6):337-42 (1978); [0006] Mishina E V et al
Pharm Res 13(1):141-5 (1996); [0007] Gonzalez-Rothi, Ricardo J et
al. Pharmaceutical Research 13(11):1699-1703 (1996); [0008] Almawi
W Y and Melemedjian O K, J Leukoc Biol 71:9-15 (2002); [0009]
Coleman R E Biotherapy 4:37-44 (1992); [0010] Folkman J, et al.
Science 221:719-725 (1983); [0011] Swain S. M., Endocrine therapies
of cancer In, Cancer Chemotherapy and Biotherapy, 2nd Ed., Eds.,
Chabner B A, and Longo D L, Lippincott-Raven, Philadelphia, 1996,
(pp 59-108); [0012] Haskell C M. In, Cancer Treatment, 4th Edition,
Edited by Haskell C M, and Berek J S. W B Saunders Co,
Philadelphia, 1995 (pp 78-80, pp 105-106, pp 151-152). [0013]
Josbert M. Metselaar, Liposomal targeting of glucocorticoids. A
novel treatment approach for inflammatory disorders. Chapter 6, pp
91-106, chapter 7, pp 107-122, 2003, Ph.D. Thesis, Utrecht
University, Faculty of Pharmaceutical Sciences, Faculty of
Veterinary Medicine ISBN 90-393-3285-1;
BACKGROUND OF THE INVENTION
[0014] Glucocorticoids (glucocorticosteroids) are a class of
steroid hormones characterized by an ability to bind with the
cortisol receptor found in the cells of almost all vertebrate
tissues and trigger similar effects. Glucocorticoids are
distinguished from other steroids such as sex steroids by the
specific receptors, target cells, and effects. Cortisol (or
hydrocortisone) is the most important natural human
glucocorticoid.
[0015] Glucocorticoids have potent anti-inflammatory and
immunosuppressive properties. This is particularly evident when
they are administered at pharmacologic doses, but also is important
in physiologic immune responses. As a consequence, glucocorticoids
are widely used as drugs to treat inflammatory conditions such as
arthritis or dermatitis, and as adjunctive therapy for conditions
such as autoimmune diseases. On the other hand, excessive
glucocorticoid levels, resulting from administration as a drug or
hyperadrenocorticism have side-effects on many systems, some
examples including inhibition of bone formation, suppression of
calcium absorption and delayed wound healing.
[0016] A variety of synthetic glucocorticoids, some far more potent
than cortisol, have been developed for therapeutic use. They differ
in the pharmacokinetics (absorption factor, half-life, volume of
distribution, clearance) and in pharmacodynamics (for example the
capacity of mineralocorticoid activity: retention of sodium
(Na.sup.+) and water). Because they are absorbed well through the
intestines, they are primarily administered per os (by mouth), but
also by other ways like topically on skin.
[0017] Methylprednisolone (pregna-1,4-diene-3,20-dione,
11,17,21-trihydroxy-6-methyl-,(6.alpha., 11.beta.).
C.sub.22H.sub.30O.sub.5, MW 374.48) is one example of a
therapeutically potent synthetic glucocorticoid drug, which, due to
its hydrophobic character, is usually taken orally. Like most
adrenocortical steroids, methylprednisolone is typically used for
its anti-inflammatory properties. However, glucocorticoids have a
wide range of effects, including changes in metabolism and immune
responses. Similar to other corticosteroids, the list of diseases
or pathological conditions for which methlyprednisolone is
effective is rather large. Common uses includes arthritis therapy,
and short-term treatment of bronchial inflammation due to various
respiratory diseases. while highly effective, their systemic
application is limited because of a high incidence of serious
adverse effects, especially related to long-term treatment.
[0018] Efficacy and safety studies of systemic administration of
glucocorticoids, revealed that in addition to the profound activity
of the drug in many different tissues, these drugs have rapid
clearance from plasma thereby requiring high and frequent dosing to
obtain effective amounts at the target site.
[0019] Thus, alternative approaches for parenteral administration
were investigated. For example, developing loco-regional
administration of glucocorticoids (e.g. by the use of inhalers in
asthma and in intraarticular injection in arthritis) enabled the
use of lower doses of the steroid while achieving sufficient drug
levels in a lesion, with minimal side effects.
[0020] A further approach included targeting of the drug to the
target tissue by the use of a suitable carrier, such as
liposomes.
[0021] First attempts to encapsulate corticosteroids in liposomes
were performed by Fildes F J et al. [J Pharm. Pharmacol.
30(6):337-42 (1978)] which included steroid encapsulation in the
liposome's lipid bilayer. This approach was based on the
understanding that corticosteroids are hydrophobic in nature.
However, such liposomal formulations turned to be unsuitable for
clinical applications.
[0022] Efforts were also made in developing "soluble"
glucocorticoids. Examples include succinate derivatized steroids
such as hydrocortisone hemisuccinate sodium salt and
Methylprednisolone hemisuccinate sodium salt. Another group of
soluble glucocorticoids include the phosphate derivatives of
steroids. While rendering the steroid water-soluble enabled the use
of the acidic steroids for injection, it was shown that these
"pro-drugs" are completely cleared from plasma in less than 6 hours
post injection. [Mishina E V et al Pharm Res 13(1):141-5
(1996)]
[0023] The combination of acidic steroids with liposomes was also
investigated. Schmidt et al. [Schmidt J et al. Brain
126(8):1895-1904 (2003)] describe a formulation of
polyethyleneglycol (PEG)-coated long-circulating sterically
stabilized liposomes encapsulating prednisolone phosphate (one of
the water soluble pro-drug steroids) and its beneficial effect in
treating multiple sclerosis as compared to the free form of the
steroid. However, attempts to similarly encapsulate
methylprednisolone hemisuccinate (a weak acid) failed, as it led to
an unstable formulation.
[0024] Further, encapsulation in liposomes of triamcinolone
acetonide phosphate, a water soluble strong acid derivative of
triamcinolone (pKa below 2) was described [Gonzalez-Rothi, Ricardo
J et al. Pharmaceutical Research 13(11):1699-1703 (1996)]. The
liposomal formulation was prepared by passive loading of the acidic
corticosteroid into the liposomes and used as an injectable dosage
form (intravenous or intratracheal) for treating pulmonary
conditions. Further, in ex vivo stability studies it was shown that
after 24 hours the liposome retained more than 75% of the acidic
corticosteroid.
SUMMARY OF THE INVENTION
[0025] The invention is based on the finding that using chemically
modified gluococorticoids (GC), in their amphipathic weak acid form
enables the effective loading in liposomes of the acidic GC.
Surprisingly, the thus formed liposomal weakly acidic GC was
stable, i.e. the majority of the substance remained within the
liposome as intact acidic GC after storage for 14 months at
4.degree. C. Once released from the liposome to water or body
fluids the acidic GC was hydrolyzed to obtain the active,
non-acidic GC.
[0026] Thus, according to a first of its aspects the invention
provides a pharmaceutical composition comprising a GC or GC
derivative encapsulated in a liposome, wherein said GC or GC
derivative is essentially retained in said liposome for 6 months,
preferably 10 months and more preferably 14 months, the GC or GC
derivative being selected from: [0027] i) an amphipathic weak base
GC or GC derivative having a pKa equal or below 11 and a logD at pH
7 in the range between about -2.5 and about 1.5, preferably, in the
range between about -1.5 and about 1.0; [0028] ii) an amphipathic
weak acid GC or GC derivative having a pKa above 3.5 and a logD at
pH 7 in the range between about -2.5 and about 1.5, preferably, in
the range between about -1.5 and about 1.0.
[0029] The GC derivative is preferably an acidic GC, i.e. an
amphipathic weak acid derivative of GC which is converted to the
non-acidic form upon release from the liposome to bodily fluids.
More specifically, the acidic GC is methylprednisolone sodium
hemisuccinate (MPS).
[0030] A preferred MPS formulation according to the invention
comprises sterically stabilized liposomes formed from a combination
of hydrogenated soybean phosphatidylcholine (HSPC),
(methyl)polyethylene glycol coated distearoyl phosphatidyl
ethanolamine (PEG-DSPE) and cholesterol at a molar ratio of
55:40:5.
[0031] The pharmaceutical composition is preferably utilized for
the treatment or prevention of any disease whose acceptable form of
treatment includes administration of glucocorticoids.
[0032] The pharmaceutical composition in accordance with the
invention in generally better than the non-encapsulated GC in at
least one of: better delivery to the target site, better
circulation time, slower clearance, reduced side effect, increased
efficacy or increased therapeutic index.
[0033] The pharmaceutical composition is preferably utilized for
the treatment or prevention of multiple sclerosis.
[0034] The pharmaceutical composition is also preferably utilized
for the treatment or prevention of cancer which are known to be
sensitive to steroids, such as cancers of haematopoeitic origin
including lymphoma, leukemia, myeloma, breast cancer and prostate
cancer.
[0035] The invention also provides the use of GC or GC derivative
for the preparation of the pharmaceutical composition of the
invention, the GC or GC derivative being encapsulated in a
liposome, wherein said GC or GC derivative is essentially retained
in said liposome for 6 months, preferably 10 months and more
preferably 14 months, the GC or GC derivative being selected from:
[0036] i) an amphipathic weak base GC or GC derivative having a pKa
equal or below 11 and a logD at pH 7 in the range between about
-2.5 and about 1.5, preferably, in the range between about -1.5 and
about 1.0; [0037] ii) an amphipathic weak acid GC or GC derivative
having a pKa above 3.5 and a logD at pH 7 in the range between
about -2.5 and about 1.5, preferably, in the range between about
-1.5 and about 1.0.
[0038] Yet further, the invention provides a method for delivery of
a glucocorticoid (GC), preferably a water immiscible GC, to a
target site within a body, comprising chemically modifying said GC
to an amphipathic weak acid derivative or amphipathic weak based
derivative thereof as defined in any one of claims 1 to 14, and
loading said amphipathic weak acid derivative or amphipathic weak
base derivative into a liposome. Specifically, the liposome is a
sterically stabilized liposome and the GC derivative is loaded into
the liposome by the formation of an ion or pH gradient across the
liposome membrane (i.e. by active loading techniques).
[0039] Yet further, the invention provides a method of the
treatment or prevention of a disease or pathological condition
comprising administering to a subject in need an amount of GC or GC
derivative encapsulated in liposomes, the amount being sufficient
to achieve a therapeutic effect.
[0040] Preferably, the method comprises injection of the liposomes
encapsulating GC or GC derivative.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] In order to understand the invention and to see how it may
be carried out in practice, a preferred embodiment will now be
described, by way of non-limiting example only, with reference to
the accompanying drawings, in which:
[0042] FIGS. 1A-1C are graphs showing chemical characteristics of
methylprednisolone sodium hemisuccinate (MPS), including turbidity
of MPS as function of pH (FIG. 1A); partition coefficient of MPS at
different pH points (FIG. 1B); and surface tension of
methylprednisolone hemisuccinate and dexamethasone phosphate as
function of GC concentration (FIG. 1C).
[0043] FIGS. 2A-2B--are Cryo-TEM (transmission elecron microscopy)
images of liposomes before (FIG. 2A) and after (FIG. 2B) active
loading of MPS.
[0044] FIG. 3A-3B are size exclusion chromatography of SSL-MPS
after 14 months of storage at 4.degree. C. with FIG. 3B being an
enlargement of the section describing fractions 8-17 of FIG. 3A
showing existence of very low amounts of free MPS and
methylprednisolone (MP).
[0045] FIG. 4 is a release profile of MPS and Ca.sup.2+ from
SSL-MPS when incubated in plasma.
[0046] FIG. 5 is a graph showing the effect of SSL-MPS treatment in
Experimental Autoimmune Encephalomyelitis (EAE).
[0047] FIG. 6A-6B are graphs showing the effect of SSL-MPS
treatment on survival rate (%) (FIG. 6A) and on mean clinical score
(FIG. 6B) in EAE induced animals.
[0048] FIG. 7 is a graph showing the effect of SSL-MPS treatment on
EAE compared to the effect of Betaferon and Copaxone, two
conventional drugs.
[0049] FIG. 8 is a graph showing the effect of SSL-MPS treatment in
a chronic EAE animal model.
[0050] FIG. 9 is a graph showing the effect of SSL-MPS treatment on
survival of BCL-1 lymphoma.
DETAILED DESCRIPTION OF THE INVENTION
[0051] Glucocorticoids (GCs) are a family of hormones that
predominantly affects the metabolism of carbohydrates and, to a
lesser extent, fats and proteins (and have other effects).
Glucocorticoids are made in the peripheral part (the cortex) of the
adrenal gland and chemically classed as steroids. Cortisol is the
major natural glucocorticoid. Nonetheless, the term glucocorticoid
also applies to equivalent hormones synthesized in the
laboratory.
[0052] A non-limiting list of glucocorticoids may be found at the
interne site http://www.steraloids.com/, incorporated herein in its
entirety by reference. Examples include prednisolone hemisuccinate,
methylprednisolone heeimisuccinate, dexamethasone hemisuccinate,
allopregnanolone hemisuccinate; beclomethasone 21-hemisuccinate;
betamethasone 21-hemisuccinate; boldenone hemisuccinate;
prednisolone hemisuccinate, sodium salt; prednisolone
21-hemisuccinate; nandrolone hemisuccinate; 19-nortestosterone
hemisuccinate; deoxycorticosterone 21-hemisuccinate; dexamethasone
hemisuccinate; dexamethasone hemisuccinate:spermine; corticosterone
hemisuccinate; cortexolone hemisuccinate.
[0053] Like with many other medicaments, administration of GC in a
free form may posses some disadvantages, such as the risk of
exposing the treated individual to side effects known to occur with
GC treatment, rapid clearance of the steroid from the plasma,
etc.
[0054] In the search to overcome such disadvantages, the inventors
have envisaged that while it is difficult to efficiently and stably
load in a vehicle the rather hydrophobic GC, by applying a rather
simple chemical modification on the glucocorticoid involving the
conversion of the steroid to a water-soluble derivate, it is
possible to load the derivate into liposomes.
[0055] Thus, the present invention provides stable pharmaceutical
compositions comprising a glucocorticoid (GC) or GC derivative
encapsulated in a liposome, wherein said GC or GC derivative is
essentially retained in said liposome for 6, preferably 10, more
preferably 14 months (when stored at 4.degree. C.), the GC or GC
derivative being selected from: [0056] i) an amphipathic weak base
GC or GC derivative having a pKa equal or above 11 and a logD at pH
7 in the range between about -2.5 and about 1.5, preferably, in the
range between about -1.5 and about 1.0; [0057] ii) an amphipathic
weak acid GC or GC derivative having a pKa above 3.5 and a logD at
pH 7 in the range between about -2.5 and about 1.5, preferably, in
the range between about -1.5 and about 1.0.
[0058] The term "GC derivative" as used herein denotes a GC
molecule which was chemically modified either by the insertion of a
chemical group or by the removal of a chemical group from the GC
molecule, the modification results in the conversion of the
molecule to an amphipathic weak base or amphipathic weak acid,
depending on the type of modification applied. As well appreciated
by those versed in the chemistry of steroids, these hydrophilic in
nature molecules posses at least one chemically reactive group
which may be conjugated with a weak acid or weak base to form a
respective amphipathic weak acid or amphipathic weak base molecule.
Non-limiting examples of chemically reactive group typically
included in the general structure of steroids are hydroxyl,
carboxyl, and the like, as known to those versed in chemistry. It
should be noted that in the context of the present invention GC
derivative may also encompass an active, non-modified, amphipathic
and weakly acid GC.
[0059] The GC derivative by one aspect is a pro-drug, i.e. it has
no pharmacological activity in the form it is present in the
liposome. Upon release from the liposome the GC pro-drug in
converted by enzymes, such as esterases, to its pharmacologically
active hydrophobic form.
[0060] In accordance with yet another aspect the GC encapsulated in
the liposome, is already in its pharmaceutically active form, and
does not have to undergo any enzymatic processing in order to
become active. In accordance with the second aspect the GC itself
is a weak amphipathic acid or base.
[0061] The teen "amphipathic weak acid" is used herein to denote a
molecule having both hydrophobic and hydrophilic groups, the
steroid backbone of the GC essentially constituting the hydrophobic
group, while the weak acid moiety linked to the GC by virtue of the
modification described above essentially constituting the
hydrophilic group. The GC amphipathic weak acid or GC derivative is
characterized by the following physical characteristics: [0062]
pKa: it has a pKa above 3.0, preferably above 3.5, more preferably,
in the range between about 3.5 and about 6.5; [0063] Partition
coefficient: in an n-octanol/buffer (aqueous phase) system having a
pH of 7.0, it has a logD in the range between about -2.5 and about
1.5 and more preferably between about -1.5 and about 1.0.
[0064] Such amphipathic weak acid derivatives of GC may be obtained
by reacting the GC with dicarbocylic or tricarboxylic acids or by
linking the GC to the amino group of the amino acid, by techniques
known to those versed in the art.
[0065] Specific examples of GC derivates include, without being
limited thereto, betamethasone 21-hemisuccinate prednisolone
hemisuccinate sodium salt; prednisolone 21-hemisuccinate;
dexamethasone hemisuccinate; dexamethasone hemisuccinate:spermine;
corticosterone hemisuccinate Prednisolone hemisuccinate;
Methylprednisolone heeimisuccinate; Dexamethasone
hemisuccinate.
[0066] The term "amphipathic weak base" is used herein to denote a
molecule having both hydrophobic and hydrophilic groups, the
steroid backbone of the GC essentially constituting the hydrophobic
group, while the weak base moiety linked to the GC by virtue of the
modification described above essentially constituting the
hydrophilic group. The GC amphipathic weak acid derivative is
characterized by the following physical characteristics: [0067]
pKa: it has a pKa below 11.0, more preferably between about 11.0
and about 7.5; [0068] Partition coefficient: in an n-octanol/buffer
(aqueous phase) system it has a logD in the range between about
-2.5 and about 1.5 and more preferably between about -1.5 and about
1.0.
[0069] Such amphipathic weak base derivatives of GC may be obtained
by reacting the GC with basic amino acids, such as arginine or
lysine or with any amino acid through its carboxy group, leaving
the amino group free or with polyamine such as spermidine or
spermine.
[0070] The term "liposome" is used herein to denote lipid based
bilayer vesicles. Liposomes are widely used as biocompatible
carriers of drugs, peptides, proteins, plasmic DNA, antisense
oligonucleotides or ribozymes, for pharmaceutical, cosmetic, and
biochemical purposes. The enormous versatility in particle size and
in the physical parameters of the lipids affords an attractive
potential for constructing tailor-made vehicles for a wide range of
applications. Different properties (size, colloidal behavior, phase
transitions, electrical charge and polymorphism) of diverse lipid
formulations (liposomes, lipoplexes, cubic phases, emulsions,
micelles and solid lipid nanoparticles) for distinct applications
(e.g. parenteral, transdermal, pulmonary, intranasal and oral
administration) are available and known to those versed in the art.
These properties influence relevant properties of the liposomes,
such as liposome stability during storage and in serum, the
bio-distribution and passive or active (specific) targeting of
cargo, and how to trigger drug release and membrane disintegration
and/or fusion.
[0071] The present invention is applicable for a variety of
liposome compositions and those versed in the art will know how to
select the constituents of the liposome depending on the various
considerations including the choice of GC or GC derivative, the
mode of administration of the final liposomal formulation and
others.
[0072] The liposomes are those composed primarily of
liposome-forming lipids which are amphiphilic molecules essentially
characterized by a packing parameter 0.74-1.0, or by a lipid
mixture having an additive packing parameter (the sum of the
packing parameters of each component of the liposome times the mole
fraction of each component) in the range between 0.74 and 1.
[0073] Liposome-forming lipids, exemplified herein by
phospholipids, form into bilayer vesicles in water. The liposomes
can also include other lipids incorporated into the lipid bilayers,
such as phosphatidyl ethanolamine (PE) and sterol, with their
hydrophobic moiety in contact with the interior, hydrophobic region
of the bilayer membrane, and the head group moiety oriented toward
the exterior, polar surface of the bilayer membrane. The type and
level of the additional, non-liposome forming lipid components will
be determined by the additive packing parameter of the entire
components of the lipid bilayer to remain in the range of
0.74-1.0.
[0074] The liposome-forming lipids are preferably those having a
glycerol backbone wherein at least one, preferably two, of the
hydroxyl groups at the head group is substituted with, preferably
an acyl chain (to form an acyl or diacyl derivative), however, may
also be substituted with an alkyl or alkenyl chain, a phosphate
group or a combination or derivatives of same and may contain a
chemically reactive group, (such as an amine, acid, ester, aldehyde
or alcohol) at the headgroup, thereby providing a polar head group.
Sphyngolipids, such as sphyngomyelins, are good alternative to
glycerophopholipids.
[0075] Typically, the substituting chain(s), e.g. the acyl, alkyl
or alkenyl chain is between 14 to about 24 carbon atoms in length,
and has varying degrees of saturation being fully, partially or
non-hydrogenated lipids. Further, the lipid may be of natural
source, semi-synthetic or fully synthetic lipid, and neutral,
negatively or positively charged. There are a variety of synthetic
vesicle-forming lipids and naturally-occurring vesicle-forming
lipids, including the phospholipids, such as phosphatidylcholine
(PC), phosphatidylinositol (PI), phosphatidylglycerol (PG),
dimyristoyl phosphatidylglycerol (DMPG); egg yolk
phosphatidylcholine (EPC), 1-palmitoyl-2-oleoylphosphatidyl choline
(POPC), distearoylphosphatidylcholine (DSPC), dimyristoyl
phosphatidylcholine (DMPC); phosphatidic acid (PA),
phosphatidylserine (PS) 1-palmitoyl-2-oleoylphosphatidyl choline
(POPC), and the sphingophospholipids such as sphingomyelins (SM)
having 12-24 carbon atom acyl or alkyl chains. The above-described
lipids and phospholipids whose hydrocarbon chain
(acyl/alkyl/alkenyl chains) have varying degrees of saturation can
be obtained commercially or prepared according to published
methods. Other suitable lipids include in the liposomes are
glyceroglycolipids and sphingoglycolipids and sterols (such as
cholesterol or plant sterol).
[0076] Preferably, the phospholipid is egg phophatidylcholine
(EPC), 1-palmitoyl-2-oleoylphosphatidyl choline (POPC),
distearoylphosphatidylcholine (DSPC) or hydrogenated soy
phosphatidylcholine (HSPC).
[0077] Cationic lipids (mono and polycationic) are also suitable
for use in the liposomes of the invention, where the cationic lipid
can be included as a minor component of the lipid composition or as
a major or sole component. Such cationic lipids typically have a
lipophilic moiety, such as a sterol, an acyl or diacyl chain, and
where the lipid has an overall net positive charge. Preferably, the
head group of the lipid carries the positive charge. Monocationic
lipids may include, for example,
1,2-dimyristoyl-3-trimethylammonium propane (DMTAP)
1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP);
N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium
bromide (DMRIE);
N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethyl-ammonium
bromide (DORIE); N-[1-(2,3-dioleyloxy)
propyl]-N,N,N-trimethylammonium chloride (DOTMA);
3.beta.[N-(N',N'-dimethylamino ethane) carbamoly] cholesterol
(DC-Chol); and dimethyl-dioctadecylammonium (DDAB).
[0078] Examples of polycationic lipids include a similar lipophilic
moiety as with the mono cationic lipids, to which polycationic
moiety is attached. Exemplary polycationic moieties include
spermine or spermidine (as exemplified by DOSPA and DOSPER), or a
peptide, such as polylysine or other polyamine lipids. For example,
the neutral lipid (DOPE) can be derivatized with polylysine to form
a cationic lipid. polycationic lipids include, without being
limited thereto,
N-[2-[[2,5-bis[3-aminopropyl)amino]-1-oxopentyl]amino]ethyl]-N,N-dimethyl-
-2,3-bis[(1-oxo-9-octadecenyl)oxy]-1-propanaminium (DOSPA), and
ceramide carbamoyl spermine (CCS).
[0079] The lipids mixture forming the liposome can be selected to
achieve a specified degree of fluidity or rigidity, to control the
stability of the liposome in serum and to control the rate of
release of the entrapped agent in the liposome.
[0080] Further, the liposomes may also include a lipid derivatized
with a hydrophilic polymer to form new entities known by the term
lipopolymers. Lipopolymers preferably comprise lipids modified at
their head group with a polymer having a molecular weight equal or
above 750 Da. The head group may be polar or apolar, however, is
preferably a polar head group to which a large (>750 Da) highly
hydrated (at least 60 molecules of water per head group) flexible
polymer is attached. The attachment of the hydrophilic polymer head
group to the lipid region may be a covalent or non-covalent
attachment, however, is preferably via the formation of a covalent
bond (optionally via a linker). The outermost surface coating of
hydrophilic polymer chains is effective to provide a liposome with
a long blood circulation lifetime in vivo. The lipopolymer may be
introduced into the liposome by two different ways: (a) either by
adding the lipopolymer to a lipid mixture forming the liposome. The
lipopolymer will be incorporated and exposed at the inner and outer
leaflets of the liposome bilayer [Uster P. S. et al. FEBBS Letters
386:243 (1996)]; (b) or by firstly prepare the liposome and then
incorporate the lipopolymers to the external leaflet of the
pre-formed liposome either by incubation at temperature above the
Tm of the lipopolymer and liposome-forming lipids, or by short term
exposure to microwave irradiation.
[0081] Preparation of vesicles composed of liposome-forming lipids
and derivatization of such lipids with hydrophilic polymers
(thereby forming lipopolymers) has been described, for example by
Tirosh et al. [Tirosh et al., Biopys. J., 74(3):1371-1379, (1998)]
and in U.S. Pat. Nos. 5,013,556; 5,395,619; 5,817,856; 6,043,094,
6,165,501, incorporated herein by reference and in WO 98/07409. The
lipopolymers may be non-ionic lipopolymers (also referred to at
times as neutral lipopolymers or uncharged lipopolymers) or
lipopolymers having a net negative or a net positive charge.
[0082] There are numerous polymers which may be attached to lipids.
Polymers typically used as lipid modifiers include, without being
limited thereto: polyethylene glycol (PEG), polysialic acid,
polylactic (also termed polylactide), polyglycolic acid (also
termed polyglycolide), apolylactic-polyglycolic acid, polyvinyl
alcohol, polyvinylpyrrolidone, polymethoxazoline,
polyethyloxazoline, polyhydroxyethyloxazoline,
polyhydroxypropyloxazoline, polyaspartamide, polyhydroxypropyl
methacrylamide, polymethacrylamide, polydimethylacrylamide,
polyvinylmethylether, polyhydroxyethyl acrylate, derivatized
celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
The polymers may be employed as homopolymers or as block or random
copolymers.
[0083] While the lipids derivatized into lipopolymers may be
neutral, negatively charged, as well as positively charged, i.e.
there is no restriction to a specific (or no) charge, the most
commonly used and commercially available lipids derivatized into
lipopolymers are those based on phosphatidyl ethanolamine (PE),
usually, distearylphosphatidylethanolamine (DSPE).
[0084] A specific family of lipopolymers employed by the invention
include monomethylated PEG attached to DSPE (with different lengths
of PEG chains, the methylated PEG referred to herein by the
abbreviation PEG) in which the PEG polymer is linked to the lipid
via a carbamate linkage resulting in a negatively charged
lipopolymer. Other lipopolymer are the neutral methyl
polyethyleneglycol distearoylglycerol (mPEG-DSG) and the neutral
methyl polyethyleneglycol oxycarbonyl-3-amino-1,2-propanediol
distearoylester (mPEG-DS) [Garbuzenko O. et al., Langmuir.
21:2560-2568 (2005)]. The PEG moiety preferably has a molecular
weight of the head group is from about 750 Da to about 20,000 Da.
More preferably, the molecular weight is from about 750 Da to about
12,000 Da and most preferably between about 1,000 Da to about 5,000
Da. One specific PEG-DSPE employed herein is that wherein PEG has a
molecular weight of 2000 Da, designated herein .sup.2000PEG-DSPE or
.sup.2kPEG-DSPE.
[0085] Preparation of liposomes including such derivatized lipids
has also been described, where typically, between 1-20 mole percent
of such a derivatized lipid is included in the liposome
formulation.
[0086] It is well established that preparation of liposomal
formulation involve the selection of an appropriate lipid
composition in addition to the aqueous phase ingredients, such as
buffers, antioxidants, metal chelators, and cryoprotectants.
Charge-inducing lipids, such as phosphatidylglycerol can be
incorporated into the liposome bilayer to decrease vesicle-vesicle
fusion, and to increase interaction with cells, while cholesterol
and sphingomyelin can be included in formulations in order to
decrease permeability and leakage of encapsulated drugs. Buffers at
neutral pH can decrease hydrolysis. Addition of an antioxidant,
such as sodium ascorbate can decrease oxidation, etc.
[0087] Variations in ratios between these liposome constituents,
dictates the pharmacological properties of the liposome, including
stability of the liposomes, which is a major concern for various
types of vesicular applications. Evidently, the stability of
liposomes should meet the same standards as conventional
pharmaceuticals. Chemical stability involves prevention of both the
hydrolysis of ester bonds in the phospholipid bilayer and the
oxidation of unsaturated sites in the lipid chain. Chemical
instability can lead to physical instability or leakage of
encapsulated drug from the bilayer and fusion and aggregation of
vesicles. Chemical instability also results in short blood
circulation time of the liposome, which affects the effective
access to and interaction with the target.
[0088] A preferred formulation according to the invention is that
comprising phosphatidylcholine (PC) such as egg PC (EPC) or
hydrogenated soy PC (HSPC) as a the liposome forming lipid,
PEGylated (2000 Da) distearoyl-phosphatidylethanolamine (PEG-DSPE)
and cholesterol. Evidently, other lipids mixtures may be utilized
in the same, similar or different mole ratio, and provided that the
final additive packing parameter of the different constituents of
the liposome is in the range of between about 0.74 and 1.0.
[0089] The pharmaceutical formulation of the invention was proven
to be highly stable. An exemplified embodiment of the invention in
which the GC derivative, Methylprednisolone succinate
(methylprednisolone modified with succinic acid) was encapsulated
in a liposome comprising the above three constituents, was shown to
have only marginal reduction (less than 20% from initial
concentration) in the GC derivative after storage at 4.degree. C.
for 14 months (FIGS. 3A-3B).
[0090] Thus, in the context of the present invention, the term
"stability" denotes a formulation which under conventional storage
conditions (4.degree. C.) retains the majority (more than 80%,
preferably more than 90%) of the GC/GC derivative in the liposome,
for 6 months, preferably for 10 months and more preferably for 14
months. Accordingly, the term "essentially retains" used herein
denotes that 80% and preferably 90% of the GC or GC derivative is
retained in the liposomes under storage conditions for about 6,
preferably 10 and more preferably 14 months. According to one
preferred embodiment, stability of the liposomes is maintained by
the use of sterically stabilized liposome (SSL), i.e. liposomes
coated with a hydrophilic component. According to a preferred
embodiment, the SSL comprises a combination of hydrogenated soy
phosphatidylcholine (HSPC), .sup.2000PEG-DSPE and cholesterol at a
molar ratio of 55:40:5.
[0091] In general, there are a variety of drug-loading methods
available for preparing liposomes with entrapped drug, including
passive entrapment and active remote loading. The passive
entrapment method is most suited for entrapping of lipophilic drugs
in the liposome membrane and for entrapping drugs having high water
solubility. In the case of ionizable hydrophilic or amphipathic
drugs, even greater drug-loading efficiency can be achieved by
loading the drug into liposomes against a transmembrane ion
gradient [Nichols, J. W., et al., Biochim. Biophys. Acta
455:269-271 (1976); Cramer, J., et al., Biochemical and Biophysical
Research Communications 75(2):295-301 (1977)]. This loading method,
generally referred to as remote loading, typically involves a drug
which is amphipathic in nature and has an ionizable group which is
loaded by adding it to a suspension of liposomes having a higher
inside/lower outside H.sup.+ and/or ion gradient.
[0092] The liposomes employed in the context of the present
invention are preferably loaded by the remote loading principle.
The resulting formulation exhibited a significantly high GC
derivative to lipid ratio. Preferably, the mole ratio between the
GC derivative and lipid is between 0.01 and 2.0, more preferably,
between 0.04 and 0.25. For high loading of the GC derivative it is
at times preferable that the concentration of the same in the
liposome be such that it precipitates in the presence of a
pre-entrapped counter ion.
[0093] Liposomes having an H.sup.+ and/or ion gradient across the
liposome bilayer for use in remote loading can be prepared by a
variety of techniques. A typical procedure comprises dissolving a
mixture of lipids at a ratio that forms stable liposomes in a
suitable organic solvent and evaporated in a vessel to form a thin
lipid film. The film is then covered with an aqueous medium
containing the solute species that will form the aqueous phase in
the liposome interior space. After liposome formation, the vesicles
may be sized to achieve a size distribution of liposomes within a
selected range, according to known methods. The liposomes utilized
in the present invention are preferably uniformly sized to a
selected size range between 70-100 nm, preferably about 80 nm.
[0094] After sizing, the external medium of the liposomes is
treated to produce an ion gradient across the liposome membrane
(typically with the same buffer used to form the liposomes), which
is typically a higher inside/lower outside ion concentration
gradient. This may be done in a variety of ways, e.g., by (i)
diluting the external medium, (ii) dialysis against the desired
final medium, (iii) gel exclusion chromatography, e.g., using
Sephadex G-50, equilibrated in the desired medium which is used for
elution, or (iv) repeated high-speed centrifugation and
resuspension of pelleted liposomes in the desired final medium. The
external medium which is selected will depend on the type of
gradient, on the mechanism of gradient formation and the external
solute and pH desired, as will now be described.
[0095] In the simplest approach for generating an ion and/or
H.sup.+ gradient, the lipids are hydrated and sized in a medium
having a selected internal-medium pH. The suspension of the
liposomes is titrated until the external liposome mixture reaches
the desired final pH, or treated as above to exchange the external
phase buffer with one having the desired external pH. For example,
the original hydration medium may have a pH of 5.5, in a selected
buffer, e.g., glutamate, citrate, succinate, fumarate buffer, and
the final external medium may have a pH of 8.5 in the same or
different buffer. The common characteristic of these buffers is
that they are formed from acids which are essentially liposome
impermeable. The internal and external media are preferably
selected to contain about the same osmolarity, e.g., by suitable
adjustment of the concentration of buffer, salt, or low molecular
weight non-electrolyte solute, such as dextrose or sucrose.
[0096] In another general approach, the gradient is produced by
including in the liposomes, a selected ionophore. To illustrate,
liposomes prepared to contain valinomycin in the liposome bilayer
are prepared in a potassium buffer, sized, then the external medium
exchanged with a sodium buffer, creating a potassium inside/sodium
outside gradient. Movement of potassium ions in an
inside-to-outside direction in turn generates a lower inside/higher
outside pH gradient, presumably due to movement of protons into the
liposomes in response to the net electronegative charge across the
liposome membranes [Deamer, D. W., et al., Biochim. et Biophys.
Acta 274:323 (1972)].
[0097] A similar approach is to hydrate the lipid and to size the
formed multilamellar liposome in high concentration of magnesium
sulfate. The magnesium sulfate gradient is created by dialysis
against 20 mM HEPPES buffer, pH 7.4 in sucrose. Then, the A23187
ionophore is added, resulting in outwards transport of the
magnesium ion in exchange for two protons for each magnesium ion,
plus establishing a inner liposome high/outer liposome low proton
gradient [Senske D B et al. (Biochim. Biophys. Acta 1414: 188-204
(1998)].
[0098] In another more preferred approach, the proton gradient used
for drug loading is produced by creating an ammonium ion gradient
across the liposome membrane, as described, for example, in U.S.
Pat. Nos. 5,192,549 and 5,316,771, incorporated herein by
reference. The liposomes are prepared in an aqueous buffer
containing an ammonium salt, such as ammonium sulfate, ammonium
phosphate, ammonium citrate, etc., typically 0.1 to 0.3 M ammonium
salt, at a suitable pH, e.g., 5.5 to 7.5. The gradient can also be
produced by including in the hydration medium sulfated polymers,
such as dextran sulfate ammonium salt, heparin sulfate ammonium
salt or sucralfate. After liposome formation and sizing, the
external medium is exchanged for one lacking ammonium ions. In this
approach, during the loading the amphipathic weak base is exchanged
with the ammonium ion.
[0099] Yet, another approach is described in U.S. Pat. No.
5,939,096, incorporated herein by reference. In brief, the method
employs a proton shuttle mechanism involving the salt of a weak
acid, such as acetic acid, of which the protonated form
trans-locates across the liposome membrane to generate a higher
inside/lower outside pH gradient. An amphipathic weak acid compound
is then added to the medium to the pre-formed liposomes. This
amphipathic weak acid accumulates in liposomes in response to this
gradient, and may be retained in the liposomes by cation (i.e.
calcium ions)-promoted precipitation or low permeability across the
liposome membrane, namely, the amphipathic weak acid is exchanges
with the acetic acid.
[0100] The liposomes loaded with the GC or GC derivative may be
administered in various ways. It may be formulated in combination
with physiologically acceptable excipients, as known in the art.
The pharmaceutically acceptable excipients employed according to
the invention generally include inert, non-toxic substances which
preferably do not react with liposomes. The excipients may be any
of those conventionally used and is limited only by
chemical-physical considerations, such as solubility and lack of
reactivity with liposomes, and by the route of administration. The
excipients may also at times have the effect of the improving the
delivery or penetration of the liposomal formulation to a target
tissue, for improving the stability of the liposomal formulation,
for slowing clearance rates, for imparting slow release properties,
for reducing undesired side effects etc. The excipient may also be
a substance that stabilizes the formulation (e. g. a preservative),
for providing the formulation with an edible flavor, etc. The
excipient may include additives, colorants, diluents, buffering
agents, disintegrating agents, moistening agents, preservatives,
flavoring agents, and pharmacologically compatible carriers. As an
example, when treating a neurodegenerative condition, the excipient
may be a molecule which is known to promote or facilitate entry
through the blood brain barrier (BBB) such as transferin
receptor-binding agents, antibodies, or any drug that by itself
transfers through the BBB.
[0101] The pharmaceutical composition of the invention may have an
advantage for the treatment of a variety of conditions typically
those are the conditions which are known to be treated (in at lease
a phase of their course by the administration of GC. Examples of
such conditions include neurodegenerative conditions and cancer, as
detailed hereinafter. To this end, the liposomal formulation of the
invention may comprise one or more active ingredients, in addition
to the GC or GC derivative. The additional active ingredients may
be in a free form or also encapsulated in liposomes (together or
separated from the liposomes containing the GC derivative). For
example, when treating cancer, the additional active ingredient may
be a cytotoxic drug, such as doxorubicin, encapsulated in the same
or different liposomes. For treating a neurodegenerative condition,
the liposomal formulation may be combined with Copaxone or
Betaferone.
[0102] The following is a non-limiting list of medical conditions
which may be treated or prevented with the liposomal formulation of
the invention additional conditions, which are known to benefit
from GC treatment, are also included in the scope of the
invention:
[0103] Endocrine Disorders including primary or secondary
adrenocortical insufficiency; Congenital adrenal hyperplasia
Hypercalcemia associated with cancer, nonsuppurative
thyroiditis.
[0104] Collagen Diseases including, for example, during an
exacerbation or as maintenance therapy in selected cases of
[0105] Dermatologic Diseases including, for example, Pemphigus
Bullous dermatitis, Severe erythema multi-herpetiformis forme
(Stevens--Severe seborrheic Johnson syndrome) dermatitis
Exfoliative dermatitis Severe psoriasis Mycosis fungoides.
[0106] Allergic States including, for example, control of severe or
incapacitating allergic conditions unresponsive to adequate trials
of conventional treatment in: Bronchial asthma, Drug
hypersensitivity Contact dermatitis reactions, Atopic dermatitis,
Urticarial transfusion, Serum sickness reactions, Seasonal or
perennial, Acute noninfectious allergic rhinitis laryngeal
edema.
[0107] Ophthalmic Diseases including, for example, severe acute and
chronic allergic and inflammatory processes involving the eye, such
as: Herpes zoster ophthalmicus, Sympathetic ophthalmia Iritis,
iridocyclitis Anterior segment Chorioretinitis inflammation Diffuse
posterior uveitis, Allergic conjunctivitis and choroiditis,
Allergic corneal marginal, Optic neuritis ulcers, Keratitis.
[0108] Respiratory Diseases, including, for example, symptomatic
sarcoidosis Loeffler's syndrome not Berylliosis manageable by other
Fulminating or disseminate, not manageable by other means,
Aspiration pneumonitis, tuberculosis optionally used concurrently
with appropriate antituberculous chemotherapy.
[0109] Hematologic Disorders, including acquired (autoimmune)
hemolytic anemia, Idiopathic thrombocytopenic purpura, secondary
thrombocytopenia, Erythroblastopenia (RBC anemia). Congenital
(erythroid) hypoplastic anemia.
[0110] Neoplastic Diseases, including, for example, for management
of: Leukemias and lymphomas, myeloma, breast cancer and prostate
cancer.
[0111] Edematous States, including, for example, to induce diuresis
or remission of proteinuria in the nephrotic syndrome, without
uremia, of the idiopathic type or that due to lupus
erythematosus.
[0112] Nervous System, including, for example, acute exacerbations
of multiple sclerosis (MS).
[0113] As well as other conditions, such as tuberculous meningitis
with sub-arachnoid block or impending block when used concurrently
with appropriate antituberculous chemotherapy; Trichinosis with
neurological or myocardial involvement.
[0114] Thus, the invention also pertains to a method of treatment
or prevention of a disease or pathological condition, the method
comprises providing a subject in need of said treatment an amount
of the liposomal formulation of the invention, the amount being
effective (hereinafter the "effective amount") to treat or prevent
the disease or pathological condition. Preferred conditions to be
treated by the present invention are cancer and neurodegenerative
conditions.
[0115] The term "treatment" as used herein denotes the
administering of an amount of the GC or GC derivative encapsulated
in a liposome effective to ameliorate undesired symptoms associated
with a disease, to prevent the manifestation of such symptoms
before they occur, to slow down the progression of the disease,
slow down the deterioration of symptoms associated with the
disease, to enhance an onset of a remission period of a disease, to
slow down any irreversible damage caused in a progressive chronic
stage of a disease, to delay the onset of said progressive stage,
to lessen the severity or cure a disease, to improve survival rate
or more rapid recovery from a disease, to prevent a disease form
occurring, or a combination of two or more of the above.
[0116] As an example, when referring to neurodegenerative
conditions, treatment denotes inhibition or slowing down of
abnormal deterioration of the nervous system as well as prevention
in subjects with high disposition of developing a neurodegenerative
condition (as determined by considerations known to those versed in
medicine) or for preventing the re-occurrence of an acute stage of
a neurodegenerative condition in a chronically ill subjects. In the
latter case, the pharmaceutical composition comprising the
liposomal GC derivative may be administered to a subject who does
not have a neurodegenerative condition but is at high-risk of
developing such a condition, e.g. as a result of exposure to an
agent which is known to cause abnormal generation of reactive
oxidative species or subjects with family history of the disease
(i.e. genetic disposition).
[0117] Further, as an example, when the disease is cancer,
treatment denotes, inter alia, inhibition or reduction of the
growth and proliferation of tumor cells: including arresting growth
of the primary tumor, or decreasing the rate of cancer related
mortality, or delaying cancer related mortality, which may result
in the reduction of tumor size or total elimination thereof from
the individual's body, or decreasing the rate of occurrence of
metastatic tumors, or decreasing the number of metastatic tumors
appearing in an individual.
[0118] The liposomal GC derivative may be provided as a single
dose, however is preferably administered to a subject in need of
treatment over an extended period or time (e.g. to produce a
cumulative effective amount) in a single daily dose, in several
doses a day, as a single dose for several days, etc. The treatment
regimen and the specific formulation to be administered will depend
on the type of disease to be treated and may be determined by
various considerations, known to those skilled in the art of
medicine, e. g. the physicians.
[0119] The term "effective amount" or "therapeutically effective
amount" is used herein to denote the amount of the GC derivative
when loaded in the liposome in a given therapeutic regimen which is
sufficient to achieve a desired effect, e.g. inhibition or
reduction of the growth and proliferation of tumor cells, or
inhibition or reduction of degradation of nerve cells and thereby
the deterioration of the nervous system. The amount is determined
by such considerations as may be known in the art and depends on
the type and severity of the condition to be treated and the
treatment regime. 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. As generally
known, an effective amount depends on a variety of factors
including the mode of administration, type of vehicle carrying the
amphipathic weak acid/base, the reactivity of the GC derivative,
the liposome's distribution profile within the body, a variety of
pharmacological parameters such as half life in the body after
being released from the liposome, on undesired side effects, if
any, on factors such as age and gender of the treated subject,
etc.
[0120] The term "administering" is used to denote the contacting or
dispensing, to delivering or applying the liposomal formulation, to
a subject by any suitable route for delivery thereof to the desired
location in the subject, these include oral, parenteral (including
subcutaneous, intramuscular and intravenous, intraarterial,
intraperitoneally) and intranasal administration as well as by as
well as intrathecal and infusion techniques.
[0121] According to one embodiment, the formulations used in
accordance with the invention are in a form suitable for injection.
The requirements for effective pharmaceutical vehicles for
injectable formulations are well known to those of ordinary skill
in the art [See Pharmaceutics and Pharmacy Practice, J.B.
Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages
238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel,
4.sup.th ed., pages 622-630 (1986)].
[0122] It is noted that humans are treated generally longer than
experimental animals as exemplified herein, which treatment has a
length proportional to the length of the disease process and active
agent effectiveness. The doses may be a single dose or multiple
doses given over a period of several days.
[0123] While the following disclosure provides experimental data
with animal model, there are a variety of acceptable approaches for
converting doses from animal models to humans. For example,
calculation of approximate body surface area (BSA) approach makes
use of a simple allometric relationship based on body weight (BW)
such that BSA is equal to body weight (BW) to the 0.67 power
[Freireich E. J. et.al. Cancer Chemother. Reports 1966, 50(4)
219-244; and as analyzed in Dosage Regimen Design for
Pharmaceutical Studies Conducted in Animals, by Mordenti, J, in J.
Pharm. Sci., 75:852-57, 1986]. Further, allometry and tables of BSA
data have been established [Extrapolation of Toxicological and
Pharmacological Data from Animals to Humans, by Chappell W &
Mordenti J, Advances in Drug Research, Vol. 20, 1-116, 1991
(published by Academic Press Ltd)]
[0124] Another approach for converting doses is a
pharmacokinetic-based approach using the area under the
concentration time curve (AUC) or Physiologically Based
PharmacoKinetic (PBPK) methods are described [Voisin E. M. et al.
Regul Toxicol Pharmacol. 12(2):107-116. (1990)].
[0125] The invention will now be described by way of non-limiting
examples showing the effect of SSL-MPS on EAE and lymphoma
cells.
DESCRIPTION OF SPECIFIC EXAMPLES
General
Materials
[0126] Hydrogenated soybean phosphatidylcholine (HSPC) was obtained
from Lipoid KG (Ludwigshafen, Germany).
[0127] N-carbamyl-poly-(ethylene glycol methyl
ether)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine triethyl
ammonium salt (PEG-DSPE) (the polyethylene moiety of this
phospholipid having a molecular mass of 2000 Da) was obtained from
Genzyme Liestale, Switzerland.
[0128] Cholesterol (>99% pure) was obtained from Sigma (St.
Louis, Mo., USA).
[0129] [.sup.3H] Cholesteryl hexadecyl ether (45 Ci per mmol) was
from NEN Life Science Products (Boston, Mass., USA). tert-Butanol
(99% pure) was purchased from BDH, Poole, UK.
[0130] The weak acid steroids, the pro-drugs methylprednisolone
sodium hemisuccinate (MPS) and hydrocortisone sodium hemisuccinate
(HYD), were obtained from Pfeizer, Belgium
[0131] All the other chemicals, including buffers were of
analytical grade or better, and were obtained from Sigma. Purified
water was obtained from WaterPro PS HPLC/Ultrafilter Hybrid model,
(Labconco, Kansas City, Mo., USA).
Methods
Liposome Preparation
[0132] A stock solution HSPC/Cholesterol/PEG-DSPE-2000 at molar
ratio of 55:40:5 was dissolved in ethanol at 70.degree. C. to a
final gel lipid concentration of 62.5% (w/v). The solution was then
incubated at 70.degree. C. until all the lipids are dissolved to a
clear solution. The stock solution was then added to a solution of
calcium acetate 200 mM at 70.degree. C. to receive 10% lipid
concentration (w/v) hence reaching a final ethanol concentration of
16% (w/v). The mixture was constantly stirred at 70.degree. C. to
receive a milky dispersion at this stage lipids were hydrated to
form multi lamellar liposome (MLV) dispersion.
[0133] The vesicles that were formed were downsized using extrusion
through a polycarbonate filter of defined pore size starting with
400-nm and ending with 50-nm pore size filters, as the last
extrusion step under low to medium pressure. This processes results
in 80.+-.15-nm liposomes. The extrusion device (Northern Lipids,
Canada) was kept in a constant temperature of 70.degree. C. during
the entire procedure.
[0134] The removal of extraliposomal Ca acetate to create the Ca
acetate gradient {[Ca acetate] in liposome >>[Ca acetate] in
medium} was created by dialysis against dextrose 5% or saline 0.9
at 4.degree. C. (4 exchanges.times.100 volume each, the final one
over night).
[0135] Liposome phospholipids concentration was determined from
organic phosphorus concentration by a modified Bartlet procedure
[Shmeeda, H., et al. In: Methods in Enzymology "Liposomes",
(Duzgunes, N., ed.), 367:272-292 (2003)]. Lipid concentration in
the resulting liposome stock solution was .about.40 mM.
[0136] The amount of calcium inside the liposomes was determined by
the use of atomic absorption spectrometry (AAS).
Preparation and Characterization of Radioactive-SSL
[0137] [.sup.3H] cholesteryl ether-labeled sterically stabilized
liposomes (SSL) composed of HSPC:Chol:.sup.200PEG-DSPE (55:40:5
mole ratio), and a trace amount of [.sup.3H] cholesteryl hexadecyl
ether (0.125 .mu.L) were prepared as described above. The liposome
size was determined by Dynamic Light Scattering (DLS) to be
87.+-.15 nm.
Loading of GC Derivative into Liposomes
[0138] A stock solution of methylprednisolone hemi succinate sodium
salt (MPS, the GC derivative) was dissolved in 5% dextrose (pH 7.2)
to a concentration of .about.9 mg/ml and added to the preformed SSL
dispersion after the calcium acetate gradient was established. MPS
concentration was .about.9 mg/ml and phospholipid .about.32 mM
phosphate.
[0139] Loading was achieved by incubation of the components above
for the desired time at 62.degree. C. (above matrix lipid T.sub.m).
Liposomes were then cooled to 4.degree. C. and dialyzed against 5%
dextrose at 4.degree. C. to remove acetate released during loading
and to remove unloaded drug or alternatively unloaded drug was
removed by the ion exchanger Dowex 1.times.400 mesh (Cl.sup.-
form).
State of Aggregation, Partition Coefficient and Surface Tension of
MPS
1. State of Aggregation of MPS
[0140] Aggregation of MPS was determined from the change in
turbidity measured as intensity of light scattered at 90.degree. to
excitation beam using a spectrofluormeter under conditions that MPS
lack absorbance (excitation and emission at the same wavelength
Ex=600 nm Em=600 nm). There is a large increase in the light
scattered by MPS solution/dispersion due to formation of
aggregates.
[0141] The intensity of scattered light (at 90.degree. to the
excitation), also defined as turbidity is proportional to
concentration and to the size of the aggregates. [Zuidam, N.J. and
Barenholz, Y., Biochim. Biophys. Acta 1368:115-128 (1998)]. The
state of aggregation of MPS was tested in the following manner: To
quartz cuvette MPS (2 ml) at concentration of .about.6.5 mg/ml MPS
was added. The solution was tittered with HCl (1.756M) and light
scattering using excitation and emission at (both at 600 nm with
attenuation of 1%) and pH of the solution was monitored.
2. Partition Coefficient
[0142] Partition coefficient (logD) of some GC derivatives (which
are amphipathic weak acids) was determined by the `shake flask` as
described [Samuni, A. M. and Barenholz, Y., Free Radicals Biol.
Med. 22:1165-1174 (1997)].
3. Surface Tension
[0143] Surface tension was measured using .mu.trouge S (Kibron
Inc., Helsinki Finland). A solution containing GC derivative (300
.mu.L) was placed in the well after calibration and zeroing of the
sensor using pure water and air. The measurement was performed at
26.degree. C.
Precipitation of MPS Inside SSL
[0144] MPS precipitation inside the intraliposomal aqueous phase of
the vesicle was visualized using Cryo TEM as described [Lasic, D.
D., Frederik, P. M., Stuart, M. C. A., Barenholz, Y. and McIntosh,
T. J., Gelation of liposome interior. A novel method for drug
encapsulation. FEBS Lett. 312, 255-258 (1992); Lasic, D. D., et al.
Biochim. Biophys. Acta 1239, 145-156 (1995)].
Precipitation Studies
[0145] To 600 mOsm Ca-acetate solution at 63.degree. C. and at
different pH points MPS was added at final concentration of 5
mg/ml, then mixed solution was incubated for 40 minutes after which
the solution was centrifuged and the supernatant was analyzed using
HPLC.
Loading Efficiency
[0146] Loading efficiency is the ratio between MPS/phospholipid
concentrations after and before loading. The quantification of MPS
was done in an HPLC apparatus as described by Anderson, and
Taphouse 1981 [Anderson B. D. and Taphouse. V. J Pharm Sci,
70:181-6 (1981)] quantification of phospholipid was done by
modified Bartlet procedure [Shmeeda, H., et al. In: Methods in
Enzymology "Liposomes", (Duzgunes, N., ed.), 367:272-292
(2003)].
Stability Determination
[0147] Determination of MPS Release from Liposome
[0148] The level of MPS released from SSL-MPS was determined by
first separating the liposomes from free MPS using gel exclusion
chromatography on Sepharose cross-linked CL-4B column using.
Liposomes were eluted at the void volume and the free MPS at the
later eluted fractions (FIG. 3A-3B).
[0149] Stability upon storage at 4.degree. C. and kinetic of
release at 37.degree. C. in 80% plasma was determined by gel
exclusion chromatography described above. Then the different column
fractions were analyzed as described above, for MPS, phospholipids
and Ca in the void volume fraction.
[0150] In addition, SSL-MPS were incubated with 80% human inflamed
synovial fluid at a ratio of 80% plasma at 37.degree. C. Then at
different time points sample were vortexed with the anion exchange
resin, DOWEX 1.times.400 mesh (Cl.sup.- form), which bind only free
MPS. The samples were analyzed for liposome encapsulated MPS and
liposome phospholipid content.
Results
[0151] Table 1 below provides logD and pKa of the tested GC
derivatives, as calculated using Advanced Chemistry Development
(ACD/Labs) [Software Solaris V4.67 ( 1994-2005 ACD/Labs) SciFinder
SCHOLAR Version 2004.2 .COPYRGT. American Chemical Society
2004].
TABLE-US-00001 TABLE 1 logD and pKa of different GC derivatives
Modified GC logD at pH 7 pKa Prednisolone phosphate -4.25 1.67 .+-.
0.10 Prednisolone hemisuccinate -0.64 4.29 .+-. 0.17
Methylprednisolone phosphate -3.76 1.67 .+-. 0.10
Methylprednisolone hemisuccinate -0.15 4.29 .+-. 0.17 Dexamethasone
phosphate -3.88 1.67 .+-. 0.10 Dexamethasone hemisuccinate -0.27
4.29 .+-. 0.17
Turbidity, Partition Coefficient and Surface Tension of MPS
[0152] The turbidity (indicating the aggregation) of MPS was
determined. The results shown in FIG. 1A indicate that at pH 7.2
the amphipathic weak acid derivative (the pro-drug) is
non-aggregated water soluble and in acidic pH, it aggregates,
therefore showing increase in turbidity. The decline in turbidity
observed at very low pH, was due to the formation of very large
aggregates which precipitated. The doted arrow indicates the point
of transition from titration with HCl (.mu.mol of H.sup.+, left
side of arrow) to titration with NaOH (.mu.mol of OH.sup.-, right
side of arrow).
[0153] Partition coefficient of MPS was determined at different pH
points. As shown in FIG. 1B, MPS is indeed an amphipathic
substance.
[0154] Further, the surface tension of MPS was determined and as
evident from FIG. 3C, MPS is surface active at all concentrations
used (0.785-30 mM) and has a CAC (critical association/aggregation
concentration) point at .about.5 mM while GC that has a phosphate
group (a strong acidic group) such as dexamethasone phosphate was
not surface active at least up to concentration of 50 mM and did
not self associate to form micelles and/or other organized
assemblies.
Precipitation of MPS by Calcium Ion
[0155] The precipitation of MPS in the presence of calcium acetate
solution was determined as described above. Table 2 shows the
percent of MPS that precipitated in the presence of calcium ions,
i.e. at different pH. As shown, precipitation already occurred at
pH 6.8. Precipitation was increased to a very large extent (97% of
the MPS) at pH around the pKa of GC (pH 4.5).
TABLE-US-00002 TABLE 2 precipitation of MPS pH % MPS precipitated
6.8 44.1 6.2 42.8 4.5 96.6
General Loading Efficiency for Different Liposomal Formulations
1. Liposome Loading Efficiency
[0156] Three separate batches (identified by dates) were used in
order to determine loading efficacy of the drug into the liposomes
(HSPC:Chol:.sup.2000PEG-DSPE (55:40:5 mole ratio), as summarized in
Table 3.
TABLE-US-00003 TABLE 3 Loading efficiency into SSL mg/ml mM
MPS/lipid MPS phosphate (mg/.mu.mole) % Encapsulation 18 JUL. 2004
Before 7.46 39.80 0.188 dialysis.sup.(a) After 7.56 40.70 0.186
99.1 dialysis.sup.(b) 06 APR. 2005 Before 6.22 33.20 0.19
dialysis.sup.(a) After 4.99 27.60 0.18 96.6 dialysis.sup.(b) 04 MAY
2005 Before 9.68 45.73 0.21 dialysis.sup.(a) After 6.35 32.19 0.20
93.2 dialysis.sup.(b) .sup.(a)MPS in liposome + MPS in the
extraliposome medium .sup.(b)MPS in liposome only
2. Loading Efficiency of MPS for Different Liposomal
Formulations
[0157] It was determined that the optimum conditions for efficient
loading include .about.600 mOsm of Calcium acetate. The loading
efficiency of MPS in HSPC:Chol:.sup.2000PEG-DSPE (55:40:5 mole
ratio) liposomes with this MPS/phospholipids ratio was obtained
when using initial pro-drug concentration between 5-10 mg/ml,
preferably 9 mg/ml. The concentration of the pro-drug in the final
formulation was .about.6.5 mg/ml which was used in the following
experiments (hereinafter termed the SSL-MPS formulation or in brief
SSL-MPS).
[0158] FIG. 2A-2B are Cryo-TEM images of liposomes before (FIG. 2A)
and after (FIG. 2B) loading clearly showing location of the
precipitate in the internal aqueous space of the liposome.
Stability of Liposomal Formulation
[0159] The concentration of MPS in SSL-MPS (i.e. intact liposomal
formulation) over 14 months was determined as described above. FIG.
3A shows that after 14 months .about.80% of MPS was retained in the
liposome. Part of the free MPS was hydrolyzed to its active form,
methylrednisolone (MP). FIG. 3B provides a Sepharose 4B
size-exclusion chromatograph of the liposomal preparation at after
14 months of storage at 4.degree. C., with an enlargement (FIG. 3C)
of the graph at fractions 8 to 17, showing the existence of free
MPS as well as free MP.
[0160] Further, stability of SSL-MPS in clinical relevant milieus
was determined. FIG. 4 shows the stability of the liposomal
formulation in human plasma. The retention of 100% of the
encapsulated calcium in the encapsulated liposome under condition
that MPS is released (with a half life in liposome of 50 hours)
indicates that the liposomes are intact for at least 66 hours in
plasma. This suggests that the release of MPS is due to its
amphiphacy. The half life of MPS release is in a similar value to
SSL half life in plasma post i.v. injection.
Example 1
Multiple Sclerosis (MS)
Induction of Acute EAE Experimental Animal Model Using Proteolipid
Protein (PLP)
[0161] 6-7 week old SJL female mice were immunized by subcutaneous
injection with an emulsion containing proteolipid protein (PLP)
139-151 peptide and complete Freund's adjuvant (CFA), containing
150 ug of peptide and 200 ug of Mycobacterium tuberculosis. In
order to boost the immune system Pertussis Toxin (PT) 150 ng were
injected intraperitoneally (i.p.) to the mice on the first day and
48 hours later.
[0162] Each mouse was examined daily for clinical signs of EAE
using the following Table 4:
TABLE-US-00004 TABLE 4 clinical signs scoring Score Signs
Description 0 Normal behavior No neurological signs 1 Distal limp
tail The distal part of the tail is limp and droops 1.5 Complete
limp tail The whole tail is loose and droops 2 Complete limp tail
The whole tail is loose and with righting reflex droops. Animal has
difficulties to return on his feet when it is laid on his back 3
Ataxia Woobly walk- when the mouse walks the hind legs are unsteady
4 Early paralysis The mouse has difficulties standing on its hind
legs but still has remnants of movement 5 Full paralysis The mouse
can't move its legs at all, it looks thinner and emaciated.
Incontinence 6 Moribund/death
[0163] The number of mice in each animal group which developed the
disease (sick) was summed and the percentage thereof was
calculated.
[0164] In addition, the mean maximal score (MMS) by summing the
maximal scores of each of the 10 mice in the group and calculating
therefrom the mean maximal score of the group according to the
following equation:
.SIGMA.maximal score of each mouse/number of mice in the group
[0165] Further, the mean duration of disease (MDD) expressed in
days was calculated according to the following equation:
.SIGMA.duration of disease of each mouse/number of mice in the
group
[0166] Further, each group's mean score (GMS) (burden of disease)
was determined by summing the scores of each of the 10 mice in the
group and calculating the mean score per day, according to the
following equation:
.SIGMA.total score of each mouse per day/number of mice in the
group.
Treatment of EAE with SSL-MPS
[0167] The EAE induced mice were divided into treatment groups
according to the following Table 5.
TABLE-US-00005 TABLE 5 treatment design No. mice/ Day of No. of
Treatment group injection injection Control 8 0 0 Free-MPS 50 mg/kg
BW 9 14 1 SSL-MPS 50 mg/kg BW 9 14 1
[0168] Follow up was conducted for a period of 3 weeks, and
clinical signs of EAE were determined at different time points. For
each group, the incidence, MMS=mean maximal score; MDD=mean disease
duration (days); MDO=mean day of onset and mean score were
determined (Table 6). Further, for each group the mean clinical
scores at each time point was determined (FIG. 5).
TABLE-US-00006 TABLE 6 observed clinical signs Group Incidence MMS
MDO MDD Mean Score Control 6/8 .sup. 3 .+-. 0.365 13.7 .+-. 0.667
18.5 .+-. 0.5 1.4 .+-. 0.104 SSL-MPS 5/9 2.2 .+-. 0.583 20.4 .+-.
2.16 6 .+-. 2.07 0.444 .+-. 0.090 Free-MPS 7/9 2.35 .+-. 0.39 16.7
.+-. 1.83 10.9 .+-. 2.7 0.861 .+-. 0.105
Treatment of Severe Disease Burden
[0169] For mice showing a severe disease burden (determined by
summing the scores of each of the 10 mice in the group and
calculating the mean score per day, according to the following
equation: .SIGMA.total score of each mouse per day/number of mice
in the group) a different treatment was applied. Specifically,
after immunization as described above, mice were treated with 50
mg/kg BW SSL-MPS (days 10, 14, 18) or with free-MPS 50 mg/kg BW
(days 10, 14, 18) or dextrose 5% (days 10, 14, 18). For each group,
the incidence, MMS=mean maximal score; MDD=mean disease duration
(days); MDO=mean day of onset and mean score were determined Table
7. Further, for each group the mean clinical scores at each time
point was determined (FIG. 6B) as well as the survival curve (FIG.
6A)
TABLE-US-00007 TABLE 7 observed clinical signs Incidence Group
(#dead) MMS MDO MDD Mean Score Control 9/9 (6) 5.56 .+-. 0.24 11.3
.+-. 0.28 7.56 .+-. 2.37 4.18 .+-. 0.203 SSL-MPS 9/9 (0) 2.61 .+-.
0.36 13.9 .+-. 0.92 6.78 .+-. 1.88 0.705 .+-. 0.09 Free-MPS 9/9 (3)
4.44 .+-. 0.50 12.67 .+-. 0.65 10.11 .+-. 1.92 2.62 .+-. 0.208
[0170] The fact that 6 out of 9 died in the control group
(untreated) and the high mean clinical score of the control group
confirm that the disease developed was severe (as compared Table 6
showing the effect in animals which developed a mild disease, where
no mice died and disease mean score of the untreated control group
was less than 2.)
[0171] The survival curve FIG. 6A, and mean clinical score in FIG.
6B, also both show that the disease developed with a severe mean
score.
[0172] Specific attention should be given to the following
observations obtained with respect to the animals which developed a
severe burden of disease:
[0173] 1. While there was mortality in the control and free-MPS
groups, all animal survived in the SSL-MPS treated group;
[0174] 2. At day 19 (FIG. 6B) treatment with SSL-MPS led to a mean
clinical score close to 0, as compared to that of the free-MPS
treated group or control group, being .about.3 and .about.4.8
respectively.
[0175] 3. Mean score of the disease (Table 5) for the SSL-MPS
treated group was 4 times lower than that of the control and
.about.2 times lower than that of the free-MPS treated group.
[0176] Thus, it was concluded that SSL-MPS has a beneficial
therapeutic effect during severe states of the disease as compared
to free MPS.
Comparison with Conventional MS Drugs in the Acute EAE Model
[0177] SJL female mice (6-7 week old) were immunized as described
above. The immunized mice were divided into groups and each group
was treated on days 8, 11, and 14 post immunization with the
following treatment formulations:
[0178] Group I--50 mg/kg BW SSL-MPS;
[0179] Group II--free-MPS 50 mg/kg BW;
[0180] Group III--Dextrose 5%;
[0181] Group IV--Coapxone 250 ug/0.1 cc;
[0182] Group VI--Betaferon human 2000 ui/0.1 cc.
[0183] For each group, the incidence, MMS=mean maximal score;
MDD=mean disease duration (days); MDO=mean day of onset and mean
score were determined Table 8. Further, for each group the mean
clinical scores at each time point was determined (FIG. 7)
TABLE-US-00008 TABLE 8 observed clinical signs Incidence Group
(#dead) MMS MDO MDD Mean Score Control 10/10 (3) 3.9 .+-. 0.526 11
.+-. 0 9.8 .+-. 1.2 2.3 .+-. 0.223 Betaferone 10/8 (3) 3.15 .+-.
0.753 10.3 .+-. 1.84 7.7 .+-. 1.51 1.8 .+-. 0.245 Compaxone 10/8
(3) 2.9 .+-. 0.69 9.9 .+-. 1.74 8.1 .+-. 1.72 1.8 .+-. 0.219
SSL-MPS 10/9 (1) 2.7 .+-. 0.578 11.3 .+-. 1.48 3.5 .+-. 1.13 0.74
.+-. 0158.sup.
[0184] FIG. 7 presents the clinical score at different time points
during the follow-up period. As observed, overall effect of SSL-MPS
on the mean burden of the disease was 3 time lower than that of the
control of free MPS treated groups. Further, SSL-MPS was effective
in lowering the mean clinical score from severe level of early
paralysis to distal limp tail. Only one mouse died in the SSL-MPS
treated group compared to 3 and 5 death incidence in the other
groups.
[0185] Thus, it was concluded that SSL-MPS has a beneficial effect
over the currently much more superior effect over currently
clinically available drugs and this formulation has the ability to
lower mean clinical score from severe state of early paralysis to
mild one.
Induction of Acute EAE Using MOG (Myelin Oligodendrocyte
Glycoprotein)
[0186] Induction of chronic EAE using MOG 35-55 peptide was
performed as described [Offen D et al J Mol Neurosci. 15(3):167-76
(2000)]. In general, female C57B1/6 mice were inoculated (s.c.
injection in the right flank) with an encephalitogenic emulsion
(MOG plus CFA enriched with MT (mycobacterium tuberculosis).
Pertussis toxin was injected i.p (250 ng/mouse) on the day of
inoculation and 48 hrs later. A boost of the MOG emulsion was
injected s.c. in the right flank one week after first
injection.
[0187] After immunization as described above, mice were treated
with 50 mg/kg BW SSL-MPS (days 12, 14, 16). The mean clinical
scores at each time point was determined (FIG. 8). As shown,
SSL-MPS was effective in reducing the clinical signs of acute
EAE.
Example 2
Cancer
[0188] Corticosteroids have proven therapeutic efficacy in a
variety of cancer types and are used extensively in cancer therapy,
particularly for hematological malignancies (leukemia, lymphoma,
myeloma) and hormone-responsive cancers (breast and prostate
carcinomas). Frequently, corticosteroids are used within the frame
of treatment protocols that include chemotherapy. [Lorraine I.
McKay and John A. Cidlowski. corticosteroids Cancer Medicine e.5
B.C. Decker Inc., SBN 1-55009-113-12000. by BC Decker Inc. First
published 1981. Fifth Edition 2000. 01 02 0 QP 9 8 7 6 5 4 3
Printed in Canada].
[0189] At day 1 of the experiment BALB/C mice were injected i.p.
with 1 million J6456 lymphoma cells (mouse T-cell lymphoma) then
mice were divided into groups and treated by i.v. injections with
free-MPS or SSL-MPS according to the following treatment schedule
(Table 9). The median of survival as determined on Day 14 of
treatment was determined and is also shown in Table 9:
TABLE-US-00009 TABLE 9 treatment schedule Median Group No. of mice
Injection day No. injection survival Control 6 0 0 16 Free-MPS 10
5, 9, 12 3 16 15 mg/kg BW Free-MPS 10 5, 9, 12 4 16 50 mg/kg BW
SSL-MPS 10 5, 9, 12, 16 4 19 15 mg/kg BW SSL-MPS 10 5, 9, 12, 16 4
21 30 mg/kg BW SSL-MPS 10 5, 9, 12, 16 4 23 50 mg/kg BW
[0190] The above results show that median survival time was
extended by SSL-MPS-treatment in a dose-dependent manner.
[0191] In a further assay, survival of BCL-1 (mouse B cell lymphoid
leukemia) tumor bearing mice was determined. According to this
assay, at day 1 of the experiment BALB/C mice were injected I.P
with 1 million BCL-1 lymphoma cells (B cell line, IC50 of MPS in
the nmolar range). Then, the mice were divided into groups and
treated at days 5, 9, 12, 16, by i.v. injections, with the
following treatment formulations:
[0192] Group I--5 mg/kg BW free-MPS;
[0193] Group II--25 mg/kg BW free-MPS;
[0194] Group III--5 mg/kg BW SSL-MPS;
[0195] Group IV--50 mg/kg BW SSL-MPS.
[0196] The survival median of the different groups was determined
and summarized in Table 10.
TABLE-US-00010 TABLE 10 Survival median Group median Control 19
Free-MPS 5 mg/kg BW 19 Free-MPS 25 mg/kg BW 19 SSL-MPS 5 mg/kg BW
35 SSL-MPS 50 mg/kg BW 47
[0197] Survival curve shown in FIG. 9 exhibited a beneficial effect
for SSL-MPS as compared to free MPS or the control group. It is
important to note that this cell line was highly sensitive to
MPS.
* * * * *
References