Title of Invention

"LIPOSOMAL COMPOSITIONS OF GLUCOCORTICOID AND GLUCOCORTICOID DERIVATIVES"

Abstract The present invention provides pharmaceutical compositions comprising a glucocorticoid or glucocorticoid derivative stably encapsulated in a liposome, the stability being exhibited by a reduction in the glucocorticoid or glucocorticoid derivative of less than 20% after 14 months of storage. 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 invention also provides the use of the above defined glucocorticoid or glucocorticoid derivative for the preparation of such pharmaceutical compositions as well as methods of treatment utilizing same. The therapeutic effect of the pharmaceutical composition of the invention was exhibited in vivo with appropriate models of multiple sclerosis and cancer.
Full Text FIELD OF THE INVENTION
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
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.
Gonzalez-Rothi, Ricardo J et al. Pharmaceutical Research 13(11):1699-1703 (1996);
Schmidt J et al. Brain 126(8):1895-1904 (2003);
Fildes FJ et al. J Pharm. Pharmacol. 30(6):337-42 (1978);
Mishina EV et al Pharm Res 13(1): 141-5 (1996);
Gonzalez-Rothi, Ricardo J et al. Pharmaceutical Research 13(11): 1699-1703 (1996);
Almawi WY and Melemedjian OK, JLeukoc Biol 71: 9 - 15 (2002);
Coleman RE Biotherapy 4:37 - 44 (1992);
Folkman J, et al. Science 221:719 - 725 (1983);
Swain S.M., Endocrine therapies of cancer In, Cancer Chemotherapy and Biotherapy, 2nd Ed., Eds., Chabner BA, and Longo DL, Lippincott-Raven, Philadelphia, 1996, (pp 59-108);
Haskell CM. In, Cancer Treatment, 4th Edition, Edited by Haskell CM, and Berek JS. WB Saunders Co, Philadelphia, 1995 (pp 78-80, pp 105-106, pp 151-152).
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
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.
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.
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*) 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.
Methylprednisolone (pregna-1,4-diene-3,20-dione, 11,17,21 -trihydroxy-6-
methyl-,(6a, 11P). C22H3oO5, 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 hightly effective, their systemic application is limited because of a high
incidence of serious adverse effects, especially related to long-term treatment.
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.
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.
A further approach included targeting of the drug to the target tissue by the use
of a suitable carrier, such as liposomes.
First attempts to encapsulate corticosteroids in liposomes were performed by
Fildes FJ 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.
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 EV et al Pharm Res 13(1): 141-5 (1996)]
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.
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
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°C. Once released from the liposome to
water or body fluids the acidic GC was hydrolyzed to obtain the active, non-acidic GC.
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:
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;
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.
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).
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.
The pharmaceutical composition is preferably utilized for the treatment or
prevention of any disease whose acceptable form of treatment includes administration
of glucocorticoids.
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.
The pharmaceutical composition is preferably utilized for the treatment or
prevention of multiple sclerosis.
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.
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:
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;
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.
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).
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.
Preferably, the method comprises injection of the liposomes encapsulating GC
or GC derivative.
BRIEF DESCRIPTION OF THE DRAWINGS
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:
Figures 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. IB);
and surface tension of methylprednisolone hemisuccinate and dexamethasone phosphate
as function of GC concentration (Fig. 1C).
Figures 2A-2B - are Cryo-TEM (transmission elecron microscopy) images of
liposomes before (Fig. 2A) and after (Fig. 2B) active loading of MPS.
Figure 3A-3B are size exclusion chromatography of SSL-MPS after 14 months
of storage at 4°C with Fig. 3B being an enlargment of the section describing fractions 8-
17 of Fig. 3A showing existance of very low amounts of free MPS and
methylprednisolone (MP).
Figure 4 is a release profile of MPS and Ca2+ from SSL-MPS when incubated in
plasma.
Figure 5 is a graph showing the effect of SSL-MPS treatment in Experimental
Autoimmune Encephalomyelitis (EAE).
Figure 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.
Figure 7 is a graph showing the effect of SSL-MPS treatment on EAE compared
to the effect of Betaferon and Copaxone, two conventional drugs.
Figure 8 is a graph showing the effect of SSL-MPS treatment in a chronic EAE
animal model.
Figure 9 is a graph showing the effect of SSL-MPS treatment on survival of
BCL-1 lymphoma.
DETAILED DESCRIPTION OF THE INVENTION
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.
A non-limiting list of glucocorticoids may be found at the internet 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.
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.
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.
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°C), the GC or GC derivative being
selected from:
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;
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;.
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.
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.
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.
The term "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:
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;
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.
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.
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.
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:
pKa: it has a pKa below 11.0, more preferably between about 11.0 and
about 7.5;
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.
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.
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.
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.
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.
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.
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
glycerophophol ipids.
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), l-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 abovedescribed
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).
Preferably, the phospholipid is egg phophatidylcholine (EPC), l-palmitoyl-2-
oleoylphosphatidyl choline (POPC), distearoylphosphatidylcholine (DSPC) or
hydrogenated soy phosphatidylcholine (HSPC).
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) l,2-dioleyloxy-3-
(trimethylamino) propane (DOTAP); N-[l-(2,3,- ditetradecyloxy)propyl]-N,N-dimethyl-
N-hydroxyethylammonium bromide (DMRIE); N-[l-(2,3,-dioleyloxy)propyl]-N,Ndimethyl-
N-hydroxy ethyl- ammonium bromide (DORIE); N-[l-(2,3-dioleyloxy)
propyl]-N,N,N- trimethylammonium chloride (DOTMA); 3(3[N-(N',N'-
dimethylaminoethane) carbamoly] cholesterol (DC-Choi); and
dimethyl-dioctadecylammonium (DDAB).
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]-loxopentyl]
amino]ethyl]-N,N-dimethyl-2,3-bis[(l -oxo-9-octadecenyl)oxy]-1 -
propanaminium (DOSPA), and ceramide carbamoyl spermine (CCS).
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.
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 750Da. The head group may be polar or apolar, however, is preferably a
polar head group to which a large (>750Da) 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 liposomeforming
lipids, or by short term exposure to microwave irradiation.
Preparation of vesicles composed of liposome-forming lipids and derivati/ation
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. Patent 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 nonionic
lipopolymers (also referred to at times as neutral lipopolymers or uncharged
lipopolymers) or lipopolymers having a net negative or a net positive charge.
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.
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).
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-l,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 750Da 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 2000Da, designated herein 2000PEGDSPE
or 2kPEG-DSPE.
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.
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. Chargeinducing
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.
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.
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 (2000Da) 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.
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°C for 14 months (Figs. 3A-3B).
Thus, in the context of the present invention, the term "stability" denotes a
formulation which under conventional storage conditions (4°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), 2000PEG-DSPE and cholesterol at a molar ratio of 55:40:5.
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+ and/or ion gradient.
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.
Liposomes having an H+ 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 70-100nm, preferably about
80nm.
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.
In the simplest approach for generating an ion and/or H+ 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.
18
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)].
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 20mM 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 DB et al. (Biochim. Biophys. Acta
1414: 188-204(1998)].
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 US Patent 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.
Yet, another approach is described in US 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.
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.
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.
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:
Endocrine Disorders including primary or secondary adrenocortical
insufficiency; Congenital adrenal hyperplasia Hypercalcemia associated with cancer,
nonsuppurative thyroiditis.
Collagen Diseases including, for example, during an exacerbation or as
maintenance therapy in selected cases of
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.
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.
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 margina,! Optic neuritis ulcers, Keratitis.
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.
Hematologic Disorders, including acquired (autoimmune) hemolytic anemia,
Idiopathic thrombocytopenic purpura, secondary thrombocytopenia, Erythroblastopenia
(RBC anemia). Congenital (erythroid) hypoplastic anemia.
Neoplastic Diseases, including, for example, for management of: Leukemias and
lymphomas, myeloma, breast cancer and prostate cancer.
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.
Nervous System, including, for example, acute exacerbations of multiple
sclerosis (MS).
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.
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.
The term "treatment" as used herein denotes the administering of a 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.
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).
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.
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.
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.
The term "administering" is used to denote the contacting or dispensing,
delivering or applying the Hposomal 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.
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, 4th ed., pages 622-630 (1986)].
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.
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)]
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)].
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
Hydrogenated soybean phosphatidylcholine (HSPC) was obtained from Lipoid
KG (Ludwigshafen, Germany).
N-carbamyl-poly-(ethylene glycol methyl ether)-l,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-
Cholesterol (>99% pure) was obtained from Sigma (St. Louis, MO, USA).
[3H] Cholesteryl hexadecyl ether (45 Ci per mmol) was from NEN Life Science
Products (Boston, MA, USA). tert-Butanol (99% pure) was purchased from BDH,
Poole, UK.
The weak acid steroids, the pro-drugs methylprednisolone sodium hemisuccinate
(MPS) and hydrocortisone sodium hemisuccinate (HYD), were obtained from Pfeizer,
Belgium
-25-
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
A stock solution HSPC/Cholesterol/PEG-DSPE-2000 at molar ratio of 55:40:5
was dissolved in ethanol at 70°C to a final gel lipid concentration of 62.5% (w/v). The
solution was then incubated at 70°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°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°C to receive a milky dispersion at
this stage lipids were hydrated to form multi lamellar liposome (MLV) dispersion.
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°C during the entire procedure.
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°C (4 exchanges x 100 volume each, the final one over
night).
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 ~40 mM.
The amount of calcium inside the liposomes was determined by the use of
atomic absorption spectrometry (AAS).
Preparation and characterization of radioactive-SSL
[3H] cholesteryl ether-labeled sterically stabilized liposomes (SSL) composed of
HSPC:Chol:2000PEG-DSPE (55:40:5 mole ratio), and a trace amount of [3H] cholesteryl
hexadecyl ether (0.125 jiCi/umol PL) were prepared as described above. The liposome
size was determined by Dynamic Light Scattering (DLS) to be 87±15nm.
Loading ofGC derivative into liposomes
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 ~9 mg/ml
and added to the preformed SSL dispersion after the calcium acetate gradient was
established. MPS concentration was ~9 mg/ml and phospholipid -32 mM phosphate.
Loading was achieved by incubation of the components above for the desired
time at 62°C (above matrix lipid Tm). Liposomes were then cooled to 4°C and dialyzed
against 5% dextrose at 4°C to remove acetate released during loading and to remove
unloaded drug or alternatively unloaded drug was removed by the ion exchanger Dowex
1x400 mesh (Cl" form).
State of aggregation, partition coefficient and surface tension of MPS
\. State of aggregation of MPS
Aggregation of MPS was determined from the change in turbidity measured as
intensity of light scattered at 90° to excitation beam using a spectrofluormeter under
conditions that MPS lack absorbance (excitation and emission at the same wavelength
Ex= 600nm Em= 600nm). There is a large increase in the light scattered by MPS
solution/dispersion due to formation of aggregates.
The intensity of scattered light (at 90°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 (2ml) at
concentration of ~6.5mg/ml MPS was added. The solution was tittered with HC1
(1.756M) and light scattering using excitation and emission at (both at 600nm with
attenuation of 1%) and pH of the solution was monitored.
2. Partition coefficient
Partition coefficient (logD) of some GC derivatives (which are amphipathic
weak acids) was determined by the 'shake flask1 as described [Samuni, A.M. and
Barenholz, Y., Free Radicals Biol. Med. 22:1165-1174 (1997)].
3. Surface Tension
Surface tension was measured using utrouge S (Kibron Inc., Helsinki Finland).
A solution containing GC derivative (SOOuJL) was placed in the well after calibration
and zeroing of the sensor using pure water and air. The measurement was performed at
26°C.
Precipitation ofMPS inside SSL
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 Mclntosh, T.J., Gelation of liposome interior. A novel method for
drug encapsulation. FEES Lett. 312, 255-258 (1992); Lasic, D.D., et al. Biochim.
Biophys. Acta 1239, 145-156 (1995)].
Precipitation studies
To 600 mOsm Ca-acetate solution at 63° C and at different pH points MPS was
added at final concentration of 5mg/ml, then mixed solution was incubated for 40
minutes after which the solution was centrifuged and the supernatant was analyzed
using HPLC.
Loading efficiency
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
Determination of MPS release from liposome
The level of MPS released from SSL-MPS was determined by first separating
the liposomes from free MPS using gel exclusion chromatography on Sepharose crosslinked
CL-4B column using. Liposomes were eluted at the void volume and the free
MPS at the later eluted fractions (Fig. 3A-3B).
Stability upon storage at 4°C and kinetic of release at 37°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.
In addition, SSL-MPS were incubated with 80% human inflamed synovial fluid
at a ratio of 80% plasma at 37°C. Then at different time points sample were vortexed
with the anion exchange resin, DOWEX 1x400 mesh (Cl" form), which bind only free
MPS. The samples were analyzed for liposome encapsulated MPS and liposome
phospholipid content.
Results
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 ©
American Chemical Society 2004].
Table 1 - logD and pKa of different GC derivatives
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 -0.1S 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 ofMPS
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 prodrug)
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 HC1 (^mol of H*, left side of arrow) to titration
with NaOH (jamol of OH", right side of arrow).
Partition coefficient of MPS was determined at different pH points. As shown in
Fig. IB, MPS is indeed an amphipathic substance.
Further, the surface tension of MPS was determined and as evident from
Fig. 3C, MPS is surface active at all concentrations used (0.785 - 30mM) and has a
CAC (critical association/aggregation concentration) point at ~5mM 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
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).
General Loading efficiency for different liposomal formulations
1. Liposome loading efficiency
Three separate batches (identified by dates) were used in order to determine
loading efficacy of the drug into the liposomes (HSPC:Chol:2000PEG-DSPE (55:40:5
mole ratio), as summarized in Table 3.
(Table Removed)
(Ta) MPS in liposome + MPS in the extraliposome medium
(b) MPS in liposome only
2. Loading efficiency of MPS for different liposomal formulations
It was determined that the optimum conditions for efficient loading include -600
mOsm of Calcium acetate. The loading efficiency of MPS in HSPC:Chol:2000PEGDSPE
(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 -6.5 mg/ml which was used
in the following experiments (hereinafter termed the SSL-MPS formulation or in brief
SSL-MPS).
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
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
-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°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.
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 fMS)
Induction of acute EAE experimental animal model using proteolipid protein (PLP)
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 150ug of peptide and 200ug of Mycobacterium
tuberculosis. In order to boost the immune system Pertussis Toxin (PT) 150ng were
injected intraperitoneally (i.p.) to the mice on the first day and 48 hours later.
Each mouse was examined daily for clinical signs of EAE using the following
(Table Removed)
The distal part of the tail is limp and droops
The whole tail is loose and droops
The whole tail is loose and droops. Animal has
difficulties to return on his feet when it is laid on his
back
Woobly walk- when the mouse walks the hind legs
are unsteady
The mouse has difficulties standing on its hind legs
but still has remnants of movement
The mouse can't move its legs at all, it looks thinner
and emaciated. Incontinence
The number of mice in each animal group which developed the disease (sick)
was summed and the percentage thereof was calculated.
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:
^maximal score of each mouse/number of mice in the group
Further, the mean duration of disease (MDD) expressed in days was calculated
according to the following equation:
^duration of disease of each mouse/number of mice in the group
Further, each group's mean score (CMS) (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:
X total score of each mouse per day/ number of mice in the group.
Treatment ofEAE with SSL-MPS
The EAE induced mice were divided into treatment groups according to the
following Table 5.
Free-MPS 50mg/kg BW 14
SSL-MPS 50mg/kg BW 14
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 Removed)
Treatment of severe disease burden
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: £ 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 50mg/kg BW SSL-MPS (days 10, 14, 18) or with free-
MPS 50mg/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 7- observed clinical signs
Control 9/9(6) 5.56±0.24 11.3±0.28 7.56±2.37 4.18±0.203
SSL-MPS 9/9^6) 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 10.1U1.92 2.62±0.208
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.)
The survival curve Fig. 6A, and mean clinical score in Fig. 6B, also both show
that the disease developed with a severe mean score.
Specific attention should be given to the following observations obtained with
respect to the animals which developed a severe burden of disease:
1. While there was mortality in the control and free-MPS groups, all animal
survived in the SSL-MPS treated group;
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 ~ 3 and -4.8 respectively.
3. Mean score of the disease (Table 5) for the SSL-MPS treated group was
4 times lower than that of the control and ~2 times lower than that of the free-MPS
treated group.
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
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:
Group I - 50mg/kg BW SSL-MPS;
Group II - free-MPS 50mg/kg BW;
Group III - Dextrose 5%;
Group IV - Coapxone 250ug/0.1 cc;
Group VI - Betaferon human 2000ui/0.1 cc.
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 8- observed clinical signs
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
SSL-MPS 10/9 (1) 2.7±0.578 11.3±1.48 3.5±1.13
1.8±0.245
Compaxone 10/8(3) 2.9±0.69 9.9±1.74 8.1±1.72 1.8±0.219
0.74±0158
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.
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 oligodendrocvte slycoprotein)
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.
After immunization as described above, mice were treated with 50mg/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
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., SEN 1-55009-113-12000. by BC
Decker Inc. First published 1981. Fifth Edition 2000. 01 02 OQP 9 8 7 6 5 4 3 Printed
in Canada].
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 Removed)
The above results show that median survival time was extended by SSLMPS-
treatment in a dose-dependent manner.
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:
Group I - 5 mg/kg BW free-MPS;
Group II - 25 mg/kg BW free-MPS;
Group III - 5 mg/kg BW SSL-MPS;
Group IV - 50 mg/kg BW SSL-MPS.
The survival median of the different groups was determined and summarized in
Table 10.
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.











We Claim:
1. A pharmaceutical composition comprising a glucocorticoid (GC) or GC
derivative encapsulated in a liposome, wherein at least 80% of said GC or GC derivative
is essentially retained in said liposome for 6 months, the GC or GC derivative being
selected from:
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 -2.5 and 1.5;
ii) an amphipathic weak acid GC or GC derivative having a pKa above 3 and a logD at pH 7 in the range between -2.5 and 1.5,
wherein said liposome comprises at least one liposome forming lipid, the mole ratio between said GC or GC derivative and said at least one liposome forming lipid is between 0.01 and 2.0; and wherein said liposome comprise in its intraliposomal aqueous phase precipitated GC or GC derivative.
2. The pharmaceutical composition as claimed in claim 1, wherein said GC or GC derivative is selected from an amphipathic weak base having a pKa equal or below 11 and a logD at pH 7 in the range between -1.5 and 1.0; 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 -1.5 and 1.0.
3. The pharmaceutical composition as claimed in claim 1, wherein said GC derivative is a pro-drug which is converted to an active GC upon release thereof from said liposome into a body fluid.
4. The pharmaceutical composition as claimed in claim 1, wherein said GC derivative is an acidic glucocorticoid.
5. The pharmaceutical composition as claimed in claim 4, wherein said acidic GC is selected from methylprednisolone sodium hemisuccinate (MPS), hydrocortisone sodium hemisuccinate (HYD), Dexamethasone hemisuccinate, Prednisolone hemisuccinate.
6. The pharmaceutical composition as claimed in claim 1, wherein said liposome comprises a phospholipid.
7. The pharmaceutical composition as claimed in claim 6, wherein said liposome comprises a combination of a phospholipid, a lipopolymer and cholesterol.
8. The pharmaceutical composition as claimed in claim 7, wherein said liposome comprises a combination hydrogenated soybean phosphatidylcholine (HSPC), polyethylene glycol coated distearoyl phosphatidyl ethanolamine (PEG-DSPE) and cholesterol.
9. The pharmaceutical composition as claimed in claim 1, wherein the liposome is a sterically stabilized liposome (SSL).
10. The pharmaceutical composition as claimed in claim 1, wherein the mole ratio between said GC or GC derivative and said phospholipid is between 0.04 and 0.25.
11. The pharmaceutical composition as claimed in claim 1, for the treatment of neurodegenerative disorders.
12. The pharmaceutical composition as claimed in claim 11, for the treatment of multiple sclerosis.
13. The pharmaceutical composition as claimed in claim 1, for the treatment of cancer.

Documents:

2301-delnp-2007-abstract.pdf

2301-DELNP-2007-Claims (11-01-2010).pdf

2301-DELNP-2007-Claims-(15-11-2011).pdf

2301-delnp-2007-claims.pdf

2301-DELNP-2007-Correspondence Others-(15-11-2011).pdf

2301-DELNP-2007-Correspondence-Others (11-1-2010).pdf

2301-DELNP-2007-Correspondence-Others-(11-03-2010).pdf

2301-DELNP-2007-Correspondence-Others-(27-07-2007).pdf

2301-DELNP-2007-Correspondence-Others-(30-03-2007).pdf

2301-delnp-2007-correspondence-others.pdf

2301-DELNP-2007-Description (Complete) (11-1-2010).pdf

2301-delnp-2007-description (complete).pdf

2301-DELNP-2007-Drawings (11-01-2010).pdf

2301-delnp-2007-drawings.pdf

2301-DELNP-2007-Form-1 (11-1-2010).pdf

2301-DELNP-2007-Form-1-(27-07-2007).pdf

2301-delnp-2007-form-1.pdf

2301-delnp-2007-form-18.pdf

2301-delnp-2007-form-2.pdf

2301-DELNP-2007-Form-26-(27-07-2007).pdf

2301-DELNP-2007-Form-3 (11-1-2010).pdf

2301-delnp-2007-form-3.pdf

2301-delnp-2007-form-5.pdf

2301-DELNP-2007-GPA-(15-11-2011).pdf

2301-DELNP-2007-Others-Document-(27-07-2007).pdf

2301-delnp-2007-pct-210.pdf

2301-delnp-2007-pct-304.pdf

2301-delnp-2007-pct-306.pdf

2301-DELNP-2007-Petition-137 (11-1-2010).pdf


Patent Number 250843
Indian Patent Application Number 2301/DELNP/2007
PG Journal Number 05/2012
Publication Date 03-Feb-2012
Grant Date 01-Feb-2012
Date of Filing 23-Mar-2007
Name of Patentee YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HERBREW UNIVERSITY OF JERUSALEM
Applicant Address HI TECH PARK, EDMOND SAFRA CAMPUS, GIVAT RAM, 91390 JERUSALEM, ISRAEL
Inventors:
# Inventor's Name Inventor's Address
1 BARENHOLZ, YECHEZKEL 18 NAVE SHAANAN STREET, 93707 JERUSALEM, ISRAEL
2 GABIZON, ALBERTO,A. 56/7 BERNSTEIN STREET, 96920 JERUSALEM, ISRAEL
3 AVNIR, YUVAL 2 NOVOMISKI STREET, RAMAT SHARET, 96908 JERUSALEM, ISRAEL
PCT International Classification Number A61K 9/127
PCT International Application Number PCT/IL2005/000963
PCT International Filing date 2005-09-11
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/608,140 2004-09-09 U.S.A.