Note: Descriptions are shown in the official language in which they were submitted.
LIPOSOME COMPRISING RAPAMYCIN OR A DERIVATIVE THEREOF AND USE
THEREOF IN THERAPY
Field of the Disclosure
[ 0 0 01] The present disclosure relates to a lipid-based formulation
comprising rapamycin and
derivatives thereof and their applications.
Background of the Disclosure
[ 0 0 0 2 ] The mammalian target of rapamycin (mTOR) is a kinase that in
humans is encoded by
the MTOR gene. The mTOR integrates the input from upstream pathways, and the
mTOR pathway
is a central regulator of mammalian metabolism and physiology, with important
roles in the
function of tissues. For example, mTOR signaling may result in diseases
related to metabolism,
immune function, brain function, aging and even cancers. Regulation, mostly
inhibition, of mTOR
may improve or treat the diseases and conditions. The first-known inhibitor of
mTOR is rapamycin,
from which mTOR's name derives:
HO
' 40
arili 0 I 'H
N
0 0
HC) 0 Cr'
44=6L
0
',,,,,.."=,,r0^^,s1"-... ,%.
Rapamycin, M.W.: 914.17 g/mol.
[ 0 0 0 3 ] Clinical applications of rapamycin include use as an
immunosuppressant, treatment of
tuberous sclerosis complex, drug-eluting coronary stent, etc. Rapamycin, as a
BCS Class II drug,
i.e., having low aqueous solubility (2.6 tig/mL) and high permeability, only
exhibits a
bioavailability of 14% to 18%, which thus precludes it from clinical
development as an anti-cancer
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Date Recue/Date Received 2022-04-28
agent. The solution to overcome the low solubility of rapamycin is to develop
analogs or
derivatives thereof, namely "rapalogs." Examples of rapalogs include
everolimus, temsirolimus,
etc. Though everolimus may exhibit ¨20% bioavailability in a tablet form and
temsirolimus may
exhibit even up to 100% bioavailability via intravenous infusion, the oral
rapalog has low
bioavailability and the injectable rapalog is highly allergenic due to
excipients. Hence, there is still
no clinically available rapamycin or rapalogs formulation with high
bioavailability, high efficacy
and low toxicity.
Summary of the Disclosure
[0004] The present invention is based in part on the development of a liposome
specific to
rapamycin or an analog thereof, or a prodrug or salt of rapamycin or its
analog, having good
stability before or after lyophilization and hydration, superior tumor
inhibition ability and reduced
toxicity of rapamycin or its analog.
[0005] The present disclosure provides a liposome comprising a lipid
ingredient encapsulating
rapamycin or an analog thereof, or a prodrug or salt of rapamycin or its
analog,
wherein:
the lipid ingredient is selected from the group consisting of: cholesterol,
phosphatidylcholine (PC),
L-a-phosphatidylcholine (EggPC), 1,2-Didecanoyl-sn-glycerol-3-phosphocholine
(DDPC), 1,2-
distearoyl-sn-glycerol-3-phosphorylethanolamine (DSPE), distearoyl
phosphatidylcholine
(DSPC), dioleoyl phosphatidylethanolamine (DOPE), dipalmitoyl
phosphatidylcholine (DPPC),
hydrogenated soy phosphatidylcholine (HSPC), 1,2-palmitoyl-phosphatidic acid
(DPPA), 1,2-
dimyristoyl phosphatidylcholine (DMPC), dioleoyl phosphatidylcholine (DOPC),
palmitoyl-
myristoyl phosphatidylcholine (PMPC), palmitoyl-oleoyl phosphatidylcholine
(POPC), dioleoyl
phosphatidylglycerol (DOPG), distearoyl phosphatidyl glycerol (DSPG),
dipalmitoyl
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Date Recue/Date Received 2022-04-28
phosphatidylglycerol (DPPG), dipalmitoyl phosphatidylethanolamine (DPPE), or a
PEG and
combinations thereof;
the rapamycin analog is able to inhibit the mammalian target of rapamycin
(mTOR);
the amount of the lipid ingredient, based on the dry weight of the total
amount of liposome, ranges
from about 30% (w/w) to 95% (w/w); and the amount of rapamycin or an analog
thereof, or a
prodrug or salt of rapamycin or its analog, based on the dry weight of the
total amount of liposome,
ranges from about 5% (w/w) to 30% (w/w).
[ 0006] In one embodiment, the liposome is a poly(ethylene glycol) (PEG)-
modified liposome.
[0007] In some embodiments, the analog of rapamycin is selected from the group
consisting
of everolimus, temsirolimus, tacrolimus, prerapamycin, zotarolimus,
ridaforolimus, 7-epi-
rapamycin, 7-thiomethyl-rapamycin, 7-epi-trimethoxyphenyl-rapamycin, 7-epi-
thiomethyl-
rapamycin, 7-demethoxy-rapamycin, 32-demethoxy-rapamycin, 2-desmethyl-
rapamycin, and 42-
0-(2-hydroxy)ethyl rapamycin, rapamycin oximes, rapamycin aminoesters,
rapamycin
dialdehydes, rapamycin 29-enols, 0-alkylated rapamycin derivatives, water-
soluble rapamycin
esters, alkylated rapamycin derivatives, rapamycin amidino carbamates, biotin
esters of rapamycin,
carbamates of rapamycin, rapamycin hydroxy esters, rapamycin 42-sulfonates, 42-
(N-carboalkoxy)
sulfamates, rapamycin oxepane isomers, imidazolidyl rapamycin derivatives,
rapamycin
alkoxyesters, rapamycin pyrazoles, acyl derivatives of rapamycin, rapamycin
amide esters,
rapamycin fluorinated esters, rapamycin acetals, oxorapamycins, and rapamycin
silyl ethers.
[ 0008] In some embodiemnts, the amount rapamycin or an analog thereof, or a
prodrug or salt
of rapamycin or its analog, based on the dry weight of the total amount of
liposome, ranges from
about 5% (w/w) to 25% (w/w), about 5% (w/w) to 20% (w/w), about 10% (w/w) to
30% (w/w),
about 10% (w/w) to 25% (w/w), about 10% (w/w) to 20% (w/w), about 15% (w/w) to
30% (w/w),
about 15% (w/w) to 25% (w/w) or about 15% (w/w) to 20% (w/w). In some further
embodiments,
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Date Recue/Date Received 2022-04-28
the amount rapamycin or an analog thereof, or a prodrug or salt of rapamycin
or its analog, based
on the dry weight of the total amount of liposome, is about 10% (w/w), about
12% (w/w), about
14% (w/w), about 15% (w/w), about 16% (w/w), about 17% (w/w), about 18% (w/w),
about 5%
(w/w) to 20% (w/w), about 10% (w/w) to 20% (w/w), about 15% (w/w) to 25% (w/w)
or about
15% (w/w) to 20% (w/w).
[0 0 0 9 ] In some embodiments, the lipid ingredient is DOPE, DDPC,
cholesterol, DSPE, EggPC,
HSPC, DPPC, DMPC, DSPC, PC, a combination of DPPC, DDPC, cholesterol and DSPE
(or
DSPE-PEG), a combination of DOPE, DDPC, cholesterol and DSPE (or DSPE-PEG), a
combination of HSPC and DDPC, a combination of DSPC and DDPC, a combination of
DOPE
and HSPC or a combination of DOPE and DSPC. In some further embodiments, the
lipid
ingredient based on the dry weight of the total amount of liposome is about
65% (w/w) to about
95% (w/w) or 70% (w/w) to about 90% (w/w) of DOPE, DDPC, cholesterol, DSPE,
EggPC, HSPC,
DMPC, DPPC, DSPC, or PC, or a combination of about 15% (w/w) to about 65%
(w/w) of DPPC,
about 20% (w/w) to about 65% (w/w) of DDPC, about 0% (w/w) to about 30% (w/w)
of cholesterol
and about 15% (w/w) to about 65% (w/w) of DSPE (or DSPE-PEG), a combination of
about 25%
(w/w) to about 60% (w/w) of DOPE, about 25% (w/w) to about 70% (w/w) of DDPC,
about 0%
(w/w) to about 20% (w/w) of cholesterol and about 0% (w/w) to about 25% (w/w)
of DSPE (or
DSPE-PEG), a combination of about 40% (w/w) to about 55% (w/w) of HSPC and
about 30%
(w/w) to about 40% (w/w) of DDPC, a combination of about 40% (w/w) to about
55% (w/w) of
DSPC and about 30% (w/w) to about 40% (w/w) of DDPC, a combination of about
35% (w/w) to
about 45% (w/w) of DOPE and about 35% (w/w) to about 50% (w/w) of HSPC or a
combination
of about 35% (w/w) to about 45% (w/w) of DOPE and about 35% (w/w) to about 50%
(w/w) of
DSPC. The amount of rapamycin or an analog thereof, or a prodrug or salt of
rapamycin or its
analog in the above embodiments is that described herebefore.
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Date Recue/Date Received 2022-04-28
[0 0 1 0] In some further embodiments, based on the dry weight of the total
amount of liposome,
the amounts of the lipid ingredient and rapamycin or an analog thereof, or a
prodrug or salt of
rapamycin or its analog are those listed below.
Formulation
(weight percent; wt% (dry weight)
Rapamycin DPPC DDPC cholesterol DSPE-PEG2000
about 10-20% about 15-65% about 20-
about 0-30% about 0-25%
65%
Rapamycin DOPE DDPC cholesterol DSPE-PEG2000
about 5-20% about 25-60% about 25-
about 0-20% about 0-25%
70%
Rapamycin EggPC
about 16% about 84%
Rapamycin HSPC
about 16% about 84%
Rapamycin DSPC
about 16% about 84%
Rapamycin DOPE
about 17% about 83%
Rapamycin DPPC
about 17% about 83%
Rapamycin DDPC
about 15-25% about 70-90%
Rapamycin DPPC
about 15-20% about 80-90%
Rapamycin HSPC DDPC
about 18% about 47% about 34%
Rapamycin DSPC DDPC
about 18% about 48% about 34%
Rapamycin DOPE HSPC
about 17% about 41% about 43%
Rapamycin DOPE DSPC
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Date Recue/Date Received 2022-04-28
about 17% 1 about 40% labout 43%1
[0011] In one embodiment, the average particle size of the liposome ranges
from about 100
nm to about 500 nm. In some embodiments, the average particle size of the
liposome can be about
100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190
nm, 200 nm,
210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300
nm, 310 nm,
320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410
nm, 420 nm,
430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, or in a range
consisting of
any two values noted above, e.g., from 100 nm to 500 nm, from 150 nm to 450
nm, from 100 nm
to 250, from 180 nm to 220 nm, etc.
[0012] The present disclosure also provides a liposome formulation comprising
a liposome of
the present disclosure and a cryoprotectant.
[0013] In one embodiment, the cryoprotectant is a disaccharide. In some
embodiments, the
cryoprotectant is sucrose or trehalose. In one embodiments, the cryoprotectant
based on the dry
weight of the total amount of liposome is about 80% (w/w), about 84% (w/w),
about 87% (VOW),
about 90% (w/w), about 94% (w/w), about 95% (w/w), about 97% (w/w) or in a
range consisting
of any two values noted above, e.g., from about 80% to about 97%, from about
84% to about 98%,
etc.
[0014] The present disclosure also provides a method for treating cancer,
diabetes, obesity,
neurological disease and genetic disorder and/or preventing an organ
transplant rejection, in a
subject, comprising administrating a therapeutically effective amount of a
liposome of the present
disclosure to the subject.
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Date Recue/Date Received 2022-04-28
Brief Description of the Drawing
[0015] Figures 1(A) and 1(B) show particle size of different ratio of
liposomal rapamycin
formulation.
[0016] Figures 2(A), 2(B), 3(A) and 3(B) show characteristics of lyophilized
products and re-
hydrated lyophilized products.
[0017] Figures 4(A) and 4(B) show in vivo toxicity results on Balb/c mice.
[0018] Figures 5(A) and 5(B) show in vivo experimental results on SKOV3
xenograft
NOD/SOID mice.
[0019] Figures 6(A) and 6(B) show in vivo experimental results on MDA-MB-231
xenograft
NOD/SOID mice.
[0020] Figures 7(A) and 7(B) show in vivo experimental results on CT26 Balb/c
mice.
Detailed Description of the Disclosure
[0021] Unless defined otherwise, all the technical and scientific terms used
herein have the
same meaning as is commonly understood by one of ordinary skill in the art.
All patents,
applications, published applications and other publications are incorporated
by reference in their
entirety. In the event that there is a plurality of definitions of a term,
those in this section prevail
unless stated otherwise.
[0022] As used herein, the term "rapalog" refers to derivatives of rapamycin
with inhibition
activity on mTOR.
[0023] As used herein, the term "liposome" refers to a microscopic closed
vesicle having an
internal phase enclosed by lipid bilayer. A liposome can be a small single-
membrane liposome
such as a small unilamellar vesicle (SUV), large single-membrane liposome such
as a large
unilamellar vesicle (LUV), a still larger single-membrane liposome such as a
giant unilamellar
vesicle (GUV), a multilayer liposome having multiple concentric membranes such
as a multi-
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Date Recue/Date Received 2022-04-28
lamellar vesicle (MLV), or a liposome having multiple membranes that are
irregular and not
concentric such as a multivesicular vesicle (MVV). According to the
disclosure, a liposome is a
generic term encompassing a variety of single- and multi-lamellar lipid
vehicles formed by the
generation of enclosed lipid bilayers or aggregates. Liposomes may be
characterized as having
vesicular structures with a bilayer membrane, generally comprising a
phospholipid, and an inner
medium. Liposomes can range in size from several nanometers to several
micrometers in diameter.
A liposome used according to the disclosure can be made with different
methods, as would be
known to one of ordinary skill in the art. Further details with respect to the
preparation of liposomes
are set forth in U.S. Pat. No. 4,342,826 and PCT International Publication No.
WO 80/01515, both
of which are incorporated by reference.
[0024] As used herein, "tumor" denotes a neoplasm, and includes both benign
and malignant
tumors. This term particularly includes malignant tumors, which can be either
solid or non-solid.
Tumors can also be further divided into subtypes, such as adenocarcinomas.
[ 0 02 5] As used herein, a "(therapeutically) effective dose/amount" is a
dose/amount sufficient
to prevent advancement or cause regression of a disease or which is capable of
relieving symptoms
caused by the disease.
[ 0026] As used herein, "mammal" or "mammalian subject" includes farm animals,
such as
cows, hogs and sheep, as well as pets or animals used in sports such as
horses, dogs, and cats.
[ 0 02 7] Rapamycin (C511-179N013) is a macrolide compound that was isolated
in 1975 from
Streptomyces hygroscopicus. Rapamycin (otherwise known as sirolimus) is an
inhibitor of mTOR
that prevents activation of T cells and B cells by inhibiting their response
to interleukin-2 (IL-2).
It is an FDA-approved drug for immunosuppression, possessing both antifungal
and antineoplastic
properties.
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Date Recue/Date Received 2022-04-28
[0028] However, rapamycin has poor solubility and pharmacokinetics and thus
its therapeutic
effect is limited. For BCS Class II or IV drugs (i.e., having low solubility),
nanocarriers or
conjugates may be a promising way to enhance their bioavailability. Examples
include lipid-based
nanocarriers, such as liposomes; polymer-based nanocarriers, such as polymeric
micelles;
inorganic nanoparticles, such as silica nanoparticles; viral nanoparticles;
drug conjugates, such as
antibody-drug conjugates, etc. Compared with traditional solubilizer or
polymer formulations,
lipids as vehicles are highly biocompatible and biodegradable. In particular,
liposomes can be
made of amphiphilic phospholipids, and amphiphilic phospholipids can form a
barrier to protect
the hydrophobic drug from exposure to aqueous or biological environments. They
also can affect
pharmacokinetics and distribution. However, the bioavailability of liposome-
based oral rapalog is
not significantly improved.
[0029] Accordingly, the present disclosure provides a liposome specifically
for encapsulating
rapamycin or an analog thereof, or a prodrug or salt of rapamycin or its
analog for cancer therapy
in order to improve the low bioavailability and allergic problem of
solubilizer. The liposome of
the present disclosure provides high bioavailability, high efficacy, and low
toxicity.
[0030] Liposomes are artificial vesicles composed of concentric lipid bilayers
separated by
water-compartments and have been extensively investigated as drug delivery
vehicles. Due to their
structure, chemical composition and colloidal size, all of which can be well
controlled by
preparation methods, liposomes exhibit several properties which may be useful
in various
applications. Liposomes are used as carriers for drugs and antigens because
they can serve several
different purposes. Liposome encapsulated drugs are inaccessible to
metabolizing enzymes.
Conversely, body components (such as erythrocytes or tissues at the injection
site) are not directly
exposed to the full dose of the drug. The duration of drug action can be
prolonged by liposomes
because of a slower release of the drug in the body. Liposomes have a direct
potential, which
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Date Recue/Date Received 2022-04-28
means that targeting options change the distribution of the drug in the body.
Cells use endocytosis
or phagocytosis mechanism to take up liposomes into the cytosol. Furthermore,
liposomes can
protect a drug against degradation (e.g. metabolic degradation). However,
liposomes have a
potential disadvantage in their relatively limited ability to adequately
release certain encapsulated
drugs (such as anti-cancer drugs).
[0031] To improve the quality of the liposome particles, several means can be
adopted, such
as submicron filtration, lyophilization, etc. In one embodiment, the liposome
particles
encapsulating rapamycin or an analog thereof, or a prodrug or salt of
rapamycin or its analog are
processed by submicro filtration to remove precipitated drug or particles of a
larger size.
[0032] In another embodiment, the liposomes encapsulating rapamycin or an
analog thereof,
or a prodrug or salt of rapamycin or its analog are processed by
lyophilization. The liposomal
lyophilization may include the following steps: (1) freezing a liquid
containing the liposome
particles by decreasing temperature to form a solid/ice form, (2) freeze
concentrating the solid/ice
form by decreasing pressure, (3) sublimating the solid/ice form by elevating
the temperature to
.. obtain a crude product, and (4) conducting final drying to obtain a final
liposomal freeze-dried
stable product (e.g., lyo-cake). Prior to the lyophilization process, a
cryoprotectant can be
introduced into the liquid containing the liposome particles to protect the
active ingredient (i.e.,
rapamycin, rapalogs). The lyophilized liposome of the present disclosure is
stable for a long time
(at least 20 weeks) and is suitable for clinical applications. After hydration
of the lyophilized
liposome, the liposome can maintain the particle size and stability before
lyophilization.
[0033] The liposome of the present disclosure demonstrates good stability
before or after
lyophilization and hydration, superior tumor inhibition ability, and reduced
toxicity of rapamycin
or its analog.
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Date Recue/Date Received 2022-04-28
[0034] The liposomes of the present disclosure may be administered by any
route that
effectively transports the liposomes to the appropriate or desirable site of
action. Preferred modes
of administration include intravenous (IV) and intra-arterial (IA). Other
suitable modes of
administration include intramuscular (IM), subcutaneous (SC), and
intraperitoneal (IP). Such
administration may be bolus injections or infusions. Another mode of
administration may be
perivascular delivery. The formulation may be administered directly or after
dilution.
Pharmaceutical compositions comprising the liposomes of the present disclosure
may be
formulated using one or more physiologically acceptable carriers comprising
excipients and
auxiliaries known in the art, which facilitate the processing of the active
ingredients into
preparations that can be used pharmaceutically.
[0035] Examples are provided below to more clearly illustrate the concept of
the present
disclosure.
EXAMPLE
Example 1 Preparation of Liposome of the Disclosure
Preparation of Liposome Containing Rapamycin
[ 0036] Rapamycin, 1,2-D io leoyl-sn-glycerol-3 -phosphoethanolamine
(DOPE), 1,2-
D ipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2-D idecanoyl-sn-glycero-3
-pho spho choline
(DDPC), cholesterol and N-(Methylpolyoxyethylene oxycarbony1)-1,2-distearoyl-
sn-glycero-3-
phosphoethanolamine (DSPE-PEG2000) were provided in different dry weight
ratios as shown in
the table in paragraph [0010] for the preparation of liposome encapsulating
rapamycin. The raw
materials are dissolved in the mixture of chloroform and methanol. Heating the
solution to 40 C
and reduced the pressure to remove organic solvent and form a thin-film. After
the organic solvent
is completely removed, adding the solution that containing cryoprotectants to
hydrate the thin-film
for 1 hour. The particle size of liposomal formulation is shown in Figures
1(A) and 1(B). These
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Date Recue/Date Received 2022-04-28
results revealed that liposomes encapsulating rapamycin with the appropriate
amount of DSPE-
PEG2000 perform a smaller particle size.
Preparation of Liposome Containing Rapamycin (RAP-P)
[ 0 0 3 7 ] Rapamycin, 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-
Didecanoyl-
sn-glycero-3-phosphocholine (DDPC) and N-(Methylpolyoxyethylene oxycarbony1)-
1,2-
distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2000) were provided in a
dry weight
ratio of!: 2.4: 1.9: 1.6 for the preparation of liposome encapsulating
rapamycin. The raw materials
are dissolved in the mixture of chloroform and methanol. Heating the solution
to 40 C and reduced
the pressure to remove organic solvent and form a thin-film. After the organic
solvent is completely
removed, adding the solution that containing cryoprotectants to hydrate the
thin-film for 1 hour.
The resulting product was lyophilized and the amounts of rapamycin, DPPC,
DDPC, DSPE-
PEG2000 based on the dry weight of the total amount of liposome are 15%, 35%,
27%, 23%,
respectively. The final product is named after the RAP-P formulation.
Preparation of Liposome Containing Rapamycin (RAP-E)
[ 0 0 3 8 ] Rapamycin, 1,2-D io leoyl-sn-
glycerol-3 -phosphoethanolamine (DOPE), 1,2-
Didecanoyl-sn-glycero-3-phosphocholine (DDPC) and N-(Methylpolyoxyethylene
oxycarbony1)-
1,2-distearoyl-sn-glycero-3-phosphoethanolamine were provided in a dry weight
ratio of 1: 2.4:
1.8: 1.6 for the preparation of liposome encapsulating rapamycin. The raw
materials are dissolved
in the mixture of chloroform and methanol. Heating the solution to 40 C and
reduced the pressure
to remove organic solvent and form a thin-film. After the organic solvent is
completely removed,
adding the solution that containing cryoprotectants to hydrate the thin-film
for 1-2 hours. The
resulting product was lyophilized and the amounts of rapamycin, DOPE, DDPC and
DSPE-
PEG2000 based on the dry weight of the total amount of liposome are 15%, 35%,
27%, 23%,
respectively. The final product is named after the RAP-E formulation.
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Date Recue/Date Received 2022-04-28
Characterization
[0039] The average particle size and polydispersity index (PI) of the particle
size of the RAP-
P formulation were 127.4 +/- 10.8 nm and 1.29 +/- 0.41, respectively. The
average particle size
and PI of the particle size of the RAP-E formulation were 348.6 +/- 30.9 nm
and 0.47 +/- 0.13,
respectively. The results show that both RAP-P and RAP-E can be used as proper
formulations for
use in administering rapamycin.
Example 2 Different Forms of Liposome Formulation
[0040] To investigate the applications of the liposome formulation, RAP-P and
RAP-E
formulations noted in Example 1 were further processed.
Submicron Filtration
[0041] The RAP-P and RAP-E formulations were filtered via a 0.45 gm membrane
to remove
free or precipitated drugs, particles and impurities of a larger size to
increase the stability and
safety of the product. To verify the validity of the data, samples with
rapamycin standard of
different concentrations (from 0.05 to 5 gg/mL) were used in UPLC-MS/MS tests
to establish a
calibration curve of signal (area) to rapamycin concentration. Results showed
that the coefficient
of determination (r2) of the said linear calibration curve was 0.9999.
[0042] The average particle size and PI of the particle size of the RAP-P
formulation after
filtration were 182.4 +/- 4.3 nm and 1.10 +/- 0.07, respectively. The average
particle size and PI
of the particle size of the RAP-E formulation after filtration were 219.0 +/-
5.1 nm and 0.58 +1-
0.09, respectively. The results showed that, after filtration, both RAP-P and
RAP-E formulations
exhibit narrower particle size distribution, and the average particle size of
RAP-E formulation is
reduced by about 37% reduction.
Lyophilization
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Date Recue/Date Received 2022-04-28
[0043] The RAP-P and RAP-E formulations were lyophilized to obtain lyophilized
products.
At first, freezing the sample at minus 45 C. Then vacuum the pressure to 200
mTorr to remove
most of the aqueous through primary drying. At last, increasing the
temperature to minus 20 C for
secondary drying in order to remove the residual aqueous. To evaluate the
effect of cryoprotectants,
sucrose and trehalose, at a final concentration (w/w) of 87% or 94%, were
introduced into the
formulation before lyophilization. The average particle size is shown in
Figures 2(A) and 2(B),
and the results show that both sucrose and trehalose, at a final concentration
(w/w) of 87% or 94%,
do not alter or impact the characterization of liposome particles after
lyophilization.
[ 004 4] Long-term storage stability of lyophilized products was also
verified. The lyophilized
products of RAP-P and RAP-E formulation, with 87% (w/w) of trehalose as the
cryoprotectant,
were stored at -20 C (minus 20 C) for 20 weeks, and parts of the products were
retrieved after
being stored for 4, 16 and 20 weeks and re-hydrated to 2 mg/mL for
characterization. The average
particle size and PI of particle size of the re-hydrated products are shown in
Figures 3(A) and 3(B).
The results revealed that the lyophilized products can be stable for at least
20 weeks when stored
at -20 C (minus 20 C).
Example 3 In vivo Studies
[0045] To study the applicability of the claimed formulation, toxicity study,
pharmacokinetic
and efficacy thereof were researched in vivo with mice.
Toxicity Study
[0046] 6-8 week old male BALB/c mice were obtained from BioLasco (Ilan,
Taiwan). A
preliminary toxicity study was conducted with mice administered a single dose
of RAP-P and
RAP-E formulations, and the body weight change was chosen as the indication.
Figures 4(A) and
4(B) show the results of the study which reveal that the maximum tolerated
dose (MTD) of RAP-
P formulation is 120 mg/kg in mice, and MTD of RAP-E is 220 mg/kg in mice. For
comparison,
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Date Recue/Date Received 2022-04-28
the lethal dosage 50% (LD50) of rapamycin in previous intravenous injection
formulation was 40
mg/kg in rats, equivalent to 80 mg/kg in mice (Baker, H.; Sidorowicz, A.;
Sehgal, S.N.; Vezina, C.
Rapamycin (ay-22,989), a new antifungal antibiotic. Iii. In vitro and in vivo
evaluation. The
Journal of antibiotics 1978, 31, 539-545). The inventive liposome formulation
of rapamycin
provides an MTD dose significantly higher than LD50 of the previous
intravenous injection
formulation, which would make it benefit in clinical applications.
Pharmacokinetic Study
[0047] 6-8 week old male BALB/c mice were obtained from BioLasco (Ilan,
Taiwan). Animals
were randomly divided into 2 groups (n=6). Blood was collected from the cheek
before injection.
After administration the drug through the tail vein, and blood was collected
at 0.083, 0.5, 1, 2, 4,
8, 10, and 24 hours. The blood was extracted with methanol and 0.1M zinc
sulfate. The drug
concentration in blood was analyzed by UPLC-MS/MS. The details of the
pharmacokinetic data
of RAP-P and RAP-E formulations are listed in Table 1 below:
[0048] Table 1:
n .-= 6 unit RAP-P RAP-E
Administration route IV IV
dose mg/kg 50 50
Cmax ng/mL 18,190 24,569
AUC0-24 hr*ng/m1 71,147 63,193
.11/2 hr 5.47 5.84
CL (clearance) mL/hr/kg 685 755
Effect on Treating Xenograft Ovarian Tumor in Mice
[0049] Akt and mTOR phosphorylation are frequently detected in ovarian cancer,
and SKOV3,
an ovarian cancer cell line, is resistant to cis-platinum and doxorubicin.
Hence, SKOV3 was
selected as a model tumor for evaluating the treatment efficacy of the
inventive liposome
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Date Recue/Date Received 2022-04-28
containing rapamycin. SKOV3 xenograft on NOD/SCFD mice was used as an animal
model,
classified as three groups: a control group (no drug), a comparative group
(administration of
docetaxel, dose: 4 mg/kg, Q3d*4) and the inventive group (administration of
RAP-E formulation,
dose: 50 mg/kg, Q3d*4). 6 week old NOD SCID immunodeficient mice (BioLasco,
Ilan, Taiwan)
were inoculated subcutaneously with 8* 105 SKOV3 cells (human ovarian cancer
cell). Following
tumor inoculation, measuring the tumor size twice a week, and starting
treatment when the tumor
grows to 80-100 mm3. Figure 5(A) shows the tumor volume on day() and day39 for
each group.
Mean inhibition rate of docetaxel and RAP-E formulation were 53% and 62%,
respectively; Day
39 tumor inhibition rate of docetaxel and RAP-E formulation were 57% and 61%;
and both groups
.. exhibited significant inhibition of tumor volume over the control group.
Figure 5(B) shows the
body weight change of mice in each group. The results reveal that the
inventive formulation (RAP-
E) can achieve comparable efficacy (inhibition of tumor size) to docetaxel but
has lower toxicity.
Effect on Treating Xenograft Breast Tumor in Mice
[0 0 5 0] In addition to ovarian cancer, research has shown PI3K/ AKT/ mTOR
and pathways
.. are frequently dysregulated in breast cancer. Upregulated mTORC1
contributes to cell growth,
proliferation and promotes tumorigenesis. Rapamycin, an inhibitor of mTOR,
also might be
promising in the treatment of breast cancer. MDA-MB-231 cells, the triple-
negative breast cancer
cell line, which targeted therapy is not available can only choose
chemotherapy. However, serious
side effects may occur. Hense, MDA-MB-231 was selected as a model for
evaluating the treatment
.. efficacy of the inventive liposome containing rapamycin. MDA-MB-231
xenograft on NOD/SCID
mice was used as an animal model, classified as three groups: a control group
(no drug), a
comparative group (administration of Lipo-Dox, dose: 2 mg/kg, Q3d*4) and the
inventive group
(administration of RAP-E formulation, dose: 25 mg/kg, Q3d*4). 6 week old NOD
SOD
immunodeficient mice (BioLasco, Ilan, Taiwan) were inoculated subcutaneously
with 1*106
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Date Recue/Date Received 2022-04-28
MDA-MB-231 cells (human breast cancer cell). Following tumor inoculation,
measuring the
tumor size twice a week, and starting treatment when the tumor grows to 80-100
mm3. Figure 6(A)
shows the tumor volume on day 0 and day 16 for each group. Mean inhibition
rate of Lipo-Dox
and RAP-E formulation were 27% and 34%, respectively; Day 16 tumor inhibition
rate of Lipo-
Dox and RAP-E formulation were 55% and 52%; and both groups exhibited
significant inhibition
of tumor volume over the control group. Figure 6(B) shows the body weight
change of mice in
each group, especially significant weight loss in the group of administration
of Lipo-Dox. The
results reveal that the inventive formulation (RAP-E) can achieve comparable
efficacy (inhibition
of tumor size) to Lipo-Dox but has lower toxicity.
Comparison of Efficacy between 5-FU and the Inventive Formulation on Mouse
Colon Cancer
[ 0051 ] CT26 model on Balb/c mice was used as another animal model,
classified as three
groups: a control group (no drug), a comparative group (administration of 5-
FU, dose: 25 mg/kg,
Q3d*4) and the inventive group (administration of RAP-E formulation, dose: 25
mg/kg, Q3d*4).
6 week old Balb/c mice (BioLasco, Ilan, Taiwan) were inoculated subcutaneously
with 1*105
CT26 cells (murine colorectal carcinoma cell). Following tumor inoculation,
measuring the tumor
size twice a week, and starting treatment when the tumor grows to 80-100 mm3.
Figure 7(A) shows
the tumor volume on day 0 and day 16 for each group. Mean inhibition rate of 5-
FU and RAP-E
formulation were 11% and 37%, respectively; Day 16 tumor inhibition rate of 5-
FU and RAP-E
formulation were 26% and 53%, respectively; and both groups exhibited greater
inhibition of
tumor volume over the control group. Figure 7(B) shows the body weight change
of mice in each
group, especially significant weight loss in the group of administration of 5-
FU. The results reveal
that the inventive formulation (RAP-E) can achieve superior efficacy
(inhibition of tumor size) to
5-FU but has lower toxicity.
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Date Recue/Date Received 2022-04-28
