U.S. patent application number 12/521357 was filed with the patent office on 2011-01-06 for liposomal pharmaceutical preparation and method for manufacturing the same.
Invention is credited to Wenmin Guo, Chunlei Li, Yanhui Li, Dongmin Shen, Caixia Wang, Jinxu Wang, Lan Zhang, Li Zhang.
Application Number | 20110002977 12/521357 |
Document ID | / |
Family ID | 39588170 |
Filed Date | 2011-01-06 |
United States Patent
Application |
20110002977 |
Kind Code |
A1 |
Li; Chunlei ; et
al. |
January 6, 2011 |
LIPOSOMAL PHARMACEUTICAL PREPARATION AND METHOD FOR MANUFACTURING
THE SAME
Abstract
The present invention relates to a liposomal pharmaceutical
preparation containing a multivalent ionic drug, a process for the
preparation of the liposomal pharmaceutical preparation, and a use
thereof in the treatment of diseases, in which the liposome has a
size of about 30-80 nm, and the phospholipid bilayer has a
phospholipid with a Tm higher than body temperature, so that the
phase transition temperature of the liposome is higher than the
body temperature.
Inventors: |
Li; Chunlei; ( Hebei
Province, CN) ; Wang; Jinxu; ( Hebei Province,
CN) ; Wang; Caixia; ( Hebei Province, CN) ;
Li; Yanhui; ( Hebei Province, CN) ; Shen;
Dongmin; (Hebei Province, CN) ; Guo; Wenmin; (
Hebei Province, CN) ; Zhang; Li; ( Hebei Province,
CN) ; Zhang; Lan; ( Hebei Province, CN) |
Correspondence
Address: |
CANTOR COLBURN LLP
20 Church Street, 22nd Floor
Hartford
CT
06103
US
|
Family ID: |
39588170 |
Appl. No.: |
12/521357 |
Filed: |
December 29, 2007 |
PCT Filed: |
December 29, 2007 |
PCT NO: |
PCT/CN2007/071403 |
371 Date: |
June 26, 2009 |
Current U.S.
Class: |
424/450 ;
514/285; 514/656 |
Current CPC
Class: |
A61K 9/1271 20130101;
A61K 31/136 20130101; A61K 31/704 20130101; A61K 9/1273 20130101;
A61P 35/00 20180101 |
Class at
Publication: |
424/450 ;
514/285; 514/656 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/437 20060101 A61K031/437; A61K 31/136 20060101
A61K031/136; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 29, 2006 |
CN |
200610102339.8 |
Claims
1.-20. (canceled)
21. A liposomal drug, wherein (1) the liposomal drug comprises a
multivalent ionic drug as active ingredient, which has two or more
dissociable groups with a dissociation constant pKa of 4.5-9.5; (2)
the liposome of the liposomal drug has a size of 30-80 nm; (3) the
bilayer of the liposome comprises a phospholipid with a phase
transition temperature (Tm) higher than the body temperature,
cholesterol, and a hydrophilic polymer-modified lipid; and (4) the
intraliposomal phase of the liposome comprises a multivalent
counter ion.
22. The liposomal drug according to claim 21, wherein the size of
the liposome is 35-75 nm.
23. The liposomal drug according to claim 21, wherein the
dissociation constant pKa of the active ingredient is 5.0-9.5.
24. The liposomal drug according to claim 21, wherein the
multivalent ionic drug is an anticancer drug selected from
mitoxantrone, vincristine, vinorelbine, vinblastine, or any
combination thereof.
25. The liposomal drug according to claim 21, wherein the
multivalent counter ion carries two or more charges opposite to the
active drug ingredient, the multivalent counter ion being selected
from a saturated or unsaturated organic acid anion, an inorganic
acid anion or an ionic form of an amino acid, and wherein the
organic acid is selected from citric acid, tartaric acid, fumaric
acid, oxalic acid, malonic acid, succinic acid, malic acid and
maleic acid; the inorganic acid is selected from sulfate anion and
phosphate anion; the amino acid is selected from cystine.
26. The liposomal drug according to claim 25, wherein the
multivalent counter ion is sulfate anion.
27. The liposomal drug according to claim 21, wherein the
phospholipid with a phase transition temperature (Tm) higher than
the body temperature in the phospholipid bilayer is selected from
phosphatidylcholine, hydrogenated soybean phosphatidylcholine,
hydrogenated egg yolk phosphatidylcholine, dipalmitoyl
phosphatidylcholine, distearoyl phosphatidylcholine, or any
combination thereof.
28. The liposomal drug according to claim 27, wherein the
phospholipid bilayer further comprises a phospholipid with a phase
transition temperature Tm not higher than the body temperature.
29. The liposomal drug according to claim 28, wherein the
phospholipid with a phase transition temperature Tm higher than the
body temperature is about 50-100 mol/mol % relative to the total
content of the phospholipids in the phospholipid bilayer.
30. The liposomal drug according to claim 21, wherein the
cholesterol is about 2-60 mol/mol % relative to the total content
of the ingredients in the phospholipid bilayer.
31. The liposomal drug according to claim 21, wherein the
hydrophilic polymer-modified lipid is selected from PEG-modified
distearoyl phosphatidyl ethanolamine (DSPE-PEG), PEG-modified
distearoyl phosphatidyl glycerol (DSPG-PEG), PEG-modified
cholesterol (chol-PEG), polyvidone-modified distearoyl phosphatidyl
ethanolamine (DSPE-PVP), polyvidone-modified distearoyl
phosphatidyl glycerol (DSPG-PVP), or polyvidone-modified
cholesterol (chol-PVP), or any combination thereof.
32. The liposomal drug according to claim 21, wherein the
hydrophilic polymer-modified lipid is in an amount of 0.1-20
mol/mol % relative to the total content of the phospholipids in the
phospholipid bilayer.
33. The liposomal drug according to claim 21, which comprises
hydrogenated soybean phosphatidylcholine, cholesterol and
PEG-modified distearoyl phosphatidyl ethanolamine in a weight ratio
of 3:1:1, in which the PEG-modified distearoyl phosphatidyl
ethanolamine is PEG2000-modified distearoyl phosphatidyl
ethanolamine.
34. The liposomal drug according to claim 21, wherein the phase
transition temperature Tm of the liposome is higher than the body
temperature.
35. A method for preparing the liposomal drug according to claim
21, which comprises the following steps: (1) preparing a liposome
with a size of 30-80 nm using a phospholipid with a Tm higher than
the body temperature, cholesterol and a hydrophilic
polymer-modified lipid; and (2) encapsulating a multivalent ionic
drug in the liposome, wherein the multivalent ionic drug has two or
more dissociable groups with a dissociation constant pKa of
4.5-9.5, and the intraliposomal phase of the liposome comprises a
multivalent counter ion.
36. A liposomal pharmaceutical preparation, which comprises the
liposomal drug according to claim 21, and optionally a
pharmaceutically acceptable carrier and/or an excipient.
37. The liposomal pharmaceutical preparation according to claim 16,
which further comprises a salt for altering osmotic pressure, a
buffering agent and/or an antioxidant.
38. A method for the treatment of a tumor in a patient, which
method comprises: administering a liposomal drug or a liposomal
pharmaceutical preparation to a patient in need of the treatment;
wherein (1) the liposomal drug comprises a multivalent ionic drug
as active ingredient, which has two or more dissociable groups with
a dissociation constant pKa of 4.5-9.5; (2) the liposome of the
liposomal drug has a size of 30-80 nm; (3) the bilayer of the
liposome comprises a phospholipid with a phase transition
temperature (Tm) higher than the body temperature, cholesterol, and
a hydrophilic polymer-modified lipid; and (4) the intraliposomal
phase of the liposome comprises a multivalent counter ion; and
wherein the liposomal pharmaceutical preparation comprises the
liposomal drug and optionally a pharmaceutically acceptable carrier
and/or an excipient.
39. The method according to claim 38, which further comprises
treating the patient by thermotherapy.
Description
FIELD OF INVENTION
[0001] The present invention relates to a liposomal preparation and
a drug-encapsulating liposomal pharmaceutical preparation,
especially to a liposomal pharmaceutical preparation of
mitoxantrone. The present invention further relates to methods for
manufacturing the liposome, liposomal pharmaceutical preparation
and uses thereof.
BACKGROUND OF THE INVENTION
[0002] Liposomes can be used as a carrier for many drugs,
especially for antitumor drugs (in particular chemotherapeutic
drugs). Liposomes can reduce the distribution of drug in normal
tissues, but increase the accumulation of drug in tumor tissues,
thereby improving the therapeutic index of drug. The reason why a
liposome can target passively to a tumor relates to the
physiological properties of tumor tissue. Tumor blood vessels may
have a pore size of up to 100-780 nm due to its rapid growth, while
normal vascular endothelial cells have a typical space of about 2
nm. Therefore, liposomes can accumulate passively in tumor region
if they can circulate for a relatively long period in blood and
have a size of less than 200 nm, because after liposomes with small
size are administered via intravenous injection, they can not enter
normal tissues but can penetrate blood vessel of tumor region and
arrive at treatment area.
[0003] However, it is not easy to achieve the therapeutic
advantages of liposome, and the following four requirements have to
be met: (1) the drug can be encapsulated in liposome in a good
encapsulation efficiency and a sufficient drug loading; (2) the
drug will not be released from the liposome during storage period
in vitro; (3) there is not a notable drug leakage during blood
circulation of liposomal drug; and (4) the drug can be released
effectively and thereby exerting its therapeutic effects when
liposomes are accumulated in the tumor region. With regard to the
current liposome techniques, the former three problems have been
solved well, therefore, the rational release in vivo of liposomal
drug draws more attentions. One critical technical problem to be
solved for developing some liposomal drugs is to effectively
control the rational release of liposomal drugs after targeting to
a tumor region. This is especially important for some drugs, such
as mitoxantrone.
[0004] It was found by a liposome study group in Canada that a
liposome formulation having a size of about 100 nm, which was
prepared by using hydrogenated soybean phosphatidylcholine (HSPC)
and cholesterol as phospholipid bilayer and loading drug by a 300
mM citric acid gradient, was not as good as free mitoxantrone. In
order to improve the therapeutic effect of liposome, the group
finally changed the composition of phospholipid bilayer into
dimyristoyl phosphatidylcholine (DMPC) and cholesterol, and
obtained a preparation with improved therapeutic indexes. However,
the leakage of drug may increase during the storage period because
the phase transition temperature of DMPC is about 21.degree. C., so
that the preparation may not be stable (Liposomal formulations of
mitoxantrone, U.S. Pat. No. 5,858,397).
[0005] Neopharm Corporation of USA used another technique to
develop a liposome formulation of mitoxantrone, in which a
cardiolipin carrying negative charge was added to phospholipid
bilayer. Due to the intensive interaction between cardiolipin and
mitoxantrone, mitoxantrone could be inserted into the phospholipid
bilayer in a passive loading mode. This passive loading technique
is different from active loading technique. By virtue of active
loading technique, a drug would deposit in the intraliposomal
aqueous phase in a form of precipitation. The Phase I clinical
study on the product of Neopharm indicated that liposome drugs
could increase the possibility of occasional infection, compared
with free drug. The development of this product was ceased in view
of safety (Liposomal preparations of mitoxantrone,
CN01817424.8).
[0006] Pacific Institute of Materia Medica (Changchou, China) also
filed a patent application for a liposomal preparation of
mitoxantrone (A liposomal injection of mitoxantrone or mitoxantrone
hydrochloride and the process for making the same,
CN200410041612.1). In this application, traditional pH value
gradient method was used to load drugs. This application seeks to
protect a formulation with a specific ratio, and does not disclose
the effects of factors such as composition of phospholipids, kinds
of buffer salts in internal aqueous phase, size of liposome,
drug/liposome ratio, etc. on the therapeutic efficacy and toxicity
of liposome.
[0007] Zhirong Zhang, et al of West China School of Pharmacy,
Sichuan University also studied liposomal preparations of
mitoxantrone. They used soybean phosphatidylcholine with a phase
transition temperature of 0.degree. C. (which is marketed under the
trade name EPIKURON 200) to prepare liposomes of about 60 nm. In
this article, only pharmacokinetics was studied without concerning
toxicity and therapeutic efficacy of the obtained liposomal
preparation. Relevant contents can be seen in "Preparation of long
circulating mitoxantrone liposomes and its pharmacokinetics",
Zhirong Zhang, Botao Yu and Yisong Duan, Acta Pharmaceutica Sinica,
2002, Vol. 37, No. 6; Studies on preparation of long circulating
mitoxantrone liposomes with transmembrane ammonium sulfate
gradients, Zhirong Zhang, Botao Yu, Yisong Duan and Yuan Huang,
Chinese Pharmaceutical Journal, 2002 Vol. 37, No. 12; and Study on
the preparation techniques of mitoxantrone liposomes, Yisong Duan,
West China Journal of Pharmaceutcal Sciences, 2001 Vol. 16, No.
02.
[0008] In the above studies, the size of liposomes is usually
controlled in the range of 80.about.150 nm, since there is a
consensus in the field of liposome that a liposome with a size of
about 100 nm would have the best targeting efficiency (Pharmacol.
Rev. 1999 51: 691-744.). However, as mentioned above, a liposome
should not only have an excellent targeting efficiency, but also a
sufficient release from liposome to exert its effect.
[0009] As indicated above, according to the prior field, the
leakage of drug during blood circulation should be essentially
avoid so that the drug could be effectively transferred to tumors,
but this requirement also results in a difficulty of releasing the
drug from the liposome when it is targeted to tumor region. In
conventional processes for making liposomes, a drug is usually
encapsulated by a active loading technique, in which the drug
encapsulated in the liposome is present in a colloid precipitate
form having no bioactivity, so that only when the drug is released
effectively from the liposome, it can change into a therapeutic
drug with bioactivity. If the release rate of drug is too slow, the
drug can hardly exert its therapeutic actions even though it has
been targeted effectively to the tumor region, and its therapeutic
effect may be even inferior to an unencapsulated drug.
[0010] Therefore, there is an urgent need in the field for a
liposomal preparation capable of delivering a drug with good
targeting ability and releasing the drug in the targeted tissues
effectively, and for a corresponding liposomal pharmaceutical
preparation.
SUMMARY OF THE INVENTION
[0011] The present inventors surprisingly found by chance that some
drugs having a plurality of dissociable groups and a liability of
forming compact precipitate with multivalent counter ion, could be
processed to form a small unilamellar liposomal preparation with an
effectively improved therapeutic index, so that the above technical
problem could be solved.
[0012] Therefore, in one aspect, the present invention provides a
liposomal preparation with a size of about 30-80 nm having a
phospholipid with a Tm higher than body temperature in phospholipid
bilayer, so that the phase transition temperature of liposome is
higher than the body temperature. Examples of said phospholipid
include but are not limited to phosphatidylcholine, hydrogenated
soybean phosphatidylcholine (HSPC), hydrogenated egg-yolk
phosphatidylcholine, dipalmitoyl phosphatidylcholine (DPPC) or
distearoyl phosphatidylcholine (DSPC) or any combination
thereof.
[0013] In one embodiment, the phospholipid with a Tm higher than
body temperature in the phospholipid bilayer represents 50-100
mol/mol %, preferably 55-95 mol/mol %, and more preferably 55-95
mol/mol % of the total content of phospholipids.
[0014] Optionally, the phospholipid bilayer of the liposomal
preparation of the present invention further comprises additional
phospholipids, for example, a phospholipid with a Tm not higher
than the body temperature, such as dimyristoyl phosphatidylcholine
(DMPC) and the like. The amount of the phospholipid in the
liposomal preparations of the present invention can be
conventionally determined by those of ordinary skilled in the
field, provided that the Tm value of the liposomal preparation is
not markedly reduced to a value lower than the body
temperature.
[0015] The liposomal preparation of the present invention can also
optionally comprise cholesterol in order to regulate the fluidity
of liposome membrane.
[0016] The liposomal preparation of the present invention can also
optionally comprise additional excipients, especially excipients
for further modifying surface characteristics of liposome to confer
the liposome better behavior in vivo. Such excipients include, for
example, lipids and the like modified with hydrophilic
polymers.
[0017] In another aspect, the present invention provides a
liposomal pharmaceutical preparation, which comprises a drug of
interest, especially a multivalent ionic drug, in a liposomal
preparation of the present invention. Therefore, the present
invention relates to a liposomal pharmaceutical preparation having
a size of 30-80 nm, wherein: (1) the liposomal pharmaceutical
preparation comprises a multivalent ionic drug as active
ingredient; (2) the phospholipid bilayer comprises a phospholipid
with a Tm higher than body temperature so that the phase transition
temperature of the liposome is higher than the body temperature;
and optionally (3) the liposomal pharmaceutical preparation
comprises additional drugs and/or additional excipients acceptable
in the liposomal pharmaceutical preparation. Preferably, the main
peaks of size of the liposomal pharmaceutical preparation are
centered around 35-75 nm, especially around 40-60 nm.
[0018] In another aspect, the present invention provides a method
for preparing the above liposomal pharmaceutical preparation, the
method comprising the following steps: (1) preparing a liposome
using a phospholipid with a Tm higher than body temperature and
optionally additional phospholipids and/or cholesterol; and (2)
encapsulating a drug of interest, especially a multivalent ionic
drug in the liposome.
[0019] The present invention also provides a method for treatment
of disease, comprising administering a liposomal pharmaceutical
preparation of the present invention to a subject in need of the
treatment. Preferably, the subject is a mammal, especially a human
being.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is the in vivo pharmacokinetics of PLM60 in Kunming
mice and the comparison thereof with the in vivo pharmacokinetics
of free mitoxantrone, in which PLM represents PEGylated
mitoxantrone liposome, FM represents free mitoxantrone, the
abscissa represents time (hour) and the ordinate represents plasma
level of mitoxantrone (.mu.g mitoxantrone/mL plasma).
[0021] FIG. 2 is the profile of PLM60 and FM in mice tumor, in
which PLM60 represents PEGylated mitoxantrone liposome, FM
represents free mitoxantrone, the abscissa represents time (hour)
and the ordinate represents the concentration of mitoxantrone in
tumor tissues (.mu.g mitoxantrone/g tumor tissue).
[0022] FIG. 3 is the comparison of in vivo pharmacokinetics in mice
of different formulations, in which the abscissa represents time
(hour) and ordinate represents the plasma level of mitoxantrone
(.mu.g mitoxantrone/mL plasma), and the dosages of different
formulations are all 4 mg/kg.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0023] Usually, liposomes are formed with phospholipids and
cholesterol as membrane materials. These two ingredients not only
are the basic materials for forming liposome bilayer, but also have
very important physiological functions.
[0024] The physical properties of liposomal membrane are closely
related to the temperature. When temperature is elevated, acyl side
chains of lipid bilayer change form ordered array into unordered
array. This kind of change can result in many changes of physical
properties of lipid membrane. For example, "gel" state may change
into "liquid crystal" state, the cross section of membrane may
increase, the thickness of bilayer may decrease, the membrane
fluidity may increase. The temperature at which such changes happen
is called phase transition temperature. The phase transition
temperature of lipid membrane can be determined by Differential
Scanning Calorimertry, Electron Spins Resonance (ESR) and the like.
The phase transition temperature of liposome membrane depends on
the kinds of phospholipids. Generally, the longer the acyl side
chain, the higher the phase transition temperature; and vice versa.
For example, the phase transition temperature of dimyristoyl
phosphatidylcholine is 24.degree. C., while those of dipalmitoyl
phosphatidylcholine and distearoyl phosphatidylcholine are
41.degree. C. and 58.degree. C., respectively. Membrane fluidity is
an important property of liposome. At phase transition temperature,
membrane fluidity will increase, and the drug encapsulated in the
liposome has the maximum release rate. Thus the membrane fluidity
affects directly the stability of liposome.
[0025] In one embodiment, the present invention provides a liposome
preparation having a size of about 30-80 nm and a phospholipid with
a Tm higher than body temperature in phospholipid bilayer, so that
the phase transition temperature of liposome is higher than the
body temperature.
[0026] Preferably, the liposomal pharmaceutical preparation of the
present invention is prepared by using phospholipids with a
relatively high phase transition temperature Tm, such as
phosphatidylcholine. If the Tm of phosphatidylcholine is higher
than the body temperature, the length of its hydrocarbon chain is
preferably not less than 16 carbons. Preferably, the phospholipids
of the present invention include but not limited to hydrogenated
soybean phosphatidylcholine, hydrogenated egg-yolk
phosphatidylcholine, dipalmitoyl phosphatidylcholine (DPPC) or
distearoyl phosphatidylcholine (DSPC), or any combination
thereof.
[0027] In the liposomal preparation of the present invention, the
phospholipids with a Tm higher than the body temperature in
phospholipid bilayer represent about 50-100 mol/mol %, preferably
about 55-95 mol/mol %, more preferably about 60-90 mol/mol %
relative to the total content of all phospholipids. Optionally, the
phospholipid bilayer may comprise additional phospholipids, for
example, phospholipids with a Tm not higher than the body
temperature, such as dimyristoyl phosphatidylcholine (DMPC) and the
like. Such phospholipids may be present in the liposome in any
suitable amount, provided that it does not render the phase
transition temperature of the liposomal preparation below the body
temperature. The suitable amount can be determined according to
conventional techniques by those of ordinary skilled in the
field.
[0028] Preferably, the liposomal preparation of the present
invention may further comprise cholesterol. Cholesterol has a
function of regulating membrane fluidity. When the liposome
membrane comprises 50% (mol/mol) cholesterol, the phase transition
of liposome membrane may disappear. Cholesterol is called "fluidity
buffer" by Papahadjopoulos et al., because the addition of
cholesterol to phospholipids under phase transition temperature can
reduce the ordered array of membrane and increase membrane
fluidity, while the addition of cholesterol to phospholipids above
the phase transition temperature can increase the ordered array of
membrane and reduce the membrane fluidity. In the liposomal
preparation of the present invention, the content of cholesterol
can be 2-60 mol/mol %, 5-55 mol/mol % or 10-50 mol/mol % relative
to the total amount of ingredients of liposome. More specifically,
the content of cholesterol can be 15-45 mol/mol %, for example
20-40 mol/mol % relative to the total amount of ingredients of
liposome. The content of cholesterol in the liposome of the present
invention can be determined easily according to conventional
techniques by those of ordinary skilled in the field.
[0029] It should be appreciated that the phospholipid bilayer in
the liposome of the present invention can also comprise additional
excipients, especially excipients for further modifying surface
characteristics of the liposome to confer better in vivo behaviors
to the liposome. Such excipients include, for example, lipid
substances modified with hydrophilic polymers, and the examples
thereof are PEG-modified distearoyl phosphatidyl ethanolamine
(DSPE-PEG), PEG-modified distearoyl phosphatidyl glycerol
(DSPG-PEG), PEG-modified cholesterol (chol-PEG),
polyvidone-modified distearoyl phosphatidyl ethanolamine
(DSPE-PVP), polyvidone-modified disteroyl phosphatidyl glycerol
(DSPG-PVP), or polyvidone-modified cholesterol (chol-PVP). Said
excipients can also be membrane materials modified with a specific
antibody or ligand. The amount of such excipients in the liposome
of the present invention can be determined according to
conventional techniques by those of ordinary skilled in the field,
for example, can be 0.1-20 mol/mol %, preferably 0.3-18 mol/mol %,
more preferably 0.5-15 mol/mol %, especially 0.8-12 mol/mol %, for
example 1-10 mol/mol %, or 2-8 mol/mol %, 2.5-7 mol/mol %, 3-6
mol/mol %, etc. relative to the mole number of phospholipids. In
the cases of using PEG-modified lipids as excipients, the molecular
weight of PEG moiety can be, for example, 400-20000 Dalton,
preferably 600-15000 Dalton, more preferably 800-10000 Dalton,
especially 1000-8000 Dalton, for example 1200-5000 Dalton. The use
of PEG in the present invention can also be determined easily
according to conventional trails by those of ordinary skilled in
the field.
[0030] The liposomal preparation of the present invention is a
small unilamellar liposomal preparation, and should have a suitable
size. Preferably, the size of the preparation is 30-80 nm, more
preferably 35-70 nm, especially preferably 40-60 nm. The size of
liposome can be determined by particle size analyzer or electron
microscope or other means. It should be understood that the
liposome particles in the present invention can not have a
completely uniform size, but span a size range, due to the nature
of liposome per se and properties of manufacture process.
Therefore, in the liposomal preparation of the present invention,
the presence of liposome particles out of the stated size range may
not be excluded, provided that they do not evidently affect the
characters of the liposomal preparation or pharmaceutical
preparation of the present invention.
[0031] The liposome in the present invention can be prepared by
various suitable methods, including, for example, film dispersion
method, injection method, ultrasonic dispersion method,
freeze-drying method, freeze-thaw method and the like. According to
the starting systems used for preparing liposome, the methods can
be divided into: (1) methods based on dry lipid membrane, lipid
powder; (2) methods based on emulsifying agents; (3) liposome
preparation methods based on mixed micelles; and (4) liposome
preparation methods based on a triple phase mixture of ethanol,
phospholipids and water. The encapsulation of drug can be
implemented by either passive loading mode or active loading mode.
These methods can be found in many review articles about
liposomes.
[0032] During or after the preparation of liposomal preparation,
many suitable methods can be used to encapsulate a drug in liposome
and form a liposomal pharmaceutical preparation. Suitable methods
include for example active loading methods and passive loading
methods. Active loading method is usually performed by gradient
methods, for example an ammonium sulfate gradient method, i.e.,
using an ammonium sulfate solution as aqueous phase to firstly
prepare a liposome comprising ammonium sulfate in both
intraliposomal and extraliposomal phase, then forming a
concentration gradient of ammonium sulfate between the
intraliposomal and extraliposomal phase by removing extraliposomal
ammonium sulfate. Intraliposomal NH.sub.4.sup.+ dissociates into
NH.sub.3 and H.sup.+ which leads to a concentration difference of
H.sup.+ (i.e. pH gradient) between intraliposomal and
extraliposomal phase, so that after an extraliposomal drug in
molecular state enters into the intraliposomal aqueous phase, it
changes into ionic state, thereby the drug can not return to the
extraliposomal aqueous phase and the liposome has less leakage of
drug and is more stable. Passive loading method can be performed by
organic solvent injection method, film dispersion method,
freeze-thaw method, and the like.
[0033] In the present invention, any suitable drug ingredients can
be used. Preferably, the active pharmaceutical ingredient in the
liposomal pharmaceutical preparation of the present invention is a
multivalent ionic drug. The term "multivalent ionic drug" means a
drug having two or more dissociable groups with a dissociation
constant pKa of 4.5.about.9.5, so that the drug has more positive
charges or more negative charges in the ranges of pKa. Preferably,
said dissociation constant is in the range of 5.0-9.5. More
preferably, said dissociation constant is in the range of 5.5-9.5.
Especially preferably, said dissociation constant is in the range
of 6.0-9.0 m, especially 6.5-9.0. The pKa value of each dissociable
group of ion drug can be determined easily according conventionally
techniques by those of ordinary skilled in the field.
[0034] In the present invention, the multivalent ionic drugs can
include but are not limited to anticancer drugs, for example, drugs
useful for prevention or treatment of the following cancers: lung
cancers (such as non-small cell lung cancer), pancreas cancer,
breast cancer, rectum cancer or multiple myeloma, liver cancer,
cervical carcinoma, gastric carcinoma, carcinoma of prostate, renal
carcinoma and/or carcinoma of bladder. Therefore, in one embodiment
of the present invention, the multivalent ionic drug is a
multivalent ion anticancer drug. Preferably, the multivalent ionic
drug is mitoxantrone, vincristine, vinorelbine or vinblastine. More
preferably, said multivalent ionic drug is mitoxantrone and can
optionally combine with at least one of additional drugs, which can
for example be an antitumor drug, such as vincristine, vinorelbine
or vinblastine, and the like.
[0035] It is necessary to specifically note that in the prevent
invention, the multivalent ionic drug can also be a combination of
any one or two or more of the above drugs, for example, a
combination of two anticancer drugs, a combination of one or more
anticancer drugs with additional drugs such as immunopotentiator,
and a combination of two or more other kinds of drugs.
[0036] It should also be noted that the liposomal drugs of the
present invention can also optionally comprise one or more of
additional non-multivalent ionic drugs besides the multivalent
ionic drugs mentioned above, which can be administered in
combination with the multivalent ionic drugs as mentioned above.
The combinatory administration comprises the administration with
all the components in one preparation, also comprises the
combinatory administration in separate unit dosage form.
[0037] It should be appreciated that the drug as active ingredient
as mentioned herein comprises not only its original form, but also
its derivatives, for example solvates (such as hydrates and alcohol
addition products), prodrugs and other physiologically acceptable
derivatives, as well as active metabolites, and the like.
Derivatives, prodrugs and other physiologically acceptable
derivatives as well as active metabolites of a drug are all well
known to those of ordinary skilled in the field.
[0038] The liposomal pharmaceutical preparation of the present
invention can further comprise two or more multivalent counter ions
with charges opposite to that of active ingredient. Examples of the
multivalent counter ions include but are not limited to organic
acid anions, such as acid anions of the following saturated or
unsaturated organic acids: citric acid, tartaric acid, fumaric
acid, oxalic acid, malonic acid, succinic acid, malic acid and
maleic acid, and the like; inorganic acid anions, such as sulfate
anion, phosphate anion and the like. Among them citrate anion,
sulfate anion or phosphate anion are preferred. Furthermore, said
multivalent counter ions can also be amino acids, such as cystine
and the like. Without being bound by any specific theory, it is
presumed that the multivalent counter ion is able to form an
insoluble precipitate with a drug of interest (e.g., multivalent
ionic drug) encapsulated in the liposome, thereby the existence of
the multivalent ionic drug in the liposome is stabilized.
[0039] The liposomal pharmaceutical preparation of the present
invention further comprises optionally additional excipients and
carriers commonly known in the pharmaceutical field, such as
sucrose, histidine, antioxidants, stabilizers, dispersants,
preservatives, diluents, solvents, salts for altering osmotic
pressure, and the like.
[0040] In one embodiment, the present invention provides a method
for preparing the liposomal pharmaceutical preparation of the
present invention, comprising: firstly preparing the liposomal
preparation of the present invention as mentioned above, and
subsequently incubating a drug of interest with the liposomal
preparation in a suitable condition. More specifically, the method
for preparing the liposomal pharmaceutical preparation of the
present invention comprises the following steps: (1) dissolving
lipid excipients suitable for preparing a liposome in a suitable
organic solvent, such as tert-butyl alcohol or cyclohexane, then
lyophilizing to obtain a lyophilized powder; (2) hydrating the
lyophilized powder with a solution containing a counter ion of the
drug active ingredient of interest to form an empty liposome; (3)
removing the extraliposomal counter ion by a suitable means such as
dialysis or column chromatography and the like in order to form a
counter ion gradient between the intraliposomal phase and
extraliposomal phase; and (4) incubating the drug with the liposome
to obtain the liposome drug. Descriptions about phospholipids,
cholesterol, excipients and the like refer to the supra for the
liposomal preparation.
[0041] Preferably, the lipid is a phospholipid, especially a lipid
with a relatively high phase transition temperature, for example,
phosphatidylcholine, hydrogenated soybean phosphatidylcholine,
hydrogenated egg yolk phosphatidylcholine, dipalmitoyl
phosphatidylcholine (DPPC) or distearoyl phosphatidylcholine
(DSPC), or any combination thereof. Optionally, said lipid can also
comprise cholesterol in an amount of, for example, 2-60 mol/mol %,
5-55 mol/mol % or 10-50 mol/mol %. More specifically, the amount of
cholesterol can be 15-45 mol/mol %, for example 20-40 mol/mol %
relative to the total mole number of all ingredients in the
liposome. Those of ordinary skilled in the field can determine the
cholesterol amount depending on specific requirements for the phase
transition temperature of liposome to be obtained and the desired
properties.
[0042] Once the liposomal pharmaceutical preparation is prepared,
the encapsulation efficiency of drug in liposome can be determined
by conventional techniques. Methods for determining the
encapsulation efficiency of liposome includes ultrafiltration,
dialysis, column chromatography, minicolumn centrifugation, and the
like. Ultrafiltration is not used due to the high requirements for
experiment device; column chromatography is not used because the
dilution requires a large amount of eluent, and the content of drug
is very low, so that it is difficult to conduct content
determination, moreover, the dilution of a large amount of eluent
can also lead to leakage of drug in liposome, it can be known from
trial data that the encapsulation efficiency for dialysis is lower
(perhaps due to the breakage of liposome after dilution) and the
time for dialysis is long, thus the method is not suitable.
Determination of encapsulation efficiency by minicolumn
centrifugation has the following advantages: short time consuming,
small dilution rate for solution of liposome, and no need for
expensive instruments.
[0043] The liposomal pharmaceutical preparation of the present
invention ensures not only sufficient encapsulation efficiency and
sufficient drug loading, but also no release of drug from liposome
during in vitro storage, no notable leakage of drug from liposome
during blood circulation to increase toxicity. An important notable
effect of the liposome drug of the present invention is that the
release rate of drug is accelerated efficiently, the therapy index
of liposome is improved, the half-life period is significantly
prolonged, the toxicity is reduced markedly in comparison with the
current products in the field, and thus the effective therapeutic
effects of drug are achieved. For example, for a liposomal
pharmaceutical preparation prepared by using hydrogenated soybean
phosphatidylcholine (HSPC) and dipalmitoyl phosphatidylcholine
(DPPC), the toxicity thereof is markedly reduced and the
therapeutic index thereof is significantly improved. On the
contrary, if the phospholipid bilayer is composed of dimyristoyl
phosphatidylcholine (DMPC), the release of drug will be too fast
and lead to a notable toxicity, even the safety will not be as good
as a free drug. Without being bound by a certain theory, it is
presumed that the small unilamellar liposomal preparation of the
present invention can accelerate the release of drug because the
small unilamellar liposomal preparation may contain more liposome
particles in which drug precipitation with a small particle size is
contained, in comparison with a larger unilamellar liposome
preparation, if the drug/lipid ratio is fixed. Drug precipitation
with a small particle size would have a relatively great specific
surface area, and thus have a more rapid dissolution rate under
same conditions.
[0044] Moreover, the liposomal pharmaceutical preparation of the
present invention should be prepared using suitable phospholipids
in order to achieve an effective release of drug in target tissues,
especially in tumors. Preferably, the phospholipid bilayer of the
liposomal pharmaceutical preparation of the present invention is
composed of phospholipids with a relatively high phase transition
temperature. During experiments, it was found that the toxicity of
preparation would decrease significantly and the therapeutic index
would be improved notably if hydrogenated soybean
phosphatidylcholine (HSPC) and dipalmitoyl phosphatidylcholine
(DPPC) or the like were employed in the preparation. If the
phospholipid bilayer is composed of dimyristoyl phosphatidylcholine
(DMPC), the release of drug would be too fast and would lead to a
notable toxicity, even the safety would not be as good as an
unencapsulated drug.
[0045] The liposomal pharmaceutical preparation of the present
invention can be administered to a patient in need thereof in an
administration route commonly used in the field. In one embodiment
of the present invention, the liposome drug is formulated into a
preparation for parenteral administration. In one preferred
embodiment of the present invention, the liposome drug is
administered by injection.
[0046] The present invention also provides a method for the
treatment of disease, especially tumors in a patient, the method
comprising administering a liposomal pharmaceutical preparation of
the present invention to the patient in need of the treatment.
Preferably, a thermotherapy method (such as a radioactive
thermotherapy method) can also be applied in combination to a tumor
patient in order to enhance the therapeutic effect of the liposomal
pharmaceutical preparation. In the present invention, the patient
can be a mammal, preferably a human.
[0047] The present invention also relates to a use of the liposomal
preparation or liposomal pharmaceutical preparation as mentioned
above in the manufacture of a medicament for treatment of a tumor
patient.
[0048] The present invention is further illustrated by the
following examples, which is only exemplary and should not be
construed as a limitation to the present invention.
Part 1: Preparation of Liposomes
Example 1
General Methods for Preparing Liposomes
1. General Method 1
[0049] Phospholipid (e.g., hydrogenated soy phosphatidylcholine
(HSPC), dipalmitoyl phosphatidylcholine (DPPC) or dimyristoyl
phosphatidylcholine (DMPC)) and cholesterol (molar ratio of 1:1 to
10:1) are dissolved in an organic solvent, such as t-butyl alcohol
or cyclohexane, to form a clear solution. The solution is treated
by conventional lyophilization to obtain a lyophilized powder. The
lyophilized powder is hydrated at 60-65.degree. C. with (50-1000
mM) ammonium sulfate solution, citric acid solution or transition
metal sulfate (e.g., nickel sulfate) solution, and shaken for about
1 hour to obtain heterogenous multilamellar vesicles. The size of
the obtained vesicles is reduced by a microfluidizer or a high
pressure extrusion apparatus to obtain liposomes. A sample of the
obtained liposomes is diluted by 200 times with 0.9% NaCl solution
and detected by NanoZS. The extraliposomal buffer solution is
removed by ultrafiltration apparatus to form a dynamic
transmembrane gradient. A mitoxantrone hydrochloride solution (10
mg/mL) is added to the empty liposomes at a suitable liposome/drug
ratio, and the loading of drug is conducted at 60-65.degree. C.
After incubation for about 1 hour, a gel exclusion chromatography
is employed to determine encapsulation efficiency (EE).
2. General Method 2
[0050] Phospholipid (e.g., hydrogenated soy phosphatidylcholine
(HSPC), dipalmitoyl phosphatidylcholine (DPPC) or dimyristoyl
phosphatidylcholine (DMPC)) and cholesterol (molar ratio of 1:1 to
10:1) are mixed, and a polyethylene glycol-modified distearoyl
phosphatidylethanolamine (DSPE-PEG) in 0.1-20% by mole of
phospholipid is added at the same time. The obtained mixture is
dissolved in an organic solvent, such as t-butyl alcohol or
cyclohexane, to form a clear solution. The solution is treated by
conventional lyophilization to obtain a lyophilized powder. The
lyophilized powder is hydrated at 60-65.degree. C. with (50-1000
mM) ammonium sulfate solution, citric acid solution or transition
metal sulfate (e.g., nickel sulfate) solution and shaken for about
1 hour to obtain heterogenous multilamellar vesicles. The size of
the obtained vesicles is reduced by a microfluidizer or a high
pressure extrusion apparatus to obtain liposomes. A sample of the
obtained liposomes is diluted by 200 times with 0.9% NaCl solution
and detected by NanoZS. The extraliposomal buffer solution is
removed by ultrafiltration apparatus to form a dynamic
transmembrane gradient. A mitoxantrone hydrochloride solution (10
mg/mL) is added to the empty liposomes at a suitable liposome/drug
ratio, and the loading of drug is conducted at 60-65.degree. C.
After incubation for about 1 hour, a gel exclusion chromatography
is employed to determine encapsulation efficiency (EE).
Example 2
Preparation of Mitoxantrone Liposome PLM60
[0051] HSPC, cholesterol and DSPE-PEG2000 at a weight ratio of
3:1:1 were dissolved in 95% t-butyl alcohol to form a clear
solution. The solution was treated by lyophilization to obtain a
lyophilized powder. The lyophilized powder was hydrated with an
ammonium sulfate solution (300 mM) at 60-65.degree. C. and shaken
for about 1 hour to obtain heterogenous multilamellar vesicles
having a final concentration of phospholipid of 96 mg/mL The size
of vesicles was reduced by a microfluidizer to obtain liposomes. A
sample of the obtained liposomes was diluted by 200 times with 0.9%
NaCl and detected by NanoZS, having an average size of about 60 nm
and a main peak between 40 nm and 60 nm. The extraliposomal
ammonium sulfate solution was removed by an ultrafiltration
apparatus and substituted by a solution with 250 mM sucrose and 50
mM glycine to form a dynamic transmembrane gradient. A mitoxatrone
hydrochloride solution (10 mg/mL) was added to the empty liposomes
at a liposome/drug ratio of 16:1, and the loading of drug was
conducted at 60-65.degree. C. After incubation for about 1 hour,
the encapsulation efficiency (EE) was determined as 100% by a gel
exclusion chromatography. The obtained liposomes were named as
PLM60.
Example 3
Preparation of Mitoxantrone Liposome PLM85
[0052] HSPC, cholesterol and DSPE-PEG2000 at a weight ratio of
3:1:1 were dissolved in 95% t-butyl alcohol to form a clear
solution. The solution was treated by lyophilization to obtain a
lyophilized powder. The lyophilized powder was hydrated with an
ammonium sulfate solution (300 mM) at 60-65.degree. C. and shaken
for about 1 hour to obtain heterogenous multilamellar vesicles
having a final concentration of phospholipid of 96 mg/mL The size
of vesicles was reduced by a high pressure extrusion apparatus to
obtain liposomes. A sample of the obtained liposomes was diluted by
200 times with NaCl solution and detected by NanoZS, having an
average size of about 85 nm The extraliposomal ammonium sulfate
solution was removed by an ultrafiltration apparatus and
substituted by a solution with 250 mM sucrose and 50 mM glycine to
form a dynamic transmembrane gradient. A mitoxatrone hydrochloride
solution (10 mg/mL) was added to the empty liposomes at a
liposome/drug ratio of 16:1, and the loading of drug was conducted
at 60-65.degree. C. After incubation for about 1 hour, the
encapsulation efficiency (EE) was determined as 100% by a gel
exclusion chromatography. The obtained liposomes were named as
PLM85.
Example 4
Preparation of Mitoxantrone Liposome PLM100
[0053] The same method as described in Example 3 was used to
prepare mitoxatrone hydrochloride liposome PLM100, in which the
formulation is identical to that of PLM60 and PLM85, but the size
of liposomes was 100 nm
Example 5
Preparation of Mitoxantrone Liposome PLM60-dppc
[0054] DPPC, cholesterol and DSPE-PEG2000 at a weight ratio of
3:1:1 were mixed, and other steps were identical to those of
Example 2. The obtained liposomes were named as PLM60-dppc.
Example 6
Preparation of Mitoxantrone Liposome PLM60-dmpc
[0055] DMPC, cholesterol and DSPE-PEG2000 at a weight ratio of
3:1:1 were mixed, and other steps were identical to those of
Example 2. The obtained liposomes were named as PLM60-dmpc.
Example 7
Preparation of Mitoxantrone Liposome PLM60-dmpc-0.1
[0056] DMPC, cholesterol and DSPE-PEG2000 at a weight ratio of
3:1:0.1 were mixed, and other steps were identical to those of
Example 2. The obtained liposomes were named as PLM60-dmpc-0.1.
Example 8
Preparation of Adriamycin Liposome PLD60
[0057] Adriamycin was substituted for mitoxantrone during the step
of loading drug, and other steps were identical to those of Example
2. The obtained liposomes were named as PLD60.
Part 2: Drug Release of Different Liposome Formulations
Example 9
Differences of Drug-Release Between Adriamycin Liposome PLD60 and
Mitoxantrone Liposome PLM60
[0058] In the present example, mitoxantrone and adriamycin were
loaded under a pH gradient. If a certain concentration of ammonium
chloride was added to a release medium, free ammonia molecules
would diffuse to the inner phase of liposome with the help of
gradient, so that the pH of inner phase would be elevated and the
protonated drug in the inner phase would be converted into its
neutral form which could diffuse across membrane. This process
could accelerate the dissolution of precipitation in the inner
phase of liposome in the meantime. The speed of drug release was
controlled by both the dissolution of precipitation and the
membrane permeability of liposome. The conditions for drug release
were as follows. Liposomes were diluted by 25 times with release
media. The release media were isotonic, had a pH of 7.4, and had a
concentration of ammonium chloride of 2, 10 and 40 mM,
respectively. The diluted liposomes were placed in dialysis
tubings, and dialysis was performed on 2 mL diluted liposome by 400
mL of release medium at 37.degree. C. Samples were taken at
different time points for analysis until 96 hours later.
[0059] The obtained data were subjected to an regression analysis.
In the release media having 2, 10 and 40 mM ammonium chloride, the
drug-release half-life periods of PLD60 were 94.3, 31.9 and 11.2
hours, respectively. With regard to PLM60, no obvious release was
observed in the three release media. Since PLD60 and PLM60 have no
difference in composition and size, the difference of drug release
kinetic characteristic could be attributed to their different
pharmaceutical features. Adriamycin and mitoxantrone are both
anthracycline antibiotics, and their differences lie in that
adriamycin contains one dissociable group at physiological pH while
mitoxantrone contains two dissociable groups (pKa=8.15) at
physiological pH. This example illustrates that a drug with
multi-dissociable groups such as mitoxantrone can form a complex
precipitation with counter-ions when an active loading method is
employed, so that the in vitro release of drug is significantly
slowed down. On the other hand, a drug with uni-dissociable group
such as adriamycin could be released too quickly even in a release
medium without plasma when a small size liposome is employed.
Example 10
Release Behaviors of Mitoxantrone Liposomes with Different
Sizes
[0060] Two release conditions were taken to compare the release
behaviors of mitoxantrone liposomes with different size.
[0061] Release condition 1: a liposome was diluted by 25 times with
a release medium. The release medium contained 50% human plasma,
was adjusted to be isotonic by glucose and had a pH of 7.4. Other
conditions were identical to those of Example 9. The obtained data
were subjected to a regression analysis. The result showed that the
release half-life period of PLM60 was 56.4 hours, while PLM85 was
not significantly released under the same conditions.
[0062] Release condition 2: a release medium containing 50% human
plasma and 20 mM ammonium chloride was used, and other conditions
were identical to those of Example 9. The obtained data were
subjected to a regression analysis. The result showed that the
release half-life period of PLM60 was 26.2 hours, while the release
half-life period of PLM85 was 36.7 hours.
[0063] This example sufficiently indicated that the release of drug
could be significantly enhanced by reducing the size of
liposome.
Example 11
Release Behaviors of Mitoxantrone Liposomes with Different Membrane
Compositions
[0064] The same release conditions as described in Example 9 were
used.
[0065] The result indicated that the release half-life of
PLM60-DPPC was 116 hours, the release half-life of PLM60-DMPC was
26 hours, and the release half-life of PLM60-DMPC-0.1 was 15 hours.
This example indicated that the use of a phospholipid with a lower
phase transition temperature Tm could accelerate the drug release.
However, if the release of drug was accelerated excessively, the
toxicity of drug could increase excessively as well, and this was
further confirmed in the following examples.
Part 3: In Vivo Pharmacokinetics
Example 12
Pharmacokinetic Behavior of PLM60 in Kunming Mice and the
Comparison Between PLM60 and Free Mitoxantrone
[0066] This example was conducted in male Kunming mice with a body
weight of about 20 g. Different dose levels of mitoxantrone were
injected through tail vein in mice. The dosages of PLM60 were 1, 2
and 4 mg/kg, and the dosage of free mitoxantrone (FM) was 2 mg/kg.
Plasma samples were taken at different time points. The methods for
processing and detecting plasma samples were described in the
document: Methods in enzymology, Vol: 391, p 176-185. The results
were shown in Table 1 and FIG. 1, in which it was clearly indicated
that the half-life period of mitoxantrone was significantly
extended by encapsulation of liposomes. At the same dosage, PLM60
had a retention time in blood circulation 32 times of that of FM,
and an AUC 3700 times of that of FM. A plot of AUC against dose
indicated that PLM60 had a linear pharmacokinetics in vivo.
TABLE-US-00001 TABLE 1 Pharmacokinetics of PLM60 and FM in Kunming
mice PLM60 PLM60 PLM60 FM 4 mg/kg 2 mg/kg 1 mg/kg 2 mg/kg
Parameters Value Value Value Value AUC 1451.666 728.398 452.709
0.198 0-48(mg/L*h) AUC 1654.543 892.437 503.078 0.199
0-.infin.(mg/L*h) AUMC 0-48 21838.034 12050.681 7049.259 0.103 AUMC
0-.infin. 36234.611 24686.917 10488.811 0.135 MRT 0-48(h) 15.043
16.544 15.571 0.517 MRT 0-.infin.(h) 21.900 27.662 20.849 0.675
T.sub.max(h) 0.083 0.083 0.250 0.083 C.sub.max(mg/L) 86.329 47.513
25.970 0.699
Example 13
Tissue Distribution of PLM60 and FM in Tumor-Bearing Mice
[0067] There were obvious differences in tissue distribution
between PLM60 and FM in tumor-bearing mice. Male Kunming mice
having a body weight about 20 g were used in the present example.
The mice were inoculated in right oxter with S-180 sarcoma cells at
a ration of 5.times.10.sup.5. Drugs were injected through vein in
mice when tumor grew to 0.4-0.7 g. After the administration of
drugs, mice were executed at various time points and their tissues
were taken out to determine the concentration of mitoxantrone. The
tissues included hearts, livers, spleens, lungs, kidneys,
intestines, bone marrow and tumors. The results showed that PLM60
had a very clear targeting to tumor tissues. The detailed data were
shown in Table 2 and FIG. 2.
TABLE-US-00002 TABLE 2 Tissue distribution data of PLM60 and FM in
tumor-bearing mice PLM-60 FM 4 mg/kg 4 mg/kg Tissue t (h) C-.mu.g/g
SD t (h) C-.mu.g/g SD Heart 1 4.01 0.38 1 5.385 0.679 4 3.39 0.38 4
3.517 0.952 8 3.48 0.64 8 3.197 0.357 16 2.83 0.57 24 2.943 0.549
24 2.06 0.48 Liver 1 6.78 0.78 1 4.770 0.997 4 5.99 0.67 4 3.556
0.543 8 6.31 0.38 8 2.659 0.439 16 6.22 0.95 24 1.937 0.346 24 4.52
0.65 Spleen 1 4.66 1.37 1 4.044 0.414 4 4.36 0.67 4 4.460 0.494 8
4.78 1.70 8 3.774 2.676 16 7.56 2.13 24 7.752 2.469 24 5.91 1.00
Lung 1 8.44 1.08 1 10.205 1.732 4 4.58 2.36 4 8.024 1.859 8 6.33
1.43 8 7.018 0.728 16 5.12 1.24 24 8.082 0.844 24 2.89 0.23 Kidney
1 7.09 0.84 1 18.243 1.238 4 7.12 1.17 4 17.192 5.010 8 7.04 0.96 8
13.409 1.251 16 6.75 1.16 24 7.463 1.209 24 5.82 0.84 Intestine 1
1.66 0.66 1 1.532 0.309 4 2.33 0.66 4 2.140 0.655 8 2.34 0.64 8
2.551 1.204 16 2.42 0.51 24 3.936 1.625 24 2.25 0.32 Bone 1 1.09
0.54 1 0.127 0.041 marrow 4 0.64 0.14 8 0.73 0.16 16 0.54 0.24 24
0.12 0.02 Tumor 1 91.28 7.41 1 0.0614 0.0078 4 63.90 8.56 4 0.0133
0.0027 8 54.01 8.04 16 38.61 9.19 24 10.41 2.67
Example 14
Pharmacokinetics Comparison of Different Liposome Formulations
[0068] The animals used in this example were similar to those of
Example 12. PLM60-DPPC, PLM60-DMPC-0.1 and PLM60-DMPC at 4 mg/kg
were injected through tail vein in mice. The data were shown in
Table 3 and FIG. 3. It was shown that pharmacokinetics of liposomal
drugs changed significantly with the change of liposome membrane
composition. The MRT values of PLM60-DPPC, PLM60-DMPC-0.1 and
PLM60-DMPC in vivo were 14.22, 7.914 and 10.123 hours,
respectively. The difference between PLM60-DPPC and PLM60-DMPC lied
in the lengths of hydrocarbon chains of phospholipids, which were
16 and 14 carbons, respectively. The length of acyl chain could
significantly influence the membrane permeability of phospholipid
bilayer. The phase transition temperature of DPPC was 41.degree. C.
and the phase transition temperature of DMPC was 23.degree. C. The
difference between PLM60-DMPC-0.1 and PLM60-DMPC lied in the degree
of PEGylation. The release of liposomal drug in plasma depends on
two factors: one is the release of liposomal drug across
phospholipid bilayer and the other is the clearance by lipoprotein
and reticuloendothelial system (RES). Since the PEGylation of
PLM60-DMPC-0.1 was not complete, the release caused by plasma
components had more influences on it.
TABLE-US-00003 TABLE 3 Comparison of in vivo pharmacokinetics of
different liposome formulations in mice PLM60-DPPC PLM60-DMPC-0.1
PLM60-DMPC 4 mg/kg 4 mg/kg 4 mg/kg Parameter Value Value Value AUC
1506.710 174.849 337.641 0-48(mg/L*h) AUC 1581.242 175.705 344.134
0-.infin.(mg/L*h) AUMC 0-48 21425.274 1383.757 3417.981 AUMC
0-.infin. 26235.613 1478.267 3818.856 MRT 0-48(h) 14.220 7.914
10.123 MRT 0-.infin.(h) 16.592 8.413 11.097 T.sub.max(h) 1.000
1.000 1.000 C.sub.max(mg/L) 81.976 19.853 39.115
Part 4: Comparison of Toxicity of Different Formulations
Example 15
Comparison of Acute Toxicity Between PLM60 and FM
[0069] 100 Kunming mice (half male and half female) with a body
weight of 18-22 g were divided into 10 groups, each group had 10
mice, half male and half female. Mice of groups 1-5 were
administrated with different dose levels of FM, while mice of
groups 6-10 were administrated with an equivalent dose level of
liposomal drug. Body weight changes were observed and the death
time of each animal was recorded. The dead animals were dissected
and autopsied. The results of all groups were shown in Table 4,
which showed that the acute toxicity of PLM60 was far lower than
that of FM.
TABLE-US-00004 TABLE 4 Acute toxicity comparison of PLM60 and FM to
Kunming mice Survival time of male mice Survival time of female
mice Liposome (day) (day) and dose mg/kg No. 1 No. 2 No. 3 No. 4
No. 5 No. 1 No. 2 No. 3 No. 4 No. 5 FM 20 7 8 8 9 6 8 8 8 9 NA FM
12 18 13 13 NA 7 12 12 13 14 NA FM 7.2 NA NA NA NA NA NA NA NA NA
NA FM 4.32 NA NA NA NA NA NA NA NA NA NA FM 2.59 NA NA NA NA NA NA
NA NA NA NA PLM60 20 17 NA 12 NA NA NA NA NA NA NA PLM60 12 NA NA
NA NA NA NA NA NA NA NA PLM60 7.2 NA NA NA NA NA NA NA NA NA NA
PLM60 4.32 NA NA NA NA NA NA NA NA NA NA PLM60 2.59 NA NA NA NA NA
NA NA NA NA NA NA: No data, i.e., No animal died at the end of
experimental observation.
Example 16
Acute Toxicity Comparison of Different Liposome Formulations
[0070] 90 male Balb/c mice with a body weight of 18-22 g were
divided into 9 groups, each group had 10 mice. The mice of group 1
were administered with FM at 6 mg/kg, while mice of other 8 groups
were administered with PLM60, PLM60-DPPC and PLM60-DMPC-0.1 and
PLM60-DMPC at 6 and 12 mg/kg, respectively. Body weight changes
were observed and the death time of each animal was recorded. The
dead animals were dissected and autopsied. The results of death of
mice of FM group and liposomal drug groups were shown in Table 5.
This experiment showed that the order of acute toxicity was:
PLM60<PLM60-DPPC<PLM60-DMPC-0.1 FM<PLM60-DMPC. This
experiment also confirmed that the release of drug could be further
accelerated by using small unilamellar vesicles and phospholipid
with a lower Tm as the composition of bilayer, such as PLM60-DMPC,
thereby resulting in more toxicity in vivo. It should be noted that
the toxicity of liposomes with incomplete PEGylation was lower than
that of liposomes with more complete PEGylation. This may be
attributed to that under the action of lipoprotein and the attack
of immune system during blood circulation, PLM60-DMPC-0.1 with
incomplete PEGylation would release drug earlier in comparison with
PLM60-DMPC and would not release suddenly in important tissues,
thereby exhibiting a lower toxicity, but the toxicity of
PLM60-DMPC-0.1 was still nearly equivalent to that of free
mitoxantrone.
TABLE-US-00005 TABLE 5 Acute toxicity comparison of different
liposomes Survival time of mice(day) Formulary and dose(mg/kg) 1 2
3 4 5 6 7 8 9 10 FM-6 NA NA NA NA NA NA NA NA NA NA PLM60-6 NA NA
NA NA NA NA NA NA NA NA PLM60-12 NA NA NA NA NA 10 NA NA NA 11
Plm60DPPC-6 NA NA NA NA NA NA NA NA NA NA Plm60DPPC-12 10 10 12 11
NA NA NA 13 NA 14 Plm60-DMPC-6 4 NA NA 3 6 7 7 6 NA NA Plm60DMPC-12
3 3 5 3 3 3 4 3 3 3 Plm60DMPC-0.1-6 NA NA NA NA NA NA NA NA NA NA
Plm60DMPC-0.1-12 10 12 10 12 10 10 10 11 10 10 NA: No animal died
at the end of experimental observation
Example 17
Toxicity Comparison of Liposome Formulations with Different
Sizes
[0071] Male C57 mice with a body weight of 18-22 g were used in
toxicity comparison of PLM60, PLM85 and PLM100. The dose was 9
mg/kg. The results indicated that body weight varieties caused by
the three liposome formulations were equivalent, which confirmed
that the three liposome formulations had no significant difference
in toxicity under the experimental conditions. In mice of FM group,
the body weight decreased over 30% and about 20% mice died.
Part 5: In Vivo Anti-Tumor Effects
Example 18
Comparison of Treatment Effects of PLM60 and FM on S-180
Sarcoma
[0072] Ascitic tumor-bearing mice which were inoculated with S180
tumor cells 7 days ago were executed by decollation, and milky
viscous ascitic fluid was extracted and diluted with RPMI 1640
medium. After dilution, the tumor cell number was adjusted to
2.5.times.10.sup.6 cells/ml. 0.2 mL of the tumor cell suspension
containing about 5.times.10.sup.5 tumor cells was inoculated into
forward limb oxter tissues of male KM mice with a body weight of
18-22 g. After inoculation, the cells in the residual tumor cell
suspension were counted under light microscope, and living tumor
cells were greater than 95%. The number of inoculated mice was
80.
[0073] Seven days after inoculation, 39 mice with clear-cut tumors
having a diameter of about 5 mm were selected and divided into 5
groups by both tumor volume and body weight, i.e., 7 mice in blank
control group, 8 mice in each of 4 mg/kg PLM60 group, 6 mg/kg PLM60
group, 4 mg/kg FM group and 6 mg/kg FM group. The mice were
administered by intravenous injection.
[0074] The mice were bred normally after administration. Tumor
diameters were measured by vernier caliper 3 times per week, and
body weights were measured at the same time. Tumor volume (TV) was
calculated with the following formula: V=1/2.times.a.times.b.sup.2,
in which a and b represent length and width, respectively. The
tumor volumes were calculated by using the measurement results.
Mice were executed by decollation on the 21.sup.st day after
inoculation, tumors were taken out and weighed. Tumor inhibition
rate (%) was calculated with the following formula: tumor
inhibition rate=(1-average tumor weight of drug group/average tumor
weight of control group).times.100%. The experimental result was
tested by t-test.
[0075] Table 6 showed that the growth of S180 solid tumor was
significantly suppressed in the 4 mg/kg PLM60 group and 6 mg/kg
PLM60 group.
TABLE-US-00006 TABLE 6 Effects of PLM60 on S180 solid tumor weight
Ratio of Group Weight of tumor (mg) tumor-inhibiting (%) Control
2813.8 .+-. 884.2 PLM60 4 mg/kg 421.9 .+-. 215.4.sup.a 85.00 PLM60
6 mg/kg 332.4 .+-. 162.5.sup.a 88.19 free mitoxantone 4 mg/kg
2828.5 .+-. 1067.8 -- free mitoxantone 6 mg/kg 2293.3 .+-. 1720.0
18.50 .sup.ain comparison with the control group, p < 0.01
Example 19
Treatment Effects of PLM60 and FM on L1210 Ascites Model
[0076] Ascitic tumor BDF1 mice which were inoculated with L1210
ascitic tumor cells 7 days ago were executed by decollation, and
milky viscous ascitic fluid was extracted under aseptic condition
and diluted by RPMI 1640 medium. After dilution, the tumor cell
number was adjusted to 2.5.times.10.sup.6 cells/ml. 0.2 mL of the
tumor cell suspension containing about 5.0.times.10.sup.5 tumor
cells was inoculated into the abdominal cavity of a 7-8 week-old
female BDF1 mouse. After inoculation, the cells in the residual
tumor cell suspension were counted under light microscope, and
living tumor cells were greater than 95%.
[0077] 24 hours later, the mice were divided into 8 groups by body
weight, and were administered with FM at 2, 4 and 8 mg/kg, and
PLM60 at 2, 4, and 6 mg/kg by injection in a volume of 20 ml/kg
through tail vein in mice, respectively. After administration, the
mice were bred normally. Their body weights were measured 3 times
per week, the death time of each mouse was observed and recorded,
and survival time was calculated. Mean survival time (MST) and
median survival time were employed to evaluate the survival time of
each group. Experimental observation was kept for 60 days after the
inoculation.
[0078] The data were analyzed by SPSS 11.5 statistics software. The
results showed that all administration groups exhibited significant
increase of survival time in comparison with the control group, and
the PLM60 (8 mg/kg) group exhibited significant improvement of
treatment in comparison with the FM group at the same dose
(P<0.05). The results were shown in Table 7.
TABLE-US-00007 TABLE 7 Effects of L1210 ascitic tumor on BDF1 mice
survival time Number Survival time (95% of Number confidence
interval) Ratio Dose animals of dead Mean Median of survival Group
(mg/kg) (N) animals (N) survival time survival time (%) Control --
13 13 9.62 .+-. 0.40 9.00 .+-. 0.25 0 (8.83-10.40) (8.51-9.49) FM60
2 12 11 20.17 .+-. 3.77 14.00 .+-. 1.15 8.33 (12.77-27.56)
(11.74-16.26) 4 12 9 36.75 .+-. 4.00 31.00 .+-. 0.85 25.00
(28.92-44.58) (29.33-32.67) 6 12 10 28.42 .+-. 4.49 25.00 .+-. 3.46
16.67 (19.63-37.21) (18.21-31.79) PLM60 2 12 9 36.42 .+-. 4.08
29.00 .+-. 1.71 25.00 (28.41-44.42) (25.65-32.35) 4 11 2 57.55 .+-.
1.60 N.sup.b 81.82 (54.40-60.69) 6 12 5 48.00 .+-. 4.38 N.sup.b
58.33 (39.42-56.58) 8 12 4 53.00 .+-. 3.71 N.sup.b 66.67
(45.72-60.28) N.sup.b: Only few animals died at the end of
experimental observation of 60 days and the median survival time
could not be calculated.
Example 20
Treatment Effects of PLM60 and FM on L1210 Liver Metastasis
Model
[0079] Ascitic tumor BDF1 mice which were inoculated L1210 ascitic
tumor cells 7 days ago were executed by decollation, and milky
viscous ascitic fluid was extracted under aseptic condition and
diluted with RPMI 1640 medium. After dilution, the tumor cell
number was adjusted to 2.5.times.10.sup.5 cells/ml. 0.2 mL of the
tumor cell suspension containing about 5.0.times.10.sup.4 tumor
cells was intravenously inoculated into a 7-8 week-old male BDF1
mouse. After inoculation, the cells in the residual tumor cell
suspension were counted under light microscope, and living tumor
cells were greater than 95%. Total 62 mice were inoculated.
[0080] 24 hours later, the mice were grouped and administered.
After administration, the mice were bred normally. The body weights
of mice were measured 3 times per week, the death time of each
mouse was observed and recorded every day, and survival time was
calculated. Experimental observation was kept for 60 days after the
inoculation.
[0081] The result showed that all mice in the control group died
between the 11.sup.th and 14.sup.th day after inoculation, all mice
in the three FM dose level groups died between the 11.sup.th and
17.sup.th day after inoculation, all mice in the 2 mg/kg PLM60
group died between the 15.sup.th and 29.sup.th day after
inoculation, only one mouse in the 6 mg/kg PLM60 group died on the
39.sup.th day after inoculation, and no mouse in the 8 mg/kg PLM60
group died during the observation.
[0082] The data were analyzed by SPSS 11.5 statistics software. The
results showed that the 6 mg/kg FM group and all liposomal drug
groups exhibited a significant increase in survival time of mice in
comparison with the blank control group. At same dose level,
liposomal mitoxantrone exhibited a significant increase in survival
time of mice in comparison with free mitoxantrone. The results were
shown in Table 8.
TABLE-US-00008 TABLE 8 Effects of intravenous inoculation of L1210
on BDF1 mice survival time Number Number Survival time (95%
confidence of of dead interval) Dose animals animals Mean survival
Median Survival Group (mg/kg) (N) (N) time survival time rate (%)
Control -- 6 6 11.83 .+-. 0.48 11.0 0 (10.90-12.77) FM60 2 8 8
12.13 .+-. 0.61 11.0 0 (10.93-13.32) 4 8 8 13.25 .+-. 0.53 13.00
.+-. 0.46 0 (12.22-14.28) (12.11-13.89) 6 8 8 14.50 .+-. 0.71 14.00
.+-. 0.91 0 (13.11-15.89) (12.21-15.79) PLM60 2 8 8 19.13 .+-. 1.57
18.00 .+-. 1.41 0 (16.04-22.21) (15.23-20.77) 4 8 5 36.50 .+-. 6.51
22.00 .+-. 5.660.61 37.50 (23.75-49.25) (10.91-33.09) 6 8 1 57.38
.+-. 2.46 N.sup.b 87.50 (52.56-62.19) 8 8 0 Na N.sup.a 100.00
N.sup.a: No animal died until at the end of 60 days experimental
observation and the median survival time was not calculated.
N.sup.b: Only one of animals died at the end of 60 days
experimental observation and the median survival time was not
calculated.
Example 21
Treatment Effects of Liposomal Mitoxantrone With Different Size on
L1210 Ascitic Tumor
[0083] The experimental scheme and data process mode were the same
as Example 19. Five groups were setup, including control group, FM
group, PLM60 group, PLM85 group and PLM100 group. The
administration dosage for mice in each group was 4 mg/kg. The
results were shown in Table 9. The results showed that liposome
with smaller size had better treatment effects.
TABLE-US-00009 TABLE 9 Effects of L1210 ascitic tumor on BDF1 mice
survival time Survival time (95% confidence interval) Number of
Number Mean Dose animal of dead survival Median Survival Group
(mg/kg) (N) animal (N) time survival time rate (%) Control -- 12 12
9.08 .+-. 0.19 9.00 .+-. 0.21 0 FM 4 12 8 38.67 .+-. 3.54 36.00
.+-. 6.06 33.33 PLM60 4 12 4 47.00 .+-. 2.88 N.sup.b 66.67 PLM85 4
12 8 39.17 .+-. 4.1 38.00 .+-. 11.26 33.33 PLM100 4 12 10 30.08
.+-. 3.59 23.00 .+-. 2.89 16.66 N.sup.b: Only few animals died at
the end of 60 days experimental observation and the median survival
time was not calculated
[0084] Some preferred examples of the present invention are
described above, but these examples are in no way intended to limit
the scope of the invention. Besides what have been described and
illustrated in the text, a skilled ordinary technician in the field
would clearly realize other modifications and variations and
changes of the present invention after reading the disclosure of
the present invention, and all of them should be covered in the
protection scope of the present invention. All patents, published
patent applications and publications cited here are incorporated by
reference, just like their full texts are incorporated in the
text.
* * * * *