U.S. patent application number 16/668754 was filed with the patent office on 2020-02-27 for purified therapeutic nanoparticles and preparation methods thereof.
The applicant listed for this patent is CSPC ZHONGQI PHARMACEUTICAL TECHNOLOGY (SHIJIAZHUANG) CO., LTD.. Invention is credited to Dongjian Chen, Chunlei Li, Yanhui Li, Yongfeng Li, Min Liang, Caixia Wang, Shixia Wang, Yajuan Wang.
Application Number | 20200060982 16/668754 |
Document ID | / |
Family ID | 52461987 |
Filed Date | 2020-02-27 |
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United States Patent
Application |
20200060982 |
Kind Code |
A1 |
Li; Chunlei ; et
al. |
February 27, 2020 |
PURIFIED THERAPEUTIC NANOPARTICLES AND PREPARATION METHODS
THEREOF
Abstract
Purified therapeutic nanoparticles are provided herein. Such
nanoparticles comprise an active pharmaceutical ingredient and
human serum albumin, wherein the weight ratio of human serum
albumin to the active ingredient in the therapeutic nanoparticles
is from 0.01:1 to 1:1, and wherein the nanoparticles are
substantially free of free human serum albumin that is not
incorporated in the nanoparticles. The present disclosure also
provides pharmaceutical compositions that comprise the purified
therapeutic nanoparticles and are also substantially free of free
human serum albumin. Methods for preparing and using the purified
therapeutic nanoparticles and compositions thereof are also
provided.
Inventors: |
Li; Chunlei; (Shijiazhuang,
CN) ; Li; Yanhui; (Shijiazhuang, CN) ; Liang;
Min; (Shijiazhuang, CN) ; Wang; Caixia;
(Shijiazhuang, CN) ; Wang; Yajuan; (Shijiazhuang,
CN) ; Wang; Shixia; (Shijiazhuang, CN) ; Chen;
Dongjian; (Shijiazhuang, CN) ; Li; Yongfeng;
(Shijiazhuang, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CSPC ZHONGQI PHARMACEUTICAL TECHNOLOGY (SHIJIAZHUANG) CO.,
LTD. |
Shijiazhuang |
|
CN |
|
|
Family ID: |
52461987 |
Appl. No.: |
16/668754 |
Filed: |
October 30, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14790646 |
Jul 2, 2015 |
10500165 |
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16668754 |
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PCT/CN2014/091277 |
Nov 17, 2014 |
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14790646 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/337 20130101;
A61P 35/00 20180101; B82Y 5/00 20130101; A61K 9/5169 20130101 |
International
Class: |
A61K 9/51 20060101
A61K009/51; B82Y 5/00 20060101 B82Y005/00; A61K 31/337 20060101
A61K031/337 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 3, 2014 |
CN |
201410314042.2 |
Claims
1. Purified therapeutic nanoparticles, comprising an active
ingredient and human serum albumin (HSA), wherein the weight ratio
of HSA to the active ingredient in the therapeutic nanoparticles is
selected from 0.01:1, 0.02:1, 0.025:1, 0.03:1, 0.04:1, 0.05:1,
0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.10:1, 0.11:1, 0.12:1, 0.13:1,
0.14:1, 0.15:1, 0.16:1, 0.17:1, 0.18:1, 0.19:1, 0.2:1, 0.21:1,
0.22:1, 0.23:1, 0.24:1, 0.25:1, 0.26:1, 0.27:1, 0.28:1, 0.29:1,
0.3:1, 0.31:1, 0.32:1, 0.33:1, 0.34:1, 0.35:1, 0.36:1, 0.37:1,
0.38:1, 0.39:1, 0.4:1, 0.41:1, 0.42:1, 0.43:1, 0.44:1, 0.45:1,
0.46:1, 0.47:1, 0.48:1, 0.49:1, 0.5:1, 0.51:1, 0.52:1, 0.53:1,
0.54:1, 0.55:1, 0.56:1, 0.57:1, 0.58:1, 0.59:1, 0.6:1, 0.65:1,
0.70:1, 0.75:1, 0.8:1, 0.85:1, 0.9:1, 0.95:1, 1:1, 1.5:1, 2:1,
2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1,
8:1, 8.5:1 or the range between any two ratios above, wherein the
active ingredient is encapsulated inside HSA and wherein the
therapeutic nanoparticles are substantially free of free HSA that
is not incorporated in the nanoparticles, and wherein the average
particle size of the therapeutic nanoparticles is from 110 to 150
nm.
2. The therapeutic nanoparticles according to claim 1, wherein the
weight ratio of human serum albumin to the active ingredient is
selected from 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1,
0.09:1, 0.10:1, 0.11:1, 0.12:1, 0.13:1, 0.14:1, 0.15:1, 0.16:1,
0.17:1, 0.18:1, 0.19:1, 0.2:1, 0.21:1, 0.22:1, 0.23:1, 0.24:1,
0.25:1, 0.26:1, 0.27:1, 0.28:1, 0.29:1, 0.3:1, 0.31:1, 0.32:1,
0.33:1, 0.34:1, 0.35:1, 0.36:1, 0.37:1, 0.38:1, 0.39:1, 0.4:1,
0.41:1, 0.42:1, 0.43:1, 0.44:1, 0.45:1, 0.46:1, 0.47:1, 0.48:1,
0.49:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, or the range between any
two ratios above.
3. The therapeutic nanoparticles according to claim 1, wherein the
nanoparticles contain at most 5% free HSA (by weight).
4. The therapeutic nanoparticles according to claim 1, wherein the
active ingredient has the properties of being insoluble or slightly
soluble in water, but soluble or free soluble in an organic
solvent.
5. The therapeutic nanoparticles according to claim 4, wherein the
active ingredient is selected from taxanes, macrolides,
camptothecins, anthracycline antibiotics, colchicine,
thiocolchicine dimer, amiodardone, liothyronine, cyclosporine,
exemestane, flutamide, fulvestrant, romidepsin, semustine, and
ibuprofen.
6. The therapeutic nanoparticles according to claim 5, wherein the
active ingredient is a taxane.
7. The therapeutic nanoparticles according to claim 5, wherein the
taxane is paclitaxel or docetaxel.
8. The therapeutic nanoparticles according to claim 4, wherein the
organic solvent is a pure solvent having low water-solubility and
low boiling point or its mixture with small molecular alcohols.
9. A pharmaceutical composition, comprising the therapeutic
nanoparticles according to claim 1, wherein the composition is
substantially free of free HSA that is not incorporated in the
nanoparticles.
10. The pharmaceutical composition according to claim 9, wherein
the pharmaceutical composition is in the form of liquid or
lyophilized powder.
11. The pharmaceutical composition according to claim 9, wherein
the pharmaceutical composition also comprises lyophilization
excipient when the pharmaceutical composition is in the form of
lyophilized powder.
12. The pharmaceutical composition according to claim 11, wherein
the lyophilization excipient is selected from one or more of
mannitol, sucrose, lactose, maltose, trehalose, and dextran.
13. The pharmaceutical composition according to claim 9, wherein
the composition contains at most 5% free HSA (by weight).
14. The pharmaceutical composition according to claim 9, wherein
the active ingredient is paclitaxel.
15. A method for treating cancer, comprising: administering a
therapeutically effective amount of the therapeutic nanoparticles
according to claim 1 to a subject in need thereof.
16. The method according to claim 15, wherein the cancer is
selected from the group consisting of liver cancer, prostatic
cancer and lung cancer.
17. The method according to claim 15, wherein the active ingredient
is selected from the group consisting of taxanes, macrolides,
camptothecins, anthracycline antibiotics, colchicine,
thiocolchicine dimer, amiodardone, liothyronine, cyclosporine,
exemestane, flutamide, fulvestrant, romidepsin, semustine, and
ibuprofen.
18. The method according to claim 15, wherein the active ingredient
is taxane.
19. The method according to claim 15, wherein the active ingredient
is paclitaxel or docetaxel.
20. The method according to claim 15, wherein the nanoparticles
contain at most 5% free HSA (by weight).
Description
CROSS REFERENCE OF RELATED APPLICATIONS
[0001] The present application is a continuation application of
U.S. patent application Ser. No. 14/790,646, filed on Jul. 2, 2015,
the entire contents of which are incorporated herein by reference
and priority to which is hereby claimed.
TECHNICAL FIELD
[0002] The present disclosure relates to the pharmaceutical field,
and more particularly, to nanoparticles comprising human serum
albumin and a hydrophobic drug, and the preparation method thereof,
and even more particularly, to albumin nanoparticles comprising
specific physical properties and the preparation method
thereof.
BACKGROUND
[0003] As an anticancer drug, paclitaxel acts as a microtubule
inhibitor in mitosis, and plays an important role in polymerization
and stabilization of intracellular microtubule. At the stage of
mitosis, paclitaxel disables the separation of microtubules, so
that cells are blocked between G2 and M phase. As a result, the
fast-dividing tumor cells are arrested at the phase of mitosis by
paclitaxel, leading to the death of the cells due to hindered
replication. Paclitaxel has important clinical activity to various
cancers (for example, breast cancer, ovarian cancer, lung cancer,
and bladder cancer, etc.).
[0004] Due to poor water-solubility, however, the application of
paclitaxel in human body is limited. In order to make paclitaxel
suitable for intravenous injection, Bristol-Myers Squibb (BMS) has
developed TAXOL.RTM., in which a surfactant polyoxyethylene castor
oil (CREMOPHOR.RTM. EL) and anhydrous alcohol are added together as
co-solvent to enhance the solubility of paclitaxel. Taxol has
significant activity to ovarian cancer, breast cancer, lung cancer,
esophagus cancer, and head and neck cancer. However, it has been
demonstrated that Taxol may induce therapy-related toxicity, and
significant acute and accumulative toxicity, such as
myelosuppression, febrile neutropenia and hypersensitivity etc.
These side effects are related to the surfactant polyoxyethylene
castor oil used (Anantbhushan et al., Asia Pac J Clin Oncol. 2013;
9:176-181). Based on clinical research reports and post-marketing
safety data, an overall incidence of hypersensitivity induced by
Taxol is about 39%. At present, antihistamines and steroids are
administrated to patients ahead of time to alleviate the side
effects due to the surfactant, when Taxol is used.
[0005] In order to improve the water solubility of paclitaxel,
structure modifications are also conducted by researchers using
functional groups which may provide higher water solubility, for
example, sulfonate derivatives (Kingston et al., U.S. Pat. No.
5,059,699 (1991)), and amino-acid ester derivatives (Mathew et al.,
J. Med. Chem. 35:145-151 (1992)). They exhibit obvious biological
activities after modification. However, these modifications may
induce undesired side effects or reduce the pharmaceutical
efficiency besides the increase of cost of the pharmaceutical
formulations.
[0006] To avoid adverse effects of CREMOPHOR.RTM. EL in paclitaxel
formulations, another drug delivery system that does not contain
any emulsifying agent or surfactant was developed. Such a system is
in the form of micro-particles or nanoparticles and contains
albumin, a portion of which forms complexes with paclitaxel.
However, this system still has many disadvantages. For example, the
formulation requires a large amount of human serum albumin (HSA),
which may cause allergy. HSA is still obtained from human blood,
and has potential safety risks due to possible contaminations
during blood collection and storage. In addition, HSA is relatively
expensive and may be in shortage in certain regions.
SUMMARY
[0007] The present disclosure provides purified therapeutic
nanoparticles, compositions comprising such nanoparticles, methods
of preparing such nanoparticles or compositions, and methods of
using such nanoparticles or compositions.
[0008] In one aspect, the present disclosure provides purified
therapeutic nanoparticles that comprise an active ingredient and
human serum albumin, wherein the weight ratio of human serum
albumin (HSA) to the active ingredient in the therapeutic
nanoparticles is selected from 0.01:1, 0.02:1, 0.04:1, 0.05:1,
0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.10:1, 0.11:1, 0.12:1, 0.13:1,
0.14:1, 0.15:1, 0.16:1, 0.17:1, 0.18:1, 0.19:1, 0.2:1, 0.21:1,
0.22:1, 0.23:1, 0.24:1, 0.25:1, 0.26:1, 0.27:1, 0.28:1, 0.29:1,
0.3:1, 0.31:1, 0.32:1, 0.33:1, 0.34:1, 0.35:1, 0.36:1, 0.37:1,
0.38:1, 0.39:1, 0.4:1, 0.41:1, 0.42:1, 0.43:1, 0.44:1, 0.45:1,
0.46:1, 0.47:1, 0.48:1, 0.49:1, 0.5:1, 0.51:1, 0.52:1, 0.53:1,
0.54:1, 0.55:1, 0.56:1, 0.57:1, 0.58:1, 0.59:1, 0.6:1, 0.65:1,
0.70:1, 0.75:1, 0.8:1, 0.85:1, 0.9:1, 0.95:1, 1:1, 1.5:1, 2:1,
2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1,
8:1, 8.5:1 or the range between any two ratios above, and wherein
the therapeutic nanoparticles are substantially free of free HSA
that is not incorporated in the nanoparticles.
[0009] In certain embodiments, the weight ratio of human serum
albumin to the active ingredient is selected from 0.03:1, 0.04:1,
0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.10:1, 0.11:1, 0.12:1,
0.13:1, 0.14:1, 0.15:1, 0.16:1, 0.17:1, 0.18:1, 0.19:1, 0.2:1,
0.21:1, 0.22:1, 0.23:1, 0.24:1, 0.25:1, 0.26:1, 0.27:1, 0.28:1,
0.29:1, 0.3:1, 0.31:1, 0.32:1, 0.33:1, 0.34:1, 0.35:1, 0.36:1,
0.37:1, 0.38:1, 0.39:1, 0.4:1, 0.41:1, 0.42:1, 0.43:1, 0.44:1,
0.45:1, 0.46:1, 0.47:1, 0.48:1, 0.49:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1,
0.9:1, or the range between any two ratios above.
[0010] In certain embodiments, the nanoparticles contain at most 5%
free HSA (by weight).
[0011] In certain embodiments, the active ingredient has the
properties of being insoluble or slightly soluble in water, but
soluble or free soluble in an organic solvent.
[0012] In certain embodiments, the active ingredient is selected
from taxanes, macrolides, camptothecins anthracycline antibiotics,
colchicine, thiocolchicine dimer, amiodardone, liothyronine,
cyclosporine, exemestane, flutamide, fulvestrant, romidepsin,
semustine, and ibuprofen.
[0013] In certain embodiments, the active ingredient is a taxane,
such as paclitaxel or docetaxel.
[0014] In certain embodiments, the organic solvent is a pure
solvent having low water-solubility and low boiling point or its
mixture with small molecular alcohols.
[0015] In certain embodiments, the active ingredient is
encapsulated inside human serum albumin.
[0016] In certain embodiments, the average particle size of the
therapeutic nanoparticles is selected from: 30, 40, 50, 60, 70, 80,
90, 100, 110, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,
130, 131, 132, 133, 134, 135, 136, 137, 138, 140, 141, 142, 143,
144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,
157, 158, 159, 160, 165, 170, 175, 180, 185, 190, 195, 200 nm, or
the range between any two numerical values above.
[0017] In another aspect, the present disclosure provides purified
therapeutic nanoparticles that comprise an active ingredient and
human serum albumin, wherein the active ingredient is encapsulated
inside human serum albumin; the weight ratio of human serum albumin
to the active ingredient is from 0.03:1 to 0.7:1; the average
particle size of the therapeutic nanoparticles is from 50 nm to 190
nm; and the nanoparticles are substantially free of free HSA that
is not incorporated in the nanoparticles.
[0018] In another aspect, the present disclosure provides a
pharmaceutical composition, comprising the purified therapeutic
nanoparticles provided herein, wherein the composition is
substantially free of free HSA that is not incorporated in the
nanoparticles.
[0019] In certain embodiments, the pharmaceutical composition is in
the form of liquid or lyophilized powder.
[0020] In certain embodiments where the pharmaceutical composition
is in the form of lyophilized powder, it comprises one or more
lyophilization excipients, such as mannitol, sucrose, lactose,
maltose, trehalose, dextran, or a mixture thereof.
[0021] In certain embodiments, the pharmaceutical composition
contains at most 5% free HSA (by weight).
[0022] In another aspect, the present disclosure provides a method
for preparing the purified therapeutic nanoparticles provided
herein. The method comprises:
[0023] 1) dissolving the active ingredient in organic solvent to
form an oil phase, and dissolving human serum albumin in water to
form an aqueous phase;
[0024] 2) forming an oil-in-water emulsion using the oil phase and
aqueous phase above;
[0025] 3) removing the organic solvent in the emulsion to obtain a
suspension containing the therapeutic nanoparticles; and
[0026] 4) removing free HSA that is not incorporated in the
nanoparticles from the suspension to obtain purified therapeutic
nanoparticles.
[0027] In certain embodiments, the organic solvent is selected from
one or more of chloroform and ethanol.
[0028] In certain embodiments, the method further comprises:
between steps 3) and 4), a step of dialyzing the suspension of step
3) with an aqueous solution to remove remaining organic solvent
from the suspension.
[0029] In certain embodiments, the aqueous solution is water.
[0030] In certain embodiments, said separating in step 4) is
conducted using a method selected from: centrifugation, dialysis,
and exclusion chromatography.
[0031] In a related aspect, the present disclosure provides method
for preparing the pharmaceutical composition provided herein,
comprising:
[0032] 1) dissolving the active ingredient in organic solvent to
form an oil phase, and dissolving human serum albumin in water to
form an aqueous phase;
[0033] 2) forming an oil-in-water emulsion using the oil phase and
aqueous phase above;
[0034] 3) removing the organic solvent in the emulsion to obtain a
suspension containing the therapeutic nanoparticles;
[0035] 4) removing free HSA that is not incorporated in the
nanoparticles to obtain purified therapeutic nanoparticles;
[0036] 5) re-suspending the purified therapeutic nanoparticles in
an excipient-containing solution; and
[0037] 6) optionally lyophilizing the re-suspension of the purified
therapeutic nanoparticles to obtain the pharmaceutical
composition.
[0038] In certain embodiments, the method further comprises:
between steps 3) and 4), a step of dialyzing the suspension of step
3) with an aqueous solution to remove remaining organic solvent
from the suspension.
[0039] In certain embodiments, the aqueous solution is water.
[0040] In another related aspect, the present disclosure provides a
method for preparing the pharmaceutical composition provided
herein, comprising:
[0041] 1) dissolving the active ingredient in organic solvent to
form an oil phase, and dissolving human serum albumin in water to
form an aqueous phase;
[0042] 2) forming an oil-in-water emulsion using the oil phase and
aqueous phase above;
[0043] 3) removing the organic solvent in the emulsion to obtain a
suspension containing the therapeutic nanoparticles;
[0044] 4) dialyzing the suspension obtained after removal of the
organic solvent by an excipient-containing solution to remove free
HSA that is not incorporated in the nanoparticles; and
[0045] 5) optionally lyophilizing the dialyzed suspension to obtain
the pharmaceutical composition.
[0046] In certain embodiments, the method further comprises:
between steps 3) and 4), a step of dialyzing the suspension of step
3) with an aqueous solution to remove remaining organic solvent
from the suspension.
[0047] In certain embodiments, the aqueous solution is water.
[0048] In another aspect, the present disclosure provides a method
for treating cancer, comprising: administrating a therapeutically
effective amount of the pharmaceutical composition provided herein
to a subject in need thereof.
[0049] In another aspect, the present disclosure provides a
pharmaceutical composition, comprising the therapeutic
nanoparticles provided herein, wherein the composition further
comprises HSA as a lyophilization excipient.
BRIEF DESCRIPTION OF THE FIGURES
[0050] FIG. 1. Scanning electron microscopic image of the sample of
Example 5.
[0051] FIG. 2. Scanning electron microscopic image of the
nanoparticles purified from the sample of Example 5.
[0052] FIG. 3. X diffraction pattern of paclitaxel.
[0053] FIG. 4. X diffraction pattern of lyophilized powder of
albumin.
[0054] FIG. 5. X diffraction pattern of the paclitaxel-albumin
nanoparticles corresponding to Example 2.
[0055] FIG. 6. X diffraction pattern of the paclitaxel-albumin
nanoparticles corresponding to Example 4.
[0056] FIG. 7. X diffraction pattern of the paclitaxel-albumin
nanoparticles corresponding to Example 6.
[0057] FIG. 8. X diffraction pattern of the paclitaxel-albumin
nanoparticles corresponding to Example 8.
[0058] FIG. 9. In vitro release profiles of purified nanoparticles
of various formulations.
[0059] FIG. 10. In vitro release profiles of purified nanoparticles
and traditional formulations.
[0060] FIG. 11. Pharmacokinetic profiles of purified nanoparticles
and traditional formulations in dogs.
[0061] FIG. 12. Albumin concentration influences inhibition effect
of drugs on MCF-7 cells.
[0062] FIG. 13. Albumin concentration influences inhibition effect
of drugs on SPC-A-1 cells.
[0063] FIG. 14. Albumin concentration influences uptake of drugs by
human vascular endothelial cell EA.hy 926.
DETAILED DESCRIPTION
[0064] The present disclosure provides purified HSA-containing
therapeutic nanoparticles, compositions comprising such
nanoparticles and methods of preparing or using such nanoparticles
and compositions.
[0065] The purified therapeutic nanoparticles or the compositions
that comprising such nanoparticles have one or more of the
following superior properties:
[0066] (1) Compared with previously known compositions that
comprise HSA-containing nanoparticles, the purified therapeutic
nanoparticles or compositions thereof reduce allergic reactions in
subjects to which the nanoparticles or compositions are
administered (see, e.g., Examples 25, 27, and 61). While not
wishing to be bound by any hypothesis, it is proposed that the
reduction in allergic reactions may be resulted from the reduced
amount of HSA polymers in the purified nanoparticles or
compositions thereof. It was discovered by the present inventors
that during the preparation of nanoparticles, a portion of HSA
monomers formed HSA polymers, causing more severe allergic
reactions (see, e.g., Examples 32 and 60). Purification of
nanoparticles from the initial preparation eliminates most free HSA
that is not incorporated into nanoparticles, including free HSA
polymers.
[0067] (2) Compared with previously known compositions that
comprise HSA-containing nanoparticles, certain compositions
comprising purified therapeutic nanoparticles provided herein are
more stable (see e.g., Examples 62 and 63). This is unexpected in
view of the significant reduction in the ratio of HSA to active
ingredients in the purified nanoparticles and compositions of the
present disclosure and in view of the belief in the art that a
large amount of HSA is important or required for stabilizing
compositions that comprise nanoparticles.
[0068] (3) While maintaining in vitro release profiles, maximum
tolerated doses, pharmacokinetic properties, and effectiveness in
animal studies (see e.g., Examples 22 and 26-31), purified
therapeutic nanoparticles or compositions thereof provided herein
are easier to be delivered to or taken up by human target cells
(e.g., human tumor cells and human vascular endothelial cells) and
achieved better desirable effects in those cells (see e.g.,
Examples 58 and 59).
[0069] Unless otherwise defined, all scientific terms used herein
have the same meaning as commonly understood by one of ordinary
skills in the art to which this disclosure belongs.
[0070] Although the number ranges and approximate parameter values
are given in a broad range of the present disclosure, all numbers
in the specific examples are described as precise as possible.
However, certain error exists in any numerical values essentially,
which may be resulted from the standard deviation during the
measurement for each of them. Additionally, it should be understood
that all ranges disclosed herein encompass any and all possible
sub-ranges contained therein. For example, it should be understood
that the range "from 1 to 10" described herein encompasses any and
all possible sub-ranges between the minimum 1 and the maximum 10
(including the endpoints); i.e., all sub-ranges started from the
minimum 1 or more, e.g., 1 to 6.1, and all sub-ranges ended at the
maximum 10 or less, e.g., 5.5 to 10. Additionally, it should be
understood that any reference referred as "incorporated herein" is
incorporated in its entirety.
[0071] Additionally, it should be noted that unless otherwise
clearly and explicitly stated, the singular form includes the
plural referent, as used in the present disclosure. The term "or"
and the term "and/or" are used interchangeably, unless otherwise
clearly indicated in the context.
[0072] The term "nanoparticle" used herein refers to the particle
with at least one dimension (for example, 1, 2, or 3 dimensions) in
nano-scale, for example, at the level of about 1 nm, about 10 nm,
or about 100 nm.
[0073] The term "about" means more or less than 10% of a particular
value. For example, "about 50 nm" refers to 45 nm to 55 nm.
[0074] The term "therapeutic nanoparticle" used herein refers to
the nanoparticles that can be used for the treatment or prevention
of diseases, wherein the diseases, for example cancers, are
preferably selected from liver cancer, prostatic cancer and lung
cancer.
[0075] The term "human serum albumin monomer" or "HSA monomer" used
herein refers to the water soluble globulin composed of 585 amino
acids and the approximate molecular weight is around 66,000
Daltons. It is the most abundant protein in human blood plasma. The
retention time for human serum albumin monomer is the longest in
size exclusion chromatography and count for the majority in normal
human albumin products. HSA has multiple hydrophobic binding sites
which can bind a diverse set of drugs, especially neutral and
negatively charged hydrophobic compounds.
[0076] The term "human serum albumin polymer" or "HSA polymer" used
herein refers to the sum of polymers polymerized by human serum
albumin monomer, including dimer, trimer and polymer. The retention
time for human serum albumin polymer in size exclusion
chromatography is shorter than HSA monomers. HSA polymers are
present in only a small amount (typically<5%) in human albumin
products.
[0077] HSA is accumulated in various growing tumors and is used as
a source of energy and amino acids uptake by tumor cells. gp60
(albondin) is hyper-expressed in the endothelium of blood vessels
and binds HSA to carry it into the underlying tissue by
transcytosis. gp60 is not expressed in tissue cells and it is not
involved in the HSA transport in tissue cells. SPARC (secreted
protein, acidic and rich in cysteine), has the homologous amino
acids sequences with gp60 and can bind HSA and gp60 antibodies.
SPARC is over-expressed in multiple tumor types and many studies
manifest that SPARC correlates with the tissue cellular uptake of
HSA. There are researches suggest that HSA-bound drugs also cross
the endothelial barrier to the tumor tissue and internalize in
tumor cells via Gp60-SPARC transmembrane transport pathway.
[0078] The term "substantially pure nanoparticles" or "purified
nanoparticles" used herein refers to nanoparticles composed of
human serum albumin and an active ingredient where less than 10%
HSA (e.g., less than 9%, less than 8%, less than 7%, less than 6%,
less than 5%, less than 4%, less than 3%, less than 2%, less than
1%, less than 0.5%, or less than 0.1%) is free HSA.
[0079] Similarly, the term "substantially free of free human serum
albumin" or "substantially free of free HSA" used herein refers to
having less than 10% free HSA (e.g., less than 9%, less than 8%,
less than 7%, less than 6%, less than 5%, less than 4%, less than
3%, less than 2%, less than 1%, less than 0.5%, or less than
0.1%)
[0080] The term "free human serum albumin" or "free HSA" used
herein refers to HSA is not incorporated in nanoparticles. The
amount of free HSA in nanoparticles or compositions thereof may be
measured by methods known in the art or a method provided in the
present disclosure, such as in Examples 11 and 17-19.
[0081] The term "active ingredient" used herein refers to the
active pharmaceutical ingredient. Particularly, the active
ingredient refers to any substance or entity that can play a
therapeutic role (for example, the treatment, prevention,
alleviation or suppression of any disease and/or disorder).
[0082] The term "dialysis fold" used herein refers to the volume
ratio of dialysate consumed to a sample solution in a dialysis
process where the volume of the sample solution is kept
constant.
[0083] The term "lyophilization excipient" used herein refers to
compounds added to a pharmaceutical composition that comprises
purified therapeutic nanoparticles to maintain such nanoparticle
during the freeze-drying process.
[0084] The terms, "treat" and "treatment," refer to medical
management of a disease, disorder, or condition of a subject (i.e.,
patient) (see, e.g., Stedman's Medical Dictionary). "Treating
cancer" refers to reducing the number of symptoms of cancer,
decreasing the severity of one or more symptoms, or delaying cancer
progression.
[0085] A "therapeutically effective dose" of a specific therapeutic
agent refers to that amount of the agent sufficient to result in
reducing the severity of, eliminating, or delaying the onset or
reoccurrence of one or more symptoms of cancer in a statistically
significant manner.
[0086] In one aspect, the present disclosure provides purified
therapeutic nanoparticles that comprise an active ingredient and
HSA.
[0087] In some embodiments, the weight ratio of human serum albumin
to the active ingredient in the therapeutic nanoparticles is
selected from the group consisting of 0.01:1, 0.02:1, 0.04:1,
0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.10:1, 0.11:1, 0.12:1,
0.13:1, 0.14:1, 0.15:1, 0.16:1, 0.17:1, 0.18:1, 0.19:1, 0.2:1,
0.21:1, 0.22:1, 0.23:1, 0.24:1, 0.25:1, 0.26:1, 0.27:1, 0.28:1,
0.29:1, 0.3:1, 0.31:1, 0.32:1, 0.33:1, 0.34:1, 0.35:1, 0.36:1,
0.37:1, 0.38:1, 0.39:1, 0.4:1, 0.41:1, 0.42:1, 0.43:1, 0.44:1,
0.45:1, 0.46:1, 0.47:1, 0.48:1, 0.49:1, 0.5:1, 0.51:1, 0.52:1,
0.53:1, 0.54:1, 0.55:1, 0.56:1, 0.57:1, 0.58:1, 0.59:1, 0.6:1,
0.65:1, 0.70:1, 0.75:1, 0.8:1, 0.85:1, 0.9:1, 0.95:1, 1:1, 1.5:1,
2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1,
7.5:1, 8:1, 8.5:1 or the range between any two ratios above.
[0088] In some specific embodiments, the weight ratio of human
serum albumin to the active ingredient is selected from the group
consisting of 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1,
0.09:1, 0.10:1, 0.11:1, 0.12:1, 0.13:1, 0.14:1, 0.15:1, 0.16:1,
0.17:1, 0.18:1, 0.19:1, 0.2:1, 0.21:1, 0.22:1, 0.23:1, 0.24:1,
0.25:1, 0.26:1, 0.27:1, 0.28:1, 0.29:1, 0.3:1, 0.31:1, 0.32:1,
0.33:1, 0.34:1, 0.35:1, 0.36:1, 0.37:1, 0.38:1, 0.39:1, 0.4:1,
0.41:1, 0.42:1, 0.43:1, 0.44:1, 0.45:1, 0.46:1, 0.47:1, 0.48:1,
0.49:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1 or the range between any
two ratios above, for example, 0.03:1 to 0.19:1, or 0.21:1 to
0.9:1.
[0089] More particularly, in certain embodiments, the weight ratio
of human serum albumin to the active ingredient is 0.043:1,
0.071:1, 0.13:1, 0.15:1, 0.16:1, 0.17:1, 0.18:1, 0.24:1, or 0.57:1,
or the range between any two ratios above, for example, 0.043:1 to
0.071:1, 0.043:1 to 0.13:1, 0.043:1 to, 0.043:1 to 0.15:1, 0.043:1
to 0.16:1, 0.043:1 to 0.17:1, 0.043:1 to 0.18:1, 0.043:1 to 0.24:1,
0.043:1 to 0.57:1, 0.071:1 to 0.13:1, 0.071:1 to 0.15:1, 0.071:1 to
0.16:1, 0.071:1 to 0.17:1, 0.071:1 to 0.18:1, 0.071:1 to 0.24:1,
0.071:1 to 0.57:1, 0.13:1 to 0.15:1, 0.13:1 to 0.16:1, 0.13:1 to
0.17:1, 0.13:1 to 0.18:1, 0.13:1 to 0.24:1, 0.13:1 to 0.57:1,
0.15:1 to 0.16:1, 0.15:1 to 0.17:1, 0.15:1 to 0.18:1, 0.15:1 to
0.24:1, 0.15:1 to 0.57:1, 0.16:1 to 0.17:1, 0.16:1 to 0.18:1,
0.16:1 to 0.24:1, 0.16:1 to 0.57:1, 0.17:1 to 0.18:1, 0.17:1 to
0.24:1, 0.17:1 to 0.57:1, 0.18:1 to 0.24:1, 0.18:1 to 0.57:1 or
0.24:1 to 0.57:1.
[0090] In some embodiments, the active ingredient suitable for
encapsulation inside human serum albumin are insoluble or slightly
soluble in water, and soluble or freely soluble in an organic
solvent. The organic solvent may be a pure solvent with low water
solubility (i.e., water solubility less than 6%) and low boiling
point (i.e., boiling point lower than 80.degree. C.) or its mixture
of the above-described pure solvent with small molecular alcohols
including ethanol, tert-butanol, isopropanol, etc. The specific
organic solvents include but are not limit to chloroform,
dichloromethane, etc.
[0091] The active ingredient suitable for the present disclosure is
taxanes, including but not limited to, paclitaxel, docetaxel,
cabazitaxel, hydrophobic derivatives of docetaxel (e.g.,
2'-O-hexanoyldocetaxel, and 2'-benzoyldocetaxel); or macrolides,
including but not limited to rapamycin and its derivatives (e.g.,
temsirolimus and everolimus), epothilone B and its derivatives,
tanespimycin and its derivatives; or camptothecins, including but
not limited to 10-hydroxy camptothecin, SN-38 and its derivatives;
or anthracycline antibiotics, including but not limited to
aclacinomycin and pirarubicin; or other active ingredients
including colchicine and its derivatives, thiocolchicine dimer,
amiodardone, liothyronine, cyclosporine, exemestane, flutamide,
fulvestrant, romidepsin, semustine, ibuprofen, cyclosporine,
propofol, vinblastine, etc. In some embodiments, the active
ingredient is selected from one or more of paclitaxel and
docetaxel. In particular embodiments, the active ingredient is
paclitaxel. In some other embodiments, the active ingredient is
selected from docetaxel, rapamycin and its derivatives, exemestane,
flutamide, fulvestrant, etc.
[0092] In certain embodiments, the active ingredient includes but
is not limited to: chemotherapeutics, radiotherapeutic agents,
immunotherapeutic agents, and thermally therapeutic agents etc. For
example, aminoglutethimide, azathioprine, bleomycin sulphate,
busulfan, carmustine, chlorambucil, cisplatin, cyclophosphamide,
cyclosporine, dacarbazine, dactinomycin, daunorubicin, amycin,
paclitaxel, etoposide, fluorouracil, interferon-.alpha., lomustine,
mercaptopurine, methoptrexate, mitotane, procarbazine
hydrochloride, thioguanine, vinblastine sulfate and vincristine
sulfate.
[0093] In some embodiments, the average particle size of the
therapeutic nanoparticles is selected from 30, 50, 70, 120, 121,
122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,
135, 136, 137, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148,
149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 165,
170, 175, 180, 185, 190, 195, 200 nm, or the range between any two
numerical values above. It should be understood by the skilled
person in the art that the particle size can be determined using
any appropriate method that exists or will appear in the future,
which includes but is not limited to settling method, sieve method,
microscopic observation or laser particle sizer. It also should be
understood that when the therapeutic nanoparticles disclosed in the
present disclosure comprise multiple particles, not all therapeutic
nanoparticles should have the same particle size, and they will
also be encompassed in the scope of the present disclosure, as long
as their average particle size (i.e., the average particle
diameter) falls in the above range. In some specific embodiments,
the particle size is determined by a laser particle sizer; and the
average particle size of the therapeutic nanoparticles is selected
from 50, 60, 70, 80, 90, 100, 110, 120, 125, 126, 127, 128, 129,
130, 131, 132, 133, 134, 135, 136, 137, 138, 140, 141, 142, 143,
144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,
157, 158, 159, 160 nm, or the range between any two numerical
values above.
[0094] In some aspects, the particle sizes of the nanoparticles are
in a specific range, for example, from 30 nm to 200 nm, preferably,
from 50 to 190 nm. Preferably, the particle size is substantially
uniform.
[0095] In one specific embodiment, purified therapeutic
nanoparticles are provided, which comprise paclitaxel and human
serum albumin, wherein paclitaxel is encapsulated in human serum
albumin; and the weight ratio of human serum albumin to paclitaxel
is 0.043:1, and the average particle size of the therapeutic
nanoparticles is 140 nm.
[0096] In another specific embodiment, purified therapeutic
nanoparticles are provided, which comprise paclitaxel and human
serum albumin, wherein paclitaxel is encapsulated in human serum
albumin; and the weight ratio of human serum albumin to paclitaxel
is 0.071:1, and the average particle size of the therapeutic
nanoparticles is 134 nm.
[0097] In another specific embodiment, purified therapeutic
nanoparticles are provided, which comprise paclitaxel and human
serum albumin, wherein paclitaxel is encapsulated in human serum
albumin; and the weight ratio of human serum albumin to paclitaxel
is 0.13:1, and the average particle size of the therapeutic
nanoparticles is 125 nm.
[0098] In another specific embodiment, purified therapeutic
nanoparticles are provided, which comprise paclitaxel and human
serum albumin, wherein paclitaxel is encapsulated in human serum
albumin; and the weight ratio of human serum albumin to paclitaxel
is 0.15:1, and the average particle size of the therapeutic
nanoparticles is 136 nm.
[0099] In another specific embodiment, purified therapeutic
nanoparticles are provided, which comprise paclitaxel and human
serum albumin, wherein paclitaxel is encapsulated in human serum
albumin; and the weight ratio of human serum albumin to paclitaxel
is 0.16:1, and the average particle size of the therapeutic
nanoparticles is 133 nm.
[0100] In another specific embodiment, purified therapeutic
nanoparticles are provided, which comprise paclitaxel and human
serum albumin, wherein paclitaxel is encapsulated in human serum
albumin; and the weight ratio of human serum albumin to paclitaxel
is 0.17:1, and the average particle size of the therapeutic
nanoparticles is 136 nm.
[0101] In another specific embodiment, purified therapeutic
nanoparticles are provided, which comprise paclitaxel and human
serum albumin, wherein paclitaxel is encapsulated in human serum
albumin; and the weight ratio of human serum albumin to paclitaxel
is 0.18:1, and the average particle size of the therapeutic
nanoparticles is 138 nm.
[0102] In another specific embodiment, purified therapeutic
nanoparticles are provided, which comprise paclitaxel and human
serum albumin, wherein paclitaxel is encapsulated in human serum
albumin; and the weight ratio of human serum albumin to paclitaxel
is 0.24:1, and the average particle size of the therapeutic
nanoparticles is 141 nm.
[0103] In another specific embodiment, purified therapeutic
nanoparticles are provided, which comprise paclitaxel and human
serum albumin, wherein paclitaxel is encapsulated in human serum
albumin; and the weight ratio of human serum albumin to paclitaxel
is 0.57:1, and the average particle size of the therapeutic
nanoparticles is 145 nm.
[0104] In another specific embodiment, purified therapeutic
nanoparticles are provided, which comprise docetaxel and human
serum albumin, wherein docetaxel is encapsulated in human serum
albumin; and the weight ratio of human serum albumin to docetaxel
is 0.1:1, and the particle size of the therapeutic nanoparticles is
in the range from 110 nm to 150 nm.
[0105] Purified therapeutic nanoparticles of the present disclosure
are substantially free of free HSA. In certain embodiments,
purified therapeutic nanoparticles contain at most 8%, 7%, 6%, 5%,
4%, 3%, 2%, 1%, 0.5% or 0.1% free HSA by weight (i.e., at most 8%,
7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of total HSA in the
purified therapeutic nanoparticles is free HSA that does not bind
an active ingredient and is not incorporated into
nanoparticles.
[0106] In certain embodiments, purified therapeutic nanoparticles
consist essentially of an active ingredient and HSA where
substantially all of the HSA (i.e., more than 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of HSA) are bound to
the active ingredient.
[0107] In certain embodiments, purified therapeutic nanoparticles
comprise a minimum amount (e.g., less than 0.05, 0.04, 0.03, 0.02,
or 0.01 mg/ml, or less than 5, 1, 0.5, 0.1, 0.05, or 0.01 .mu.g/ml)
of one or more organic solvents used in preparing such
nanoparticles.
[0108] In certain embodiments, purified therapeutic nanoparticles
do not comprise any surfactants.
[0109] In certain embodiments, purified nanoparticles provided
herein have a relatively high zeta potential, such as from -20 to
-45 or from -25 to -40.
[0110] In another aspect, the present disclosure provides a
pharmaceutical composition that comprises purified therapeutic
nanoparticles provided herein and is substantially free of free HSA
that is not incorporated in the nanoparticles. In certain
embodiments, the pharmaceutical composition comprises less than
10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% free
HSA.
[0111] In some embodiments, the pharmaceutical composition is
provided in the form of liquid, including but not limited to, the
form suitable for injection to a subject. In one specific
embodiment, the pharmaceutical composition is prepared into an
injection.
[0112] In some other embodiments, the pharmaceutical composition is
provided in the form of solid, including, but not limited to, dry
powder or lyophilized powder.
[0113] In certain specific embodiments, the nanoparticle or the
pharmaceutical composition of the present disclosure is free of
deferoxamine or the salt thereof, for example, deferoxamine
mesylate.
[0114] In certain specific embodiments, the purified nanoparticles
or the pharmaceutical compositions of the present disclosure are
free of additional stabilizer.
[0115] When provided in a liquid form, the pharmaceutical
composition comprises the therapeutic nanoparticles of the present
disclosure and a pharmaceutically acceptable carrier. The
therapeutic nanoparticles are suspended in the pharmaceutically
acceptable carrier. The pharmaceutically acceptable carrier
includes but is not limited to a buffer solution, a preservative
agent, water for injection, normal saline, and an isotonic
solution. In some specific embodiments, the concentration of the
active ingredient of therapeutic nanoparticles is 1-10 mg/ml (e.g.,
5 mg/ml) in the pharmaceutical composition in a liquid form.
[0116] When provided in a solid form, the pharmaceutical
composition comprises, consists essentially of, or consists of: the
therapeutic nanoparticles of the present disclosure and a
lyophilization excipient. In some specific embodiments, the
lyophilization excipient is selected from one or more of mannitol,
sucrose, lactose, maltose, trehalose, and dextran. In certain
specific embodiments, the therapeutic nanoparticles are suspended
in a solution of lyophilization excipient with the concentration
from 5% to 10%, and subsequently the solution is lyophilized to
obtain a pharmaceutical composition in a form of lyophilized
powder. In particular embodiments, the solution of lyophilization
excipient is selected from one or more of 5% mannitol solution, 10%
sucrose solution, 5% dextran solution, 10% lactose solution, 10%
trehalose solution, and 10% maltose solution. In some specific
embodiments, the content of the therapeutic nanoparticles in the
pharmaceutical composition in a solid form is from 4.8% to 10% by
weight.
[0117] The method for preparing purified therapeutic nanoparticles
of the present disclosure is also provided, comprising: (1)
preparing a suspension containing therapeutic nanoparticles by
mixing an active ingredient with human serum albumin, and (2)
purifying nanoparticles from the suspension to obtain substantially
pure therapeutic nanoparticles.
[0118] In another aspect, the present disclosure also provides
therapeutic nanoparticles obtained by the method provided
herein.
[0119] In another aspect, a method for preparing purified
therapeutic nanoparticles is provided. The method comprises:
[0120] 1) dissolving the active ingredient in organic solvent to
form an oil phase, and dissolving human serum albumin in water to
form an aqueous phase;
[0121] 2) forming an oil-in-water emulsion using the oil phase and
aqueous phase;
[0122] 3) removing the organic solvent in the emulsion to obtain a
suspension containing the therapeutic nanoparticles;
[0123] 4) removing free HSA that is not incorporated in the
nanoparticles from the suspension to obtain purified therapeutic
nanoparticles.
[0124] Appropriate organic solvent(s) can be selected by the
skilled person based on the properties of the active ingredient. In
some specific embodiments, the suitable organic solvent is
chloroform, ethanol or a mixture of chloroform and ethanol when the
active ingredient is taxanes. More particularly, the suitable
organic solvent is a mixture of chloroform and ethanol when the
active ingredient is paclitaxel or docetaxel. In some specific
embodiments, the volume ratio between chloroform and ethanol is in
the range from 1:1 to 20:1, and for example, it is selected from
1:1, 4:1, 9:1 and 11:1. In some specific embodiments, the
concentration of human serum albumin in the aqueous phase is in the
range from 2% to 10% (w/v); for example, it is selected from 2%,
4%, 5% and 10%. In some embodiments, the ratio between the active
ingredient and the organic solvent in the oil phase is in the range
from 0.3-7.5 g/15-20 ml. In some specific embodiments, the ratio
between the active ingredient and the organic solvent in the oil
phase is selected from: 0.3 g/15 ml, 0.6 g/15 ml, 1 g/20 ml, 1.25
g/15 ml, 1.8 g/15 ml, 2 g/15 ml, 2.5 g/15 ml, 3 g/20 ml, 3 g/15 ml,
5 g/15 ml, 7.5 g/15 ml, or the range between any two numerical
values above.
[0125] When an oil-in-water emulsion is formed with the oil phase
and the aqueous phase, the volume ratio between the two phases is
selected from 1:10 to 1:100. In some embodiments, the mixing ratio
of the oil phase and the aqueous phase is 3:100 or 1:25. An
oil-in-water emulsion is formed using the method known in the art,
which includes, but not limited to, homogenization. In particular
embodiments, the mixture of the oil phase and the aqueous phase is
emulsified using a high shear disperser, and subsequently, it is
homogenized using a high pressure homogenizer, so as to obtain an
oil-in-water emulsion. In particular embodiments, the mixture of
the oil phase and the aqueous phase is emulsified for 2-10 min
using a high shear disperser, and subsequently, it is homogenized
using a high pressure homogenizer under the pressure of 10000-20000
psi, so as to obtain an oil-in-water emulsion.
[0126] The organic solvent can be removed from the emulsion using
any appropriate method. In some embodiments, the organic solvent is
removed from the emulsion using rotary vacuum evaporation. In
particular embodiments, the organic solvent is removed from the
emulsion using a rotary evaporator at 40.degree. C. under the
pressure of 40 mbar. After removal of the organic solvent, the
resulting suspension comprises the therapeutic nanoparticles of the
present disclosure. However, excessive albumin, which does not
participate in the formation of the nanoparticle, is also contained
in the suspension.
[0127] Excessive free albumin is further removed from the
suspension to obtain purified therapeutic nanoparticles of the
present disclosure that are substantially free of free HSA. In some
embodiments, this is conducted using centrifugation, dialysis, or
exclusion chromatography. In particular embodiments, the suspension
can be directly used for removing free HSA after removal of the
organic solvent. Alternatively, it can be stored for further
use.
[0128] Since intravenous infusion is the desirable administration
route for the therapeutic nanoparticles of the present disclosure,
the product must be sterile. Heat sterilizing is not applicable in
the present disclosure, since both the nanoparticles and human
serum albumin are sensitive to temperature. Thus feasible
sterilization methods include aseptic production or sterilization
filtration. In such cases, after removal of the organic solvent,
the suspension is sterilized by passing through a filter, and
subsequently, it is lyophilized to obtain a solid. The solid
obtained is re-suspended in sodium chloride solution. In some
embodiments, the solid is re-suspended in 0.9% sodium chloride
solution to reach a paclitaxel concentration of about 5 mg/ml.
[0129] When the therapeutic nanoparticles are purified using
centrifugation, removal of free HSA is performed at 21000.times.g
for 60 min or at equivalent conditions.
[0130] When the therapeutic nanoparticles are purified by dialysis,
the nanoparticle-containing suspension obtained in step 3) is
dialyzed using an ultrafiltration membrane to remove free HSA. In
particular embodiments, the nanoparticle liquid obtained according
to the method of the present disclosure is dialyzed in equal volume
of 5% mannitol solution using a regenerated cellulose
ultrafiltration membrane with the cutoff molecular weight of 300K,
and the dialysis fold is 5.
[0131] When therapeutic nanoparticles are purified using exclusion
chromatography, therapeutic nanoparticles are separated from free
HSA using an exclusion chromatographic column. In particular
embodiments, nanoparticle-containing suspension obtained in step 3)
is applied onto a sepharose column, and the eluting peak
corresponding to therapeutic nanoparticles is collected.
[0132] In certain embodiments, the method further comprises between
steps 3) and 4) a step of dialyzing the suspension of step 3) with
an aqueous solution (e.g., water or 5% mannitol solution, 10%
sucrose solution, 5% dextran solution, 10% lactose solution, 10%
trehalose solution, and 10% maltose solution, etc.) to remove the
remaining organic solvent from the suspension. For example, the
suspension may be dialyzed in water or an aqueous solution using an
ultrafiltration membrane that allows the organic solvent to pass
but not HSA or nanoparticles (e.g., a cellulose ultrafiltration
membrane with a cutoff molecular weight of 30K). While not wishing
to be bound by any theory, the removal or reduction of the
remaining organic solvent prior to removal of free HSA from the
nanoparticle-containing suspension may improve stability of
purified nanoparticles or compositions thereof.
[0133] In another aspect, a method for preparing a pharmaceutical
composition comprising the therapeutic nanoparticles is also
provided. The method comprises:
[0134] 1) dissolving the active ingredient in organic solvent to
form an oil phase, and dissolving human serum albumin in water to
form an aqueous phase;
[0135] 2) forming an oil-in-water emulsion using the oil phase and
aqueous phase;
[0136] 3) removing the organic solvent in the emulsion to obtain a
suspension containing the therapeutic nanoparticles;
[0137] 4) removing free HSA that is not incorporated in the
nanoparticles to obtain purified therapeutic nanoparticles;
[0138] 5) re-suspending the purified therapeutic nanoparticles in a
pharmaceutically acceptable carrier-containing solution to obtain
the pharmaceutical composition; and
[0139] 6) optionally lyophilizing the re-suspension of the purified
therapeutic nanoparticles where the pharmaceutical composition is
in the form of solid.
[0140] Steps 1) and 4) of this method are the same as described
with respect to the method of preparing purified therapeutic
nanoparticles. In addition, in certain embodiments, the method
comprises between steps 3) and 4) a step of dialyzing the
suspension of step 3) with an aqueous solution to remove the
remaining organic solvent from the suspension also as described
with respect to the method of preparing purified therapeutic
nanoparticles.
[0141] In some embodiments, the pharmaceutically acceptable
carrier-containing solution is the solution containing
lyophilization excipient. In some specific embodiments, the
lyophilization excipient is selected from one or more of mannitol,
sucrose, lactose, maltose, trehalose, and dextran. In some other
specific embodiments, the lyophilization excipient is HSA. In
specific embodiments, the therapeutic nanoparticles are suspended
in a solution of lyophilization excipient at the concentration from
5% to 10%.
[0142] Optionally, the pharmaceutical composition is prepared into
lyophilized powder after lyophilization. In particular embodiments,
the solution of lyophilization excipient is selected from one or
more of 5% mannitol solution, 10% sucrose solution, 5% dextran
solution, 10% lactose solution, 10% trehalose solution, 10% maltose
solution, and 10% human serum albumin solution.
[0143] In some specific embodiments, the content of the therapeutic
nanoparticles in the pharmaceutical composition in liquid form is
from 0.1% to 30%, and preferably, from 0.2% to 10%, and more
preferably, from 0.5% to 5%, for example, 1%. In some specific
embodiments, the content of the active ingredient (for example,
paclitaxel) in the pharmaceutical composition in liquid form of the
present disclosure is from 0.1 to 100 mg/ml, and preferably, from
0.5 to 50 mg/ml, and more preferably, from 1 to 20 mg/ml, for
example, 5 mg/ml.
[0144] In certain embodiments, the content of the therapeutic
nanoparticles in the pharmaceutical composition in solid form is
from 0.1% to 80% by weight, and preferably, from 0.5% to 50%, and
more preferably, from 1% to 30%, for example, from 2% to 10%.
[0145] In some specific embodiments, the content of the active
pharmaceutical ingredient (for example, paclitaxel) in the
pharmaceutical composition in liquid form is from 0.1% to 80% by
weight, and preferably, from 0.5% to 50%, and more preferably, from
1% to 30%, for example, from 2% to 10%.
[0146] Alternatively, the pharmaceutical composition of the present
disclosure can be prepared using another procedure of dialysis. The
method comprises:
[0147] 1) dissolving the active ingredient in organic solvent to
form an oil phase, and dissolving human serum albumin in water to
form a aqueous phase;
[0148] 2) forming an oil-in-water emulsion using the oil phase and
aqueous phase above;
[0149] 3) removing the organic solvent in the emulsion to obtain a
suspension containing the therapeutic nanoparticles;
[0150] 4) dialyzing the suspension obtained after removal of the
organic solvent by a pharmaceutically acceptable carrier-containing
solution to remove free HSA that is not incorporated in the
nanoparticles; and
[0151] 5) optionally lyophilizing the dialyzed suspension when the
pharmaceutical composition is prepared in the form of solid.
[0152] Steps 1) and 4) of this method are the same as described
with respect to the method of preparing purified therapeutic
nanoparticles when dialysis is used to remove free HSA. In
addition, in certain embodiments, the method comprises between
steps 3) and 4) a step of dialyzing the suspension of step 3) with
an aqueous solution to remove the remaining organic solvent from
the suspension also as described with respect to the method of
preparing purified therapeutic nanoparticles.
[0153] In some embodiments, a pharmaceutically acceptable
carrier-containing solution is used as the dialysate, which is the
lyophilization excipient-containing solution. In some specific
embodiments, the lyophilization excipient is selected from one or
more of mannitol, sucrose, lactose, maltose, trehalose, and
dextran.
[0154] In specific embodiments, the therapeutic nanoparticles are
suspended in a solution of lyophilization excipient with the
concentration from 5% to 10%. Optionally, the pharmaceutical
composition is prepared into lyophilized powder after
lyophilization. In particular embodiments, the solution of
lyophilization excipient is selected from one or more of 5%
mannitol solution, 10% sucrose solution, 5% dextran solution, 10%
lactose solution, 10% trehalose solution, 10% maltose solution, and
10% human serum albumin solution. In some embodiments, dialysis is
performed using an ultrafiltration membrane with cutoff molecular
weight of 300 k.
[0155] In another aspect, the present disclosure provides methods
for using the purified therapeutic nanoparticles or compositions
thereof. Because the present purified nanoparticles or compositions
are effective means for delivering various active ingredients, they
may be used for treating any diseases or disorders that are
responsive to the active ingredients. For example, the purified
therapeutic nanoparticles or compositions thereof may be used in
treating cancer, such as liver cancer, prostatic cancer and lung
cancer. Additional diseases or disorders that may be treated
include breast cancer, multiple myeloma, transplant rejection,
colon cancer, lymphoma, fever, etc.
[0156] In a particular aspect, the present disclosure provides a
method for treating cancer that comprises administering a
therapeutically effective amount of a pharmaceutical composition
provided herein to a subject in need thereof. In specific
embodiments, the subject is a mammal, including but not limited to
human, canine, mouse, and rat.
[0157] A therapeutically effective amount of a pharmaceutical
composition may be determined or adjusted depending on various
factors including the specific therapeutic agents or pharmaceutical
compositions, the routes of administration, the subject's
condition, that is, stage of the disease, severity of symptoms
caused by the disease, general health status, as well as age,
gender, and weight, and other factors apparent to a person skilled
in the medical art. Similarly, the dose of the therapeutic for
treating a disease or disorder may be determined according to
parameters understood by a person skilled in the medical art.
Optimal doses may generally be determined using experimental models
and/or clinical trials.
[0158] The pharmaceutical composition may be administered via
through any suitable routes, for example, oral, nasal,
intracutaneous, subcutaneous, intramuscular or intravenous
administration.
[0159] In another aspect, a pharmaceutical kit is also provided in
the present disclosure, which comprises purified therapeutic
nanoparticles or a pharmaceutical composition thereof provided
herein. If required, the pharmaceutical kit also comprises
instruction, package, and a container holding the therapeutic
nanoparticles or the pharmaceutical composition.
EXAMPLES
[0160] The Examples below were intended to better illustrate the
therapeutic nanoparticles and the pharmaceutical composition
disclosed herein, and not to limit any aspect of the present
disclosure.
Example 1
[0161] 3 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech Co.,
Ltd) was dissolved into 20 ml chloroform/ethanol (9:1, v/v), and
added into 500 ml human serum albumin solution (5% w/v) (CAS:
70024-90-7, Guangdong Shuanglin Biopharmaceutical. Co., Ltd.). The
mixture was emulsified for 2 min using a high shear disperser
(Model F22E, Fluko Co., Ltd., Shanghai) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer (Model M110-EH30K, MFIC Company, USA) under
pressure of 10000-20000 psi to obtain a nano-emulsion.
Subsequently, the nano-emulsion was transferred to a rotatory
evaporator (Model R-210, Buchi Company, Switzerland) to remove the
organic solvent in the solution by vacuum evaporation at 40 mbar
and at 40.degree. C. in a water-bath. The paclitaxel-albumin
nanoparticles were thus generated with an average diameter of 136
nm, and the suspension was translucent.
[0162] The suspension can be smoothly filtered through a 0.22 .mu.m
sterile filter (Sartorius AG, Germany). There was no significant
variation of the particle size after filtration, and no significant
change was observed after storage for 48 h at room temperature. The
suspension was aliquoted and lyophilized for 24 h in a lyophilizer
(Model LD-85S, Millrock, USA) to obtain a stable off-white
cake.
Example 2
[0163] 0.32 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech
Co., Ltd) was dissolved into 15 ml chloroform/ethanol (11:1, v/v),
and added into which 500 ml human serum albumin solution (4% w/v)
(CAS: 70024-90-7, Guangdong Shuanglin Biopharmaceutical. Co.,
Ltd.). The mixture was emulsified for 2 min using a high shear
disperser (Model F22E, Fluko Co., Ltd., Shanghai) to obtain a
primary emulsion. The primary emulsion was then homogenized using a
high pressure homogenizer (Model M110-EH30K, MFIC Company, USA)
under a pressure of 10000-20000 psi to obtain a nano-emulsion.
Subsequently, the nano-emulsion was transferred to a rotatory
evaporator (Model R-210, Buchi Company, Switzerland) to remove the
organic solvent in the solution by vacuum evaporation at 40 mbar
and at 40.degree. C. in a water-bath. The paclitaxel-albumin
nanoparticles were thus generated with an average diameter of 145
nm, and the suspension was translucent.
[0164] The suspension can be smoothly filtered through a 0.22 .mu.m
sterile filter (Sartorius AG, Germany). There was no significant
variation of the particle size after filtration, and no significant
change was observed after storage for 48 h at room temperature. The
suspension was aliquoted and lyophilized for 24 h in a lyophilizer
(Model LD-85S, Millrock, USA) to obtain a stable off-white
cake.
Example 3
[0165] 0.63 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech
Co., Ltd) was dissolved into 15 ml chloroform/ethanol (11:1, v/v),
and added into 500 ml human serum albumin solution (4% w/v) (CAS:
70024-90-7, Guangdong Shuanglin Biopharmaceutical. Co., Ltd.). The
mixture was emulsified for 2 min using a high shear disperser
(Fluko FZ-20) to obtain a primary emulsion. The primary emulsion
was then homogenized using a high pressure homogenizer (Model
M110-EH30K, MFIC Company, USA) under a pressure of 10000-20000 psi
to obtain a nano-emulsion. Subsequently, the nano-emulsion was
transferred to a rotatory evaporator (Model R-210, Buchi Company,
Switzerland) to remove the organic solvent in the solution by
vacuum evaporation at 40 mbar and at 40.degree. C. in a water-bath.
The paclitaxel-albumin nanoparticles were thus generated with an
average diameter of 141 nm, and the suspension was translucent.
[0166] The suspension can be smoothly filtered through a 0.22 .mu.m
sterile filter (Sartorius AG, Germany). There was no significant
variation of the particle size after filtration, and no significant
change was observed after storage for 48 h at room temperature. The
suspension was aliquoted and lyophilized for 24 h in a lyophilizer
(Model LD-85S, Millrock, USA) to obtain a stable off-white
cake.
Example 4
[0167] 1.25 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech
Co., Ltd) was dissolved into 15 ml chloroform/ethanol (11:1, v/v),
and added into which 500 ml human serum albumin solution (4% w/v)
(CAS: 70024-90-7, Guangdong Shuanglin Biopharmaceutical. Co.,
Ltd.). The mixture was emulsified for 2 min using a high shear
disperser (Model F22Z, Fluko Co., Ltd., Shanghai) to obtain a
primary emulsion. The primary emulsion was then homogenized using a
high pressure homogenizer (Model M110-EH30K, MFIC Company, USA)
under a pressure of 10000-20000 psi to obtain a nano-emulsion.
Subsequently, the nano-emulsion was transferred to a rotatory
evaporator (Model R-210, Buchi Company, Switzerland) to remove the
organic solvent in the solution by vacuum evaporation at 40 mbar
and at 40.degree. C. in a water-bath. The paclitaxel-albumin
nanoparticles were thus generated with an average diameter of 138
nm, and the suspension was translucent.
[0168] The suspension can be smoothly filtered through a 0.22 .mu.m
sterile filter (Sartorius AG, Germany). There was no significant
variation of the particle size after filtration, and no significant
change was observed after storage for 48 h at room temperature. The
suspension was aliquoted and lyophilized for 24 h in a lyophilizer
(Model LD-85S, Millrock, USA) to obtain a stable off-white
cake.
Example 5
[0169] 1.88 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech
Co., Ltd) was dissolved into 15 ml chloroform/ethanol (11:1, v/v),
and added into which 500 ml human serum albumin solution (4% w/v)
(CAS: 70024-90-7, Guangdong Shuanglin Biopharmaceutical. Co.,
Ltd.). The mixture was emulsified for 2 min using a high shear
disperser (Fluko FZ-20) to obtain a primary emulsion. The primary
emulsion was then homogenized using a high pressure homogenizer
(Model M110-EH30K, MFIC Company, USA) under a pressure of
10000-20000 psi to obtain a nano-emulsion. Subsequently, the
nano-emulsion was transferred to a rotatory evaporator (Model
R-210, Buchi Company, Switzerland) to remove the organic solvent in
the solution by vacuum evaporation at 40 mbar and at 40.degree. C.
in a water-bath. The paclitaxel-albumin nanoparticles were thus
generated with an average diameter of 133 nm, and the suspension
was translucent.
[0170] The suspension can be smoothly filtered through a 0.22 .mu.m
sterile filter (Sartorius AG, Germany). There was no significant
variation of the particle size after filtration, and no significant
change was observed after storage for 48 h at room temperature. The
suspension was aliquoted and lyophilized for 24 h in a lyophilizer
(Model LD-85S, Millrock, USA) to obtain a stable off-white
cake.
Example 6
[0171] 2.5 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech
Co., Ltd) was dissolved into 15 ml chloroform/ethanol (11:1, v/v),
and added into which 500 ml human serum albumin solution (4% w/v)
(CAS: 70024-90-7, Guangdong Shuanglin Biopharmaceutical. Co.,
Ltd.). The mixture was emulsified for 2 min using a high shear
disperser (Fluko FZ-20) to obtain a primary emulsion. The primary
emulsion was then homogenized using a high pressure homogenizer
(Model M110-EH30K, MFIC Company, USA) under a pressure of
10000-20000 psi to obtain a nano-emulsion. Subsequently, the
nano-emulsion was transferred to a rotatory evaporator (Model
R-210, Buchi Company, Switzerland) to remove the organic solvent in
the solution by vacuum evaporation at 40 mbar and at 40.degree. C.
in a water-bath. The paclitaxel-albumin nanoparticles were thus
generated with an average diameter of 125 nm, and the suspension
was translucent.
[0172] The suspension can be smoothly filtered through a 0.22 .mu.m
sterile filter (Sartorius AG, Germany). There was no significant
variation of the particle size after filtration, and no significant
change was observed after storage for 48 h at room temperature. The
suspension was aliquoted and lyophilized for 24 h in a lyophilizer
(Model LD-85S, Millrock, USA) to obtain a stable off-white
cake.
Example 7
[0173] 5 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech Co.,
Ltd) was dissolved into 15 ml chloroform/ethanol (11:1, v/v), and
added into which 500 ml human serum albumin solution (4% w/v) (CAS:
70024-90-7, Guangdong Shuanglin Biopharmaceutical. Co., Ltd.). The
mixture was emulsified for 2 min using a high shear disperser
(Fluko FZ-20) to obtain a primary emulsion. The primary emulsion
was then homogenized using a high pressure homogenizer (Model
M110-EH30K, MFIC Company, USA) under a pressure of 10000-20000 psi
to obtain a nano-emulsion. Subsequently, the nano-emulsion was
transferred to a rotatory evaporator (Model R-210, Buchi Company,
Switzerland) to remove the organic solvent in the solution by
vacuum evaporation at 40 mbar and at 40.degree. C. in a water-bath.
The paclitaxel-albumin nanoparticles were thus generated with an
average diameter of 134 nm, and the suspension was translucent.
[0174] The suspension can be smoothly filtered through a 0.22 .mu.m
sterile filter (Sartorius AG, Germany). There was no significant
variation of the particle size after filtration, and no significant
change was observed after storage for 48 h at room temperature. The
suspension was aliquoted and lyophilized for 24 h in a lyophilizer
(Model LD-85S, Millrock, USA) to obtain a stable off-white
cake.
Example 8
[0175] 7.5 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech
Co., Ltd) was dissolved into 15 ml chloroform/ethanol (11:1, v/v),
and added into which 500 ml human serum albumin solution (4% w/v)
(CAS: 70024-90-7, Guangdong Shuanglin Biopharmaceutical. Co.,
Ltd.). The mixture was emulsified for 2 min using a high shear
disperser (Fluko FZ-20) to obtain a primary emulsion. The primary
emulsion was then homogenized using a high pressure homogenizer
(Model M110-EH30K, MFIC Company, USA) under a pressure of
10000-20000 psi to obtain a nano-emulsion. Subsequently, the
nano-emulsion was transferred to a rotatory evaporator (Model
R-210, Buchi Company, Switzerland) to remove the organic solvent in
the solution by vacuum evaporation at 40 mbar and at 40.degree. C.
in a water-bath. The paclitaxel-albumin nanoparticles were thus
generated with an average diameter of 140 nm, and the suspension
was translucent.
[0176] The suspension can be smoothly filtered through a 0.22 .mu.m
sterile filter (Sartorius AG, Germany). There was no significant
variation of the particle size after filtration, and no significant
change was observed after storage for 48 h at room temperature. The
suspension was aliquoted and lyophilized for 24 h in a lyophilizer
(Model LD-85S, Millrock, USA) to obtain a stable off-white
cake.
Example 9
[0177] 1 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech Co.,
Ltd) was dissolved into 20 ml chloroform/ethanol (4:1, v/v), and
added into which 500 ml human serum albumin solution (2% w/v) (CAS:
70024-90-7, Guangdong Shuanglin Biopharmaceutical. Co., Ltd.). The
mixture was emulsified for 2 min using a high shear disperser
(Fluko FZ-20) to obtain a primary emulsion. The primary emulsion
was then homogenized using a high pressure homogenizer (Model
M110-EH30K, MFIC Company, USA) under a pressure of 10000-20000 psi
to obtain a nano-emulsion. Subsequently, the nano-emulsion was
transferred to a rotatory evaporator (Model R-210, Buchi Company,
Switzerland) to remove the organic solvent in the solution by
vacuum evaporation at 40 mbar and at 40.degree. C. in a water-bath.
The paclitaxel-albumin nanoparticles were thus generated with an
average diameter of 136 nm, and the suspension was translucent.
[0178] The suspension can be smoothly filtered through a 0.22 .mu.m
sterile filter (Sartorius AG, Germany). There was no significant
variation of the particle size after filtration, and no significant
change was observed after storage for 48 h at room temperature. The
suspension was aliquoted and lyophilized for 24 h in a lyophilizer
(Model LD-85S, Millrock, USA) to obtain a stable off-white
cake.
Example 10
Determination Methods for the Content of Human Serum Albumin and
Paclitaxel
[0179] The content of human serum albumin was determined by HPLC.
The human serum albumin was determined at a wavelength of 228 nm in
an HPLC equipped with a Tosohaas TSK G3000 SWXL gel column and a
UV-detector (1260VWD G1314B, Agilent technologies), with a mobile
phase of 0.1 mol/L dipotassium hydrogen phosphate solution and an
injection volume of 10 .mu.l. The albumin content was calculated
using the external standard method.
[0180] Preparation of the test solutions: the test solutions were
prepared by diluting the solution for determination using 0.9%
sodium chloride solution to an albumin concentration lower than 3
mg/ml.
[0181] The content of paclitaxel was determined by HPLC. The
paclitaxel was determined at a wavelength of 228 nm in an HPLC
equipped with a C18 reverse phase column and a UV-detector (1260VWD
G1314B, Agilent technologies), with a mobile phase of
acetonitrile-water (1:1, v/v) and an injection volume of 10 .mu.l.
The paclitaxel content was calculated using the external standard
method.
[0182] Preparation of the test solutions: the test solutions were
prepared by diluting the solution for determination using
acetonitrile until full dissolution of paclitaxel, with the
concentration of 20-200 .mu.g/ml.
Example 11
[0183] The product obtained in Example 1 was reconstituted in a
0.9% sodium chloride solution to obtain sample 1, with the
paclitaxel content of 5 mg/ml. Subsequently, sample 1 was diluted
using simulated blood plasma containing 5% albumin, so that the
paclitaxel content may reach 20 .mu.g/ml to obtain sample 2
(particles were completely disintegrated under such condition, and
no paclitaxel-human serum albumin bound particles exist). 1 ml
sample 1 and 1 ml sample 2 were centrifuged at 21000.times.g for
various durations, respectively. The concentrations of paclitaxel
and albumin in the supernatant were determined using the methods
mentioned in Example 10, and the results are listed in Table 1.
TABLE-US-00001 TABLE 1 The concentrations of paclitaxel and albumin
in the supernatant under different centrifugation durations Sample
1 Sample 2 Centrifugation 5 mg/ml solution 20 .mu.g/ml solution
duration Albumin Paclitaxel Albumin Paclitaxel (min) (mg/ml) %
(mg/ml) % (mg/ml) % (mg/ml) % 0 42.8 100.0* 5.01 100.0* 52.7 100.0*
0.0217 100.0* 20 44.2 103.2 0.78 15.6 53.1 100.8 0.0218 99.5 40
45.2 105.6 0.38 7.7 52.2 99.0 0.0220 101.3 80 44.9 104.9 0.11 2.1
53.5 101.6 0.0218 100.6 80 45.3** 105.8 0.11 2.1 53.5 101.5 0.0219
100.9 Note*: the percentage contents of albumin and paclitaxel in
supernatant were both calculated based on the concentration at 0
min (100%). Note**: After reconstitution, the concentration of
albumin increased since the solution volume was reduced to 90% of
its initial volume due to precipitation after centrifugation of the
suspension.
[0184] It was suggested that no precipitation was generated after
centrifugation for the sample fully disintegrated (Sample 2). No
significant variations of the paclitaxel and albumin concentrations
in supernatant were observed for different centrifugation
durations, i.e., there was no paclitaxel crystal or heavy particle
in the solution.
[0185] Precipitate occurred at the bottom after centrifugation for
the non-disintegrated sample (Sample 1). The paclitaxel
concentration in supernatant decreased with centrifugation time,
and finally reached equilibrium. The albumin concentration slightly
increased with time, and finally reached equilibrium (the volume of
the supernatant was about 90% of the total volume). In conclusion,
the nanoparticles in the sample can be isolated and purified by
centrifugation.
[0186] After centrifugation for 60 min, the paclitaxel
concentration in supernatant reached equilibrium. Thus,
paclitaxel-albumin particles can be isolated at 21000.times.g for
60 min.
Example 12
Preparation of the Nanoparticles
[0187] Using the centrifugation method mentioned in Example 11,
particles were isolated from the samples obtained in Example 1-9.
After centrifugation, the supernatants were discarded, and the
precipitates thus obtained were the paclitaxel-albumin
nanoparticles, which were referred as particle 1, 2, 3, 4, 5, 6, 7,
8, and 9, corresponding to Example 1-9.
Example 13
Scanning Electron Microscopic Observation on Morphology of the
Particles Before and after Separation
[0188] Lyophilized powder of the sample obtained in Example 5, and
the precipitate from Example 5 obtained in Example 12 (particle 5)
were observed under a scanning electron microscope (S-4800,
Hitachi). It can be seen from the result in FIG. 1 that a small
amount of particles in the sample from Example 5 were embedded in
the support agent formed by a great amount of albumin. While for
Particle 5, these particles existed independently (See FIG. 2). It
can thus be confirmed that the precipitate obtained using the
separation method in Example 11 was pure or substantially pure
nanoparticles.
Example 14
The Ratio Between Albumin and Paclitaxel in the Particle
[0189] The paclitaxel-albumin nanoparticles (corresponding to
Example 1-9) obtained in Example 12 were re-suspended respectively
in 1 ml 0.9% sodium chloride solution. The contents of paclitaxel
and albumin in the samples were each determined using the method in
Example 10, and the results are as follows:
TABLE-US-00002 TABLE 2 The ratio between albumin and paclitaxel in
various purified nanoparticles obtained after centrifugation
Concen- tration in Albumin Paclitaxel oil phase content content
(mg/ml) (mg/ml) (mg/ml) Albumin:Paclitaxel Example 8 500 0.24 5.58
0.043:1 Example 7 333 0.38 5.34 0.071:1 Example 6 167 0.65 5.03
0.13:1 Example 1 150 0.76 5.04 0.15:1 Example 5 125 0.83 5.16
0.16:1 Example 9 50 0.86 5.03 0.17:1 Example 4 83 0.82 4.56 0.18:1
Example 3 42 1.11 4.63 0.24:1 Example 2 21 2.10 3.69 0.57:1
[0190] It has been suggested by the results above that the ratios
between albumin and paclitaxel in the purified nanoparticles from
the products obtained using different formulation process were
different, and they have certain regularity. It also can be seen
that increased concentration of paclitaxel in oil phase was
inversely proportional to the albumin content.
Example 15
Preparation of Purified Nanoparticles Using Dialysis
[0191] Dialysis was conducted in equal volume to the samples
prepared in Example 2, Example 5, and Example 8 after reconstituted
by water for injection against the dialysis solution of 5% mannitol
using a regenerated cellulose ultrafiltration membrane (PXC300C50,
Millipore) with the cut-off molecular weight of 300K, and the
dialysis fold was 5. The contents of paclitaxel and albumin in the
samples were determined after dialysis using the method in Example
10, and the results are as follows:
TABLE-US-00003 TABLE 3 The ratios between albumin and paclitaxel in
the purified nanoparticles from various formulations obtained by
dialysis Albumin Paclitaxel content content (mg/ml) (mg/ml)
Albumin:Paclitaxel Example 8 0.25 5.12 0.048:1 Example 5 0.73 4.85
0.15:1 Example 2 2.19 3.71 0.59:1
[0192] The above results have indicated a substantially similar
weight ratio between albumin and paclitaxel in the particles
obtained by dialysis using an ultrafiltration membrane, as compared
to the particles obtained by centrifugation. As a result, dialysis
using an ultrafiltration membrane also can be used for isolating
the particles in the suspension from excessive albumin, and for
replacement of the solution surrounding the particles.
Example 16
Preparation of Purified Nanoparticles on Chromatographic Column
[0193] 20 ml Sepharose 4B gel was packed in a glass column with
diameter of 10 mm (.PHI. 10 mm*230 mm, Beijing Mancang Technology
Ltd.). The column was equilibrated with 0.9% sodium chloride
solution to 3 times of the column volumes. 1 ml samples prepared in
Example 2, Example 5, and Example 8 were loaded on the top of the
gel column, and eluted using 0.9% sodium chloride solution,
respectively. The effluent was on-line monitored using a
UV-detector at the wavelength of 280 nm. The effluent corresponding
to the first peak on the chromatogram was a slightly cloudy
suspension, which was collected for determination of paclitaxel and
albumin. Recording was continued until the second peak was
finished. Both peaks were well separated, indicating that the
particles and free albumin can be effectively separated using the
Sepharose 4B gel column. The contents of paclitaxel and albumin in
the particles were determined using the method in Example 10, and
the results are as follows:
TABLE-US-00004 TABLE 4 The ratios between albumin and paclitaxel in
the purified nanoparticles from various formulations obtained by
separation on a chromatographic column Albumin Paclitaxel content
content (mg/ml) (mg/ml) Albumin:Paclitaxel Example 8 0.10 2.51
0.041:1 Example 5 0.33 2.33 0.14:1 Example 2 0.68 1.23 0.55:1
[0194] The above results indicated a substantially similar weight
ratio between albumin and paclitaxel in the particles obtained by
separation on a gel column, as compared to the particles obtained
by centrifugation and dialysis. As a result, gel column separation
also can be used for isolating the particles in the suspension from
excessive albumin.
Example 17
Determination of the Free Albumin Content in the Purified
Nanoparticles Prepared by Dialysis Using Centrifugation
[0195] The particle suspension obtained by dialysis in Example 15
was further isolated by centrifugation under the conditions of
Example 11. After all particles were precipitated at the bottom of
the centrifuge tube, the albumin concentration in the supernatant
was determined, and the results are listed in Table 5.
TABLE-US-00005 TABLE 5 The variation of albumin concentration in
the purified nanoparticles from various formulations obtained by
dialysis before and after centrifugation Concentration
Concentration in the before supernatant after centrifugation
centrifugation (mg/ml) (mg/ml) Example 2 2.19 0.02 Example 5 0.73
0.01 Example 8 0.25 ND ND stands for lower than the limit of
determination, i.e., not detected.
[0196] It can be seen from the above results that in the
paclitaxel-albumin nanoparticle suspension obtained by dialysis,
the proportion of free albumin was quite low, and most albumin was
bound to paclitaxel to form nanoparticles.
Example 18
Determination of the Free Albumin Content in the Purified
Nanoparticles Prepared by Chromatographic Column Separation Using
Centrifugation
[0197] The particle suspension obtained by chromatographic column
separation in Example 16 was further subjected to centrifugation
under the conditions of Example 11. After all particles were
precipitated at the bottom of the centrifuge tube, the albumin
concentration in the supernatant was determined, and the results
are listed in Table 6.
TABLE-US-00006 TABLE 6 The variation of albumin concentration in
the purified nanoparticles from various formulations obtained by
chromatographic column separation before and after centrifugation
Concentration Concentration in the before supernatant after
centrifugation centrifugation (mg/ml) (mg/ml) Example 2 0.68 ND
Example 5 0.33 ND Example 8 0.10 ND ND stands for lower than the
limit of determination, i.e., not detected.
[0198] It can be seen from the above results that in the
paclitaxel-albumin nanoparticle suspension obtained by
chromatographic column separation, there was almost no free
albumin, and most albumin was bound to paclitaxel to form
nanoparticles.
Example 19
Determination of the Free Albumin Content in the Purified
Nanoparticles Prepared by Centrifugation Using Chromatographic
Column Separation
[0199] A particle suspension was obtained by re-suspending the
paclitaxel-albumin nanoparticles obtained in Example 12 in 0.9%
sodium chloride solution, and the purified nanoparticles and free
albumin (if any) were separated from each other using Sepharose 4B
column as mentioned in Example 16. During elution, the UV
absorbance curve was monitored. After the particles were completely
eluted, the following eluent was collected, in which the albumin
concentration was determined. The results are listed in Table
7.
TABLE-US-00007 TABLE 7 The variation of albumin concentration in
the purified nanoparticles from various formulations obtained by
centrifugation before and after chromatographic column separation
Concentration Albumin before column concentration after separation
column separation (mg/ml) (mg/ml) Example 2 0.57 ND Example 5 0.16
ND Example 8 0.043 ND ND stands for lower than the limit of
determination, i.e., not detected.
[0200] It can be seen from the above results that in the
paclitaxel-albumin nanoparticle suspension obtained by
centrifugation, there was almost no free albumin, and most albumin
was bound to paclitaxel to form nanoparticles. In Example 17, 18,
and 19, it can be demonstrated by the evidences observed in three
isolation measures that purified nanoparticles can be obtained by
all three measures with almost no free albumin.
Example 20
Determination of Particle Size and Potential
[0201] Paclitaxel-albumin nanoparticles obtained in Example 12
(corresponding to Example 1-9) were each re-suspended in purified
water. The particle size and the zeta potential of the purified
nanoparticles were detected using Malven NANO-ZS laser particle
sizer, and the results are listed below:
TABLE-US-00008 TABLE 8 The particle size and the potential of the
purified nanoparticles after centrifugation Average Zeta Initial
particle size potential average of the pure of the pure particle
size particle particle (nm) (nm) (mV) Example 6 125 126.6 -28.7
Example 5 133 132 -27.1 Example 7 134 127.5 -37 Example 1 136 137
-28.1 Example 9 136 137.4 -33.6 Example 4 138 138.7 -34.2 Example 8
140 139 -39.4 Example 3 141 151.2 -27.5 Example 2 145 145 -33
[0202] It can be seen from the results that the average particle
size of the purified nanoparticles obtained after centrifugation
was substantially the same as its initial average particle size,
and the zeta potential was still relatively high, so that the
charge can be employed to stabilize the particle suspension.
Example 21
X Diffraction Pattern
[0203] The crystal form was determined in an X-ray diffraction
instrument for the drug substance of paclitaxel and lyophilized
albumin powder, and the results are shown in FIG. 3 and FIG. 4. As
shown, the drug substance of paclitaxel was crystalline powder, and
the lyophilized albumin powder was amorphous powder. Representative
purified nanoparticles obtained in Example 12 (corresponding to
Example 2, 4, 6, and 8) were lyophilized in a lyophilizer to obtain
solid powder, the crystal form of which was detected in an X-ray
diffraction instrument, and the results are shown in FIGS. 5, 6, 7,
and 8. It can be seen from the X diffraction pattern that the ratio
of albumin to paclitaxel in the particles was in the range from
0.043:1 to 0.57:1, and both albumin and paclitaxel were in
amorphous form, which was significantly different from the drug
substance of paclitaxel, and slightly different from the
lyophilized albumin powder.
Example 22
Release of Paclitaxel from the Particles
[0204] Using the separation method for particles established in
Example 11, free component can be effectively separated from the
component in nanoparticle form. As a consequence, the release of
paclitaxel from particles can be detected at different
concentrations using such a method, and specific procedure was
shown below:
[0205] For the paclitaxel-albumin nanoparticles obtained in Example
12 (corresponding to Example 1-9, respectively), representative
purified nanoparticles (corresponding to Example 2, 4, 6, and 8,
and referred as Particle 2, 4, 6, and 8) were selected and 0.9%
sodium chloride solution was added based on the ratio between
albumin and paclitaxel, to form a stock solution with paclitaxel
concentration of 5 mg/ml. The stock solution and simulated blood
plasma solution were kept in 37.degree. C. The stock solution was
diluted using simulated blood plasma solution to obtain a series of
paclitaxel solutions with concentrations of 5000, 1000, 200, 150,
100, 80, 50, 30, and 10 .mu.g/ml.
[0206] Determination of release rate: the paclitaxel concentrations
of the prepared sample solutions were determined, and at the same
time, the paclitaxel concentrations of the supernatants were also
determined after centrifugation at 21000.times.g for 60 min using 1
ml of each sample. The release ratio of paclitaxel at each
concentration was calculated by dividing the paclitaxel
concentration in the supernatant by the concentration before
centrifugation, and the release curves were plotted and shown in
FIG. 9 and FIG. 10. It can be seen from the release curve that the
ratio of albumin to paclitaxel in the particles was in the range
from 0.043:1 to 0.057:1, and the release behaviors of paclitaxel
for the particles were substantially the same, all of which were
correlated to the concentration.
Example 23
Preparation of Compositions Containing Therapeutic
Nanoparticles
[0207] Method 1 (Centrifugation-Re-suspension): therapeutic
nanoparticles obtained in Example 12 corresponding to Example 1-9
(referred as Particle 1, 2, 3, 4, 5, 6, 7, 8, and 9) were
re-suspended in 5% mannitol solution, 10% sucrose solution, 5%
dextran solution, 10% lactose solution, 10% trehalose solution, 10%
maltose solution, 10% human serum albumin solution, respectively,
to establish paclitaxel concentrations of 5 mg/ml. The
re-suspensions were filtered through a 0.22 .mu.m sterile filter,
and no significant variation of particle size was observed for the
particles in the filtrate. The suspensions were lyophilized in a
lyophilizer respectively to obtain the compositions containing the
pharmaceutical nanoparticles.
[0208] Method 2 (Dialysis): the samples obtained in Example 1-9
were each reconstituted to prepare suspensions with the paclitaxel
concentration of 5 mg/ml. Subsequently, the suspensions were
dialyzed using a 300 kDa ultrafiltration membrane respectively by
5% mannitol solution, 10% sucrose solution, 5% dextran solution,
10% lactose solution, 10% maltose solution, 10% trehalose solution,
and 10% human serum albumin solution. After dialyzing to 5-time of
the initial volume, free human serum albumin in the suspensions was
all replaced. The suspensions were filtered through a 0.22 .mu.m
sterile filter, and no significant variation of particle size was
observed for the particles in the filtrate. The suspensions were
lyophilized in a lyophilizer respectively to obtain the
compositions containing the therapeutic nanoparticles.
Example 24
[0209] The compositions containing the therapeutic nanoparticles
obtained in Method 1 (Centrifugation-Re-suspension) and Method 2
(Dialysis) in Example 23 were each reconstituted in water for
injection to establish a paclitaxel concentration of 5 mg/ml.
Stability for each solution was observed, and 12 h stability at
room temperature was determined. As shown, conventional
lyoprotectants can play a protective role for purified
paclitaxel-albumin nanoparticles. The results are shown in Table 9
and Table 10:
TABLE-US-00009 TABLE 9 Stability of the compositions obtained by
Centrifugation-Re-suspension Particles contained in the composition
Particle Particle Particle Particle Particle Particle Particle
Particle Particle 1 2 3 4 5 6 7 8 9 5% mannitol solution 10%
sucrose solution 5% dextran solution 10% lactose solution 10%
trehalose solution 10% maltose solution 10% human serum albumin
solution Note: stands for a clear solution, with no obvious
precipitate, and no variation of particle size as compared to its
initial result.
TABLE-US-00010 TABLE 10 Stability of the compositions obtained by
Dialysis Particles contained in the composition Particle Particle
Particle Particle Particle Particle Particle Particle Particle 1 2
3 4 5 6 7 8 9 5% mannitol solution 10% sucrose solution 5% dextran
solution 10% lactose solution 10% maltose solution 10% trehalose
solution 10% human serum albumin solution Note: stands for a clear
solution, with no obvious precipitate, and no variation of particle
size as compared to its initial result.
[0210] As shown, the stability of particles can be maintained by
replacing the albumin surrounding the particles using
Centrifugation-Re-suspension (Method 1) and Dialysis (Method
2).
Example 25
Sensitization Study in Guinea Pig
[0211] In this experiment, the product from Example 6 and 5%
mannitol formulation comprising Particle 6 from Example 24 were
selected as the test drug, wherein the paclitaxel concentrations in
the formulations are listed in the table below. The sensitization
dosages were selected as 3 mg per guinea pig and 1.25 mg per guinea
pig. Sensitization was conducted by intraperitoneal injection once
every other day, for a total of 3 injections. At the same time, a
positive control group (0.2% ovalbumin) and a negative control
group (0.9% sodium chloride injection) were also established.
Detailed dosage regimen is listed in the table below.
TABLE-US-00011 TABLE 11 Protocol for sensitization study in guinea
pig Sensitization Excitation Number Concentration Administration
Administration Administration Administration of the of the drug
volume dosage volume dosage Group animal (mg/ml) (ml/guinea pig)
(ml/guinea pig) (ml/guinea pig) (ml/guinea pig) Negative 6 -- 0.5
-- 1 -- control group Positive 6 2 0.5 1 1 2 control group Low
dosage of 6 2.5 0.5 1.25 1 2.5 product from Example 6 High dosage
of 6 6 0.5 3 1 6 product from Example 6 Low dosage 6 2.5 0.5 1.25 1
2.5 of Particle 6 Low dosage 6 6 0.5 3 1 6 of Particle 6
[0212] Administration method for sensitization: the back of guinea
pig was firmly held by cup-shape left hand, allowing the abdominal
skin stretched when the guinea pig was fixed. The abdomen of the
guinea pig was lifted, and the head was lowered down. After
disinfecting of the injection site by an alcohol wipe, the needle
of a 2 ml disposable syringe held by the right hand was punctured
into the skin of the guinea pig. The needle was inserted at the
site 1 mm left to the midline of lower abdomen. When arriving at
the subcutaneous part, the needle was inserted forward for further
5 mm to 10 mm, and subsequently punctured into the abdominal cavity
at an angle of 45.degree.. After fixing the needle, the
pharmaceutical solution was injected slowly. After the injection, a
dry cotton wipe was pressed on the pinprick in order to prevent the
outflow of the pharmaceutical. After 3 times of sensitization, the
guinea pigs in the group of the formulation from Example 6 (3
mg/guinea pig) became emaciated, and died. No abnormality was
observed in other groups.
[0213] Allergy excitation: excitation was conducted by intravenous
injection, and the excitation was performed 10 d after the last
sensitization with the dosages of 6 mg/animal and 2.5
mg/animal.
[0214] Administration method for excitation: injection was
performed to the lateral metatarsal vein of the guinea pig fixed by
an assistant. The stifles were grasped by the operator to fix the
body of the animal. Its vein was compressed, and the legs were in a
stretched state. The hair at the injection site was shaved (or the
skin at the injection site was cut). After sterilization by alcohol
wipes, thick lateral metatarsal vein can be seen. The needle of a 1
ml disposable syringe was punctured into the blood vessel along the
direction to the heart by the right hand. After the injection, a
dry cotton wipe was pressed on the pinprick in order to prevent
bleeding.
[0215] The reaction of each animal and the time when allergy
symptoms appeared or disappeared were observed immediately after
the excitation for 30 min. Maximal observation duration was 3 h.
The results of allergic reaction are listed in Table 14.
TABLE-US-00012 TABLE 12 Symptoms of allergic reaction 0 Normal 1
Dysphoria 2 Piloerection 3 Trembling 4 Nose scratching 5 Sneezing 6
Coughing 7 Tachypnea 8 Urination 9 Defecation 10 Lacrimation 11
Dyspnea 12 Wheezing 13 Peliosis 14 Gait disturbance 15 Jumping 16
Panting 17 Convulsion 18 Rotation 19 Cheyne-stokes respiration 20
Death
TABLE-US-00013 TABLE 13 Evaluation criteria for systemic allergic
reaction Symptom Degree Result 0 - Negative allergic reaction 1-4 +
Weakly positive allergic reaction 5-10 ++ Positive allergic
reaction 11-19 +++ Strongly positive allergic reaction 20 ++++
Extremely positive allergic reaction
TABLE-US-00014 TABLE 14 Results for active anaphylaxis of guinea
pig Number Posi- of the Reaction level tive Group animal 1 2 3 4 5
6 rate Negative 6 - - - - - - - control group Positive 6 ++++ ++++
++++ ++++ ++++ ++++ ++++ control group Low dosage 6 + +++ +++ ++++
++ ++ +++ of product from Example 6 High dosage 6 / / / / / / NA*
of product from Example 6 Low dosage 6 - - - - - - - of Particle 6
High dosage 6 + - - + - + + of Particle 6 *stands for the fact that
the animal became emaciated and dead after sensitization.
[0216] It has been suggested by the results of active anaphylaxis
in guinea pigs that the nanoparticle formulation containing
excessive albumin has stronger sensitization, whereas the
formulation containing purified nanoparticles can significantly
decrease the allergic reaction.
Example 26
Pharmacokinetic Study in Dogs
[0217] In this experiment, the product from Example 6 and 5%
mannitol formulation comprising Particle 6 from Example 24 were
selected as the test drug, wherein the paclitaxel concentration in
the formulations was 5 mg/ml. Beagle dog was employed for the in
vivo pharmacokinetic studies of both formulations. In each group, 3
beagle dogs were tested with administration dosage of 5 mg/kg, and
administration time length of 30 min. 2 ml blood was collected from
the cephalic vein of the forelimb of beagle dogs immediately before
administration (0 h), 15 min in the infusion process (0.25 h since
administration), the time point of needle withdrawal (0.5 h since
administration), and 0.58, 0.75, 1.0, 1.5, 2.5, 3.5, 4.5, 6.5, 8.5,
24.5 h post administration, and each blood samples were placed into
a heparin tube, shaken until homogenous, and centrifuged at 3000
rpm for 10 min to obtain the plasma. The paclitaxel concentration
in plasma was determined by HPLC/MS/MS, and the drug concentration
was plotted versus time (See FIG. 11). It can be seen from the drug
concentration-time plot that both formulations had almost the same
in vivo behaviors, so that there was no impact by excessive albumin
on the in vivo behaviors of the particles.
Example 27
Allergic Symptoms During the Pharmacokinetic Study in Dogs
[0218] In the pharmacokinetic study in dogs conducted in Example
26, adverse reactions were observed during the administration of
both formulations. In the sample group of Example 6, various
degrees of obvious allergic reactions appeared in 3 dogs, mainly
including violently struggling, excessive salivation, erythema
around mouth, and in individual experimental animals, vomiting and
urinary incontinence occurred during administration. While for the
sample group of Particle 6, only slightly struggling, salivation,
and erythema around mouth appeared in part of the animals and the
symptoms are mild. Consequently, the formulation of purified
nanoparticles was capable of significantly alleviating the allergic
reactions of the drug.
Example 28
[0219] The following samples were investigated for the maximum
tolerated dose, the samples including the composition comprising
the therapeutic nanoparticles using mannitol as the lyoprotectant
(obtained from Method 1 of Example 24, corresponding to the
nanoparticles of Example 2, 6, and 8, and referred as Particle 2,
Particle 6, and Particle 8 below), product from Example 6, and
Taxol.RTM. prepared with Cremophor.
[0220] Based on the recommendation of NCI of USA, modifications
were made to the experimental method for determining the maximum
tolerated dose of acute toxicity by single dose administration. For
Particle 2, Particle 6 and Particle 8, and lyophilized powder from
Example 6, 400 mg/kg was selected as the maximum administration
dose, and the administration dose was reduced in a ratio of 1.2,
i.e., a series of doses of 400, 333, 278, 231 and 193 mg/kg. For
TAXOL.RTM., 48 mg/kg was selected as the maximum administration
dose, and the administration dose was also reduced in a ratio of
1.2, i.e., a series of doses of 48, 40, 33.3, 27.8, 23.1 and 19.3
mg/kg. In each dose group, 3 KM male mice were assigned. The
general conditions and variations of body weight of the animals
were observed for 10 days after administration. If no animal died,
no irreversible toxic response occurred, or no more than 15% of
body weight loss appeared for 3 continuous days as compared to that
of before experiment during observation, the maximum administration
dose was considered as the maximum tolerated dose for the single
administration.
[0221] The results indicated that the maximum tolerated dose of
TAXOL.RTM. was 33.3 mg/kg. In all administration groups, during
administration of TAXOL.RTM., the mice struggled violently, and
coma and accelerated breathing occurred after administration. In
the dose groups of 40 mg/kg and 48 mg/kg, cases of death occurred.
In the rest dose groups, mice recovered gradually after
administration. The severity of toxicity symptoms and the time
demanded for recovery were correlated to the administration dose.
The maximum tolerated doses for Example 6 and Particle 2, Particle
6 and Particle 8 were all 193 mg/kg. Adverse symptoms of the mice
in the groups of doses above 193 mg/kg mainly included body weight
loss, dirt around anus, rear limb weakness/paralysis, and death.
The severity of adverse symptoms was enhanced with increased
administration dose. There was no obvious difference among the
toxicities to the mice by Particle 2, Particle 6, Particle 8 and
the product from Example 6. However, all the above toxicities were
lower than that of TAXOL.RTM. prepared with Cremophor.
Example 29
Inhibitory Effect on H22 Tumor of Liver Cancer in Mice
[0222] Particle 2, Particle 6, Particle 8, the product from Example
6, and Taxol.RTM. prepared with Cremophor were studied for liver
cancer H22 tumor inhibitory effect in mice.
[0223] The animals were divided based on body weight. Ascitic cells
of H22 liver cancer were subcutaneously inoculated to the
subcutaneous tissue in the axillary region of the forelimbs of male
KM mice with the inoculation volume of 0.2 ml, which contained
about 1.0.times.10.sup.6 cells. The animals were equally divided
into 6 groups according to the inoculation time.
[0224] Single intravenous administrations of Particle 2, Particle
6, Particle 8, the product from Example 6 and Taxol.RTM. were
performed at the maximum tolerated dose of mice 24 h after
inoculation, i.e., the administration doses of Particle 2, Particle
6, Particle 8, and the product from Example 6 were 193 mg/kg, and
the administration dose of Taxol.RTM. was 33.3 mg/kg. In the blank
control group, 0.9% sodium chloride injection was used for the
single-intravenous administration. On the 12.sup.th day after
administration, the mice were sacrificed by CO.sub.2 asphyxia, and
the tumor mass was taken out and weighed. The tumor inhibitory rate
(%) can be calculated according to the following equation: Tumor
inhibitory rate=(1-average tumor weight in the test groups/average
tumor weight in the control group).times.100%. The experimental
results were statistically analyzed by One-way ANOVA using
statistical software of SPSS 19.0.
[0225] The experimental results are listed in Table 15. For single
intravenous administration at the maximum tolerated dose of mice,
the growth of H22 tumor was significantly inhibited in all groups.
There was no inter-group difference in the tumor inhibitory effect
of Particle 2, Particle 6, Particle 8 and the product from Example
6 on H22 tumor. The tumor inhibitory rate of Particle 2, Particle 8
and the product from Example 6 was significantly higher than
TAXOL.RTM..
TABLE-US-00015 TABLE 15 Effect on the body weight of mice bearing
H22 tumor(n = 10, x .+-. sd) Tumor Initial Final Growth of Tumor
inhibitory Dosage weight weight body weight weight rate Group
(mg/kg) (g) (g) (%) (g) (%) Particle 2 193 22.8 .+-. 29.3 .+-. 28.9
0.7168 .+-. 70.9 1.6 2.7**.sup..tangle-solidup..tangle-solidup.
0.5081**.sup..tangle-solidup. Particle 6 193 23.9 .+-. 29.6 .+-.
24.0 0.9965 .+-. 59.6 1.5
2.1**.sup..tangle-solidup..tangle-solidup. 0.6347** Particle 8 193
23.8 .+-. 29.0 .+-. 22.1 0.6090 .+-. 75.3 1.4
4.3**.sup..tangle-solidup..tangle-solidup.
0.4129**.sup..tangle-solidup..tangle-solidup. The product 193 23.7
.+-. 27.4 .+-. 15.8 0.7213 .+-. 70.7 from 1.4
2.3**.sup..tangle-solidup..tangle-solidup.
0.4604**.sup..tangle-solidup. Example 6 Taxol .RTM. 33.3 23.2 .+-.
35.1 .+-. 51.4 1.2789 .+-. 48.1 2.2 2.8* 0.5556* Control -- 22.0
.+-. 38.3 .+-. 74.1 2.4644 .+-. 2.7 2.8 1.1753 **p < 0.01, *p
< 0.05, as compared to the blank control group;
.sup..tangle-solidup..tangle-solidup.p < 0.01,
.sup..tangle-solidup.p < 0.05, as compared to Taxol .RTM.
Example 30
Inhibitory Effect on the Prostatic Cancer RM-1 Tumor of Mice
[0226] Particle 2, Particle 6, Particle 8, the product from Example
6, and TAXOL.RTM. prepared with Cremophor were studied for
prostatic cancer RM-1 tumor inhibitory effect in mice.
[0227] The RM-1 tumor cells at logarithmic growth phase were
collected, and the cell number was adjusted to 2.5.times.10.sup.6
cells/ml. The suspension of tumor cells was inoculated to the
subcutaneous tissue in the axillary region of the forelimbs of 7 to
8-week-old C75 male mice with the inoculation volume of 0.2 ml,
which contained about 5.times.10.sup.5 cells. After inoculation,
the remained suspension of tumor cells was counted under an optical
microscope, with the number of live tumor cells >95%. The mice
were divided into 6 groups based on inoculation time.
[0228] Single intravenous administrations of Particle 2, Particle
6, Particle 8, the product from Example 6 and Taxol.RTM. were
performed at the maximum tolerated dose of mice 72 h after
inoculation, i.e., the administration doses of Particle 2, Particle
6, Particle 8, and the product from Example 6 were 193 mg/kg, and
the administration dose of Taxol.RTM. was 33.3 mg/kg. In the blank
control group, 0.9% sodium chloride injection was used for the
single-intravenous administration. After the outgrowth of tumor,
the diameter was measured using a vernier caliper, and the
anti-tumor effect of the pharmaceutical was observed dynamically.
The tumor volume (TV) can be calculated as follows:
V=1/2.times.a.times.b.sup.2, wherein a and b refer to the length
and width of tumor, respectively. The tumor volume can be
calculated based on the results. The tumor volume inhibitory rate
(%) can be calculated according to the following equation: Tumor
inhibitory rate=(1-average tumor volume in the administration
groups/average tumor volume in the control group).times.100%. The
experimental results were analyzed by statistical software SPSS
19.0. The inter-measure tumor volume variation with time was
analyzed by Repeated Measure Analysis, and the inter-group tumor
volume variation among each measure was analyzed by
Multivariate.
[0229] The experimental results are listed in Table 16. Single
intravenous administrations were performed at the maximum tolerated
dose 3 days after inoculation of prostatic cancer RM-1 tumor cells.
As compared to the blank control group, significant inhibitory
effect on the growth of the prostatic cancer RM-1 tumor of mice was
observed for Particle 2, Particle 6, Particle 8 and the product
from Example 6, and no inhibitory effect on the tumor was seen for
Taxol.RTM. prepared with Cremophor. No significant difference in
the inhibitory effect of Particle 2, Particle 6, Particle 8 and the
product from Example 6 was observed on RM-1 tumor of mice. However,
as compared to Taxol.RTM., all the above formulations had
significant inhibitory effect on the growth of tumor.
TABLE-US-00016 TABLE 16 Inhibitory effect on prostatic cancer RM-1
tumor of mice(n = 10, .+-.sd) Initial Final Growth Tumor volume
(mm.sup.3)/Tumor inhibitory rate % Dosage weight weight of body 7 d
after 10 d after 12 d after 14 d after (mg/kg) (g) (g) weight
inoculation inoculation inoculation inoculation Particle 2 193 27.6
.+-. 32.3 .+-. 17.0% 107.9 .+-. 210.6 .+-. 471.6 .+-. 715.5 .+-.
1.8 2.3 25.8**.sup..tangle-solidup. 128 6*.sup..tangle-solidup.
305.0 367 1.sup..tangle-solidup. 59.7% 49.9% 28.9% 40.7% Particle 6
193 29.4 .+-. 34.2 .+-. 16.3% 96.2 .+-. 122.6 .+-. 331.7 .+-. 600.3
.+-. 2.0 3.9* 23.3**.sup..tangle-solidup..tangle-solidup.
45.9**.sup..tangle-solidup..tangle-solidup.
142.2*.sup..tangle-solidup. 302
2*.sup..tangle-solidup..tangle-solidup. 64.1% 70.8% 50.0% 50.2%
Particle 8 193 28.2 .+-. 32.6 .+-. 15.8% 107.1 .+-. 148.7 .+-.
385.1 .+-. 736.4 .+-. 2.1 3.2 28.7**.sup..tangle-solidup.
56.6**.sup..tangle-solidup..tangle-solidup. 138.4* 472.9 60.0%
64.6% 41.9% 38.9% The product 193 28.1 .+-. 33.7 .+-. 19.9% 110.6
.+-. 187.1 .+-. 395.9 .+-. 590.0 .+-. from 2.0
2.6**.sup..tangle-solidup. 18.2**.sup..tangle-solidup.
60.0**.sup..tangle-solidup. 147.8*
171.2*.sup..tangle-solidup..tangle-solidup. Example 6 58.7% 55.5%
40.3% 51.1% Taxol .RTM. 33.3 26.8 .+-. 31.1 .+-. 16 2% 198.6 .+-.
430.3 .+-. 563.7 .+-. 1113.2 .+-. 1.9 2.7 103 9 272.7 311.3 460.0
25.8% -2.5% 15.0% 7.7% Control 26.6 .+-. 30.5 .+-. 14.6% 267.6 .+-.
420.0 .+-. 663.2 .+-. 1205.7 .+-. 1.5 2.3 169.1 217.7 338.4 735.1
**p < 0.01, *p < 0.05, as compared to the blank control
group; .sup..tangle-solidup..tangle-solidup.p < 0.01,
.sup..tangle-solidup.p < 0.05, as compared to Taxol .RTM.
Example 31
Inhibitory Effect on the Tumor of the Lewis Lung Cancer Mice
[0230] Particle 2, Particle 6, Particle 8, the product from Example
6, and TAXOL.RTM. prepared with Cremophor were studied for the
inhibitory effect on tumor in Lewis lung cancer mice.
[0231] The Lewis tumor cells at logarithmic growth phase were
collected, and the cell number was adjusted to 2.5.times.10.sup.6
cells/ml. The suspension of tumor cells was inoculated to the
subcutaneous tissue in the axillary region of the forelimbs of 7 to
8-week-old C57 male mice with the inoculation volume of 0.2 ml,
which contained about 5.times.10.sup.5 cells.
[0232] The grouping, administration, indicator observation and
statistical method were the same as those in Example 18.
[0233] The experimental results are listed in Table 17. Single
intravenous administrations were performed at the maximum tolerated
dose 3 days after inoculation of tumor cells of Lewis lung cancer
mice. As compared to the blank control group, significant
inhibitory effect on the growth of the tumor of the Lewis lung
cancer mice was observed for Particle 2, Particle 6, Particle 8 and
the product from Example 6, and no inhibitory effect on the tumor
was seen for Taxol.RTM. prepared with Cremophor. No significant
difference in the inhibitory effect of Particle 2, Particle 6,
Particle 8 and the product from Example 6 was observed on the tumor
of the Lewis lung cancer mice. However, as compared to Taxol.RTM.,
all the above formulations had significant inhibitory effect on the
growth of tumor.
TABLE-US-00017 TABLE 17 Inhibitory effect on the tumor of the Lewis
lung cancer mice(n = 10, x .+-. sd) Initial weight g Growth Tumor
volume (mm.sup.3)/Tumor inhibitory rate % Dosage Final of body 7 d
after 10 d after 12 d after 14 d after 17 d after 19 d after 21 d
after Group mg/kg weight g weight inoculation inoculation
inoculation inoculation inoculation inoculation inoculation
Particle 2 193 25.7 .+-. 16.4% 45.5 .+-. 172.4 .+-. 166.0 .+-.
239.2 .+-. 566.6 .+-. 856.9 .+-. 1608.1 .+-. 1.7
33.4**.sup..tangle-solidup..tangle-solidup.
37.4**.sup..tangle-solidup.
39.9**.sup..tangle-solidup..tangle-solidup.
88.8**.sup..tangle-solidup..tangle-solidup.
273.9*.sup..tangle-solidup. 436.5* 714.4 29.9 .+-. 67.7% 32.9%
48.1% 52.6% 48.5% 45.9% 34.7% 2.3 Particle 6 193 26.4 .+-. 13.7%
11.7 .+-. 153.8 .+-. 177.7 .+-. 282.8 .+-. 640.1 .+-. 940.0 .+-.
1536.4 .+-. 1.2 25.1**.sup..tangle-solidup..tangle-solidup.
70.1**.sup..tangle-solidup.
119.0*.sup..tangle-solidup..tangle-solidup.
278.3.sup..tangle-solidup. 701.1 788.6 1288.9 30.1 .+-. 91.7% 40.1%
44.5% 44.0% 41.9% 40.6% 37.6% 1.8.sup..tangle-solidup. Particle 8
193 26.7 .+-. 16.6% 0.0 .+-. 161.1 .+-. 157.4 .+-. 256.7 .+-. 461.6
.+-. 850.7 .+-. 1278.3 .+-. 1.5
0.0**.sup..tangle-solidup..tangle-solidup.
41.0**.sup..tangle-solidup.
61.7**.sup..tangle-solidup..tangle-solidup.
146.3*.sup..tangle-solidup..tangle-solidup.
244.3**.sup..tangle-solidup. 475.8*
507.3*.sup..tangle-solidup..tangle-solidup. 31.1 .+-. 100.0% 37.3%
50.8% 49.2% 58.1% 46.3% 48.1%
2.0.sup..tangle-solidup..tangle-solidup. The product 193 26.1 .+-.
21.2% 98.9 .+-. 196.5 .+-. 210.7 .+-. 360.9 .+-. 671.7 .+-. 1030.0
.+-. 1539.8 .+-. from 1.5 41.2*.sup..tangle-solidup. 46.8
72.8.sup..tangle-solidup..tangle-solidup. 271.4 521.0 863.5 1342.5
Example 6 31.6 .+-. 29.7% 23.5% 34.1% 28.6% 39% 34.9% 37.5%
2.0.sup..tangle-solidup..tangle-solidup. Taxol .RTM. 33.3 25.6 .+-.
9.6% 137.1 .+-. 254.2 .+-. 373.8 .+-. 616.0 .+-. 1111.5 .+-. 1373.7
.+-. 2486.8 .+-. 1.8 35.3 108.6 139.5 303.1 698.4 839.7 1148.3 28.0
.+-. 2.5% 1.0% -16.9% -21.9% -0.9% 13.2% -1.0% 2.2 Control 26.2
.+-. 14.3% 140.6 .+-. 256.8 .+-. 319.9 .+-. 505.2 .+-. 1101.2 .+-.
1582.7 .+-. 2462.8 .+-. 2.2 39.1 83.1 149.3 251.2 555.6 833.5
1275.3 30.0 .+-. 3.2 **p < 0.01, *p < 0.05, as compared to
the blank control group; .sup..tangle-solidup..tangle-solidup.p
< 0.01, .sup..tangle-solidup.p < 0.05, as compared to Taxol
.RTM.
Example 32
[0234] As a commercially available pharmaceutical product, human
serum albumin should contain no more than 5% of the polymer
according to its quality standard, since the albumin polymer may
induce allergic reaction. The determination method for albumin in
Example 10 can distinguish albumin monomer from the polymer. The
albumin polymer and paclitaxel content were detected in the
compositions from Example 1-9 (referred as initial formulation) and
the compositions obtained from Method 1 of Example 24, respectively
using mannitol and human serum albumin as a lyoprotectant
(corresponding to Example 1-9), based on the determination method
for albumin and paclitaxel in Example 10, and the albumin polymer
content was calculated per 1 mg paclitaxel. The results are listed
in Table 18.
TABLE-US-00018 TABLE 18 Relative amount of albumin polymer and
paclitaxel in various formulations Initial Mannitol as Albumin as
formulation lyoprotectant lyoprotectant (mg/mg) (mg/mg) (mg/mg)
Example 1 1.7 0.03 0.18 Example 2 12.5 0.11 0.20 Example 3 6.3 0.05
0.19 Example 4 3.2 0.04 0 17 Example 5 2.1 0.03 0.18 Example 6 1.6
0.03 0.18 Example 7 0.8 ND 0.17 Example 8 0.9 ND 0.16 Example 9 2.0
0.03 0.17 Note: ND stands for lower than the limit of
determination, i.e., not detected
[0235] It has been suggested by the results that the content of
albumin polymer in the product prepared directly using the method
of prior art (Homogenization) was significantly higher than the
content in the post-added albumin of the composition by the
preparation method of the present disclosure. This was resulted
from albumin polymer newly generated in the homogenization process
or evaporation process of the prior art. However, the content of
albumin polymer in the formulation comprising mannitol protectant
was lower, since only very small amount of albumin was comprised in
the formulation, so that the total amount was lower than the amount
of the polymer in initial formulation.
Example 33
Recovering Albumin for Preparation of the Nanoparticles
[0236] The dialysate resulted from the dialysis in Example 15 was
collected, and concentrated to an albumin content of about 4% using
a regenerated cellulose ultrafiltration membrane with a cut-off
molecular weight of 10K. The concentrate was dialyzed in equal
volume by purified water. After dialyzed to a 5-time volume, 4%
albumin solution can be recovered.
[0237] According to the method in Example 5, paclitaxel-albumin
nanoparticles were prepared using recovered albumin solution.
Consequently, the average diameter of the prepared
paclitaxel-albumin nanoparticles was 134 nm, and the suspension was
translucent, which was similar to the product obtained in Example
5.
[0238] The suspension can be smoothly filtered through a 0.22 .mu.m
sterile filter. There was no significant variation of the particle
size after filtration, and no significant change was observed for
the suspension after storage for 48 h at room temperature. The
suspension was aliquoted and lyophilized for 24 h in a lyophilizer
to obtain a stable off-white cake.
Example 34
Preparation of Purified Docetaxel-Albumin Particles
[0239] 3 g docetaxel was dissolved into 15 ml chloroform/ethanol
(1:1, v/v), and added into 500 ml human serum albumin solution (4%
w/v). The mixture was emulsified for 2 min using a high shear
disperser (Fluko FZ-20) to obtain a primary emulsion. The primary
emulsion was then homogenized using a high pressure homogenizer
under a pressure of 10000-20000 psi to obtain a nano-emulsion.
Subsequently, the nano-emulsion was transferred to a rotatory
evaporator to remove the organic solvent in the solution by vacuum
evaporation at 40 mbar and at 40.degree. C. in a water-bath. The
docetaxel-albumin nanoparticles were thus generated with an average
diameter of 110-150 nm, and the suspension was translucent.
[0240] The nanoparticles were isolated from the resultant
docetaxel-albumin nanoparticle suspension by centrifugation method
referred in Example 11 at 21000.times.g for 60 min. The supernatant
was discarded, and the purified nanoparticles were collected.
Purified nanoparticles were re-suspended in 5% mannitol solution.
The re-suspension was filtered through a 0.22 .mu.m sterile filter,
and there was no significant variation of the particle size in the
filtrate. The content of docetaxel in the solution was determined
using HPLC. Based on the content, the solution was subsequently
aliquoted into vials at an amount of 50 mg per vial. The vials were
placed in a lyophilizer and lyophilized for 48 h.
[0241] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
docetaxel content in the product, the ratio between albumin and
docetaxel can be calculated as 0.1:1.
Example 35
Preparation of Purified 2'-O-Hexanoyldocetaxel Albumin
Particles
[0242] 695 mg 2'-O-hexanoyldocetaxel was dissolved into 6 ml
chloroform/ethanol (10:1, v/v), and added into 102 ml human serum
albumin solution (10% w/v). The mixture was emulsified for 2 min
using a high shear disperser (Fluko FZ-20) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer under a pressure of 10000-20000 psi to obtain
a nano-emulsion. Subsequently, the nano-emulsion was transferred to
a rotatory evaporator to remove the organic solvent in the solution
by vacuum evaporation at 40 mbar and at 40.degree. C. in a
water-bath. The 2'-O-hexanoyldocetaxel albumin nanoparticles were
thus generated with an average diameter of 75-100 nm, and the
suspension was translucent.
[0243] The nanoparticles were isolated from the resultant
2'-O-hexanoyldocetaxel albumin nanoparticle suspension by dialysis
method against 5% mannitol solution referred in Example 15. The
obtained purified nanoparticles suspension was filtered through a
0.22 .mu.m sterile filter, and there was no significant variation
of the particle size in the filtrate. The content of
2'-O-hexanoyldocetaxel in the solution was determined using HPLC.
Based on the content, the solution was subsequently aliquoted into
vials at an amount of 50 mg per vial. The vials were placed in a
lyophilizer and lyophilized for 48 h.
[0244] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
2'-O-hexanoyldocetaxel content in the product, the ratio between
albumin and 2'-O-hexanoyldocetaxel can be calculated as 0.27:1.
Example 36
Preparation of Purified 2'-Benzoyl Docetaxel Albumin Particles
[0245] 226 mg 2'-benzoyl docetaxel was dissolved into 2 ml
chloroform/ethanol (9:1, v/v), and added into 35 ml human serum
albumin solution (5% w/v). The mixture was emulsified for 2 min
using a high shear disperser (Fluko FZ-20) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer under a pressure of 18000-20000 psi to obtain
a nano-emulsion. Subsequently, the nano-emulsion was transferred to
a rotatory evaporator to remove the organic solvent in the solution
by vacuum evaporation at 40 mbar and at 40.degree. C. in a
water-bath. The 2'-benzoyl docetaxel albumin nanoparticles were
thus generated with an average diameter of 30-60 nm, and the
suspension was translucent.
[0246] The nanoparticles were isolated from the resultant
2'-benzoyl docetaxel albumin nanoparticle suspension by
chromatographic column separation method referred in Example 16.
10% lactose solution was used as dialysate. The obtained purified
nanoparticles suspension was filtered through a 0.22 .mu.m sterile
filter, and there was no significant variation of the particle size
in the filtrate. The content of 2'-benzoyl docetaxel in the
solution was determined using HPLC. Based on the content, the
solution was subsequently aliquoted into vials at an amount of 50
mg per vial. The vials were placed in a lyophilizer and lyophilized
for 65 h.
[0247] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
2'-benzoyl docetaxel content in the product, the ratio between
albumin and 2'-benzoyl docetaxel can be calculated as 0.95:1.
Example 37
Preparation of Purified Rapamycin Albumin Particles
[0248] 166 mg rapamycin was dissolved into 1 ml chloroform/ethanol
(11:1, v/v), and added into 37 ml human serum albumin solution (5%
w/v). The mixture was emulsified for 2 min using a high shear
disperser (Fluko FZ-20) to obtain a primary emulsion. The primary
emulsion was then homogenized using a high pressure homogenizer
under a pressure of 10000-20000 psi to obtain a nano-emulsion.
Subsequently, the nano-emulsion was transferred to a rotatory
evaporator to remove the organic solvent in the solution by vacuum
evaporation at 40 mbar and at 40.degree. C. in a water-bath. The
rapamycin nanoparticles were thus generated with an average
diameter of 50-85 nm, and the suspension was translucent.
[0249] The nanoparticles were isolated from the resultant rapamycin
albumin nanoparticle suspension by chromatographic column
separation method referred in Example 16. 5% dextrane solution was
used as dialysate. The obtained purified nanoparticles suspension
was filtered through a 0.22 .mu.m sterile filter, and there was no
significant variation of the particle size in the filtrate. The
content of rapamycin in the solution was determined using HPLC.
Based on the content, the solution was subsequently aliquoted into
vials at an amount of 10 mg per vial. The vials were placed in a
lyophilizer and lyophilized for 48 h.
[0250] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
rapamycin content in the product, the ratio between albumin and
rapamycin can be calculated as 0.63:1.
Example 38
Preparation of Purified Temsirolimus Albumin Particles
[0251] 101 mg temsirolimus was dissolved into 0.5 ml
chloroform/ethanol (7:1, v/v), and added into 19.5 ml human serum
albumin solution (5% w/v). The mixture was emulsified for 2 min
using a high shear disperser (Fluko FZ-20) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer under a pressure of 10000-20000 psi to obtain
a nano-emulsion. Subsequently, the nano-emulsion was transferred to
a rotatory evaporator to remove the organic solvent in the solution
by vacuum evaporation at 40 mbar and at 40.degree. C. in a
water-bath. The temsirolimus nanoparticles were thus generated with
an average diameter of 80-115 nm, and the suspension was
translucent.
[0252] The nanoparticles were isolated from the resultant
temsirolimus albumin nanoparticles by centrifugation method
referred in Example 11 at 21000.times.g for 60 min. The supernatant
was discarded, and the purified nanoparticles were collected.
Purified nanoparticles were re-suspended in 5% mannitol solution.
The re-suspension was filtered through a 0.22 .mu.m sterile filter,
and there was no significant variation of the particle size in the
filtrate. The content of temsirolimus in the solution was
determined using HPLC. Based on the content, the solution was
subsequently aliquoted into vials at an amount of 10 mg per vial.
The vials were placed in a lyophilizer and lyophilized for 48
h.
[0253] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
temsirolimus content in the product, the ratio between albumin and
temsirolimus can be calculated as 0.16:1.
Example 39
Preparation of Purified Everolimus Albumin Particles
[0254] 78 mg everolimus was dissolved into 1 ml chloroform/ethanol
(9:1, v/v), and added into 22 ml human serum albumin solution (8%
w/v). The mixture was emulsified for 2 min using a high shear
disperser (Fluko FZ-20) to obtain a primary emulsion. The primary
emulsion was then homogenized using a high pressure homogenizer
under a pressure of 10000-20000 psi to obtain a nano-emulsion.
Subsequently, the nano-emulsion was transferred to a rotatory
evaporator to remove the organic solvent in the solution by vacuum
evaporation at 40 mbar and at 40.degree. C. in a water-bath. The
everolimus nanoparticles were thus generated with an average
diameter of 80-110 nm, and the suspension was translucent.
[0255] The nanoparticles were isolated from the resultant
everolimus albumin nanoparticle suspension by dialysis method
referred in Example 15 against 5% dextrane. The obtained purified
nanoparticles suspension was filtered through a 0.22 .mu.m sterile
filter, and there was no significant variation of the particle size
in the filtrate. The content of everolimus in the solution was
determined using HPLC. Based on the content, the solution was
subsequently aliquoted into vials at an amount of 10 mg per vial.
The vials were placed in a lyophilizer and lyophilized for 60
h.
[0256] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
everolimus content in the product, the ratio between albumin and
everolimus can be calculated as 0.32:1.
Example 40
Preparation of Purified Romidepsin Albumin Particles
[0257] 113 mg romidepsin was dissolved into 2 ml chloroform/ethanol
(8:1, v/v), and added into 35 ml human serum albumin solution (10%
w/v). The mixture was emulsified for 2 min using a high shear
disperser (Fluko FZ-20) to obtain a primary emulsion. The primary
emulsion was then homogenized using a high pressure homogenizer
under a pressure of 10000-20000 psi to obtain a nano-emulsion.
Subsequently, the nano-emulsion was transferred to a rotatory
evaporator to remove the organic solvent in the solution by vacuum
evaporation at 40 mbar and at 40.degree. C. in a water-bath. The 2
romidepsin nanoparticles were thus generated with an average
diameter of 120-150 nm, and the suspension was translucent.
[0258] The nanoparticles were isolated from the resultant
romidepsin albumin nanoparticle suspension by chromatographic
column separation method referred in Example 16. 10% sucrose
solution was used as dialysate. The obtained purified nanoparticles
suspension was filtered through a 0.22 .mu.m sterile filter, and
there was no significant variation of the particle size in the
filtrate. The content of romidepsin in the solution was determined
using HPLC. Based on the content, the solution was subsequently
aliquoted into vials at an amount of 50 mg per vial. The vials were
placed in a lyophilizer and lyophilized for 48 h.
[0259] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
romidepsin content in the product, the ratio between albumin and
romidepsin can be calculated as 0.25:1.
Example 41
Preparation of Purified Pirarubicin Albumin Particles
[0260] 147 mg pirarubicin was dissolved into 0.75 ml
chloroform/ethanol (5:1, v/v), and added into 19.5 ml human serum
albumin solution (5% w/v). The mixture was emulsified for 2 min
using a high shear disperser (Fluko FZ-20) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer under a pressure of 10000-20000 psi to obtain
a nano-emulsion. Subsequently, the nano-emulsion was transferred to
a rotatory evaporator to remove the organic solvent in the solution
by vacuum evaporation at 40 mbar and at 40.degree. C. in a
water-bath. The 2 pirarubicin nanoparticles were thus generated
with an average diameter of 100-120 nm, and the suspension was
translucent.
[0261] The nanoparticles were isolated from the resultant
pirarubicin albumin nanoparticle suspension by dialysis method
against 5% mannitol solution referred in Example 15. The obtained
purified nanoparticles were filtered through a 0.22 .mu.m sterile
filter, and there was no significant variation of the particle size
in the filtrate. The content of pirarubicin in the solution was
determined using HPLC. Based on the content, the solution was
subsequently aliquoted into vials at an amount of 10 mg per vial.
The vials were placed in a lyophilizer and lyophilized for 48
h.
[0262] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
pirarubicin content in the product, the ratio between albumin and
pirarubicin can be calculated as 0.47:1.
Example 42
Preparation of Purified Aclacinomycin Albumin Particles
[0263] 135 mg aclacinomycin was dissolved into 2 ml
chloroform/ethanol (8:1, v/v), and added into 25 ml human serum
albumin solution (8% w/v). The mixture was emulsified for 2 min
using a high shear disperser (Fluko FZ-20) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer under a pressure of 10000-20000 psi to obtain
a nano-emulsion. Subsequently, the nano-emulsion was transferred to
a rotatory evaporator to remove the organic solvent in the solution
by vacuum evaporation at 40 mbar and at 40.degree. C. in a
water-bath. The aclacinomycin nanoparticles were thus generated
with an average diameter of 80-150 nm, and the suspension was
translucent.
[0264] The nanoparticles were isolated from the resultant
aclacinomycin albumin nanoparticle suspension by chromatographic
column separation method referred in Example 16. 5% mannitol
solution was used as dialysate. The re-suspension was filtered
through a 0.22 .mu.m sterile filter, and there was no significant
variation of the particle size in the filtrate. The content of
aclacinomycin in the solution was determined using HPLC. Based on
the content, the solution was subsequently aliquoted into vials at
an amount of 10 mg per vial. The vials were placed in a lyophilizer
and lyophilized for 48 h.
[0265] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
aclacinomycin content in the product, the ratio between albumin and
aclacinomycin can be calculated as 0.13:1.
Example 43
Preparation of Purified Cabazitaxel Albumin Particles
[0266] 289 mg cabazitaxel was dissolved into 3 ml
chloroform/ethanol (10:1, v/v), and added into 67 ml human serum
albumin solution (5% w/v). The mixture was emulsified for 2 min
using a high shear disperser (Fluko FZ-20) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer under a pressure of 10000-20000 psi to obtain
a nano-emulsion. Subsequently, the nano-emulsion was transferred to
a rotatory evaporator to remove the organic solvent in the solution
by vacuum evaporation at 40 mbar and at 40.degree. C. in a
water-bath. The cabazitaxel nanoparticles were thus generated with
an average diameter of 65-85 nm, and the suspension was
translucent.
[0267] The nanoparticles were isolated from the resultant
cabazitaxel albumin nanoparticle suspension by equal volume
dialysis referred in Example 15 against 5% mannitol. The purified
nanoparticles suspension obtained were filtered through a 0.22
.mu.m sterile filter, and there was no significant variation of the
particle size in the filtrate. The content of cabazitaxel in the
solution was determined using HPLC. Based on the content, the
solution was subsequently aliquoted into vials at an amount of 50
mg per vial. The vials were placed in a lyophilizer and lyophilized
for 55 h.
[0268] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
cabazitaxel content in the product, the ratio between albumin and
cabazitaxel can be calculated as 0.27:1.
Example 44
Preparation of Purified Amiodardone Albumin Particles
[0269] 90 mg amiodardone was dissolved into 2 ml chloroform/ethanol
(11:1, v/v), and added into 58 ml human serum albumin solution (8%
w/v). The mixture was emulsified for 2 min using a high shear
disperser (Fluko FZ-20) to obtain a primary emulsion. The primary
emulsion was then homogenized using a high pressure homogenizer
under a pressure of 10000-20000 psi to obtain a nano-emulsion.
Subsequently, the nano-emulsion was transferred to a rotatory
evaporator to remove the organic solvent in the solution by vacuum
evaporation at 40 mbar and at 40.degree. C. in a water-bath. The 2
amiodardone nanoparticles were thus generated with an average
diameter of 70-105 nm, and the suspension was translucent.
[0270] The nanoparticles were isolated from the resultant
amiodardone albumin nanoparticle suspension by centrifugation
method referred in Example 11 at 21000.times.g for 60 min. The
supernatant was discarded, and the purified nanoparticles were
collected. Purified nanoparticles were re-suspended in 5% dextran
solution. The re-suspension was filtered through a 0.22 .mu.m
sterile filter, and there was no significant variation of the
particle size in the filtrate. The content of amiodardone in the
solution was determined using HPLC. Based on the content, the
solution was subsequently aliquoted into vials at an amount of 10
mg per vial. The vials were placed in a lyophilizer and lyophilized
for 57 h.
[0271] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
amiodardone content in the product, the ratio between albumin and
amiodardone can be calculated as 0.43:1.
Example 45
Preparation of Purified Liothyronine Albumin Particles
[0272] 93 mg liothyronine was dissolved into 2 ml
chloroform/ethanol (9:1, v/v), and added into 46 ml human serum
albumin solution (8% w/v). The mixture was emulsified for 2 min
using a high shear disperser (Fluko FZ-20) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer under a pressure of 10000-20000 psi to obtain
a nano-emulsion. Subsequently, the nano-emulsion was transferred to
a rotatory evaporator to remove the organic solvent in the solution
by vacuum evaporation at 40 mbar and at 40.degree. C. in a
water-bath. The 2 liothyronine nanoparticles were thus generated
with an average diameter of 85-125 nm, and the suspension was
translucent.
[0273] The nanoparticles were isolated from the resultant
liothyronine albumin nanoparticle suspension by centrifugation
method referred in Example 11 at 21000.times.g for 60 min. The
supernatant was discarded, and the purified nanoparticles were
collected. Purified nanoparticles were re-suspended in 5% mannitol
solution. The re-suspension was filtered through a 0.22 .mu.m
sterile filter, and there was no significant variation of the
particle size in the filtrate. The content of liothyronine in the
solution was determined using HPLC. Based on the content, the
solution was subsequently aliquoted into vials at an amount of 10
mg per vial. The vials were placed in a lyophilizer and lyophilized
for 50 h.
[0274] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
liothyronine content in the product, the ratio between albumin and
liothyronine can be calculated as 0.39:1.
Example 46
Preparation of Purified Epothilone B Albumin Particles
[0275] 127 mg Epothilone B was dissolved into 2 ml
chloroform/ethanol (10:1, v/v), and added into 52 ml human serum
albumin solution (5% w/v). The mixture was emulsified for 2 min
using a high shear disperser (Fluko FZ-20) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer under a pressure of 10000-20000 psi to obtain
a nano-emulsion. Subsequently, the nano-emulsion was transferred to
a rotatory evaporator to remove the organic solvent in the solution
by vacuum evaporation at 40 mbar and at 40.degree. C. in a
water-bath. The Epothilone B nanoparticles were thus generated with
an average diameter of 80-115 nm, and the suspension was
translucent.
[0276] The nanoparticles were isolated from the resultant
Epothilone B albumin nanoparticle suspension by equal volume
dialysis against 5% mannitol. The purified nanoparticles suspension
obtained was filtered through a 0.22 .mu.m sterile filter, and
there was no significant variation of the particle size in the
filtrate. The content of Epothilone B in the solution was
determined using HPLC. Based on the content, the solution was
subsequently aliquoted into vials at an amount of 10 mg per vial.
The vials were placed in a lyophilizer and lyophilized for 65
h.
[0277] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
Epothilone B content in the product, the ratio between albumin and
Epothilone B can be calculated as 0.14:1.
Example 47
Preparation of Purified 10-Hydroxy Camptothecin Albumin
Particles
[0278] 93 mg 10-hydroxy camptothecin was dissolved into 2 ml
chloroform/ethanol (10:1, v/v), and added into 48 ml human serum
albumin solution (5% w/v). The mixture was emulsified for 2 min
using a high shear disperser (Fluko FZ-20) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer under a pressure of 10000-20000 psi to obtain
a nano-emulsion. Subsequently, the nano-emulsion was transferred to
a rotatory evaporator to remove the organic solvent in the solution
by vacuum evaporation at 40 mbar and at 40.degree. C. in a
water-bath. The 10-hydroxy camptothecin nanoparticles were thus
generated with an average diameter of 100-135 nm, and the
suspension was translucent.
[0279] The nanoparticles were isolated from the resultant
10-hydroxy camptothecin albumin nanoparticle suspension by
centrifugation method referred in Example 11 at 21000.times.g for
60 min. The supernatant was discarded, and the purified
nanoparticles were collected. Purified nanoparticles were
re-suspended in 5% mannitol solution. The re-suspension was
filtered through a 0.22 .mu.m sterile filter, and there was no
significant variation of the particle size in the filtrate. The
content of 10-hydroxy camptothecin in the solution was determined
using HPLC. Based on the content, the solution was subsequently
aliquoted into vials at an amount of 20 mg per vial. The vials were
placed in a lyophilizer and lyophilized for 72 h.
[0280] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
10-hydroxy camptothecin content in the product, the ratio between
albumin and 10-hydroxy camptothecin can be calculated as
0.26:1.
Example 48
Preparation of Purified Cyclosporine Albumin Particles
[0281] 148 mg cyclosporine was dissolved into 1.5 ml
chloroform/ethanol (11:1, v/v), and added into 18.5 ml human serum
albumin solution (4% w/v). The mixture was emulsified for 2 min
using a high shear disperser (Fluko FZ-20) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer under a pressure of 10000-20000 psi to obtain
a nano-emulsion. Subsequently, the nano-emulsion was transferred to
a rotatory evaporator to remove the organic solvent in the solution
by vacuum evaporation at 40 mbar and at 40.degree. C. in a
water-bath. The cyclosporine nanoparticles were thus generated with
an average diameter of 100-135 nm, and the suspension was
translucent.
[0282] The nanoparticles were isolated from the resultant
cyclosporine albumin nanoparticle suspension by chromatographic
column separation method referred in Example 16. 10% lactose was
use as the dialysate. The purified nanoparticles suspension
obtained was filtered through a 0.22 .mu.m sterile filter, and
there was no significant variation of the particle size in the
filtrate. The content of cyclosporine in the solution was
determined using HPLC. Based on the content, the solution was
subsequently aliquoted into vials at an amount of 50 mg per vial.
The vials were placed in a lyophilizer and lyophilized for 72
h.
[0283] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
cyclosporine content in the product, the ratio between albumin and
cyclosporine can be calculated as 0.26:1.
Example 49
Preparation of Purified Tanespimycin Albumin Particles
[0284] 88 mg tanespimycin was dissolved into 2 ml
chloroform/ethanol (8:1, v/v), and added into 18.5 ml human serum
albumin solution (10% w/v). The mixture was emulsified for 2 min
using a high shear disperser (Fluko FZ-20) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer under a pressure of 10000-20000 psi to obtain
a nano-emulsion. Subsequently, the nano-emulsion was transferred to
a rotatory evaporator to remove the organic solvent in the solution
by vacuum evaporation at 40 mbar and at 40.degree. C. in a
water-bath. The tanespimycin nanoparticles were thus generated with
an average diameter of 85-105 nm, and the suspension was
translucent.
[0285] The nanoparticles were isolated from the resultant
tanespimycin albumin nanoparticle suspension by chromatographic
column separation method referred in Example 16. 5% mannitol was
use as the dialysate. The purified nanoparticles suspension was
filtered through a 0.22 .mu.m sterile filter, and there was no
significant variation of the particle size in the filtrate. The
content of tanespimycin in the solution was determined using HPLC.
Based on the content, the solution was subsequently aliquoted into
vials at an amount of 50 mg per vial. The vials were placed in a
lyophilizer and lyophilized for 72 h.
[0286] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
tanespimycin content in the product, the ratio between albumin and
tanespimycin can be calculated as 0.62:1.
Example 50
Preparation of Purified Propofol Albumin Particles
[0287] 603 mg propofol was dissolved into 6 ml chloroform, and
added into 73 ml human serum albumin solution (10% w/v). The
mixture was emulsified for 2 min using a high shear disperser
(Fluko FZ-20) to obtain a primary emulsion. The primary emulsion
was then homogenized using a high pressure homogenizer under a
pressure of 10000-20000 psi to obtain a nano-emulsion.
Subsequently, the nano-emulsion was transferred to a rotatory
evaporator to remove the organic solvent in the solution by vacuum
evaporation at 40 mbar and at 40.degree. C. in a water-bath. The
propofol nanoparticles were thus generated with an average diameter
of 70-115 nm, and the suspension was translucent.
[0288] The nanoparticles were isolated from the resultant propofol
albumin nanoparticle suspension by centrifugation method referred
in Example 11 at 21000.times.g for 60 min. The supernatant was
discarded, and the purified nanoparticles were collected. Purified
nanoparticles were re-suspended in 10% lactose solution. The
re-suspension was filtered through a 0.22 .mu.m sterile filter, and
there was no significant variation of the particle size in the
filtrate. The content of propofol in the solution was determined
using HPLC. Based on the content, the solution was subsequently
aliquoted into vials at an amount of 50 mg per vial. The vials were
placed in a lyophilizer and lyophilized for 72 h.
[0289] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
propofol content in the product, the ratio between albumin and
propofol can be calculated as 0.58:1.
Example 51
Preparation of Purified Vinblastine Sulfate Albumin Particles
[0290] 1.25 g vinblastine sulfate was dissolved into 10 ml
chloroform/isopropanol (10:1, v/v), and added into 202 ml human
serum albumin solution (5% w/v). The mixture was emulsified for 2
min using a high shear disperser (Fluko FZ-20) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer under a pressure of 10000-20000 psi to obtain
a nano-emulsion. Subsequently, the nano-emulsion was transferred to
a rotatory evaporator to remove the organic solvent in the solution
by vacuum evaporation at 40 mbar and at 40.degree. C. in a
water-bath. The vinblastine sulfate nanoparticles were thus
generated with an average diameter of 90-130 nm, and the suspension
was translucent.
[0291] The nanoparticles were isolated from the resultant
vinblastine sulfate albumin nanoparticle suspension by equal volume
dialysis against 5% mannitol. The purified nanoparticles suspension
obtained was filtered through a 0.22 .mu.m sterile filter, and
there was no significant variation of the particle size in the
filtrate. The content of vinblastine sulfate in the solution was
determined using HPLC. Based on the content, the solution was
subsequently aliquoted into vials at an amount of 50 mg per vial.
The vials were placed in a lyophilizer and lyophilized for 72
h.
[0292] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
vinblastine sulfate content in the product, the ratio between
albumin and vinblastine sulfate can be calculated as 0.23:1.
Example 52
Preparation of Purified Exemestane Albumin Particles
[0293] 155 mg exemestane was dissolved into 3 ml chloroform/ethanol
(6:1, v/v), and added into 27 ml human serum albumin solution (5%
w/v). The mixture was emulsified for 2 min using a high shear
disperser (Fluko FZ-20) to obtain a primary emulsion. The primary
emulsion was then homogenized using a high pressure homogenizer
under a pressure of 10000-20000 psi to obtain a nano-emulsion.
Subsequently, the nano-emulsion was transferred to a rotatory
evaporator to remove the organic solvent in the solution by vacuum
evaporation at 40 mbar and at 40.degree. C. in a water-bath. The
exemestane nanoparticles were thus generated with an average
diameter of 70-100 nm, and the suspension was translucent.
[0294] The nanoparticles were isolated from the resultant
exemestane albumin nanoparticle suspension by equal volume dialysis
against 5% mannitol. The purified nanoparticles suspension obtained
was filtered through a 0.22 .mu.m sterile filter, and there was no
significant variation of the particle size in the filtrate. The
content of exemestane in the solution was determined using HPLC.
Based on the content, the solution was subsequently aliquoted into
vials at an amount of 50 mg per vial. The vials were placed in a
lyophilizer and lyophilized for 72 h.
[0295] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
exemestane content in the product, the ratio between albumin and
exemestane can be calculated as 0.19:1.
Example 53
Preparation of Purified Flutamide Albumin Particles
[0296] 189 mg flutamide was dissolved into 2 ml
chloroform/tert-butanol (6:1, v/v), and added into 38 ml human
serum albumin solution (8% w/v). The mixture was emulsified for 2
min using a high shear disperser (Fluko FZ-20) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer under a pressure of 10000-20000 psi to obtain
a nano-emulsion. Subsequently, the nano-emulsion was transferred to
a rotatory evaporator to remove the organic solvent in the solution
by vacuum evaporation at 40 mbar and at 40.degree. C. in a
water-bath. The flutamide nanoparticles were thus generated with an
average diameter of 100-120 nm, and the suspension was
translucent.
[0297] The nanoparticles were isolated from the resultant flutamide
albumin nanoparticle suspension by chromatographic column
separation method referred in Example 16. 5% dextran solution was
used as dialysate. The purified nanoparticles suspension obtained
was filtered through a 0.22 .mu.m sterile filter, and there was no
significant variation of the particle size in the filtrate. The
content of flutamide in the solution was determined using HPLC.
Based on the content, the solution was subsequently aliquoted into
vials at an amount of 50 mg per vial. The vials were placed in a
lyophilizer and lyophilized for 60 h.
[0298] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
flutamide content in the product, the ratio between albumin and
flutamide can be calculated as 0.35:1.
Example 54
Preparation of Purified Fulvestrant Albumin Particles
[0299] 350 mg fulvestrant was dissolved into 3 ml
dichloromethane/ethanol (6:1, v/v), and added into 67 ml human
serum albumin solution (5% w/v). The mixture was emulsified for 2
min using a high shear disperser (Fluko FZ-20) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer under a pressure of 10000-20000 psi to obtain
a nano-emulsion. Subsequently, the nano-emulsion was transferred to
a rotatory evaporator to remove the organic solvent in the solution
by vacuum evaporation at 40 mbar and at 40.degree. C. in a
water-bath. The fulvestrant nanoparticles were thus generated with
an average diameter of 105-150 nm, and the suspension was
translucent.
[0300] The nanoparticles were isolated from the resultant
fulvestrant albumin nanoparticle suspension by chromatographic
column separation method referred in Example 16. 10% lactose was
used as dialysate. The purified nanoparticles suspension obtained
was filtered through a 0.22 .mu.m sterile filter, and there was no
significant variation of the particle size in the filtrate. The
content of fulvestrant in the solution was determined using HPLC.
Based on the content, the solution was subsequently aliquoted into
vials at an amount of 20 mg per vial. The vials were placed in a
lyophilizer and lyophilized for 60 h.
[0301] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
fulvestrant content in the product, the ratio between albumin and
fulvestrant can be calculated as 0.29:1.
Example 55
Preparation of Purified Semustine Albumin Particles
[0302] 650 mg semustine was dissolved into 5 ml chloroform/ethanol
(6:1, v/v), and added into 89 ml human serum albumin solution (4%
w/v). The mixture was emulsified for 2 min using a high shear
disperser (Fluko FZ-20) to obtain a primary emulsion. The primary
emulsion was then homogenized using a high pressure homogenizer
under a pressure of 10000-20000 psi to obtain a nano-emulsion.
Subsequently, the nano-emulsion was transferred to a rotatory
evaporator to remove the organic solvent in the solution by vacuum
evaporation at 40 mbar and at 40.degree. C. in a water-bath. The
semustine nanoparticles were thus generated with an average
diameter of 85-120 nm, and the suspension was translucent.
[0303] The nanoparticles were isolated from the resultant semustine
albumin nanoparticle suspension by centrifugation method referred
in Example 11 at 21000.times.g for 60 min. The supernatant was
discarded, and the purified nanoparticles were collected. Purified
nanoparticles were re-suspended in 5% mannitol solution. The
re-suspension was filtered through a 0.22 .mu.m sterile filter, and
there was no significant variation of the particle size in the
filtrate. The content of semustine in the solution was determined
using HPLC. Based on the content, the solution was subsequently
aliquoted into vials at an amount of 20 mg per vial. The vials were
placed in a lyophilizer and lyophilized for 70 h.
[0304] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
semustine content in the product, the ratio between albumin and
semustine can be calculated as 0.58:1.
Example 56
Preparation of Purified Thiocolchicine Dimer Albumin Particles
[0305] 456 mg thiocolchicine dimer was dissolved into 5 ml
chloroform/ethanol (9:1, v/v), and added into 58 ml human serum
albumin solution (8% w/v). The mixture was emulsified for 2 min
using a high shear disperser (Fluko FZ-20) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer under a pressure of 10000-20000 psi to obtain
a nano-emulsion. Subsequently, the nano-emulsion was transferred to
a rotatory evaporator to remove the organic solvent in the solution
by vacuum evaporation at 40 mbar and at 40.degree. C. in a
water-bath. The thiocolchicine dimer nanoparticles were thus
generated with an average diameter of 65-90 nm, and the suspension
was translucent.
[0306] The nanoparticles were isolated from the resultant
thiocolchicine dimer albumin nanoparticle suspension by
chromatographic column separation method referred in Example 16.
10% lactose was used as dialysate. The purified nanoparticles
suspension obtained was filtered through a 0.22 .mu.m sterile
filter, and there was no significant variation of the particle size
in the filtrate. The content of thiocolchicine dimer in the
solution was determined using HPLC. Based on the content, the
solution was subsequently aliquoted into vials at an amount of 20
mg per vial. The vials were placed in a lyophilizer and lyophilized
for 70 h.
[0307] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
thiocolchicine dimer content in the product, the ratio between
albumin and thiocolchicine dimer can be calculated as 0.37:1.
Example 57
Preparation of Purified Ibuprofen Dimer Albumin Particles
[0308] 348 mg ibuprofen was dissolved into 3 ml
dichloromethane/ethanol (11:1, v/v), and added into 62 ml human
serum albumin solution (5% w/v). The mixture was emulsified for 2
min using a high shear disperser (Fluko FZ-20) to obtain a primary
emulsion. The primary emulsion was then homogenized using a high
pressure homogenizer under a pressure of 10000-20000 psi to obtain
a nano-emulsion. Subsequently, the nano-emulsion was transferred to
a rotatory evaporator to remove the organic solvent in the solution
by vacuum evaporation at 40 mbar and at 40.degree. C. in a
water-bath. The ibuprofen nanoparticles were thus generated with an
average diameter of 65-95 nm, and the suspension was
translucent.
[0309] The nanoparticles were isolated from the resultant ibuprofen
albumin nanoparticle suspension by chromatographic column
separation method referred in Example 16. 5% dextran was used as
dialysate. The purified nanoparticles suspension obtained was
filtered through a 0.22 .mu.m sterile filter, and there was no
significant variation of the particle size in the filtrate. The
content of ibuprofen in the solution was determined using HPLC.
Based on the content, the solution was subsequently aliquoted into
vials at an amount of 20 mg per vial. The vials were placed in a
lyophilizer and lyophilized for 50 h.
[0310] When the lyophilized product was reconstituted in water for
injection, the resultant cake was dissolved rapidly, the suspension
was translucent, and no significant variation of the particle size
was observed. After determination of the human serum albumin and
ibuprofen content in the product, the ratio between albumin and
ibuprofen can be calculated as 0.19:1.
Example 58
Albumin Concentration Influences Inhibition Effect of Drugs on
Human Tumor Cells
[0311] Human lung cancer cell SPC-A-1 and breast cancer cell MCF-7
were seeded to 96-well plate, cultured overnight and made them
adherent to the wells. Then, the medium was changed to serum-free
medium in order to starve the cells. Example 6, particle 6 and
Abraxane were added to the cells at final concentrations of 160,
320, and 640 .mu.g/ml (paclitaxel concentration) for 24 hours at
37.degree. C. Cell inhibition rate of different dosing groups was
compared by using MTT method, and the results were shown in the
following table and FIGS. 12 and 13. Moreover, inhibition rate-drug
concentration curve were drew too. As showing in the inhibition
rate-drug concentration curve, the inhibition rate of particle 6
was significantly higher than other two groups, and no significant
difference between Example 6 and Abraxane. That means redundant
albumin will reduce the inhibition effect of paclitaxel on tumor
cells.
TABLE-US-00019 TABLE 19 Cells inhibition rate of different
treatment Inhibition Particle 6 Exemple 6 Abraxane rate % 640
.mu.g/ml 320 .mu.g/ml 160 .mu.g/ml 640 .mu.g/ml 320 .mu.g/ml 160
.mu.g/ml 640 .mu.g/ml 320 .mu.g/ml 160 .mu.g/ml MCF-7 cells No. 1
60.12 74.97 24.00 43.23 22.50 -3.81 25.90 10.70 6.20 No. 2 69.59
71.12 36.91 30.74 11.14 1.71 42.30 15.78 -8.14 No. 3 68.46 58.84
37.87 46.65 6.63 20.53 39.88 11.30 -1.85 No. 4 69.78 70.09 38.65
52.02 23.66 5.73 45.50 2.42 -0.08 No. 5 71.69 72.93 43.91 46.87
22.18 13.55 47.26 5.39 -4.86 No. 6 71.97 57.52 50.47 25.65 30.17
-7.71 31.19 22.88 -1.64 mean 68.60 67.58 38.63 40.86 19.38 5.00
38.67 11.41 -1.73 SD 3.98 6.83 8.02 9.43 7.99 9.70 7.68 6.69 4.41
SPC-A-1 cells No. 1 66.49 25.07 16.29 30.99 5.24 9.25 31.75 11.40
-3.20 No. 2 62.32 31.29 -3.14 20.50 -11.75 -3.19 24.09 7.84 -12.89
No. 3 64.18 25.75 0.91 29.13 3.97 -11.90 13.08 9.58 4.11 No. 4
70.26 32.73 -12.03 32.69 7.03 -5.39 18.25 2.98 -4.37 No. 5 67.98
33.58 9.87 25.88 19.83 12.75 30.02 6.42 -1.04 No. 6 77.24 44.83
7.26 28.46 10.29 29.57 18.94 14.17 12.62 mean 68.08 32.21 3.19
27.94 5.77 5.18 22.69 8.73 -0.80 SD 4.82 6.52 9.22 3.94 9.40 13.80
6.63 3.57 7.84
Example 59
Albumin Concentration Influences Uptake of Drugs by Human Vascular
Endothelial Cells
[0312] Human umbilical vascular endothelial cells EA.hy 926 were
seeded at 8.times.10.sup.5 to 6-well plate. Before adding drugs,
the medium was changed into serum-free medium. Drugs was divided
into 5 groups, group A was particle 6, B was Example 6, C was
Abraxane, D was particle 6+0.5% HSA monomer, E was particle 6+0.5%
HSA polymer. Different groups of drug were added to the cells at
final concentrations of 50, 100, and 200 .mu.g/ml (paclitaxel
concentration) for 2 hours at 37.degree. C. After 2 hours, the
cells were washed by PBS for 3 times, then 500 ul 5% TritonX-100
was added to each wells to lyse the cells. The paclitaxel
concentration in cell lysis was detected by the method mentioned in
example 10, and the results were shown in the following table and
FIG. 14. The results show that cellular uptake of paclitaxel of
group A was higher than group B and group C, and of group B and C
had no significant difference. Furthermore, group D was higher than
group E. That means higher HSA concentration hindered drug uptake
to vascular endothelial cells, and compared to monomer, the HSA
polymer would be more impede the cellular uptake.
TABLE-US-00020 TABLE 20 Drug concentration in cells of different
treatment .mu.g/ml 50 100 200 Particle 6 concentration 2.367 4.102
5.864 value 2.165 3.873 6.227 2.668 4.217 6.531 mean 2.400 4.064
6.207 SD 0.253 0.175 0.334 Example 6 concentration 1.362 2.415
3.374 value 1.626 2.517 3.668 1.654 2.688 3.941 mean 1.547 2.540
3.661 SD 0.161 0.138 0.284 Abraxane concentration 1.462 2.246 3.861
value 1.623 2.522 3.521 1.711 2.613 4.031 mean 1.599 2.460 3.804 SD
0.126 0.191 0.260 Particle 6 + concentration 1.221 2.168 3.055 0.5%
HSA value 1.254 2.517 3.251 monomer 1.021 2.231 3.461 mean 1.165
2.305 3.256 SD 0.126 0.186 0.203 Particle 6 + concentration 0.764
1.533 1.988 0.5% HSA value 0.966 1.687 1.845 polymer 0.878 1.921
2.356 mean 0.869 1.714 2.063 SD 0.101 0.195 0.264
Example 60
[0313] The contents of albumin and paclitaxel (PTX) and the ratio
of polymer in the human serum albumin used, product obtained in
Example 6, and the corresponding nanoparticle 6 were determined
using the method in Example 10. The results are listed in the
following table.
TABLE-US-00021 TABLE 21 Contents of albumin polymer (Poly-HSA)
Albumin/ PolyHSA/ Monomer Polymer palictaxel PTX (%) (%) (mg/mg)
(mg/mg) HSA 95.8 4.2 -- -- Example 6 80.7 19.3 8.8 1.7 Particle 6
73.5 26.5 0.13 0.034
[0314] It has been revealed by the results that the ratio of
poly-HSA was significantly increased during the preparation process
of paclitaxel albumin nanoparticles. However, in the product
prepared by the preparation method of the present disclosure, the
amount of poly-HSA in unit weight is significantly reduced because
of the decrease of total albumin content.
[0315] The amount of polymer in an albumin solution was increased
by dialysis using a regenerated cellulose ultrafiltration membrane
(PXC100C50, Millipore) with the cut-off molecular weight of 100K.
The ratio of albumin polymer was detected using the method in
Example 10, and the content of albumin was determined by Kjeldahl
method to avoid the deviation produced by the different responses
to potential polymers of HPLC method.
TABLE-US-00022 TABLE 22 The content of albumin polymer in HSA
before and after dialysis Monomer (%) Polymer (%) HSA before
dialysis 95.8 4.2 HSA after dialysis 25.1 74.9
[0316] It can be seen from the result that the albumin solution
with a majority amount of polymer can be obtained by dialysis
process.
Example 61
Sensitization Study of poly-HSA in Guinea Pigs
[0317] In this experiment, the HSA and poly-HSA obtained from
Example 60 were selected as the test drug. The dosages were
selected as 1 mg per guinea pig and 0.3 mg per guinea pig.
Sensitization was conducted by intraperitoneal injection once every
other day, for a total of 3 injections. At the same time, a
positive control group (0.2% ovalbumin) and a negative control
group (0.9% sodium chloride injection) were also established.
Detailed dosage regimen is listed in the table below.
TABLE-US-00023 TABLE 23 Protocol for sensitization study in guinea
pig sensitization Excitation Number Concentration Administration
Administration Administration Administration of of the drug volume
dosage volume dosage Group animals (mg/ml) (ml/animal) (mg/animal)
(ml/animal) (mg/animal) Negative 6 -- 0.5 -- 1 -- control group
Positive 6 2 0.5 1 1 2 control group Low dosage 6 0.6 0.5 0.3 1 0.6
of HSA High dosage 6 2 0.5 1 1 2 of HSA Low dosage 6 0.6 0.5 1.3 1
0.6 of Poly-HSA High dosage 6 2 0.5 1 1 2 of Poly-HSA
[0318] Administration method for sensitization: the back of guinea
pig was firmly held by cup-shape left hand, allowing the abdominal
skin stretched when the guinea pig was fixed. The abdomen of the
guinea pig was lifted, and the head was lowered down. After
disinfecting of the injection site by an alcohol wipe, the needle
of a 2 ml disposable syringe held by the right hand was punctured
into the skin of the guinea pig. The needle was inserted at the
site 1 mm left to the midline of lower abdomen. When arriving at
the subcutaneous part, the needle was inserted forward for further
5 mm to 10 mm, and subsequently punctured into the abdominal cavity
at an angle of 45.degree.. After fixing the needle, the
pharmaceutical solution was injected slowly. After the injection, a
dry cotton wipe was pressed on the pinprick in order to prevent the
outflow of the pharmaceutical.
[0319] Allergy excitation: excitation was conducted by intravenous
injection, and the excitation was performed 10 d after the last
sensitization with the dosages of 2 mg/animal and 0.6
mg/animal.
[0320] Administration method for excitation: injection was
performed to the lateral metatarsal vein of the guinea pig fixed by
an assistant. The stifles were grasped by the operator to fix the
body of the animal. Its vein was compressed, and the legs were in a
stretched state. The hair at the injection site was shaved (or the
skin at the injection site was cut). After sterilization by alcohol
wipes, thick lateral metatarsal vein can be seen. The needle of a 1
ml disposable syringe was punctured into the blood vessel along the
direction to the heart by the right hand. After the injection, a
dry cotton wipe was pressed on the pinprick in order to prevent
bleeding.
[0321] The reaction of each animal and the time when allergy
symptoms appeared or disappeared were observed immediately after
the excitation for 30 min. Maximal observation duration was 3 h.
The results of allergic reaction are listed in the following
table.
TABLE-US-00024 TABLE 24 Symptoms of allergic reaction 0 Normal 1
Dysphoria 2 Piloerection 3 Trembling 4 Nose scratching 5 Sneezing 6
Coughing 7 Tachypnea 8 Urination 9 Defecation 10 Lacrimation 11
Dyspnea 12 Wheezing 13 Peliosis 14 Gait disturbance 15 Jumping 16
Panting 17 Convulsion 18 Rotation 19 Cheyne-stokes respiration 20
Death
TABLE-US-00025 TABLE 25 Evaluation criteria for systemic allergic
reaction Symptom Degree Result 0 - Negative allergic reaction 1-4 +
Weakly positive allergic reaction 5-10 ++ Positive allergic
reaction 11-19 +++ Strongly positive allergic reaction 20 ++++
Extremely positive allergic reaction
TABLE-US-00026 TABLE 26 Results for active anaphylaxis of guinea
pig Number Posi- of the Reaction level tive Group animal 1 2 3 4 5
6 rate Negative 6 - - - - - - - control group Positive 6 ++++ ++++
++++ ++++ ++++ ++++ ++++ control group Low dosage 6 + - - - + - +
of HSA High dosage 6 + + ++ ++ + + ++ of HSA Low dosage 6 +++ ++++
+++ ++ +++ +++ +++ of Poly-HSA High dosage 6 ++++ ++++ ++++ ++++
++++ ++++ ++++ of Poly-HSA
[0322] The results show that the samples containing more poly-HSA
had stronger sensitization at same level of total HSA content.
Example 62
[0323] 2.5 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech
Co., Ltd) was dissolved into 15 ml chloroform/ethanol (11:1, v/v),
and added into which 500 ml human serum albumin solution (4% w/v)
(CAS: 70024-90-7, Guangdong Shuanglin Biopharmaceutical. Co.,
Ltd.). The mixture was emulsified for 2 min using a high shear
disperser (Fluko FZ-20) to obtain a primary emulsion. The primary
emulsion was then homogenized using a high pressure homogenizer
(Model M110-EH30K, MFIC Company, USA) under a pressure of
10000-20000 psi to obtain a nano-emulsion. Subsequently, the
nano-emulsion was transferred to a rotatory evaporator (Model
R-210, Buchi Company, Switzerland) to remove the organic solvent in
the solution by vacuum evaporation at 40 mbar and at 40.degree. C.
in a water-bath. The paclitaxel-albumin nanoparticles were thus
generated with an average diameter of 129 nm, and the suspension
was translucent. The suspension can be smoothly filtered through a
0.22 .mu.m sterile filter (Sartorius AG, Germany). There was no
significant variation of the particle size after filtration, and no
significant change was observed after storage for 48 h at room
temperature.
[0324] The purified nanoparticles were isolated respectively by
centrifugation in Example 11, by dialysis in Example 15 and by
chromatographic column separation in Example 16. The results showed
that the suspension became turbid and precipitated during dialysis.
Thus purified nanoparticles couldn't be obtained though this
method. While the purified nanoparticles obtained by centrifugation
and chromatographic column separation were turbid and precipitated
in 60 min.
Discussion
[0325] Prior art formulations have a high ratio of albumin to an
active ingredient (for example, 9:1). It has been unexpectedly
discovered by the present inventors that in such a formulation,
most albumin acts only as a protective or support agent in the
lyophilized product. Most drug molecules (for example, paclitaxel)
are encapsulated in the nanoparticles, and albumin that does not
form particles is substantially free of drug molecules. In prior
art formulations, most human serum albumin molecules are not bound
to the drug molecules.
[0326] After administration, particles in prior art formulations
are disintegrated rapidly, and complexes are formed between the
drug molecules and the endogenous human serum albumin.
Consequently, excessive human serum albumin in the prior art
formulations does not contribute to the efficacy of the
formulations (see Example 26), but instead causes safety risks due
to its aggregation and immunogenicity (see Examples 32, 25 and 27).
Furthermore, extra albumin may compete with the drug-albumin
complex to bind to the gp60 receptor, thus decrease the drug uptake
by the vascular endothelial cells (see Example 59). Moreover, after
binding to the poly-HSA formed in preparation process, the gp60
receptor may be unable to be transcytosed effectively, thus
preventing the receptor from being released. This may further
inhibit the drug uptake by the endothelial cells, and thus decrease
the effective concentration of drugs.
[0327] Based on the discoveries above, purified nanoparticles of
the present disclosure have a lower albumin: active ingredient
ratio. It has been confirmed by the present inventors that only a
small amount of human serum albumin was required for forming stable
nanoparticles with drug molecules. The reduction in human serum
albumin in purified nanoparticles and compositions thereof provided
herein reduces adverse reactions associated with aggregation and
immunogenicity of human serum albumin.
[0328] After administration, purified nanoparticles provided herein
were able to be disintegrated rapidly, and bound to endogenous
albumin for circulation in vivo. There were no significantly
difference observed in terms of safety and efficacy in animal
studies compared with prior art formulation. However, purified
nanoparticles and compositions thereof provided herein improved
uptake by human vascular endothelial cells as well as delivery and
effectiveness of active ingredients in the nanoparticles to or in
human target cells.
[0329] The various embodiments described above can be combined to
provide further embodiments. All of the U.S. patents, U.S. patent
application publications, U.S. patent applications, foreign
patents, foreign patent applications and non-patent publications
referred to in this specification and/or listed in the Application
Data Sheet are incorporated herein by reference, in their entirety.
Aspects of the embodiments can be modified, if necessary to employ
concepts of the various patents, applications and publications to
provide yet further embodiments.
[0330] These and other changes can be made to the embodiments in
light of the above-detailed description. In general, in the
following claims, the terms used should not be construed to limit
the claims to the specific embodiments disclosed in the
specification and the claims, but should be construed to include
all possible embodiments along with the full scope of equivalents
to which such claims are entitled. Accordingly, the claims are not
limited by the disclosure.
* * * * *