U.S. patent application number 17/174291 was filed with the patent office on 2021-07-08 for liposomal anticancer compositions.
The applicant listed for this patent is IriSys, LLC. Invention is credited to Robert Giannini, Igor Nikoulin, Yevgeniya Plekhov, Gerald Yakatan.
Application Number | 20210205245 17/174291 |
Document ID | / |
Family ID | 1000005464378 |
Filed Date | 2021-07-08 |
United States Patent
Application |
20210205245 |
Kind Code |
A1 |
Nikoulin; Igor ; et
al. |
July 8, 2021 |
LIPOSOMAL ANTICANCER COMPOSITIONS
Abstract
Provided herein includes methods and compositions for the
treatment of cancer. Described herein are liposome encapsulated
chemotherapeutic agents and methods for preparing and utilizing the
same.
Inventors: |
Nikoulin; Igor; (San Diego,
CA) ; Yakatan; Gerald; (San Diego, CA) ;
Plekhov; Yevgeniya; (San Diego, CA) ; Giannini;
Robert; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IriSys, LLC |
San Diego |
CA |
US |
|
|
Family ID: |
1000005464378 |
Appl. No.: |
17/174291 |
Filed: |
February 11, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16331258 |
Mar 7, 2019 |
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PCT/US2017/049968 |
Sep 1, 2017 |
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17174291 |
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62385763 |
Sep 9, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/127 20130101;
A61K 9/0019 20130101; A61K 47/10 20130101; A61P 35/00 20180101;
A61K 31/4745 20130101; A61K 9/10 20130101; A61K 31/122 20130101;
A61K 31/194 20130101; A61K 31/704 20130101; A61K 9/19 20130101;
A61K 31/375 20130101; A61K 47/24 20130101; A61K 47/28 20130101;
A61K 31/136 20130101; A61K 31/198 20130101 |
International
Class: |
A61K 31/194 20060101
A61K031/194; A61P 35/00 20060101 A61P035/00; A61K 9/00 20060101
A61K009/00; A61K 9/127 20060101 A61K009/127; A61K 9/19 20060101
A61K009/19; A61K 31/122 20060101 A61K031/122; A61K 31/136 20060101
A61K031/136; A61K 31/198 20060101 A61K031/198; A61K 31/375 20060101
A61K031/375; A61K 31/4745 20060101 A61K031/4745; A61K 31/704
20060101 A61K031/704; A61K 47/10 20060101 A61K047/10; A61K 47/24
20060101 A61K047/24; A61K 47/28 20060101 A61K047/28; A61K 9/10
20060101 A61K009/10 |
Claims
1. A pharmaceutical composition comprising a liposome, wherein the
liposome comprises a plurality of lipids; a weakly basic anticancer
compound; and oxalic acid or a salt of oxalic acid; wherein the
weight ratio of the plurality of lipids to the weakly basic
anticancer compound is from about 20:1 to about 50:1.
2. The pharmaceutical composition of claim 1, wherein the weight
ratio of the plurality of lipids to the weakly basic anticancer
compound is from 20:1 to 50:1.
3. The pharmaceutical composition of claim 1, wherein the weight
ratio of the plurality of lipids to the weakly basic anticancer
compound is from about 30:1 to about 50:1.
4. The pharmaceutical composition of claim 1, wherein the weight
ratio of the plurality of lipids to the weakly basic anticancer
compound is from 30:1 to 50:1.
5. The pharmaceutical composition of claim 1, wherein the weight
ratio of the plurality of lipids to the weakly basic anticancer
compound is from about 30:1 to about 40:1.
6. The pharmaceutical composition of claim 1, wherein the weight
ratio of the plurality of lipids to the weakly basic anticancer
compound is from about 40:1 to about 50:1.
7. The pharmaceutical composition of claim 1, wherein the weight
ratio of the plurality of lipids to the weakly basic anticancer
compound is about 40:1.
8. The pharmaceutical composition of claim 1, wherein the plurality
of lipids comprise a plurality of free cholesterols or a plurality
of phospholipids.
9. The pharmaceutical composition of claim 1, wherein the plurality
of lipids comprise a plurality of free cholesterols and a plurality
of phospholipids.
10. The pharmaceutical composition of claim 9, wherein the molar
ratio of the plurality of phospholipids to the plurality of free
cholesterols is at least 0.5 to 1 to about 4 to 1.
11. The pharmaceutical composition of claim 9, wherein the molar
ratio of the plurality of phospholipids to the plurality of free
cholesterols is from about 0.86:1 to about 3.68:1.
12. The pharmaceutical composition of claim 1, wherein the weakly
basic anticancer compound is doxorubicin, irinotecan, mitoxantrone,
or a combination of two or more thereof.
13. The pharmaceutical composition of claim 1, wherein the weakly
basic anticancer compound has a pKa from about 7.5 to about
9.0.
14. The pharmaceutical composition of claim 1, wherein the liposome
comprises about 500 .mu.g/mL to about 1,000 .mu.g/mL of the weakly
basic anticancer compound.
15. The pharmaceutical composition of claim 14, wherein the
liposome comprises about 750 .mu.g/mL to about 850 .mu.g/mL of the
weakly basic anticancer compound.
16. The pharmaceutical composition of claim 1, wherein the
plurality of lipids comprise a poloxamer.
17. The pharmaceutical composition of claim 16, wherein the
poloxamer is poloxamer 188.
18. The pharmaceutical composition of claim 1, further comprising
ascorbic acid, N-acetylcysteine, ascorbyl palmitate, ubiquinone, or
ethylenediaminetetraacetic acid.
19. The pharmaceutical composition of claim 1, wherein: (i) at
least 40% to 80% of the weakly basic anticancer compound is
released from the liposome at pH 5 under standard assay conditions;
wherein the standard assay conditions comprise 20 times or 50 times
dilution in phosphate buffered saline at pH 5.0 for 2 hours, 4
hours, 6 hours, or 8 hours at 37.degree. C.; (ii) at least 10% to
50% of the weakly basic anticancer compound is released from the
liposome at pH 6.0 under standard assay conditions; wherein the
standard assay conditions comprise 20 times or 50 times dilution in
phosphate buffered saline at pH 6.0 for 2 hours, 4 hours, 6 hours,
or 8 hours at 37.degree. C.; (iii) at least 7% to 30% of the weakly
basic anticancer compound is released from the liposome at pH 6.7
under standard assay conditions; wherein the standard assay
conditions comprise 20 times or 50 times dilution in phosphate
buffered saline at pH 6.7 for 2 hours, 4 hours, 6 hours, or 8 hours
at 37.degree. C.; or (iv) less than 5% of the weakly basic
anticancer compound is released from the liposome at pH 7.4 under
standard assay conditions; wherein the standard assay conditions
comprise 20 times or 50 times dilution in phosphate buffered saline
at pH 7.4 for 2 hours, 4 hours, 6 hours, or 8 hours at 37.degree.
C.
20. The pharmaceutical composition of claim 1, wherein the
pharmaceutical composition comprises a plurality of the liposome
having a mean longest dimension of about 40 nm to about 100 nm
determined by the intensity-averaged particle diameters measured by
dynamic light scattering; or wherein the pharmaceutical composition
comprises a plurality of the liposome having a mean longest
dimension from about 1 nm to about 50 nm determined by
cryo-transmission electron microscopy.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/331,258 filed Mar. 7, 2019, allowed, which is a Section 371
US national phase of International Application No.
PCT/US2017/049968 filed Sep. 1, 2017, which claims priority to U.S.
Application No. 62/385,763 filed Sep. 9, 2016, the content of which
is incorporated herein in its entirety and for all purposes.
BACKGROUND
[0002] The lack of an adequate drug concentration at the tumor site
as a result of dose limiting toxicity and poor local accumulation
is often the reason why an in vitro promising anticancer agent
fails when applied in vivo. Over the years many strategies have
been developed to improve intratumoral drug delivery including
passive drug targeting, active targeting to tumor cells and
triggered drug release by nanocarriers [2-12]. However,
incorporating drugs in a nanocarrier may greatly change their
kinetics and interaction characteristics with cells. The
anthracycline antibiotic doxorubicin is an effective anticancer
agent for various malignancies but its application is severely
limited due to its cardiotoxic side effects. Encapsulating
doxorubicin into pegylated liposomal nanocarriers lowers the
typical doxorubicin related side-effects [4, 5, 7-8]. Also, the
circulation time is prolonged [2] and tumor specific delivery is
improved due to the enhanced permeability and retention effect
[3].
[0003] Stability of liposomes containing anticancer compounds
depends on both the stability of liposomes in blood and the
stability of drugs inside liposomes. The stable form of drugs
inside liposomes would attribute to the prolongation of liposomes
containing anti-tumor drugs in blood and lower toxicity. It has
been demonstrated that doxorubicin can form aggregates with several
counter ions, such as sulfate [1, 13-15], phosphate [13-14], and
citrate [1, 10, 13-15]. The leakage behavior of doxorubicin from
liposomes containing different doxorubicin salts confirms that
doxorubicin-salt aggregate determines rate of the free doxorubicin
release from the liposomes.
[0004] Doxorubicin is a chemotherapy drug. In liposomal
doxorubicin, the molecules of the drug are enclosed (encapsulated)
in a fatty coating known as a liposome. The liposome allows the
doxorubicin to remain in the body for longer [2]. This means more
of the chemotherapy is delivered to the cancer cells while having
fewer side effects on healthy tissue [4, 5, 7-8]. Two liposomal
formulations of doxorubicin approved for human use are currently on
the market, MYOCET.RTM. and CAELYX.RTM. (EU) respectively
DOXIL.RTM. (US). These two liposomal doxorubicin drugs currently
work in slightly different ways and are used to treat different
types of cancer.
[0005] The nanodrug DOXIL.RTM. has been approved for the treatment
of AIDS-related Kaposi sarcoma, advanced ovarian and breast cancer
and multiple myeloma [4] DOXIL.RTM. is composed of PEGylated
phospholipids and Cholesterol and is loaded via an ammonium sulfate
gradient. Encapsulating doxorubicin into pegylated liposomal
nanocarriers lowers the typical doxorubicin related side-effects
[4-7]. Although encapsulation decreases cardiotoxicity and
increases circulation time [2] and intratumoral delivery [3],
therapeutic efficacy compared to the free formulation is rather
modest [6-9, 11-12]. To create a longer circulation time and high
stability, the carrier is composed of a robust bilayer of
phospholipids and PEG-coating. Subsequently, after entering the
tumor interstitium most of the drug remains encapsulated and the
intracellular bioavailability (i.e. the presence of drug at its
intracellular target site) is limited [9]. Thus, the liposomal
DOXIL.RTM. formulation delivers more doxorubicin to the tumor but
does not release the active doxorubicin molecule effectively. This
perpetual state with very slow drug release can account for the
unsatisfactory tumor responses compared to free drug administration
and the need for repeated administration. Indeed, the antitumor
efficacy of DOXIL.RTM. is hindered by the poor release of the
active drug from the liposome at the tumor sites [11]. Numerous
published reports demonstrate that although a significant increase
in intratumoral doxorubicin delivered by DOXIL.RTM. is observed
compared to free doxorubicin administration, this significant
increase does not necessarily correlate with an increase in
intracellular bioavailability and therefore therapeutic doxorubicin
levels [9, 11-12]. Investigation of the kinetics of free
doxorubicin versus liposomal doxorubicin (DOXIL.RTM.) in vitro as
well as in vivo demonstrated that within 8 h after administration
of free doxorubicin, 26% of the drug translocated to the nucleus
and when reaching a specific concentration killed the cell. Unlike
free doxorubicin, only 0.4% of the doxorubicin added as liposomal
formulation entered the nucleus [12]. It has been also demonstrated
that sequestering of liposomal doxorubicin in the lysosomal
compartment resulting in limited delivery to the nucleus. This
entrapment makes the bioavailable concentration of
DOXIL.RTM.-delivered doxorubicin significantly lower and therefore
ineffective as compared to free doxorubicin in killing tumor cells
[12]. Additionally, this drug is associated with a local
inflammatory tissue reaction, called palmoplantar
erythrodysesthesia (PPE) [10] which is a drawback of PEGylated
doxorubicin-loaded liposomes. The major side effects of DOXIL.RTM.
were stomatitis and skin toxicity [7, 10]. In fact, although the
cardiotoxicity is reduced by PEGylation, the long circulation time
often results in skin toxicity referred as Palmar Plantar
Erythrodysthesia (PPE) [7, 10, 38-39], which is a drawback of
PEGylated doxorubicin-loaded liposomes and remains to be
overcome.
[0006] MYOCET.RTM. includes egg phosphatidylcholine/cholesterol,
and is loaded with doxorubicin via a citrate gradient. Although
formation of doxorubicin-citrate aggregates results in relatively
high drug release rate at pH 4.5-5.5 (desirable outcome in the
acidic lysosomal environment) [1, 13-15], this also results in
extra leakage of doxorubicin from the liposomes at physiological pH
and 37.degree. C. [14]. Such release profile will lead to loss of
doxorubicin from the liposomes while liposomes are circulating in
the blood, and decrease of the efficacious concentration of
incorporated Dox [14]. In a long run (depending on administration
regimen) this might result in higher toxicity and lower efficacy of
formulated doxorubicin and shorter product shelf life as liposomal
suspension. To overcome the stability issue MYOCET.RTM. is supplied
as a three vials system that requires compounding pharmacy to
follow multistep protocol for preparation of doxorubicin loaded
liposomes prior to administration to patients. Indeed, the
procedure involves heating and vigorous shaking, and consists of
multiple steps that include separate reconstitution of the
liposomal material and doxorubicin in different medias, adjusting
pH of the empty liposomes, heating material to 55-60.degree. C.,
loading of doxorubicin into liposomes, and cooling material to RT
before use. This is a very inconvenient formulation to prepare at
the bedside.
[0007] Thus, existing therapies have room for improvement. Although
DOXIL.RTM. has excellent stability as liposomal suspension, and is
therefore in an attractive one vial presentation format, the
antitumor efficacy of DOXIL.RTM. is hindered by its decreased or
limited release of active drug from the liposome at the tumor site.
Compared to DOXIL.RTM., MYOCET.RTM. demonstrates markedly higher
release of doxorubicin at acidic pH (tumor site) but exhibits
leakage of doxorubicin from the liposomes at physiological pH
resulting in decreased safety and efficacy of the drug. The
MYOCET.RTM. product also requires the pharmacy to follow a
difficult multistep protocol for preparation of doxorubicin loaded
liposomes prior to administration to patients.
[0008] Thus, there is a need for development of novel liposomal
nanocarriers with improved drug release profiles relative to the
known marketed products (for example, MYOCET.RTM. and DOXIL.RTM.)
and with a simple, efficient, and attractive formulation
preparation system. Provided here are solutions to these and other
needs in the art.
SUMMARY
[0009] The disclosure herewith provides, inter alia, anticancer
compositions and methods for their production. In certain aspects,
the composition is a pharmaceutical composition including a
liposome. The liposome encompasses a weakly basic anticancer
compound and an acid or salt thereof. In embodiments, the acid is
oxalic acid or tartaric acid. In embodiments, the weakly basic
anticancer compound is doxorubicin, irinotecan, mitoxantrone or a
combination thereof.
[0010] In embodiments, the liposome comprises a poloxamer. In
embodiments, the poloxamer is poloxamer 188.
[0011] In embodiments, the liposome includes a plurality of lipid
compounds. The weight ratio of the plurality of lipids to the
weakly basic anticancer agent may be at least 20/1. In embodiments,
liposome includes a plurality of lipid compounds and the weight
ratio of the plurality of lipids to the weakly basic anticancer
agent is about 20/1 to about 100/1. In embodiments, the liposome
includes a plurality of lipid compounds and the weight ratio of the
plurality of lipids to the weakly basic anticancer agent is 20/1 to
about 50/1.
[0012] In embodiments, the weakly basic anticancer compound is
substantially released from the liposome only at acidic pH. In
embodiments, at least 10-100% of the weakly basic anticancer
compound is released from the liposome at pH 5.0 to 6.7 under
standard assay conditions. In embodiment, less than 5% of the
weakly basic anticancer compound is released from the liposome at
pH 7.4 under standard assay conditions. In embodiments, the
standard assay conditions include 20.times. and/or 50.times.
dilution in PBS buffer pH 5.0 or higher, e.g. pH 5.0, pH 5.5, pH
6.0, pH 6.5, pH 6.7, pH 7.0, or pH 7.4 or any intervening numbers
of the foregoing pHs or human serum, or human blood and incubation
at 37.degree. C. for 2, 4, or 8 hours. In embodiments, the standard
assay conditions include 20.times. and/or 50.times. dilution in PBS
buffer. In embodiments, the standard assay conditions include
incubation in PBS buffer having about pH 5.0. In embodiments, the
standard assay conditions include incubation in PBS buffer having
about pH 5.5. In embodiments, the standard assay conditions include
incubation in PBS buffer having about pH 6.0. In embodiments, the
standard assay conditions include incubation in PBS buffer having
about pH 6.5. In embodiments, the standard assay conditions include
incubation in PBS buffer having about pH 6.7. In embodiments, the
standard assay conditions include incubation in PBS buffer having
about pH 7.0. In embodiments, the standard assay conditions include
incubation in PBS buffer having about pH 7.4. In embodiments, the
standard assay conditions include incubation in human serum or
human blood. In embodiments, the standard assay conditions include
incubation at 37.degree. C. for about 2 hours. In embodiments, the
standard assay conditions include incubation at 37.degree. C. for
about 4 hours. In embodiments, the standard assay conditions
include incubation at 37.degree. C. for about 6 hours.
[0013] In embodiments, the liposome is substantially spherical. In
embodiments, the pharmaceutical composition includes a plurality of
liposomes with a mean longest dimension of about 60-80 nm
determined by the intensity-averaged particle diameters (Z-average)
measured by Dynamic Light Scattering. In embodiments, the
pharmaceutical composition includes a plurality of liposomes with a
mean longest dimension of about 10-30 nm determined by the
number-based particle diameters measured by Dynamic Light
Scattering. In embodiments, the pharmaceutical composition includes
s a plurality of liposomes having a mean longest dimension from
10-30 nm determined by Cryo-Transmission Electron Microscopy.
[0014] In embodiments, the liposome comprises about 500-1000
.mu.g/mL of the weakly basic anticancer compound and optionally an
acid or salt thereof. In embodiments, the liposome includes about
700-850 .mu.g/mL of the weakly basic anticancer compound and, as
applicable, an acid or salt thereof. In embodiments, the liposome
includes a plurality of the weakly basic anticancer compounds
forming an aggregate. The aggregate may be non-crystalline. The
non-crystalline aggregate may be partially or fully disorganized
(non-ordered). In embodiments, the liposome includes a plurality of
the weakly basic anticancer compound and retains greater than 90%
of the plurality of weakly basic anticancer compound after 40 days
when stored at 2-8.degree. C. under standard storage
conditions.
[0015] In embodiments, the liposome does not includes a cholesterol
or a poloxamer 188. In embodiments, the liposome does not include
an acidic organic compound other than oxalic acid, tartaric acid,
or salts thereof. In embodiments, the liposome does not include an
active agent other than the weakly basic anticancer compound. In
embodiments, the liposome does not include a drug other than the
weakly basic anticancer compound. In embodiments, the liposome does
not include a pharmaceutically active compound other than the
weakly basic anticancer compound. In embodiments, the liposome does
not include any anticancer compound other than the weakly basic
anticancer compound (e.g. the liposome includes a weakly basic
anticancer compound of only one chemical structure including salts
thereof).
[0016] In embodiments, the liposome is formed by loading the weakly
basic anticancer compound into an unloaded liposome followed by
incubation at a room temperature. In some embodiments, the unloaded
liposomes are stored for about 30 days. In embodiments, the
unloaded liposomes are stored for about 60 days. In embodiments,
the unloaded liposomes are stored for about 90 days. In
embodiments, the unloaded liposomes are stored for about 120 days.
In embodiments, the unloaded liposomes are stored for about 150
days. In embodiments, the unloaded liposomes are stored for about
180. In embodiments, the unloaded liposomes are stored for about
210 days. In embodiments, the unloaded liposomes are stored for
about 240 days. In embodiments, the unloaded liposomes are stored
for about 270 days. In embodiments, the unloaded liposomes are
stored for about 300 days. In embodiments, the unloaded liposomes
are stored for about 330 days. In embodiments, the unloaded
liposomes are stored for about 360 days. In embodiments, the
unloaded liposomes are stored for about 390 days. In embodiments,
the unloaded liposomes are stored for about 420 days. In
embodiments, the unloaded liposomes are stored for about 450 days.
In embodiments, the unloaded liposomes are stored for about 480
days. In embodiments, the unloaded liposomes are stored for about
510 days. In embodiments, the unloaded liposomes are stored for
about 540 days. In embodiments, the unloaded liposomes are stored
at 2-8.degree. C. under standard storage conditions. In
embodiments, the unloaded liposomes, stored in any conditions
above, retain greater than 90% of the weakly basic anticancer
compound upon loading of the weakly basic anticancer compound. In
embodiments, at least 40-80% of the weakly basic anticancer
compound is released from the liposome at pH 5.0 under standard
assay conditions. In embodiments, at least 20-60% of the weakly
basic anticancer compound is released from the liposome at pH 6.0.
In embodiments, at least 7-30% of the weakly basic anticancer
compound is released from the liposome at pH 6.7 under standard
assay conditions. In some embodiments, less than 5% of the weakly
basic anticancer compound is released from the liposome at pH 7.4
under standard assay conditions.
[0017] In further aspects, the disclosures provided herewith
include a method for preparing a liposome encompassing (i.e.
comprising or encapsulating) the weakly basic anticancer compound.
a weakly basic anticancer compound and an acid or salt thereof. In
embodiments, the acid is oxalic acid or tartaric acid. The method
includes: mixing a solution of the weakly basic anticancer compound
thereof with a suspension including the liposomes containing an
encapsulated acid or salt; and incubating the solution of the
weakly basic anticancer compound thereof with the suspension
including the liposomes containing an encapsulated acid or salt. In
embodiments, about 85-100% of the weakly basic anticancer compound
thereof used in mixing with the suspension of liposomes containing
an encapsulated acid or salt is retained within the liposomes. In
embodiments, about 90-100% of the weakly basic anticancer compound
thereof used in mixing with the suspension of liposomes containing
an encapsulated acid or salt is retained within the liposomes. In
embodiments, the incubating step occurs at room temperature. In the
incubating step is about 10-30 minutes. In embodiments, the
incubating step is about 5-25 minutes.
[0018] In further aspects, the disclosures provided herewith
include a kit comprising a first vial including a weakly basic
anticancer compound thereof, and a second vial including a
suspension of liposomes containing an encapsulated acid or salt. In
embodiments, the weakly basic anticancer compound thereof of the
first vial is a lyophilized weakly basic anticancer compound a
thereof. In embodiments, the suspension of liposomes containing an
encapsulated acid or salt of the second vial is an aqueous liposome
suspension.
[0019] In further aspects, the disclosures provided herewith
include a method of using the kit described above, the method
including mixing the contents of the first vial with the contents
of the second vial. In embodiments, the mixing is at room
temperature.
[0020] In an embodiment, the disclosures provided herewith include
a method for preparing a liposome encompassing a weakly basic
anticancer compound and an acid or salt thereof. In embodiments,
the acid is citric acid. The method includes mixing a solution of
the weakly basic anticancer compound and an acid or salt thereof
with a suspension including the liposomes containing an
encapsulated acid or salt thereof; and incubating the solution of
the weakly basic anticancer compound and an acid or salt thereof
with the suspension including the liposomes containing an
encapsulated acid or salt thereof.
[0021] In an embodiment, the disclosures provided herewith include
a pharmaceutical composition including a liposome, the liposome
encompassing a weakly basic anticancer compound and an acid or salt
thereof. In embodiments, the acid is citric acid. In embodiments,
the liposome includes a plurality of lipid compounds and the weight
ratio of the plurality of lipids to the weakly basic anticancer
agent is at least 20/1.
[0022] In further aspects, the disclosures herewith provide a
method of treating a cancer in a subject. The method includes
administering an effective amount of a pharmaceutical composition
to the subject in need of the treatment. The pharmaceutical
composition contains a liposome encompassing a weakly basic
anticancer compound and an acid or salt thereof. In embodiments,
the acid is oxalic acid or tartaric acid. The weakly basic
anticancer compound has anticancer activity to the cancer.
[0023] Each of the aspects and embodiments described herein are
capable of being used together, unless excluded either explicitly
or clearly from the context of the embodiment or aspect.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIGS. 1A-1E show a schematic drawing showing pH gradients in
(FIG. 1A) systematic circulation, (FIG. 1B) tumoral and local
environment and (FIG. 1C and FIG. 1D) intracellular environment.
(FIG. 1E) shows a desirable release profiles over the range of pH
8.0-5.0.
[0025] FIG. 2 is a schematic of the experimental design for the in
vivo study.
[0026] FIG. 3 is a bar graph showing the percent of intraliposomal
doxorubicin release into dissolution media after 8 hrs incubation
at 37.degree. C. Doxorubicin loading into liposomes was performed
at 70.degree. C. Left-side bars on each pair--release at pH 5;
right-side bars on each pair--release at pH 7.4. Each point on the
curves represents mean.+-.STD of data obtained in 2-6 independent
experiments. For each experiment all the measurements were
performed in sixtiplicate.
[0027] FIG. 4 is a (C-TEM) micrograph of EPC/Chol liposomes loaded
with doxorubicin via a (NH.sub.4).sub.2HPO.sub.4 gradient [13].
[0028] FIG. 5 is a (C-TEM) image of doxorubicin loaded into
liposomes buffered by citrate [15].
[0029] FIGS. 6A-6B show cryo-transmission electron microscopy
(cTEM) images of (FIG. 6A) liposomes prepared in citrate [15], and
(FIG. 6B) liposomes prepared in (NH.sub.4).sub.2SO.sub.4 [15,
25].
[0030] FIGS. 7A-7C show cryotransmission electron microscopy
characterization of doxorubicin-Oxalate-containing liposomes (lot
#647-2-157). FIG. 7A has a magnification of 27K and FIGS. 7B and 7C
have magnification of 50K.
[0031] FIG. 8 is a graph showing effect of lipid to drug (or
equally referred to lipid/drug or lipid:drug) ratio on percent of
release of intraliposomal doxorubicin into dissolution media at 8
hrs. Doxorubicin loading into Oxalate-containing liposomes was
performed at 70.degree. C. Release experiment was carried out at
37.degree. C. Release at pH 5.0--the top line. Release at pH
7.4--the bottom line. Each point on the curves represents
mean.+-.STD of data obtained in two to three independent
experiments. For each experiment all the measurements were
performed in sixtiplicate.
[0032] FIG. 9 is a graph showing effect of placebo, free
doxorubicin, DOXIL.RTM., and doxorubicin-Oxalate containing
liposomes on mouse death rate. Mice were divided into 4 groups (8
mice in each Rx group; 6 mice in Placebo group). All test articles
were administered to mice for three consecutive days via
intravenous (iv) injections. Each Rx treated group received 3 mg/kg
of doxorubicin per injection. Group #1 (Placebo) received drug free
lipid formulation. Group 2 received doxorubicin oxalate liposomes
(lot #647-2-13). Group 3 received DOXIL.RTM.. Group 4 received free
doxorubicin. The treatment is also indicated in the graph. In the
graph the Day of B-lymphoma cells administration is defined as Day
0
[0033] FIG. 10 is a graph showing effect of lipid/drug ratio on
percent of release of intraliposomal doxorubicin into dissolution
media at 8 hrs. Doxorubicin loading into tartrate-containing
liposomes was performed at 70.degree. C. Release experiment was
carried out at 37.degree. C. Release at pH 5.0--the top line.
Release at pH 7.4--the bottom line. Each point on the curves
represents mean.+-.STD of data obtained in two to three independent
experiments. For each experiment all the measurements were
performed in sixtiplicate.
[0034] FIG. 11 is a graph showing effect of lipid/drug ratio on
percent of release of intraliposomal doxorubicin into dissolution
media at 8 hrs. Doxorubicin loading into oxalate-containing
liposomes was performed at room temperature. Release experiment was
carried out at 37.degree. C. Release at pH 5.0--the top line.
Release at pH 7.4--the bottom line. Each point on the curves
represents mean.+-.STD of data obtained in two to three independent
experiments. For each experiment all the measurements were
performed in sixtiplicate.
[0035] FIG. 12 is a graph showing effect of lipid/drug ratio on
percent of release of intraliposomal doxorubicin into dissolution
media at 8 hrs. Doxorubicin loading into Tartrate-containing
liposomes was performed at room temperature. Release experiment was
carried out at 37.degree. C. Release at pH 5.0--the top line.
Release at pH 7.4--the bottom line. Each point on the curves
represents mean.+-.STD of data obtained in two to three independent
experiments. For each experiment all the measurements were
performed in sixtiplicate.
[0036] FIG. 13 is a graph showing effect of different loading
temperature on percent of intraliposomal doxorubicin release into
dissolution media determined at 37.degree. C. Doxorubicin loading
into oxalate-containing liposomes was performed at 70.degree. C.:
a) Release at pH 5.0 (the top solid line); b) Release at pH 7.4
(the bottom solid line); each point on the curves represents
mean.+-.STD of data obtained in four independent experiments.
Doxorubicin loading oxalate-containing liposomes was performed at
room temperature: c) Release at pH 5.0 (the top dotted line);
Release at pH 7.4 (the bottom dotted line); each point on the
curves represents mean.+-.STD of data obtained in two independent
experiments. For each experiment all the measurements were
performed in sixtiplicate.
[0037] FIG. 14 is a graph showing effect of different loading
temperature on percent of intraliposomal doxorubicin release into
dissolution media determined at 37.degree. C. Doxorubicin loading
into tartrate-containing liposomes was performed at 70.degree. C.:
a) Release at pH 5.0 (the top solid line); b) Release at pH 7.4
(the bottom solid line); each point on the curves represents
mean.+-.STD of data obtained in two independent experiments.
Doxorubicin loading into Tartrate-containing liposomes was
performed at room temperature. c) Release at pH 5.0 (the top dotted
line); Release at pH 7.4 (the bottom dotted line); each point on
the curves represents mean.+-.STD of data obtained in two
independent experiments. For each experiment all the measurements
were performed in sixtiplicate.
[0038] FIG. 15 is a graph showing percent of intraliposomal
doxorubicin release into dissolution media after 8 hrs incubation
at 37.degree. C. Doxorubicin loading into liposomes was performed
at room temperature. Left-side bars of each pair--release at pH 5;
right-side bars of each pair--release at pH 7.4. Each point on the
curves represents mean.+-.STD of data obtained in 2-3 independent
experiments. For each experiment all the measurements were
performed in sixtiplicate.
[0039] FIG. 16 is a graph showing the effect of various counter
ions on pH dependent doxorubicin release from liposomes. Comparison
with DOXIL.RTM. and "MYOCET". Liposomal material was diluted in
dissolution media 20.times. and release experiments were carried
out at 37.degree. C. for 8 hrs at pH 7.4, 6.7, 6.0, and 5.0. Each
point on the curves represents mean.+-.STD of data obtained in 2-3
independent experiments. For each experiment all the measurements
were performed in sixtiplicate. Formulation composition, lipid to
drug (i.e. lipid/drug) and phospholipid to free cholesterol (i.e.
phospholipid/free cholesterol or PL/FC) ratios are shown in the
Tables 28e-28g.
[0040] FIG. 17 is a graph showing the effect of various counter
ions on pH dependent doxorubicin release from liposomes. Comparison
with DOXIL.RTM. and "MYOCET". Liposomal material was diluted in
dissolution media 50.times. and release experiments were carried
out at 37.degree. C. for 8 hrs at pH 7.4, 6.7, 6.0, and 5.0. Each
point on the curves represents mean.+-.STD of data obtained in 2-3
independent experiments. For each experiment all the measurements
were performed in sixtiplicate. Formulation composition, lipid/drug
and phospholipid/free cholesterol (PL/FC) ratios are shown in the
Tables 28e-28g.
[0041] FIG. 18 is a graph showing the effect of lipid/drug and
phospholipid/free cholesterol (PL/FC) ratios on pH dependent
doxorubicin release from liposomes. Comparison with DOXIL.RTM. and
"MYOCET". Liposomal material was diluted in dissolution media
20.times. and release experiments were carried out at 37.degree. C.
for 8 hrs at pH 7.4, 6.7, 6.0, and 5.0. Each point on the curves
represents mean.+-.STD of data obtained in 2-3 independent
experiments. For each experiment all the measurements were
performed in sixtiplicate. Formulation composition, lipid/drug and
phospholipid/free cholesterol (PL/FC) ratios are shown in the
Tables 28e-28g.
[0042] FIG. 19 is a graph showing the effect of lipid/drug and
phospholipid/free cholesterol (PL/FC) ratios on pH dependent
doxorubicin release from liposomes. Comparison with DOXIL.RTM. and
"MYOCET". Liposomal material was diluted in dissolution media
20.times. and release experiments were carried out at 37.degree. C.
for 8 hrs at pH 7.4, 6.7, 6.0, and 5.0. Each point on the curves
represents mean.+-.STD of data obtained in 2-3 independent
experiments. For each experiment all the measurements were
performed in sixtiplicate. Formulation composition, lipid/drug and
phospholipid/free cholesterol (PL/FC) ratios are shown in the
Tables 28e-28g.
[0043] FIG. 20 is a graph showing the effect of lipid/drug ratio on
serum stability of liposomes. Comparison with DOXIL.RTM. and
"MYOCET". Liposomal material was diluted 50.times. in human serum
and stability of the liposomes was monitored at 37.degree. C. for
2, 4, and 8 hrs. Each point on the curves represents mean.+-.STD of
data obtained in 2-3 independent experiments. For each experiment
all the measurements were performed in sixtiplicate. Formulation
composition, lipid/drug and phospholipid/free cholesterol (PL/FC)
ratios are shown in the Tables 28e and 28h.
[0044] FIG. 21 is a graph showing the effect of phospholipid/free
cholesterol (PL/FC) ratios on serum stability of liposomes.
Comparison with DOXIL.RTM. and "MYOCET". Liposomal material was
diluted 50.times. in human serum and stability of the liposomes was
monitored at 37.degree. C. for 2, 4, and 8 hrs. Each point on the
curves represents mean.+-.STD of data obtained in 2-3 independent
experiments. For each experiment all the measurements were
performed in sixtiplicate. Formulation composition, lipid/drug and
phospholipid/free cholesterol (PL/FC) ratios are shown in the
Tables 28e and 28h.
[0045] FIG. 22 is a graph showing percent of intraliposomal
Irinotecan release into dissolution media (pH 5) after 2 hrs of
incubation at 37.degree. C. Each point on the curves represents
mean.+-.STD of data obtained in 2-6 independent experiments. For
each experiment all the measurements were performed in
sixtiplicate.
[0046] FIGS. 23A-23B are images of mitoxantrone solutions at pH 7.4
and pH 5.0. FIG. 23A shows the solution at time 0. FIG. 23B shows
the solution at time 24 hours. Mitoxantrone loading into liposomes
was performed at 2-8.degree. C.
DETAILED DESCRIPTION OF THE DISCLOSURES
[0047] The present disclosures describe compositions and processes
used to create stable anticancer compounds and acids or salts
thereof and lipid rich submicron particles (nanoparticles with
liposomes) suitable for drug delivery. Compositions and methods of
the present disclosures can be similarly applied to other drug
salts in liposomes. The composition of the doxorubicin salt and
structure of the liposomes prepared according to the methods
disclosed herein results in desirable biological and
physicochemical performance (pH dependent drug release
profile).
[0048] In embodiments of the present disclosures utilizing the
anticancer compound doxorubicin, the therapeutic utility of the
present disclosures are improved compared to known liposome
encapsulated doxorubicin therapies in their more desirable release
profile which includes: low rate of doxorubicin release from the
liposomes at physiological pH-7.4 (while in circulation), and
significantly higher release rate at more acidic pH 5.0-6.7 (e.g.
after exposure to local tumor environment or to endosomal/lysosomal
environment upon internalization of the doxorubicin loaded
liposomes by the cancer cells).
[0049] There are numerous published reports indicating the
existence and importance of the pH gradient between normal tissues
and the tumor site, as well as the effect of pH dependent drug
release from liposomes for not just delivery but for making the
free drug available (via efficient release) to the cancer cells [1,
10, 13-15, 25, 40-42]. One major difference between many solid
tumors and surrounding normal tissue is the nutritional and
metabolic environment. The functional vasculature of tumors is
often inadequate to supply the nutritional needs of the expanding
population of tumor cells, leading to deficiency of oxygen and many
other nutrients. The production of lactic acid under anaerobic
conditions and the hydrolysis of ATP in an energy-deficient
environment contribute to the acidic microenvironment found in many
tumor types [35]. The pH in human and rodent normal tissues ranges
from 7.00 to 8.06, whereas a wider range of pH values was
determined in malignant tissue, from about pH 5.8 to pH 7.6 in both
human and rodent tumors [1, 41-42]
[0050] Thus, the desirable release profile of doxorubicin from the
liposomes would be the following: maximized release rate at acidic
pH 5.0-6.7 (i.e. after exposure to local tumor environment or to
endosomal/lysosomal environment upon internalization of liposomes
by the cancer cells), while suppressing release from the liposomes
at physiological pH-7.4 (while in circulation).
Definitions
[0051] As used herein "liposomes" are largely spherical
nanoparticles made up of a lipid bilayer. In embodiments, liposomes
can encapsulate therapeutic agents for delivery. The lipid content
of liposomes can vary altering liposome size, stability,
solubility, curvature, etc. Examples of lipids include, but are not
limited to, cholesterol, phosphatidylcholine (PC)
products/derivatives (various carbon chain length fatty acids,
saturated, multi-unsaturated and mixed acid PC's);
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE);
1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA);
1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA);
1,2-dioleoyl-sn-glycero-3-phosphate (DOPA);
1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG);
1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG);
1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG);
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC);
1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS);
1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS);
1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPC);
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (DSPE-mPEG-2000);
1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[folate(polyethylene
glycol)-5000] (DSPE-mPEG-5000);
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene
glycol)-2000] (DSPE-PEG-2000);
1,2-stearoyl-3-trimethylammonium-propane (DOTAP);
L-.alpha.-phosphatidylcholine, hydrogenated (Hydro Soy PC); and
2-stearoyl-sn-glycero-3-phosphocholine (Lyso PC).
[0052] The term "encompassing," "encapsulating" or "retaining" as
used herein means to include within, for example, a liposome. This
may also be referred to as "comprising" in the context of a claim
herein. For example, a liposome may encompass a therapeutic agent
such as an anticancer compound, or acid or salt thereof. In
embodiments, when encompassed, a therapeutic agent may dissipate
from, or diffuse out of, a liposome over time.
[0053] The term "weakly basic anticancer compound" as used herein
includes any therapeutic agent useful in the treatment of a cancer
or neoplastic disease with a weakly basic pKa. In embodiments, a
weakly basic pH is indicated by a pKa of about pH 7.0-10.0. In
embodiments, a weakly basic pH is indicated by a pKa of about pH
7.0 to 9.0. In embodiments, a weakly basic pH is indicated by a pKa
of about pH 7.5 to 9.0. In embodiments, a weakly basic pH is
indicated by a pKa of about pH 8.0 to 9.0. In embodiments, an
anticancer agent has more than one pKa, any one of which can fall
within the ranges of weakly basis as described above.
[0054] The term "doxorubicin" is used according to its plain and
ordinary meaning. doxorubicin is an anticancer compound originally
obtained from Streptomyces peucetius. Doxorubicin may also be
referred to as Adriamycin, DOXIL, Rubex, Adriablastin, and
doxorubicine. In embodiments, doxorubicin is a weakly basic
anticancer compound. The structure of doxorubicin is shown
herein.
[0055] The term "irinotecan" is used according to its plain and
ordinary meaning. Irinotecan is an anticancer compound that may be
used for colorectal cancer that has metastasized (spread to other
parts of the body), including metastatic cancer that has recurred
(come back) or has not gotten better with other chemotherapy and
for the treatment of patients with metastatic adenocarcinoma of the
pancreas after disease progression following gemcitabine-based
therapy. Irinotecan may also be referred to as Camptosar;
97682-44-5; (+)-Irinotecan; Irinotecanum; and Irinotecanum. In
embodiments, irinotecan is a weakly basic anticancer compound. The
structure of irinotecan is shown herein.
[0056] The term "mitoxantrone" is used according to its plain and
ordinary meaning. Mitoxantrone is an anticancer compound that may
be used for acute myeloid leukemia (AML) and prostate cancer. It
also may be used as palliative treatment in advanced disease that
is hormone-refractory (does not respond to hormone treatment).
Mitoxantrone may also be referred to as 65271-80-9; Mitoxanthrone;
Mitozantrone; DHAQ; and Mitoxantron. In embodiments, mitoxantrone
is a weakly basic anticancer compound. The structure of
mitoxantrone is shown herein.
[0057] As used herein, the term "an acid or salt thereof" refers to
any pharmaceutically acceptable acid or salt of a stated compound.
Example acids or salts suitable for use with anticancer compounds
include, but are not limited to, 1-hydroxy-2-naphthoic acid,
2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid,
2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid,
acetic acid, adipic acid, ascorbic acid (L), aspartic acid (L),
benzenesulfonic acid, benzoic acid, camphoric acid (+),
camphor-10-sulfonic acid (+), capric acid (decanoic acid), caproic
acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid,
cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid,
ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid,
fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid
(D), gluconic acid (D), glucuronic acid (D), glutamic acid,
glutaric acid, glycerophosphoric acid, glycolic acid, hippuric
acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic
acid (DL), lactobionic acid, lauric acid, maleic acid, malic acid
(-L)malonic acid, mandelic acid (DL), methanesulfonic acid,
naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid,
nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic
acid, pamoic acid, phosphoric acid, proprionic acid, pyroglutamic
acid (-L), salicylic acid, sebacic acid, stearic acid, succinic
acid, sulfuric acid, tartaric acid (+L), thiocyanic acid,
toluenesulfonic acid (p), and undecylenic acid
[0058] As used herein, the term "disorganized" or "non-crystalline"
refers to the disordered polymorphic state of a compound. This
disorganized state may resemble an amorphous state. In
disorganized, non-crystalline aggregates the molecules do not form
an ordered crystal lattice. In embodiments, a disorganized
aggregate may also include any non-crystalline structures, and
colloids. In embodiments, some disorganized and non-crystalline
aggregates may contain some fibroid structures.
[0059] As used herein, the term "substantially released" indicates
that a majority of contents are released, e.g., from a liposome. In
embodiments, substantially released means greater than about 5% or
more of encompassed contents are released. In embodiments,
substantially released means greater than about 10% or more of
encompassed contents are released. In embodiments, substantially
released means greater than about 20% or more of encompassed
contents are released. In embodiments, substantially released means
greater than about 30% or more of encompassed contents are
released. In embodiments, substantially released means greater than
about 40% or more of encompassed contents are released. In
embodiments, substantially released means greater than about 50% or
more of encompassed contents are released. In embodiments,
substantially released means greater than about 60% or more of
encompassed contents are released. In embodiments, substantially
released means greater than about 70% or more of encompassed
contents are released. In embodiments, substantially released means
greater than about 80% or more of encompassed contents are
released. In embodiments, substantially released means greater than
about 90% or more of encompassed contents are released. In
embodiments, substantially released means greater than about 95% or
more of encompassed contents are released. In embodiments,
substantially released means greater than about 97% or more of
encompassed contents are released. In embodiments, substantially
released means greater than about 98% or more of encompassed
contents are released. In embodiments, substantially released means
greater than about 99% or more of encompassed contents are
released. In embodiments, substantial release is pH drive, e.g., an
anticancer agent is substantially released only at acidic pH.
[0060] As used herein, the terms "a standard assay condition" or
"standard assay conditions" refer to controlled set of assay
conditions, including standard measures of time, temperature, pH,
etc. In embodiments, the standard assay conditions include
20.times. and/or 50.times. dilution in PBS. In embodiments, the
standard assay conditions include 20.times. and/or 50.times.
dilution in serum or blood. In embodiments, standard assay
conditions for release assays described herein are at about
25.degree. C. In embodiments, standard assay conditions for release
assays described herein are at about 37.degree. C. In embodiments,
standard assay conditions for release assays described herein
include incubations of about 2, 4, 8 hours or any intervening
period of the foregoing or more than about 8 hours. In embodiments,
standard assay conditions for release assays described herein
include incubations of about 2 hours. In embodiments, standard
assay conditions for release assays described herein include
incubations of about 4 hours. In embodiments, standard assay
conditions for release assays described herein include incubations
of about 8 hours. In embodiments, standard assay conditions for
release assays described herein include incubations of more than 8
hours. In embodiments, standard assay conditions for release assays
described herein include incubation at about pH 7.4. In
embodiments, standard assay conditions for release assays described
herein include incubation at about pH 6.7. In embodiments, standard
assay conditions for release assays described herein include
incubation at about pH 6.0. In embodiments, standard assay
conditions for release assays described herein include incubation
at about pH 5.0.
[0061] Therefore, in some embodiments, standard assay conditions
for release assay are at 25.degree. C. or at about 25.degree. C.
and include incubation in 20.times. dilution in PBS having about pH
7.4 for about 2 hours. In some embodiments, standard assay
conditions for release assay are at 25.degree. C. or at about
25.degree. C. and include incubation in 20.times. dilution in PBS
having about pH 7.4 for about 4 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 20.times. dilution
in PBS having about pH 7.4 for about 8 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 20.times. dilution
in PBS having about pH 7.4 for more than 8 hours.
[0062] Therefore, in some embodiments, standard assay conditions
for release assay are at 25.degree. C. or at about 25.degree. C.
and include incubation in 20.times. dilution in PBS having about pH
6.7 for about 2 hours. In some embodiments, standard assay
conditions for release assay are at 25.degree. C. or at about
25.degree. C. and include incubation in 20.times. dilution in PBS
having about pH 6.7 for about 4 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 20.times. dilution
in PBS having about pH 6.7 for about 8 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 20.times. dilution
in PBS having about pH 6.7 for more than 8 hours.
[0063] Therefore, in some embodiments, standard assay conditions
for release assay are at 25.degree. C. or at about 25.degree. C.
and include incubation in 20.times. dilution in PBS having about pH
6.0 for about 2 hours. In some embodiments, standard assay
conditions for release assay are at 25.degree. C. or at about
25.degree. C. and include incubation in 20.times. dilution in PBS
having about pH 6.0 for about 4 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 20.times. dilution
in PBS having about pH 6.0 for about 8 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 20.times. dilution
in PBS having about pH 6.0 for more than 8 hours.
[0064] Therefore, in some embodiments, standard assay conditions
for release assay are at 25.degree. C. or at about 25.degree. C.
and include incubation in 20.times. dilution in PBS having about pH
5.0 for about 2 hours. In some embodiments, standard assay
conditions for release assay are at 25.degree. C. or at about
25.degree. C. and include incubation in 20.times. dilution in PBS
having about pH 5.0 for about 4 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 20.times. dilution
in PBS having about pH 5.0 for about 8 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 20.times. dilution
in PBS having about pH 5.0 for more than 8 hours.
[0065] Therefore, in some embodiments, standard assay conditions
for release assay are at 25.degree. C. or at about 25.degree. C.
and include incubation in 50.times. dilution in PBS having about pH
7.4 for about 2 hours. In some embodiments, standard assay
conditions for release assay are at 25.degree. C. or at about
25.degree. C. and include incubation in 50.times. dilution in PBS
having about pH 7.4 for about 4 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 50.times. dilution
in PBS having about pH 7.4 for about 8 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 50.times. dilution
in PBS having about pH 7.4 for more than 8 hours.
[0066] Therefore, in some embodiments, standard assay conditions
for release assay are at 25.degree. C. or at about 25.degree. C.
and include incubation in 50.times. dilution in PBS having about pH
6.7 for about 2 hours. In some embodiments, standard assay
conditions for release assay are at 25.degree. C. or at about
25.degree. C. and include incubation in 50.times. dilution in PBS
having about pH 6.7 for about 4 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 50.times. dilution
in PBS having about pH 6.7 for about 8 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 50.times. dilution
in PBS having about pH 6.7 for more than 8 hours.
[0067] Therefore, in some embodiments, standard assay conditions
for release assay are at 25.degree. C. or at about 25.degree. C.
and include incubation in 50.times. dilution in PBS having about pH
6.0 for about 2 hours. In some embodiments, standard assay
conditions for release assay are at 25.degree. C. or at about
25.degree. C. and include incubation in 50.times. dilution in PBS
having about pH 6.0 for about 4 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 50.times. dilution
in PBS having about pH 6.0 for about 8 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 50.times. dilution
in PBS having about pH 6.0 for more than 8 hours.
[0068] Therefore, in some embodiments, standard assay conditions
for release assay are at 25.degree. C. or at about 25.degree. C.
and include incubation in 50.times. dilution in PBS having about pH
5.0 for about 2 hours. In some embodiments, standard assay
conditions for release assay are at 25.degree. C. or at about
25.degree. C. and include incubation in 50.times. dilution in PBS
having about pH 5.0 for about 4 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 50.times. dilution
in PBS having about pH 5.0 for about 8 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 50.times. dilution
in PBS having about pH 5.0 for more than 8 hours.
[0069] Therefore, in some embodiments, standard assay conditions
for release assay are at 37.degree. C. or at about 37.degree. C.
and include incubation in 20.times. dilution in PBS having about pH
7.4 for about 2 hours. In some embodiments, standard assay
conditions for release assay are at 37.degree. C. or at about
37.degree. C. and include incubation in 20.times. dilution in PBS
having about pH 7.4 for about 4 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 20.times. dilution
in PBS having about pH 7.4 for about 8 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 20.times. dilution
in PBS having about pH 7.4 for more than 8 hours.
[0070] Therefore, in some embodiments, standard assay conditions
for release assay are at 37.degree. C. or at about 37.degree. C.
and include incubation in 20.times. dilution in PBS having about pH
6.7 for about 2 hours. In some embodiments, standard assay
conditions for release assay are at 37.degree. C. or at about
37.degree. C. and include incubation in 20.times. dilution in PBS
having about pH 6.7 for about 4 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 20.times. dilution
in PBS having about pH 6.7 for about 8 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 20.times. dilution
in PBS having about pH 6.7 for more than 8 hours.
[0071] Therefore, in some embodiments, standard assay conditions
for release assay are at 37.degree. C. or at about 37.degree. C.
and include incubation in 20.times. dilution in PBS having about pH
6.0 for about 2 hours. In some embodiments, standard assay
conditions for release assay are at 37.degree. C. or at about
37.degree. C. and include incubation in 20.times. dilution in PBS
having about pH 6.0 for about 4 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 20.times. dilution
in PBS having about pH 6.0 for about 8 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 20.times. dilution
in PBS having about pH 6.0 for more than 8 hours.
[0072] Therefore, in some embodiments, standard assay conditions
for release assay are at 37.degree. C. or at about 37.degree. C.
and include incubation in 20.times. dilution in PBS having about pH
5.0 for about 2 hours. In some embodiments, standard assay
conditions for release assay are at 37.degree. C. or at about
37.degree. C. and include incubation in 20.times. dilution in PBS
having about pH 5.0 for about 4 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 20.times. dilution
in PBS having about pH 5.0 for about 8 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 20.times. dilution
in PBS having about pH 5.0 for more than 8 hours.
[0073] Therefore, in some embodiments, standard assay conditions
for release assay are at 37.degree. C. or at about 37.degree. C.
and include incubation in 50.times. dilution in PBS having about pH
7.4 for about 2 hours. In some embodiments, standard assay
conditions for release assay are at 37.degree. C. or at about
37.degree. C. and include incubation in 50.times. dilution in PBS
having about pH 7.4 for about 4 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 50.times. dilution
in PBS having about pH 7.4 for about 8 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 50.times. dilution
in PBS having about pH 7.4 for more than 8 hours.
[0074] Therefore, in some embodiments, standard assay conditions
for release assay are at 37.degree. C. or at about 37.degree. C.
and include incubation in 50.times. dilution in PBS having about pH
6.7 for about 2 hours. In some embodiments, standard assay
conditions for release assay are at 37.degree. C. or at about
37.degree. C. and include incubation in 50.times. dilution in PBS
having about pH 6.7 for about 4 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 50.times. dilution
in PBS having about pH 6.7 for about 8 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 50.times. dilution
in PBS having about pH 6.7 for more than 8 hours.
[0075] Therefore, in some embodiments, standard assay conditions
for release assay are at 37.degree. C. or at about 37.degree. C.
and include incubation in 50.times. dilution in PBS having about pH
6.0 for about 2 hours. In some embodiments, standard assay
conditions for release assay are at 37.degree. C. or at about
37.degree. C. and include incubation in 50.times. dilution in PBS
having about pH 6.0 for about 4 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 50.times. dilution
in PBS having about pH 6.0 for about 8 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 50.times. dilution
in PBS having about pH 6.0 for more than 8 hours.
[0076] Therefore, in some embodiments, standard assay conditions
for release assay are at 37.degree. C. or at about 37.degree. C.
and include incubation in 50.times. dilution in PBS having about pH
5.0 for about 2 hours. In some embodiments, standard assay
conditions for release assay are at 37.degree. C. or at about
37.degree. C. and include incubation in 50.times. dilution in PBS
having about pH 5.0 for about 4 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 50.times. dilution
in PBS having about pH 5.0 for about 8 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 50.times. dilution
in PBS having about pH 5.0 for more than 8 hours.
[0077] Therefore, in some embodiments, standard assay conditions
for release assay are at 25.degree. C. or at about 25.degree. C.
and include incubation in 20.times. dilution in serum or blood
having about pH 7.4 for about 2 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 20.times. dilution
in serum or blood having about pH 7.4 for about 4 hours. In some
embodiments, standard assay conditions for release assay are at
25.degree. C. or at about 25.degree. C. and include incubation in
20.times. dilution in serum or blood having about pH 7.4 for about
8 hours. In some embodiments, standard assay conditions for release
assay are at 25.degree. C. or at about 25.degree. C. and include
incubation in 20.times. dilution in serum or blood having about pH
7.4 for more than 8 hours.
[0078] Therefore, in some embodiments, standard assay conditions
for release assay are at 25.degree. C. or at about 25.degree. C.
and include incubation in 20.times. dilution in serum or blood
having about pH 6.7 for about 2 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 20.times. dilution
in serum or blood having about pH 6.7 for about 4 hours. In some
embodiments, standard assay conditions for release assay are at
25.degree. C. or at about 25.degree. C. and include incubation in
20.times. dilution in serum or blood having about pH 6.7 for about
8 hours. In some embodiments, standard assay conditions for release
assay are at 25.degree. C. or at about 25.degree. C. and include
incubation in 20.times. dilution in serum or blood having about pH
6.7 for more than 8 hours.
[0079] Therefore, in some embodiments, standard assay conditions
for release assay are at 25.degree. C. or at about 25.degree. C.
and include incubation in 20.times. dilution in serum or blood
having about pH 6.0 for about 2 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 20.times. dilution
in serum or blood having about pH 6.0 for about 4 hours. In some
embodiments, standard assay conditions for release assay are at
25.degree. C. or at about 25.degree. C. and include incubation in
20.times. dilution in serum or blood having about pH 6.0 for about
8 hours. In some embodiments, standard assay conditions for release
assay are at 25.degree. C. or at about 25.degree. C. and include
incubation in 20.times. dilution in serum or blood having about pH
6.0 for more than 8 hours.
[0080] Therefore, in some embodiments, standard assay conditions
for release assay are at 25.degree. C. or at about 25.degree. C.
and include incubation in 20.times. dilution in serum or blood
having about pH 5.0 for about 2 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 20.times. dilution
in serum or blood having about pH 5.0 for about 4 hours. In some
embodiments, standard assay conditions for release assay are at
25.degree. C. or at about 25.degree. C. and include incubation in
20.times. dilution in serum or blood having about pH 5.0 for about
8 hours. In some embodiments, standard assay conditions for release
assay are at 25.degree. C. or at about 25.degree. C. and include
incubation in 20.times. dilution in serum or blood having about pH
5.0 for more than 8 hours.
[0081] Therefore, in some embodiments, standard assay conditions
for release assay are at 25.degree. C. or at about 25.degree. C.
and include incubation in 50.times. dilution in serum or blood
having about pH 7.4 for about 2 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 50.times. dilution
in serum or blood having about pH 7.4 for about 4 hours. In some
embodiments, standard assay conditions for release assay are at
25.degree. C. or at about 25.degree. C. and include incubation in
50.times. dilution in serum or blood having about pH 7.4 for about
8 hours. In some embodiments, standard assay conditions for release
assay are at 25.degree. C. or at about 25.degree. C. and include
incubation in 50.times. dilution in serum or blood having about pH
7.4 for more than 8 hours.
[0082] Therefore, in some embodiments, standard assay conditions
for release assay are at 25.degree. C. or at about 25.degree. C.
and include incubation in 50.times. dilution in serum or blood
having about pH 6.7 for about 2 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 50.times. dilution
in serum or blood having about pH 6.7 for about 4 hours. In some
embodiments, standard assay conditions for release assay are at
25.degree. C. or at about 25.degree. C. and include incubation in
50.times. dilution in serum or blood having about pH 6.7 for about
8 hours. In some embodiments, standard assay conditions for release
assay are at 25.degree. C. or at about 25.degree. C. and include
incubation in 50.times. dilution in serum or blood having about pH
6.7 for more than 8 hours.
[0083] Therefore, in some embodiments, standard assay conditions
for release assay are at 25.degree. C. or at about 25.degree. C.
and include incubation in 50.times. dilution in serum or blood
having about pH 6.0 for about 2 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 50.times. dilution
in serum or blood having about pH 6.0 for about 4 hours. In some
embodiments, standard assay conditions for release assay are at
25.degree. C. or at about 25.degree. C. and include incubation in
50.times. dilution in serum or blood having about pH 6.0 for about
8 hours. In some embodiments, standard assay conditions for release
assay are at 25.degree. C. or at about 25.degree. C. and include
incubation in 50.times. dilution in serum or blood having about pH
6.0 for more than 8 hours.
[0084] Therefore, in some embodiments, standard assay conditions
for release assay are at 25.degree. C. or at about 25.degree. C.
and include incubation in 50.times. dilution in serum or blood
having about pH 5.0 for about 2 hours. In some embodiments,
standard assay conditions for release assay are at 25.degree. C. or
at about 25.degree. C. and include incubation in 50.times. dilution
in serum or blood having about pH 5.0 for about 4 hours. In some
embodiments, standard assay conditions for release assay are at
25.degree. C. or at about 25.degree. C. and include incubation in
50.times. dilution in serum or blood having about pH 5.0 for about
8 hours. In some embodiments, standard assay conditions for release
assay are at 25.degree. C. or at about 25.degree. C. and include
incubation in 50.times. dilution in serum or blood having about pH
5.0 for more than 8 hours.
[0085] Therefore, in some embodiments, standard assay conditions
for release assay are at 37.degree. C. or at about 37.degree. C.
and include incubation in 20.times. dilution in serum or blood
having about pH 7.4 for about 2 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 20.times. dilution
in serum or blood having about pH 7.4 for about 4 hours. In some
embodiments, standard assay conditions for release assay are at
37.degree. C. or at about 37.degree. C. and include incubation in
20.times. dilution in serum or blood having about pH 7.4 for about
8 hours. In some embodiments, standard assay conditions for release
assay are at 37.degree. C. or at about 37.degree. C. and include
incubation in 20.times. dilution in serum or blood having about pH
7.4 for more than 8 hours.
[0086] Therefore, in some embodiments, standard assay conditions
for release assay are at 37.degree. C. or at about 37.degree. C.
and include incubation in 20.times. dilution in serum or blood
having about pH 6.7 for about 2 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 20.times. dilution
in serum or blood having about pH 6.7 for about 4 hours. In some
embodiments, standard assay conditions for release assay are at
37.degree. C. or at about 37.degree. C. and include incubation in
20.times. dilution in serum or blood having about pH 6.7 for about
8 hours. In some embodiments, standard assay conditions for release
assay are at 37.degree. C. or at about 37.degree. C. and include
incubation in 20.times. dilution in serum or blood having about pH
6.7 for more than 8 hours.
[0087] Therefore, in some embodiments, standard assay conditions
for release assay are at 37.degree. C. or at about 37.degree. C.
and include incubation in 20.times. dilution in serum or blood
having about pH 6.0 for about 2 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 20.times. dilution
in serum or blood having about pH 6.0 for about 4 hours. In some
embodiments, standard assay conditions for release assay are at
37.degree. C. or at about 37.degree. C. and include incubation in
20.times. dilution in serum or blood having about pH 6.0 for about
8 hours. In some embodiments, standard assay conditions for release
assay are at 37.degree. C. or at about 37.degree. C. and include
incubation in 20.times. dilution in serum or blood having about pH
6.0 for more than 8 hours.
[0088] Therefore, in some embodiments, standard assay conditions
for release assay are at 37.degree. C. or at about 37.degree. C.
and include incubation in 20.times. dilution in serum or blood
having about pH 5.0 for about 2 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 20.times. dilution
in serum or blood having about pH 5.0 for about 4 hours. In some
embodiments, standard assay conditions for release assay are at
37.degree. C. or at about 37.degree. C. and include incubation in
20.times. dilution in serum or blood having about pH 5.0 for about
8 hours. In some embodiments, standard assay conditions for release
assay are at 37.degree. C. or at about 37.degree. C. and include
incubation in 20.times. dilution in serum or blood having about pH
5.0 for more than 8 hours.
[0089] Therefore, in some embodiments, standard assay conditions
for release assay are at 37.degree. C. or at about 37.degree. C.
and include incubation in 50.times. dilution in serum or blood
having about pH 7.4 for about 2 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 50.times. dilution
in serum or blood having about pH 7.4 for about 4 hours. In some
embodiments, standard assay conditions for release assay are at
37.degree. C. or at about 37.degree. C. and include incubation in
50.times. dilution in serum or blood having about pH 7.4 for about
8 hours. In some embodiments, standard assay conditions for release
assay are at 37.degree. C. or at about 37.degree. C. and include
incubation in 50.times. dilution in serum or blood having about pH
7.4 for more than 8 hours.
[0090] Therefore, in some embodiments, standard assay conditions
for release assay are at 37.degree. C. or at about 37.degree. C.
and include incubation in 50.times. dilution in serum or blood
having about pH 6.7 for about 2 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 50.times. dilution
in serum or blood having about pH 6.7 for about 4 hours. In some
embodiments, standard assay conditions for release assay are at
37.degree. C. or at about 37.degree. C. and include incubation in
50.times. dilution in serum or blood having about pH 6.7 for about
8 hours. In some embodiments, standard assay conditions for release
assay are at 37.degree. C. or at about 37.degree. C. and include
incubation in 50.times. dilution in serum or blood having about pH
6.7 for more than 8 hours.
[0091] Therefore, in some embodiments, standard assay conditions
for release assay are at 37.degree. C. or at about 37.degree. C.
and include incubation in 50.times. dilution in serum or blood
having about pH 6.0 for about 2 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 50.times. dilution
in serum or blood having about pH 6.0 for about 4 hours. In some
embodiments, standard assay conditions for release assay are at
37.degree. C. or at about 37.degree. C. and include incubation in
50.times. dilution in serum or blood having about pH 6.0 for about
8 hours. In some embodiments, standard assay conditions for release
assay are at 37.degree. C. or at about 37.degree. C. and include
incubation in 50.times. dilution in serum or blood having about pH
6.0 for more than 8 hours.
[0092] Therefore, in some embodiments, standard assay conditions
for release assay are at 37.degree. C. or at about 37.degree. C.
and include incubation in 50.times. dilution in serum or blood
having about pH 5.0 for about 2 hours. In some embodiments,
standard assay conditions for release assay are at 37.degree. C. or
at about 37.degree. C. and include incubation in 50.times. dilution
in serum or blood having about pH 5.0 for about 4 hours. In some
embodiments, standard assay conditions for release assay are at
37.degree. C. or at about 37.degree. C. and include incubation in
50.times. dilution in serum or blood having about pH 5.0 for about
8 hours. In some embodiments, standard assay conditions for release
assay are at 37.degree. C. or at about 37.degree. C. and include
incubation in 50.times. dilution in serum or blood having about pH
5.0 for more than 8 hours.
[0093] As used herein, the terms "a standard storage condition" or
"standard storage conditions" refer to a condition controlled for
proper storage of compound, e.g. a pharmaceutical compound. In
embodiments, certain standard measures such as time, temperature,
humidity and others can be controlled. In embodiments, the standard
storage conditions include storage under 2-8.degree. C., ambient
relative humidity. In embodiments, the ambient relative humidity
includes any range between about 10% to about 90% relative
humidity. In embodiments, the ambient relative humidity includes
about 10% relative humidity. In embodiments, the ambient relative
humidity includes about 20% relative humidity. In embodiments, the
ambient relative humidity includes about 30% relative humidity. In
embodiments, the ambient relative humidity includes about 40%
relative humidity. In embodiments, the ambient relative humidity
includes about 50% relative humidity. In embodiments, the ambient
relative humidity includes about 60% relative humidity. In
embodiments, the ambient relative humidity includes about 70%
relative humidity. In embodiments, the ambient relative humidity
includes about 80% relative humidity. In embodiments, the ambient
relative humidity includes about 90% relative humidity.
[0094] As used herein "substantially spherical" means an average
tendency towards a spherical shape, e.g., a diameter through any
axis is roughly equivalent. In embodiments, no diameter differs in
length. In embodiments, no diameter differs in length more than
about 20% or less from a diameter at any other axis. In
embodiments, no diameter differs in length more than about 20% or
less from a diameter at any other axis within a substantially
spherical shape. In embodiments, no diameter differs in length more
than about 15% or less from a diameter at any other axis within a
substantially spherical shape. In embodiments, no diameter differs
in length more than about 10% or less from a diameter at any other
axis within a substantially spherical shape. In embodiments, no
diameter differs in length more than about 9% or less from a
diameter at any other axis within a substantially spherical shape.
In embodiments, no diameter differs in length more than about 8% or
less from a diameter at any other axis within a substantially
spherical shape. In embodiments, no diameter differs in length more
than about 7% or less from a diameter at any other axis within a
substantially spherical shape. In embodiments, no diameter differs
in length more than about 6% or less from a diameter at any other
axis within a substantially spherical shape. In embodiments, no
diameter differs in length more than about 5% or less from a
diameter at any other axis within a substantially spherical shape.
In embodiments, no diameter differs in length more than about 4% or
less from a diameter at any other axis within a substantially
spherical shape. In embodiments, no diameter differs in length more
than about 3% or less from a diameter at any other axis within a
substantially spherical shape. In embodiments, no diameter differs
in length more than about 3% or less from a diameter at any other
axis within a substantially spherical shape. In embodiments, no
diameter differs in length more than about 1% or less from a
diameter at any other axis within a substantially spherical
shape.
[0095] As used herein, the term "mean longest dimension" refers to
an average of the longest dimension of a substantially spherical
object. In some embodiments, the mean longest dimension can be
measured by the intensity-averaged particle diameters (Z-average).
In some embodiments, the intensity-averaged particle diameters
(Z-average) are calculated from the cumulants analysis as defined
in ISO 13321 (International Organization for Standardization 1996).
In some embodiments, the mean longest dimension can be measured by
the number-based particle diameters. In some embodiments, particle
size distribution by number is computed from the intensity
distribution and the optical properties of the material. There is
first-power relationship between particle size and contribution to
the distribution.
[0096] As used herein, the term "room temperature" or "RT" refers
to the temperature of an assay conducted at standard indoor
temperature. In embodiments, room temperature refers to an assay
conduct without any additional heating or cooling. In embodiments,
room temperature is a controlled temperature of about 22-25.degree.
C. In embodiments, room temperature is 25.degree. C.
[0097] As used herein, "liposome solution," "an aqueous solution of
liposomes," "liposome suspension" or "an aqueous suspension of
liposomes" refer to a liquid solution or suspension of liposomes.
Liposomes may be suspended in a variety of solvents, buffers or
solutions. In embodiments, liposomes are suspended in aqueous
phase. In embodiments, liposomes are suspended in a physiological
buffer having pH 7.0-7.4.
[0098] As used herein, the term "poloxamer" is a nonionic
copolymer. Poloxamers include a central hydrophobic chain of
polyoxypropylene and two flanking chains of polyoxyethylene. In
embodiments, a poloxamer is Poloxamer 188. Additional names for
poloxamer 188 are LUTROL.RTM. F68, P188, KOLLIPHOR.RTM. P188, and
Poly(ethylene glycol)-block-poly(propylene
glycol)-block-poly(ethylene glycol). The CAS Number for P188 is
9003-11-6. P188 has the following structure wherein X is 80, Z is
80 and Y is 27:
##STR00001##
[0099] As used herein, the term "cholesterol" or "cholesterol
compound" refers to a sterols or a modified steroids. Non-limiting
examples include: cholesterol, hydroxycholesterols, cholestans,
cholestanes, ketocholestanols, campesterol, cholesterol epoxides,
cholesterol-peg, lanosterol, esterified cholesterols. The term
"free cholesterol" refers to unesterified cholesterol with the
following general formula C27H460 and structure:
##STR00002##
[0100] As used herein, the term "phospholipid" or "phospholipids",
also called phosphatide or phosphatides, refer to an member of a
class of phosphorus- and/or fatty acid-containing substances. In
general, phospholipids are composed of a phosphate group, two
alcohols, and one or two fatty acids. On one end of the molecule
are the phosphate group and one alcohol; this end is polar, i.e.,
has an electric charge, and is attracted to water (hydrophilic).
The other end, which consists of the fatty acids, is neutral; it is
hydrophobic and water-insoluble but is fat-soluble. Some
non-limiting and illustrative examples of phospholipids include,
but not limited to, phosphatidic acid (phosphatidate),
phosphatidylcholine (pc) products/derivatives (various carbon chain
length fatty acids, saturated, multi-unsaturated and mixed acid
PC's); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE);
1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA);
1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA);
1,2-dioleoyl-sn-glycero-3-phosphate (DOPA);
1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG);
1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG);
1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG);
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC);
1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS);
1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS);
1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPC);
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (DSPE-mPEG-2000);
1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[folate(polyethylene
glycol)-5000] (DSPE-mPEG-5000);
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene
glycol)-2000] (DSPE-PEG-2000);
1,2-stearoyl-3-trimethylammonium-propane (DOTAP);
L-.alpha.-phosphatidylcholine, hydrogenated (Hydro Soy PC); and
2-stearoyl-sn-glycero-3-phosphocholine (Lyso PC),
phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine,
phosphatidylinositol (PI) phosphatidylinositol phosphate (PIP)
phosphatidylinositol bisphosphate (PIP2), phosphosphingolipids and
any derivatives thereof.
[0101] As used herein, the terms "lyophilize," "lyophilizing,"
"lyophilized" or "lyophilization", also know as freeze-drying or
cryodesiccation, refer to a dehydration process that can be used to
preserve a material or compound. Lyophilization can involve
freezing the material or compound and then reducing the surrounding
pressure to allow the frozen water or liquid component in the
freezed material or compound to the gas phase.
[0102] "Treatment," "treating," and "treat" are defined as acting
upon a disease, disorder, or condition with an agent to reduce or
ameliorate harmful or any other undesired effects of the disease,
disorder, or condition and/or its symptoms. "Treating" or
"treatment of" a condition or subject in need thereof refers to (1)
taking steps to obtain beneficial or desired results, including
clinical results such as the reduction of symptoms; (2) inhibiting
the disease, for example, arresting or reducing the development of
the disease or its clinical symptoms; (3) relieving the disease,
for example, causing regression of the disease or its clinical
symptoms; or (4) delaying the disease. For example, beneficial or
desired clinical results include, but are not limited to, reduction
and/or elimination of cancer cells and prevention and/or reduction
of metastasis of cancer cells.
[0103] As used herein, "administering" refers to the physical
introduction of a composition to a subject, using any of the
various methods and delivery systems known to those skilled in the
art. Routes of administration for the composition described herein
include intravenous, intraperitoneal, intramuscular, subcutaneous,
spinal or other parenteral routes of administration, for example by
injection or infusion. The phrase "parenteral administration" as
used herein means modes of administration other than enteral and
topical administration, usually by injection, and includes, without
limitation, intravenous, intraperitoneal, intramuscular,
intraarterial, intrathecal, intralymphatic, intralesional,
intracapsular, intraorbital, intracardiac, intradermal,
transtracheal, subcutaneous, subcuticular, intraarticular,
subcapsular, subarachnoid, intraspinal, epidural and intrasternal
injection and infusion, as well as in vivo electroporation.
Alternatively, the composition described herein can be administered
via a non-parenteral route, such as a topical, epidermal or mucosal
route of administration, for example, intranasally, orally,
vaginally, rectally, sublingually or topically. Administering can
also be performed, for example, once, a plurality of times, and/or
over one or more extended periods.
[0104] An "anticancer agent" or "anticancer compound" is a
therapeutic having an anticancer activity that can be used in the
treatment or prevention of cancer. An anticancer agent can be a
large or small molecule. Example anti-cancer agents include
antibodies, small molecules, and large molecules or combinations
thereof. Examples of "anticancer activity" include, but are not
limited to, reduction of cancer cell number, reduction of cancer
size, killing of cancer cells, reductions and/or inhibition of
metastasis and reduction of cancer cell growth and/or
proliferation.
[0105] The term "subject" or "subject in need thereof" refers to a
living organism suffering from a disease or condition or having a
possibility to have a disease or condition in the future. A term
"patient" refers to a living organism that already has a disease or
condition, e.g. a patient who has been diagnosed with a disease or
condition or has one or more symptoms associated with a disease or
condition. Non-limiting examples include humans, other mammals,
bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and
other non-mammalian animals. In some embodiments, a patient is
human.
[0106] According to the methods provided herein, the subject can be
administered an effective amount of one or more of agents,
compositions or complexes, all of which are interchangeably used
herein, (e.g. a pharmaceutical composition comprising a liposome,
the liposome encompassing an anticancer compound and an acid or
salt thereof) provided herein. The terms "effective amount" and
"effective dosage" are used interchangeably. The term "effective
amount" is defined as any amount necessary to produce a desired
effect (e.g., treatment of a disease such as cancer). Effective
amounts and schedules for administering the agent can be determined
empirically by one skilled in the art. The dosage ranges for
administration are those large enough to produce the desired
effects. The dosage should not be so large as to cause substantial
adverse side effects, such as unwanted cross-reactions,
anaphylactic reactions, and the like. Generally, the dosage can
vary with the age, condition, sex, type of disease, the extent of
the disease or disorder, route of administration, or whether other
drugs are included in the regimen, and can be determined by one of
skill in the art. The dosage can be adjusted by the individual
physician in the event of any contraindications. Dosages can vary
and can be administered in one or more dose administrations daily,
for one or several days. Guidance can be found in the literature
for appropriate dosages for given classes of pharmaceutical
products. For example, for the given parameter, an effective amount
can show an increase or decrease of at least 5%, 10%, 15%, 20%,
25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Efficacy can
also be expressed as "-fold" increase or decrease. For example, a
therapeutically effective amount can have at least a 1.2-fold,
1.5-fold, 2-fold, 5-fold, or more effect over a control. The exact
dose and formulation can depend on the purpose of the treatment,
and can be ascertainable by one skilled in the art using known
techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms
(vols. 1-3, 1992); Lloyd, The Art, Science and Technology of
Pharmaceutical Compounding (1999); Remington: The Science and
Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and
Pickar, Dosage Calculations (1999)).
[0107] As used herein, the term "cancer" refers to all types of
cancer, neoplasm or malignant tumors found in mammals, including
leukemias, lymphomas, melanomas, neuroendocrine tumors, carcinomas
and sarcomas. Exemplary cancers that may be treated with a
compound, pharmaceutical composition, or method provided herein
include lymphoma, sarcoma, bladder cancer, bone cancer, brain
tumor, cervical cancer, colon cancer, esophageal cancer, gastric
cancer, head and neck cancer, kidney cancer, myeloma, thyroid
cancer, leukemia, prostate cancer, breast cancer (e.g. triple
negative, ER positive, ER negative, chemotherapy resistant,
herceptin resistant, HER2 positive, doxorubicin resistant,
tamoxifen resistant, ductal carcinoma, lobular carcinoma, primary,
metastatic), ovarian cancer, pancreatic cancer, liver cancer (e.g.
hepatocellular carcinoma), lung cancer (e.g. non-small cell lung
carcinoma, squamous cell lung carcinoma, adenocarcinoma, large cell
lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma),
glioblastoma multiforme, glioma, melanoma, prostate cancer,
castration-resistant prostate cancer, breast cancer, triple
negative breast cancer, glioblastoma, ovarian cancer, lung cancer,
squamous cell carcinoma (e.g., head, neck, or esophagus),
colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B
cell lymphoma, or multiple myeloma. Additional examples include,
cancer of the thyroid, endocrine system, brain, breast, cervix,
colon, head & neck, esophagus, liver, kidney, lung, non-small
cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus
or Medulloblastoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma,
multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme,
ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary
macroglobulinemia, primary brain tumors, cancer, malignant
pancreatic insulinoma, malignant carcinoid, urinary bladder cancer,
premalignant skin lesions, testicular cancer, lymphomas, thyroid
cancer, neuroblastoma, esophageal cancer, genitourinary tract
cancer, malignant hypercalcemia, endometrial cancer, adrenal
cortical cancer, neoplasms of the endocrine or exocrine pancreas,
medullary thyroid cancer, medullary thyroid carcinoma, melanoma,
colorectal cancer, papillary thyroid cancer, hepatocellular
carcinoma, Paget's Disease of the Nipple, Phyllodes Tumors, Lobular
Carcinoma, Ductal Carcinoma, cancer of the pancreatic stellate
cells, cancer of the hepatic stellate cells, or prostate
cancer.
[0108] Liposomal Compositions
[0109] In embodiments, the compositions of the present disclosures
are chemically and physically stable in a manufactured drug product
to allow a commercially adequate shelf life.
[0110] In embodiments, particle size reduction can result in
significant increases in drug solubility. Materials in a
nanoparticle have a much higher tendency to leave the particle and
go into the surrounding solution than those in a larger particle of
the same composition. This phenomenon can increase the availability
of drug for transport across a biological membrane, but it can also
create physical instability of the nanoparticle itself. The
physical stability of nanoparticles may be improved by the use of
appropriate surface active agents and excipients at the right
levels to reduce the interfacial energy, controlling the surface
charge of the particles to maintain the dispersion, and
manufacturing the particles in a narrow size distribution.
[0111] In embodiments, the advantageous disposition of the
compositions of the present disclosures may be attributed to the
particle's size, shape, composition and charge. In embodiments, the
particles may be substantially spherical to move smoothly through
the capillaries. In embodiments, the size distribution range is
about 10 to 160 nm. In embodiments, the size distribution range is
about 20 to 150 nm. In embodiments, the size distribution range is
about 30 to 140 nm. In embodiments, the size distribution range is
about 40 to 130 nm. In embodiments, the size distribution range is
about 50 to 120 nm. In embodiments, the size distribution range is
about 60 to 110 nm. In embodiments, the size distribution range is
about 70 to 100 nm. In embodiments, the size distribution range is
a mean of about 10 nm. In embodiments, the size distribution range
is a mean of about 20 nm. In embodiments, the size distribution
range is a mean of about 30 nm. In embodiments, the size
distribution range is a mean of about 40 nm. In embodiments, the
size distribution range is a mean of about 50 nm. In embodiments,
the size distribution range is a mean of about 60 nm. In
embodiments, the size distribution range is a mean of about 70 nm.
In embodiments, the size distribution range is a mean of about 80
nm. In embodiments, the size distribution range is a mean of about
90 nm. In embodiments, the size distribution range is a mean of
about 100 nm. In embodiments, the size distribution range is a mean
of about 110 nm. In embodiments, the size distribution range is a
mean of about 120 nm. In embodiments, the size distribution range
is a mean of about 130 nm. In embodiments, the size distribution
range is a mean of about 140 nm. In embodiments, the size
distribution range is a mean of about 150 nm. In embodiments, the
size distribution range is a mean of about 160 nm. The composition
may include cholesterol, other lipids and surface-active agents
with or without the addition of polymers used to define particle
structure.
[0112] Particle size can be determined by multiple techniques.
Example techniques include dynamic light scattering (DLS) and
cryo-transmission electron microscopy. DLS can be used to assess
particle size by measuring of intensity, e.g., the
intensity-averaged particle diameters (Z-average) are calculated
from the cumulants analysis as defined in ISO 13321 (International
Organization for Standardization 1996). In embodiments, a mean
longest dimension of a liposome when measured by DLS by intensity
is in the range of about 40-100 nm, 50-90 nm, or 60-80 nm.
[0113] DLS can also be used to assess particle size by measurement
by number, e.g., there is first-power relationship between particle
size and contribution to the distribution. Particle size
distribution by number is computed from the intensity distribution
and the optical properties of the material. In embodiments, a mean
longest dimension of a liposomal particle when measured by DLS by
number is in the range of about 1-50 nm, 5-40 nm, or 10-30 nm
[0114] Cryotransmission electron microscopy can also be used to
assess a liposome size. In embodiments, the mean longest dimension
of a liposome as measured by cryotransmission electron microscopy
is in the range of about 1-50 nm, 5-40 nm, or 10-30 nm.
[0115] In embodiments, liposomes of the present disclosures are
unilamellar. Examples lipids that can be included within the
bilayer of a liposome include, but are not limited to, cholesterol,
phosphatidylcholine (PC) products/derivatives (various carbon chain
length fatty acids, saturated, multi-unsaturated and mixed acid
PC's); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE);
1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA);
1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA);
1,2-dioleoyl-sn-glycero-3-phosphate (DOPA);
1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG);
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC);
1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG);
1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG);
1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS);
1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS);
1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPC);
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (DSPE-mPEG-2000);
1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[folate(polyethylene
glycol)-5000] (DSPE-mPEG-5000);
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene
glycol)-2000] (DSPE-PEG-2000);
1,2-stearoyl-3-trimethylammonium-propane (DOTAP);
L-.alpha.-phosphatidylcholine, hydrogenated (Hydro Soy PC); and
2-stearoyl-sn-glycero-3-phosphocholine (Lyso PC).
[0116] Salts and Counter Ions
[0117] The present disclosures include compositions of anticancer
compounds and salts thereof encapsulated in liposomes.
Physicochemical properties of the counter ions that determine
performance and physical state of the salt aggregates include pKa,
valence, size, stereochemistry, dipole moment, polarizability. The
present disclosures provides optimal lipid to drug ratio(s) along
with proper counter ion(s) to take full advantage of both pKa
properties of the counter ion and physical state of anticancer
compounds aggregates to achieve target release properties (e.g.
highest possible .DELTA.pH 7.4 to 5.0 release differential).
[0118] Tables 1 provides example salts including applicable pKa's.
Table 2 provides example weakly basic anticancer compounds, an in
particular those that form salts or aggregates with oxalate or
tartrate.
TABLE-US-00001 TABLE 1 Pharmaceutical salts and counter ions.
Reagents Counter Ion pKa 1 pKa 2 pKa 3 Sulfate ##STR00003## -3 1.92
NA Picolinate ##STR00004## 1.07 NA NA Oxalate ##STR00005## 1.27
4.27 NA Maleate ##STR00006## 1.92 6.23 NA Phosphate ##STR00007##
1.96 7.12 12.32 Cysteinate ##STR00008## 1.96 8.18 NA Malonate
##STR00009## 2.83 5.70 NA Tartrate ##STR00010## 3.03 4.36 NA
Fumarate ##STR00011## 3.03 4.38 NA Citrate ##STR00012## 3.13 4.76
6.40 Formate ##STR00013## 3.75 NA NA N-Acetyl-L- cysteine
##STR00014## 3.82 9.52 NA Succinate ##STR00015## 4.21 5.64 NA
Ascorbate ##STR00016## 4.17 11.57 NA Acetate ##STR00017## 4.76 NA
NA
TABLE-US-00002 TABLE 2 Examples of weak bases chemotherapeutic
agents that form salts/aggregates with Oxalate or Tartrate and
demonstrate desirable pH discriminative drug release profile.
Doxorubicin hydrochloride ##STR00018## Approved for: Acute
lymphoblastic leukemia (ALL). Acute myeloid leukemia (AML). Breast
cancer. It is also used as adjuvant therapy for breast cancer that
has spread to the lymph nodes after surgery. Gastric (stomach)
cancer. Hodgkin lymphoma. Neuroblastoma. Non-Hodgkin lymphoma.
Ovarian cancer. Small cell lung cancer. Soft tissue and bone
sarcomas. Thyroid cancer. Transitional cell bladder cancer. Wilms
tumor Irinotecan hydrochloride ##STR00019## Approved for:
Colorectal cancer that has metastasized (spread to other parts of
the body), including metastatic cancer that has recurred (come
back) or has not gotten better with other chemotherapy Mitoxantrone
hydrochloride ##STR00020## Approved for: Acute myeloid leukemia
(AML). Prostate cancer. It is used as palliative treatment in
advanced disease that is hormone-refractory (does not respond to
hormone treatment).
[0119] In embodiments, a composition of the present disclosures
include a doxorubicin salt. In embodiments, the doxorubicin salt is
present in a liposome. In embodiments, the doxorubicin salt may be
formed within a liposome upon encapsulation of doxorubicin. In
embodiments, the composition provided herein exhibits desirable
physical performance and optimal pH-dependent drug release profile
which may be extremely effective in tumor tissues while exhibiting
low off target toxicity. In embodiments, without being bound by any
particular theory, at physiological pH, while circulating in the
blood, doxorubicin is mostly retained by the liposomes, whereas
strikingly higher drug release is achieved at lower pH
(.about.5.0-6.7) that occurs in intracellular lysosomal compartment
and local extracellular space of the tumor site that due to poor
vasculature tends to retain liposomes.
[0120] In embodiments, the composition of doxorubicin salt is
determined by the selection of the physical-chemical properties of
the anions and corresponding acids, and of the processing steps
used to create liposomes incorporated doxorubicin salt aggregates
with desirable physicochemical properties.
[0121] In embodiments, the optimal lipid to drug ratio(s) within a
proper counter ion(s) containing liposome encapsulating an
anticancer compound functions to take advantage of both pKa
properties of the counter ion and physical state of doxorubicin
aggregates to achieve target release properties (e.g. highest
possible .DELTA.pH 7.4/6.7/6.0/5.0 release differential--.DELTA.pH
7.4/5.0). In embodiments, doxorubicin is stabilized with suitable
counter ions inside of the liposomes at the proper lipid/drug ratio
to maximize safety and efficacy.
[0122] When a ratio between two parts, i.e. ratio between A and B,
is mentioned, it can be referred to A/B, A:B or A to B. For
instance, if the value referenced to each part A and B is 1 and 10,
respectively, it can be indicated that the A/B ratio (or the ratio
of A to B or the ratio A:B) is 1:10, 1/10 or 1 to 10.
[0123] For example, the pKa1 of sulfuric acid is -3
(doxorubicin-sulfate--DOXIL.RTM.), while the pKa1 of citric acid is
+3.13 (doxorubicin-citrate--MYOCET.RTM.) (Table 1). Sulfate can
form a strong salt with doxorubicin that may result in lower
doxorubicin release in DOXIL.RTM. liposomes at pH 7.4 to 5.0 range.
In contrast, doxorubicin-citrate is a weaker salt that may result
in higher doxorubicin release from the MYOCET.RTM. liposomes at pH
7.4 to 5.0 range.
[0124] Thus, provided herein are embodiments in which doxorubicin
optimum release from liposomes is achieved via selection of the
appropriate counter ions with pKa (e.g. pKa1) values higher than -3
(sulfuric acid) and less than +3.13 (citric acid). Thus, in
embodiments, the acid employed herein has a pKa (e.g. pKa1) higher
than -3 and less than 3. In embodiments, the acid employed herein
has a pKa (e.g. pKa1) higher than -2.9 and less than 2.9. In
embodiments, the acid employed herein has a pKa (e.g. pKa1) higher
than -2.8 and less than 2.8. In embodiments, the acid employed
herein has a pKa (e.g. pKa1) higher than -2.5 and less than
2.5.
[0125] A variety of counter ions were tested (Table 1) with
different pKa values for their ability to facilitate doxorubicin
loading into liposomes and to provide pH dependent doxorubicin
release from the liposomes. A counter ion(s) that provides the
highest differential between doxorubicin release at pH 7.4 to pH
5.0 (.DELTA.pH 7.4/5.0) has a preferred release profile.
[0126] In embodiments, an example release profile assay liposomal
samples are diluted 20.times. (i.e. 100 .mu.L of sample+1.9 mL of
diluent) or 50.times. (i.e. 50 .mu.L of sample+2.45 mL of diluent)
in PBS pH 7.4 buffers at 25.degree. C. or pre-warmed to 37.degree.
C. (to simulate in vivo temperature) and incubated for 2, 4, and 8
hrs at 25.degree. C. or 37.degree. C., respectively. In
embodiments, an example release profile assay liposomal samples are
diluted 20.times. (i.e. 100 .mu.L of sample+1.9 mL of diluent) or
50.times. (i.e. 50 .mu.L of sample+2.45 mL of diluent) in PBS pH
6.7 buffers at 25.degree. C. or pre-warmed to 37.degree. C. (to
simulate in vivo temperature) and incubated for 2, 4, and 8 hrs at
25.degree. C. or 37.degree. C., respectively. In embodiments, an
example release profile assay liposomal samples are diluted
20.times. (i.e. 100 .mu.L of sample+1.9 mL of diluent) or 50.times.
(i.e. 50 .mu.L of sample+2.45 mL of diluent) in PBS pH 6.0 buffers
at 25.degree. C. or pre-warmed to 37.degree. C. (to simulate in
vivo temperature) and incubated for 2, 4, and 8 hrs at 25.degree.
C. or 37.degree. C., respectively. In embodiments, an example
release profile assay liposomal samples are diluted 20.times. (i.e.
100 .mu.L of sample+1.9 mL of diluent) or 50.times. (i.e. 50 .mu.L
of sample+2.45 mL of diluent) in PBS pH 5.0 buffers at 25.degree.
C. or pre-warmed to 37.degree. C. (to simulate in vivo temperature)
and incubated for 2, 4, and 8 hrs at 25.degree. C. or 37.degree.
C., respectively.
[0127] In embodiments, for T0 time point determination, liposomal
formulations were diluted in PBS pH 7.4 at .about.25.degree. C. In
embodiments, for T0 time point determination, liposomal
formulations were diluted in PBS pH 6.7 at .about.25.degree. C. In
embodiments, for T0 time point determination, liposomal
formulations were diluted in PBS pH 6.0 at .about.25.degree. C. In
embodiments, for T0 time point determination, liposomal
formulations were diluted in PBS pH 5.0 at .about.25.degree. C. The
plate reader temperature may be set to 25.degree. C. and excitation
and emission wavelengths were set at 478 nm and 594 nm,
respectively. At each time point fluorescence of intact liposomes
(Fi) and total fluorescence of liposomes ruptured with Triton X-100
(Ft) was measured. The percent of drug release was quantified as
[(Fi_n-Fi_t)/Ft_avrg)]*100%, where Fi_n-Fi measured at 2, 4, or 8
hrs, Fi_t0-Fi measured at T0, and Ft_avrg-average of Ft values
determined for all time points.
[0128] In embodiments, and not to be bound by theory, the
crystalline state of various anticancer compound salts may be
selected for favorable properties. For example, cryotransmission
electron microscopy (cTEM) reveals doxorubicin precipitates as
fibrous-bundle aggregates in both citrate- and sulfate-containing
liposomes [1, 13-14]. The planar aromatic anthracycline rings are
thought to stack longitudinally to form linear fibers. These fibers
are aligned in a hexagonal arrangement to form bundles, with
approximately 12-60 fibers per bundle [25]. Doxorubicin aggregates
in the presence of sulfate typically have rigid linear fiber
bundles (interfiber spacing is approximately 27 A.degree.) compared
with the doxorubicin-citrate aggregates in the presence of citrate,
which appear mostly linear or curved (interfiber spacing is
approximately 30-35 A.degree.) [15, 25]. In embodiments, the
sulfate anion, being smaller than the citrate anion, may allow a
tighter packing arrangement, resulting in a decreased flexibility
of fiber bundles. In embodiments, doxorubicin-sulfate (e.g.
doxorubicin-sulfate aggregates) results in slower drug release at
physiological and acidic pH compare to doxorubicin-phosphate
[13-14], and citrate (e.g. citrate aggregates) [1, 13-15].
[0129] In embodiments, compositions of the present disclosures are
designed to take an advantage of tumor biology by employing proper
counter ions (oxalate and tartrate), optimized lipid/drug ratio and
optimized anticancer compound (e.g. doxorubicin) loading
conditions, strong pH dependence of drug release and preferential
targeting of chemotherapeutic agent(s) to the tumor site. In
embodiments, chelators are employed to complement counter ion(s)
and/or antioxidants.
[0130] In embodiment, using oxalate or tartrate as counter ions,
lipid/drug ratio in the optimized range, and proper loading
conditions will allow achieving targeted (pH dependent) drug
release, and therefore improve safety and efficacy of number weak
bases chemotherapeutic agents with various molecular targets (DNA
intercalating/damaging agents, topoisomerase inhibitors, kinase
inhibitors, etc.; Table 3).
TABLE-US-00003 TABLE 3 Other examples of weak bases
chemotherapeutic agents anticipated to form salts/aggregates with
Oxalate or Tartrate. Daunorubicin hydrochloride ##STR00021##
Approved for: Acute lymphoblastic leukemia in adults and children.
Acute myeloid leukemia in adults Epirubicin hydrochloride
##STR00022## Approved for: Breast cancer. It is used after surgery
in patients with early- stage breast cancer that has spread to the
lymph nodes under the arm. Idarubicin hydrochloride ##STR00023##
Approved for: Acute myeloid leukemia (AML). Bendamustine
hydrochloride ##STR00024## Approved for: B-cell non-Hodgkin
lymphoma (NHL) in patients whose disease has not gotten better with
other chemotherapy or has recurred (come back). Chronic lymphocytic
leukemia (CLL) Indimitecan hydrochloride ##STR00025## Clinical
trials. Solid tumors and lymphomas. Indotecan hydrochloride
##STR00026## Clinical trials. Solid tumors and lymphomas. Erlotinib
hydrochloride ##STR00027## Approved for: Non-small cell lung cancer
(NSCLC). It is used as first-line treatment for metastatic NSCLC in
patients with tumors that have certain epidermal growth factor
receptor (EGFR) mutations. It is used for locally advanced or
metastatic NSCLC in patients who have already been treated with
chemotherapy. Pancreatic cancer. It is used with gemcitabine
hydrochloride in patients whose disease cannot be removed by
surgery or has metastasized. Raloxifene hydrochloride ##STR00028##
Approved for: Breast cancer. It is used to decrease the chance of
invasive breast cancer in postmenopausal women who have a high risk
for developing the disease or who have osteoporosis. Raloxifene
hydrochloride is also approved to prevent and treat: Osteoporosis
in postmenopausal women Topotecan hydrochloride ##STR00029##
Approved for: Cervical cancer that has not gotten better with other
treatment or has recurred (come back). It is used with another
drug, called cisplatin. Ovarian cancer in patients whose disease
has not gotten better with other chemotherapy. Small cell lung
cancer in patients whose disease has not gotten better with other
chemotherapy. Ponatinib hydrochloride ##STR00030## Approved for:
Acute lymphoblastic leukemia that is Philadelphia chromosome
positive and has the T315I mutation. Chronic myelogenous leukemia
that has the T315I mutation. For these types of leukemia without
the T315I mutation, ponatinib hydrochloride is used when other
tyrosine kinase inhibitors cannot be used. Tipiracil hydrochloride
##STR00031## Approved for: Colorectal cancer that has metastasized
(spread to other parts of the body). It is used in patients who
have already been treated with certain chemotherapy and biologic
therapy. Procarbazine hydrochloride ##STR00032## Approved for:
Hodgkin lymphoma that is advanced. Mechlorethamine hydrochloride
##STR00033## Approved for: Bronchogenic carcinoma. Chronic
lymphocytic leukemia (CLL). Chronic myelogenous leukemia (CML).
Hodgkin lymphoma that is advanced. Malignant pleural effusion,
malignant pericardial effusion, and malignant peritoneal effusion.
Mycosis fungoides. Non-Hodgkin lymphoma (NHL). Pazopanib
hydrochloride ##STR00034## Approved for: Renal cell carcinoma (a
type of kidney cancer) that is advanced. Soft tissue sarcoma that
is advanced. It is used in patients who have already been treated
with chemotherapy. Ponatinib hydrochloride ##STR00035## Approved
for: Acute lymphoblastic leukemia that is Philadelphia chromosome
positive and has the T315I mutation. Chronic myelogenous leukemia
that has the T315I mutation. Gemcitabine hydrochloride ##STR00036##
Approved for: Breast cancer that has metastasized (spread to other
parts of the body) and has not gotten better with other
chemotherapy. It is used with paclitaxel. Non-small cell lung
cancer that is advanced or has metastasized. It is used in patients
whose disease cannot be removed by surgery. It is used with
cisplatin. Ovarian cancer that is advanced and has not gotten
better with other chemotherapy. It is used with carboplatin.
Pancreatic cancer that is advanced or has metastasized. It is used
in patients whose disease cannot be removed by surgery and who have
already been treated with other chemotherapy. It is used with
paclitaxel albumin-stabilized nanoparticle formulation. AZD7762
##STR00037## Approved for: AZD7762 is a Chk1 kinase inhibitor which
increases sensitivity to DNA-damaging agents, including
gemcitabine. Development discontinued due to unpredictable cardiac
toxicity. Chk1 kinase remains an important therapeutic target.
Vincristine sulfate ##STR00038## Approved for: Acute lymphoblastic
leukemia that is Philadelphia chromosome negative. It is used in
patients whose disease has relapsed two or more times or has not
gotten better with two or more types of treatment. Vinblastine
sulfate ##STR00039## Approved for: Breast cancer that has not
gotten better with other treatment. Choriocarcinoma that has not
gotten better with other chemotherapy. Choriocarcinoma is a type of
gestational trophoblastic disease. Hodgkin lymphoma. Kaposi
sarcoma. Mycosis fungoides. Non-Hodgkin lymphoma (NHL). Testicular
cancer Sunitinib malate ##STR00040## Approved for: Gastrointestinal
stromal tumor (a type of stomach cancer). It is used in patients
whose condition has become worse while taking another drug called
imatinib mesylate or who are not able to take imatinib mesylate.
Pancreatic cancer. It is used in patients with progressive
neuroendocrine tumors that cannot be removed by surgery, are
locally advanced, or have metastasized (spread to other parts of
the body). Renal cell carcinoma (a type of kidney cancer) that has
metastasized. Lanreotide acetate ##STR00041## Approved for:
Gastroenteropancreatic neuroendocrine tumors. It is used for some
tumors that cannot be removed by surgery, are locally advanced, or
have metastasized (spread to other parts of the body). Tamoxifen
citrate ##STR00042## Approved for: Breast cancer in women and men.
Tamoxifen citrate is also approved to prevent: Breast cancer in
women who are at high risk for the disease. Leuprolide acetate
##STR00043## Approved for: Prostate cancer that is advanced.
[0131] In embodiments, targeting the liposomal drug release
according to the blood--tumor site--lysosomes pH gradient improves
the safety and efficacy of weak bases chemotherapeutic agents
compare to their non- or less-pH discriminative liposomal and/or
free forms.
[0132] Lipid to Drug (i.e. Lipid/Drug) and Phospholipid to Free
Cholesterol (i.e. PL/FC) Ratios
[0133] In embodiments, the optimal drug load in the particles of
the present application is achieved with the proper counter ion
selection. In addition, lipophilic inactive components and surface
active agents may be selected. In addition, proper drug to lipid
ratio may be selected. In embodiments, selections are made in order
to decrease release neutral pH while in circulation and increase
extracellular/intracellular release at more acidic pH at the target
site.
[0134] In embodiments, the lipid to drug (lipid/drug) and PL/FC
ratio is related to desirable physicochemical and biological
performance.
[0135] In embodiments, the structure of the particles is determined
by the selection of the formulation components and lipid/drug and
PL/FC ratio, and of the processing steps used to create the
particles. Structural and quantitative elements that determine
particle performance include lipid/drug and PL/FC ratio, counter
ions, particle size (and size distribution in the population),
particle shape, particle charge and the distribution of individual
components in the particle, especially those at the particle
surface.
[0136] In embodiments, the structured lipid rich
nanoparticles/liposomes of the present application are designed to
carry a useful drug load in a parenterally administered drug
product. Drugs of interest with respect to this delivery system
includes those drugs/salts complexes which have low solubility at
physiological pH and significantly higher solubility at more acidic
(local tumor extracellular and endosomal/lysosomal environment) pH.
The liposomes included in the present application may have unique
physical-chemical performance. In embodiments, high lipid/drug and
.ltoreq.4.0 but .gtoreq.1.0 PL/FC ratios may result in a stable
lipid layer that restricts release of liposomal content via
concentration gradient and in serum or blood but allow
discriminative drug release in response to change of the outside
pH, and therefore to take the advantage of the particular counter
ions and pKa(s) of the corresponding acid(s).
[0137] In embodiments, the doxorubicin may be a pegylated liposomal
doxorubicin, such as that used in DOXIL.RTM.. In embodiments the
doxorubicin is substituted with amphiphilic block copolymer rather
than polyethylene glycol.
[0138] In embodiments, the lipids used in the liposome formulations
herein are PEGylated lipids. In embodiments, rather than pegylated
lipids, poloxamer lipids are used (e.g. P188 lipids). Poloxamers
are non-ionic poly (ethylene oxide) (PEO)-poly (propylene oxide)
(PPO) copolymers. They may be used in pharmaceutical formulations
as surfactants. Their surfactant property has been useful for
detergency, dispersion, stabilization, foaming, and emulsification.
Poloxamers are broadly used in clinical applications [16]. In
embodiments, liposomes coated (e.g. intercalated) with p188 are
formed by dissolving lipids and P188 in DCM, evaporating
DCM--forming lipid film, and hydration of lipid film. This example
process results in intercalation (insertion) of p188 hydrophobic
hydrocarbon chain between the lipid hydrocarbon chain) and exposure
of more hydrophilic part in aqueous phase, and in modified lipid
surface of the liposomes that prevents their opsonization and
recognition by the macrophage system.
[0139] In embodiments, the compositions used herein having
liposomes coated with poloxamer (e.g p188) are used to treat
diseases requiring the active (e.g. the weakly basic anticancer
compound) to cross the blood brain barrier. In embodiments, the
compositions used herein having liposomes coated with poloxamer
(e.g p188) are used to treat diseases requiring the active (e.g.
the weakly basic anticancer compound) are used where Apolipoprotein
E interference is not desired.
[0140] In embodiments, lower lipid/drug ratios lead are used to
increased surface tension and compromised lipid layer integrity
that upon dilution could result in increased leakage of liposomal
content into dissolution media at neutral pH due to concentration
gradient, and could offset the pH driven release of drug. In
embodiments, higher lipid to drug ratios are used to lower surface
tension and achieve higher integrity lipid layer(s) that are
capable of preventing "off target" leakage of intraliposomal
material into dissolution media, and release the drug only in
response to pH transition.
[0141] In embodiments, liposomes include a plurality of lipids and
the ratio of the plurality of lipids to a drug (e.g. a weakly basic
anticancer agent and/or an acid or salt thereof) can be considered.
In embodiments, lipid to drug (lipid to drug) ratio represents a
weight to weight (w to w) ratio of total lipids to a drug (e.g.
doxorubicin free base) in final suspension of drug-loaded liposomes
and has a mean of about 0.5 to 1 (weight to weight), about 1 to 1,
about 5 to 1, about 10 to 1, about 20 to 1, about 30 to 1, about 40
to 1, about 50 to 1, about 60 to 1, about 70 to 1, about 80 to 1,
about 90 to 1, about 100 to 1 or any intervening number of the
foregoing or higher than about 100 to 1. In embodiments, lipid to
drug (lipid to drug) ratio represents a mole to mole (mol to mol)
ratio of total lipids to a drug in final suspension of drug-loaded
liposomes and has a mean of 0.5 to 1 (mol to mol), about 1 to 1,
about 5 to 1, about 10 to 1, about 20 to 1, about 30 to 1, about 40
to 1, about 50 to 1, about 60 to 1, about 70 to 1, about 80 to 1,
about 90 to 1 or about 100 to 1 or any intervening number of the
foregoing or higher than about 100 to 1.
[0142] In embodiments, lipid to drug (i.e. lipid/drug) ratio (mol
to mol or weight to weight) has a mean in a range of about 0.5 to 1
to 1 to 1, about 0.5 to 1 to 5 to 1, about 0.5 to 1 to 10 to 1,
about 0.5 to 1 to 20 to 1, about 0.5 to 1 to 30 to 1, about 0.5 to
1 to 40 to 1, about 0.5 to 1 to 50 to 1, about 0.5 to 1 to 60 to 1,
about 0.5 to 1 to 70 to 1, about 0.5 to 1 to 80 to 1, about 0.5 to
1 to 90 to 1 or about 0.5 to 1 to 100 to 1. In embodiments, lipid
to drug ratio (mol to mol or weight to weight) has a mean in a
range of about 1 to 1 to 5 to 1, about 1 to 1 to 10 to 1, about 1
to 1 to 20 to 1, about 1 to 1 to 30 to 1, about 1 to 1 to 40 to 1,
about 1 to 1 to 50 to 1, about 1 to 1 to 60 to 1, about 1 to 1 to
70 to 1, about 1 to 1 to 80 to 1, about 1 to 1 to 90 to 1 or about
1 to 1 to 100 to 1. In embodiments, lipid to drug ratio (mol to mol
or weight to weight) has a mean in a range of about 5 to 1 to 10 to
1, about 5 to 1 to 20 to 1, about 5 to 1 to 30 to 1, about 5 to 1
to 40 to 1, about 5 to 1 to 50 to 1, about 5 to 1 to 60 to 1, about
5 to 1 to 70 to 1, about 5 to 1 to 80 to 1, about 5 to 1 to 90 to 1
or about 5 to 1 to 100 to 1. In embodiments, lipid to drug ratio
(mol to mol or weight to weight) has a mean in a range of about 10
to 1 to 20 to 1, about 10 to 1 to 30 to 1, about 10 to 1 to 40 to
1, about 10 to 1 to 50 to 1, about 10 to 1 to 60 to 1, about 10 to
1 to 70 to 1, about 10 to 1 to 80 to 1, about 10 to 1 to 90 to 1 or
about 10 to 1 to 100 to 1. In embodiments, lipid to drug ratio (mol
to mol or weight to weight) has a mean in a range of about 20 to 1
to 30 to 1, about 20 to 1 to 40 to 1, about 20 to 1 to 50 to 1,
about 20 to 1 to 60 to 1, about 20 to 1 to 70 to 1, about 20 to 1
to 80 to 1, about 20 to 1 to 90 to 1 or about 20 to 1 to 100 to 1.
In embodiments, lipid to drug ratio (mol to mol or weight to
weight) has a mean in a range of about 30 to 1 to 40 to 1, about 30
to 1 to 50 to 1, about 30 to 1 to 60 to 1, about 30 to 1 to 70 to
1, about 30 to 1 to 80 to 1, about 30 to 1 to 90 to 1 or about 30
to 1 to 100 to 1. In embodiments, lipid to drug ratio (mol to mol
or weight to weight) has a mean in a range of about 40 to 1 to 50
to 1, about 40 to 1 to 60 to 1, about 40 to 1 to 70 to 1, about 40
to 1 to 80 to 1, about 40 to 1 to 90 to 1 or about 40 to 1 to 100
to 1. In embodiments, lipid to drug ratio (mol to mol or weight to
weight) has a mean in a range of about 50 to 1 to 60 to 1, about 50
to 1 to 70 to 1, about 50 to 1 to 80 to 1, about 50 to 1 to 90 to 1
or about 50 to 1 to 100 to 1. In embodiments, lipid to drug ratio
(mol to mol or weight to weight) has a mean in a range of about 60
to 1 to 70 to 1, about 60 to 1 to 80 to 1, about 60 to 1 to 90 to 1
or about 60 to 1 to 100 to 1. In embodiments, lipid to drug ratio
(mol to mol or weight to weight) has a mean in a range of about 70
to 1 to 80 to 1, about 70 to 1 to 90 to 1 or about 70 to 1 to 100
to 1. In embodiments, lipid to drug ratio (mol to mol or weight to
weight) has a mean in a range of about 80 to 1 to 90 to 1 or about
80 to 1 to 100 to 1. In embodiments, lipid to drug ratio (mol to
mol or weight to weight) has a mean in a range of about 90 to 1 to
100 to 1.
[0143] In embodiments, drug mol % (e.g. the number of moles of drug
relative to total number of moles of all formulation constitutions
including phospholipids, cholesterol, poloxamers, anticancer drug,
salts, etc.) has a mean of about 0.5%, about 1%, about 2%, about
3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%,
about 10%, about 15%, about 20%, about 30% or any intervening
number of the foregoing or higher than about 30%.
[0144] In embodiments, drug mol % has a range of about 0.5-1%,
about 0.5-2%, about 0.5-3%, about 0.5-4%, about 0.5-5%, about
0.5-6%, about 0.5-7%, about 0.5-8%, about 0.5-9%, about 0.5-10%,
about 0.5-15%, about 0.5-20% or about 0.5-30%. In some embodiments,
drug mol % has a range of about 1-2%, about 1-3%, about 1-4%, about
1-5%, about 1-6%, about 1-7%, about 1-8%, about 1-9%, about 1-10%,
about 1-15%, about 1-20% or about 1-30%. In some embodiments, drug
mol % has a range of about 2-3%, about 2-4%, about 2-5%, about
2-6%, about 2-7%, about 2-8%, about 2-9%, about 2-10%, about 2-15%,
about 2-20% or about 2-30%. In some embodiments, drug mol % has a
range of about 3-4%, about 3-5%, about 3-6%, about 3-7%, about
3-8%, about 3-9%, about 3-10%, about 3-15%, about 3-20% or about
3-30%. In some embodiments, drug mol % has a range of about 4-5%,
about 4-6%, about 4-7%, about 4-8%, about 4-9%, about 4-10%, about
4-15%, about 4-20% or about 4-30%. In some embodiments, drug mol %
has a range of about 5-6%, about 5-7%, about 5-8%, about 5-9%,
about 5-10%, about 5-15%, about 5-20% or about 5-30%. In some
embodiments, drug mol % has a range of about 6-7%, about 6-8%,
about 6-9%, about 6-10%, about 6-15%, about 6-20% or about 6-30%.
In some embodiments, drug mol % has a range of about 7-8%, about
7-9%, about 7-10%, about 7-15%, about 7-20% or about 7-30%. In some
embodiments, drug mol % has a range of about 8-9%, about 8-10%,
about 8-15%, about 8-20% or about 8-30%. In some embodiments, drug
mol % has a range of about 9-10%, about 9-15%, about 9-20% or about
9-30%. In some embodiments, drug mol % has a range of about 10-15%,
about 10-20% or about 10-30%. In some embodiments, drug mol % has a
range of about 15-20% or about 15-30%. In some embodiments, drug
mol % has a range of about 20-30%.
[0145] In embodiments, liposomes include a plurality of free
cholesterol (FC). In embodiments, unloaded liposomes and/or drug
loaded into the liposomes have a plurality of phospholipids (PL).
Therefore, in certain embodiments, liposomes loaded with a drug
have free cholesterol (FC) and phospholipids (PL). In embodiments,
a ratio of PL to FC (i.e. "PL to FC" or "PL/FC" ratio) represents a
weight to weight (w to w) ratio of phospholipids to free
cholesterols in final suspension of drug-loaded liposomes and has a
mean of 0.5 to 1 (w to w), about 1 to 1, about 2 to 1, about 3 to
1, about 4 to 1, about 5 to 1, about 10 to 1, about 20 to 1, about
30 to 1, about 40 to 1, about 50 to 1, about 60 to 1, about 70 to
1, about 80 to 1, about 90 to 1, about 100 to 1 or any intervening
number of the foregoing or higher than about 100 to 1. In
embodiments, a ratio of PL to FC (i.e. "PL to FC" ratio) represents
a mole to mole (mol to mol) ratio of phospholipids to free
cholesterols in final suspension of drug-loaded liposomes and has a
mean of about 0.5 to 1 (mol to mol), about 1 to 1, about 2 to 1,
about 3 to 1, about 4 to 1, or about 5 to 1, about 10 to 1, about
20 to 1, about 30 to 1, about 40 to 1, about 50 to 1, about 60 to
1, about 70 to 1, about 80 to 1, about 90 to 1, about 100 to 1 or
any intervening number of the foregoing or higher than about 100 to
1.
[0146] In embodiments, "PL to FC (i.e. PL/FC)" ratio (mol to mol or
w to w) has a mean in a range of about 0.5 to 1, about 0.55 to 1,
about 0.6 to 1, about 0.65 to 1, about 0.7 to 1, about 0.75 to 1,
about 0.8 to 1, about 0.85 to 1, about 0.9 to 1, about 0.95 to 1,
about 1 to 1, about 1.05 to 1, about 1.1 to 1, about 1.15 to 1,
about 1.2 to 1, about 1.25 to 1, about 1.3 to 1, about 1.35 to 1,
about 1.4 to 1, about 1.45 to 1, about 1.5 to 1, about 1.55 to 1,
about 1.6 to 1, about 1.65 to 1, about 1.7 to 1, about 1.75 to 1,
about 1.8 to 1, about 1.85 to 1, about 1.9 to 1, about 2 to 1,
about 2.05 to 1, about 2.1 to 1, about 2.15 to 1, about 2.2 to 1,
about 2.25 to 1, about 2.3 to 1, about 2.35 to 1, about 2.4 to 1,
about 2.45 to 1, about 2.5 to 1, about 2.55 to 1, about 2.6 to 1,
about 2.65 to 1, about 2.7 to 1, about 2.75 to 1, about 2.8 to 1,
about 2.85 to 1, about 2.9 to 1, about 3 to 1, about 3.05 to 1,
about 3.1 to 1, about 3.15 to 1, about 3.2 to 1, about 3.25 to 1,
about 3.3 to 1, about 3.35 to 1, about 3.4 to 1, about 3.45 to 1,
about 3.5 to 1, about 3.55 to 1, about 3.6 to 1, about 3.65 to 1,
about 3.7 to 1, about 3.75 to 1, about 3.8 to 1, about 3.85 to 1,
about 3.9 to 1, about 4 to 1, about 4.05 to 1, about 4.1 to 1,
about 4.15 to 1, about 4.2 to 1, about 4.25 to 1, about 4.3 to 1,
about 4.35 to 1, about 4.4 to 1, about 4.45 to 1, about 4.5 to 1,
about 4.55 to 1, about 4.6 to 1, about 4.65 to 1, about 4.7 to 1,
about 4.75 to 1, about 4.8 to 1, about 4.85 to 1, about 4.9 to 1,
about 5 to 1 or any intervening number of the foregoing.
[0147] In embodiments, "PL to FC (i.e. PL/FC)" ratio (mol to mol or
w to w) has a mean in a range of about 0.86 to 1, about 1.22 to 1,
about 1.29 to 1, about 1.62 to 1, about 1.72 to 1, about 3.68 to 1
or any intervening number of the foregoing. In embodiments, "PL to
FC" ratio (mol to mol or w to w) has a mean in a range of about
0.86 to 1. In embodiments, "PL to FC" ratio (mol to mol or w to w)
has a mean in a range of about 1.22 to 1. In embodiments, "PL to
FC" ratio (mol to mol or w to w) has a mean in a range of about
1.29 to 1. In embodiments, "PL to FC" ratio (mol to mol or w to w)
has a mean in a range of about 1.62 to 1. In embodiments, "PL to
FC" ratio (mol to mol or w to w) has a mean in a range of about
1.72 to 1. In embodiments, "PL to FC" ratio (mol to mol or w to w)
has a mean in a range of about 3.68 to 1. In embodiments, "PL to
FC" ratio (mol to mol or w to w) has a mean in a range of about
0.86 to 1 to 1.22 to 1, about 0.86 to 1 to 1.29 to 1, about 0.86 to
1 to 1.62 to 1, about 0.86 to 1 to 1.72 to 1 or about 0.86 to 1 to
3.68 to 1. In embodiments, "PL to FC" ratio (mol to mol or w to w)
has a mean in a range of about 1.22 to 1 to 1.29 to 1, about 1.22
to 1 to 1.62 to 1, about 1.22 to 1 to 1.72 to 1 or about 1.22 to 1
to 3.68 to 1. In embodiments, "PL to FC" ratio (mol to mol or w to
w) has a mean in a range of about 1.29 to 1 to 1.62 to 1, about
1.29 to 1 to 1.72 to 1 or about 1.29 to 1 to 3.68 to 1. In
embodiments, "PL to FC" ratio (mol to mol or w to w) has a mean in
a range of about 1.62 to 1 to 1.72 to 1 or about 1.62 to 1 to 3.68
to 1. In embodiments, "PL to FC" ratio (mol to mol or w to w) has a
mean in a range of about 1.72 to 1 to 3.68 to 1.
[0148] In embodiments, "PL to FC (i.e. PL/FC)" ratio (mol to mol or
w to w) has a mean in a range of about 0.5 to 1 to 1 to 1, about
0.5 to 1 to 2 to 1, about 0.5 to 1 to 3 to 1, about 0.5 to 1 to 4
to 1, about 0.5 to 1 to 5 to 1, about 0.5 to 1 to 10 to 1, about
0.5 to 1 to 20 to 1, about 0.5 to 1 to 30 to 1, about 0.5 to 1 to
40 to 1, about 0.5 to 1 to 50 to 1, about 0.5 to 1 to 60 to 1,
about 0.5 to 1 to 70 to 1, about 0.5 to 1 to 80 to 1, about 0.5 to
1 to 90 to 1 or about 0.5 to 1 to 100 to 1. In embodiments, lipid
to drug ratio (mol to mol or w to w) has a mean in a range of about
1 to 1 to 2 to 1, about 1 to 1 to 3 to 1, about 1 to 1 to 4 to 1,
about 1 to 1 to 5 to 1, about 1 to 1 to 10 to 1, about 1 to 1 to 20
to 1, about 1 to 1 to 30 to 1, about 1 to 1 to 40 to 1, about 1 to
1 to 50 to 1, about 1 to 1 to 60 to 1, about 1 to 1 to 70 to 1,
about 1 to 1 to 80 to 1, about 1 to 1 to 90 to 1 or about 1 to 1 to
100 to 1. In embodiments, lipid to drug ratio (mol to mol or w to
w) has a mean in a range of about 2 to 1 to 3 to 1, about 2 to 1 to
4 to 1, about 2 to 1 to 5 to 1, about 2 to 1 to 10 to 1, about 2 to
1 to 20 to 1, about 2 to 1 to 30 to 1, about 2 to 1 to 40 to 1,
about 2 to 1 to 50 to 1, about 2 to 1 to 60 to 1, about 2 to 1 to
70 to 1, about 2 to 1 to 80 to 1, about 2 to 1 to 90 to 1 or about
1 to 1 to 100 to 1. In embodiments, lipid to drug ratio (mol to mol
or w to w) has a mean in a range of about 3 to 1 to 4 to 1, about 3
to 1 to 5 to 1, about 3 to 1 to 10 to 1, about 3 to 1 to 20 to 1,
about 3 to 1 to 30 to 1, about 3 to 1 to 40 to 1, about 3 to 1 to
50 to 1, about 3 to 1 to 60 to 1, about 3 to 1 to 70 to 1, about 3
to 1 to 80 to 1, about 3 to 1 to 90 to 1 or about 3 to 1 to 100 to
1. In embodiments, lipid to drug ratio (mol to mol or w to w) has a
mean in a range of about 4 to 1 to 5 to 1, about 4 to 1 to 10 to 1,
about 4 to 1 to 20 to 1, about 4 to 1 to 30 to 1, about 4 to 1 to
40 to 1, about 4 to 1 to 50 to 1, about 4 to 1 to 60 to 1, about 4
to 1 to 70 to 1, about 4 to 1 to 80 to 1, about 4 to 1 to 90 to 1
or about 4 to 1 to 100 to 1. In embodiments, lipid to drug ratio
(mol to mol or w to w) has a mean in a range of about 5 to 1 to 10
to 1, about 5 to 1 to 20 to 1, about 5 to 1 to 30 to 1, about 5 to
1 to 40 to 1, about 5 to 1 to 50 to 1, about 5 to 1 to 60 to 1,
about 5 to 1 to 70 to 1, about 5 to 1 to 80 to 1, about 5 to 1 to
90 to 1 or about 5 to 1 to 100 to 1. In embodiments, lipid to drug
ratio (mol to mol or w to w) has a mean in a range of about 10 to 1
to 20 to 1, about 10 to 1 to 30 to 1, about 10 to 1 to 40 to 1,
about 10 to 1 to 50 to 1, about 10 to 1 to 60 to 1, about 10 to 1
to 70 to 1, about 10 to 1 to 80 to 1, about 10 to 1 to 90 to 1 or
about 10 to 1 to 100 to 1. In embodiments, lipid to drug ratio (mol
to mol or w to w) has a mean in a range of about 20 to 1 to 30 to
1, about 20 to 1 to 40 to 1, about 20 to 1 to 50 to 1, about 20 to
1 to 60 to 1, about 20 to 1 to 70 to 1, about 20 to 1 to 80 to 1,
about 20 to 1 to 90 to 1 or about 20 to 1 to 100 to 1. In
embodiments, lipid to drug ratio (mol to mol or w to w) has a mean
in a range of about 30 to 1 to 40 to 1, about 30 to 1 to 50 to 1,
about 30 to 1 to 60 to 1, about 30 to 1 to 70 to 1, about 30 to 1
to 80 to 1, about 30 to 1 to 90 to 1 or about 30 to 1 to 100 to 1.
In embodiments, lipid to drug ratio (mol to mol or w to w) has a
mean in a range of about 40 to 1 to 50 to 1, about 40 to 1 to 60 to
1, about 40 to 1 to 70 to 1, about 40 to 1 to 80 to 1, about 40 to
1 to 90 to 1 or about 40 to 1 to 100 to 1. In embodiments, lipid to
drug ratio (mol to mol or w to w) has a mean in a range of about 50
to 1 to 60 to 1, about 50 to 1 to 70 to 1, about 50 to 1 to 80 to
1, about 50 to 1 to 90 to 1 or about 50 to 1 to 100 to 1. In
embodiments, lipid to drug ratio (mol to mol or w to w) has a mean
in a range of about 60 to 1 to 70 to 1, about 60 to 1 to 80 to 1,
about 60 to 1 to 90 to 1 or about 60 to 1 to 100 to 1. In
embodiments, lipid to drug ratio (mol to mol or w to w) has a mean
in a range of about 70 to 1 to 80 to 1, about 70 to 1 to 90 to 1 or
about 70 to 1 to 100 to 1. In embodiments, lipid to drug ratio (mol
to mol or w to w) has a mean in a range of about 80 to 1 to 90 to 1
or about 80 to 1 to 100 to 1. In embodiments, lipid to drug ratio
(mol to mol or w to w) has a mean in a range of about 90 to 1 to
100 to 1.
[0149] In embodiments, phospholipid mol % (e.g. the number of moles
of phospholipid relative to total number of moles of all
formulation constitutions including phospholipids, cholesterol,
poloxamers, anticancer drug, salts, etc.) has a mean of about 10%,
about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,
about 80%, about 90% or any intervening number of the foregoing or
higher that about 90%.
[0150] In embodiments, free cholesterol (FC) mol % (e.g. the number
of moles of FC relative to total number of moles of all formulation
constitutions including phospholipids, cholesterol, poloxamers,
anticancer drug, salts, etc.) has a mean of about 5%, about 10%,
about 20%, about 30%, about 40%, about 50%, about 60% or any
intervening number of the foregoing or higher that about 60%.
[0151] In embodiments, lower PL/FC ratios (w/w or mol/mol) lead to
increased lipid bilayer rigidity that in its turn negatively
impacts pH dependent drug release. In embodiments, higher PL/FC
ratios (w/w or mol/mol) compromise the stability of the liposomes
in serum or blood. Thus, in some embodiments, optimal range of
PL/FC is determined to lie in 1/1 to 4/1 range.
[0152] Obtained data on Irinotecan containing liposomes are in a
good agreement with results obtained for doxorubicin and support
the effect of oxalate and tartrate counter ions and preferred
lipid/drug ratio.
[0153] Method of Loading Anticancer Compounds within Liposomes
[0154] In embodiments, the effect of the loading conditions can
alter the volume of drug contained within a liposome, release
kinetics, liposome size, etc. In embodiments, loading the liposomes
under cold (e.g. without a heated step, or at room temperature)
conditions produces favorable release kinetics.
[0155] In embodiments, an anticancer compound and its paired
liposomes containing encapsulated counter ion can be stored in
separate container (e.g. vials) for mixing in a medical setting
prior to use. In embodiments, weakly basic anticancer compound of
the present disclosures is lyophilized and readily reconstitutable
in sterile water for injection. In embodiments, lyophilized and
reconstituted anticancer compound--can be mixed with a liposome
suspension. In embodiments, this mixing occurs at room temperature.
The compositions of the present disclosures allow for a short
incubation time upon mixing. In embodiments, an incubation time is
about 0-60 minutes. In embodiments, an incubation time is about
0-45 minutes. In embodiments, an incubation time is about 0-30
minutes. In embodiments, an incubation time is about 0-25 minutes.
In embodiments, an incubation time is about 0-20 minutes. In
embodiments, an incubation time is about 0-15 minutes. In
embodiments, an incubation time is about 0-10 minutes. In
embodiments, an incubation time is about 10-30 minutes. In
embodiments, an incubation time is about 5-25 minutes. In
embodiments, liposomes are suspended in aqueous buffer,
pH.about.7.4
[0156] Lyophilization of water solution of doxorubicin in presence
of lactose and/or mannitol resulted in lyophilized material that is
readily reconstitutable in sterile water for injection at room
temperature to the final concentration 6 mg/mL. Mixing of
lyophilized and reconstituted doxorubicin of the present
disclosures with oxalate and -tartrate containing liposomes results
in efficient and rapid encapsulation of the doxorubicin.
EXAMPLES
[0157] In embodiments, the liposomal compositions of the present
disclosures have unique biological performance. Upon
administration, these particles may not be recognized as foreign,
e.g., they are not labeled with proteins which trigger clearance
processes in the tissues of the reticulo-endothelial system. In
embodiments, the liposomal compositions are coated with a component
that inhibits opsonization and phagocytosis. Furthermore, the
liposomal compositions may allow for optimized drug release under
certain conditions, e.g., pH dependent release.
[0158] In embodiments, pH dependent drug release profile may be
optimized via selection of proper counter ions, lipid composition,
and fine tuning lipid/drug ratio in consideration of systemic and
tumor biology (FIG. 1). For example, the following principles may
be considered during parameter optimization:
[0159] While in systemic circulation--restricting drug release at
neutral pH (pH of blood is 7.4) (FIG. 1A).
[0160] Upon accumulation at the tumor site--propelling drug release
at more acidic local extracellular space (FIG. 1B). Tumors may have
an acidic local environment (.about.pH 6.5-7.2) compare to the
blood [1, 40-42]. Moreover, poor vasculature of the tumor may
result in preferred accumulation of liposomal carrier of the drug.
Thus, both accumulation of liposomes and more acidic local
environment may propel local drug release in extracellular space of
the tumor site.
[0161] Upon internalization by the cancerous cells--maximal release
of the drug during liposome residency at more acidic pH (e.g.
6-6.5) of the endosomal environment (FIG. 1C) thereby requiring
less drug to be trapped in lysosomes (FIG. 1 D) [50-51]. Local
accumulation/entrapment of liposomal drug carrier at the tumor site
may also result in enhanced internalization of the liposomes by the
cancerous cells. Upon internalization and entering acidic endosomal
environment (.about.pH 6.0-6.5) liposomes may readily release the
drug, and therefore significantly improve its cytoplasmic
bioavailability.
[0162] Thus the desirable drug release profile (FIG. 1E) would
facilitate release of the majority of encapsulated drug at pH range
from 6.9 to 6.0 representing the local tumor and endosomal
environment at least in some embodiments.
Example 1: Materials and Methods: Doxorubicin
[0163] HPLC Quantification of Doxorubicin
[0164] All HPLC was performed using an Agilent 1260 Infinity
system, equipped with a G13110B pump, G1329B autosampler, G1316A
column compartment, and a G1315D diode-array detector. OpenLab CDS
(EZChrom edition) software controlled all modules and was used for
analysis and reporting of chromatography. A Phenomenex Luna C18
column (5.mu., 150.times.4.6 mm; part #00G-4252-E0) was used for
all analyses.
[0165] Sodium acetate was cGMP grade from Macco Organiques Inc.
(Valleyfield, P.Q., Canada) and hydrochloric acid (used to adjust
pH) was ACS grade from EMD Millipore (Billerica, Mass.). All water
used was purified.
[0166] Chromatographic analysis of doxorubicin (DOX) was performed
on the Agilent 1260 Infinity system using a C18 column (see above)
with a column temperature of 40.degree. C. and sample temperature
at ambient conditions (.about.25.degree. C.). All mobile phase
reagents were filtered with a 0.45 .mu.m filter membrane prior to
use. HPLC grade acetonitrile was from EMD Millipore. An isocratic
mobile phase containing 0.05 M sodium acetate (pH 4.0) and
acetonitrile (72:28, v/v) was used. Mobile phase flow rate was set
to 1.0 mL/min with a run time of 15 minutes. The diode array
detector was operated at 487 nm with a bandwidth of 4 nm. Injection
volume was set to 10 .mu.L.
[0167] Standard stock solution of doxorubicin was prepared in a
0.9% saline, or methanol, or water solution (1 mg/mL). Calibration
standards were prepared by diluting the stock solution in anhydrous
methanol to bracket the target concentration for analysis. For this
study, the doxorubicin solution was diluted with anhydrous methanol
or IPA to the final concentration 50 .mu.g/mL, 100 .mu.g/mL and 200
.mu.g/mL; respectively. Samples of liposomal suspension were also
diluted with anhydrous methanol or IPA by a factor of 8 or 10 times
prior to analysis.
[0168] Fluorometry
[0169] All analyses were performed using a Molecular Devices
SpectraMax Gemini EM Fluorescence Plate Reader. SoftMax Pro
software controlled the device and was used for analysis and
reporting of values.
[0170] Standard stock solution of doxorubicin hydrochloride was
prepared in a 0.9% saline solution (6 mg/mL). Calibration standards
were prepared by diluting the stock solution in phosphate buffered
saline, pH 7.4 and 5.0 to bracket the target concentration for
analysis. The plate reader temperature was set to 25.degree. C.,
and excitation and emission wavelengths were set at 478 nm and 594
nm, respectively. The linear response range was determined to be
0.5-4 .mu.g/mL of doxorubicin hydrochloride. To remain in the
linear response range, the doxorubicin hydrochloride calibration
standards and samples were diluted accordingly.
[0171] To determine total content of doxorubicin in liposomal
formulation (Ft), the liposomes were ruptured by addition of Triton
X-100 to the final concentration 1%, mixed by inversion, and
incubated for prior to quantification.
[0172] To determine free doxorubicin the liposomal formulation was
loaded into an ultrafiltration unit (Pierce concentrator,
ThermoScientific, Rockford, Ill.) with a molecular weight cutoff of
100,000 D. After centrifugation at 2500 rpm for 1 to 2 hours, the
filtrate was analyzed using the SpectraMax Gemini EM Fluorescence
plate reader and quantified.
[0173] To determine fluorescence of intraliposomal content of
doxorubicin the liposomal formulation was subjected to fluorometric
analysis without pretreatment with Triton X-100.
[0174] Quantification of Doxorubicin Release from Liposomal
Formulations
[0175] The method of Lee et al. [20], which employs a fluorescence
dequenching technique and relays it to 100% fluorescence (liposomes
ruptured with Triton X-100) has been used for determination of
doxorubicin release. This approach is based on the fact that
fluorescence of doxorubicin is quenched upon encapsulation into
liposomes and markedly increases upon doxorubicin release from
liposomes. Therefore, increase of fluorescence of intact liposomes
(Fi) during the incubation of sample in dissolution media
represents release of doxorubicin into the media. The difference
between Fi values at different time points and T0 relayed to Ft
(100% fluorescence of ruptured liposomes), and represents percent
of released drug.
[0176] The study was carried out at 25.degree. C. and 37.degree. C.
(to mimic in vivo conditions) at the following time points: T0, T2
hrs, T4 hrs, and T8 hrs. Individual samples were diluted in up to 4
separate diluents/dissolution medias; PBS pH 7.4 and/or PBS/pH 6.7
and/or PBS pH 6.0 and/or PBS pH 5.0 by a factor of 20 times (e.g.
100 .mu.L of sample+1.9 mL of diluent), or 50.times. (e.g. 50 .mu.L
of sample+2.45 mL of diluent). For T0 time point determination,
liposomal formulations were diluted in PBS pH 7.4 and/or pH 6.7
and/or pH 6.0 and/or pH 5.0 buffers at .about.25.degree. C. The
fluorescence of intact liposomes (Fi) and total fluorescence of
liposomes ruptured with Triton X-100 (Ft) were measured
immediately. The plate reader temperature was set to 25.degree. C.
and excitation and emission wavelengths were set at 478 nm and 594
nm, respectively.
[0177] Other liposomal samples were diluted 20.times. or 50.times.
in PBS pH 7.4, 6.7, 6.0, and pH 5.0 buffers pre-warmed to
37.degree. C. (to simulate in vivo temperature) and incubated for
2, 4, and 8 hrs at 37.degree. C.
[0178] Other liposomal samples were diluted 20.times. or 50.times.
in human serum or human blood pre-warmed to 37.degree. C. (to
simulate in vivo temperature) and incubated for 2, 4, and 8 hrs at
37.degree. C. At each time point fluorescence of intact liposomes
(Fi) and total fluorescence of liposomes ruptured with Triton X-100
(Ft) was measured. The percent of drug release was quantified as
[(Fi_n-Fi_t0)/Ft_avrg)]*100%, where Fi_n-Fi measured at 2, 4, or 8
hrs, Fi_t0-Fi measured at T0, and Ft_avrg-average of Ft values
determined for all time points. It is worth mentioning that there
was no significant change of Ft values observed at different time
points and pH.
[0179] Particle Size Determination
[0180] All analyses were performed using a Malvern Zetasizer Nano
ZS with 4 mW He--Ne laser operating at a wavelength of 633 nm and a
detection angle of 173.degree.. Zetasizer software controlled the
device and was used for analysis and reporting of values.
[0181] Particle size distribution by Intensity. The
intensity-averaged particle diameters (Z-average) were calculated
from the cumulants analysis as defined in ISO 13321 (International
Organization for Standardization 1996).
[0182] Particle size distribution by Number. In this distribution,
there is first-power relationship between particle size and
contribution to the distribution. Particle size distribution by
Number is computed from the intensity distribution and the optical
properties of the material. Typical, high-quality DLS results
usually see a decrease in diameter when going from Intensity Mean
to Number Mean values [35].
[0183] In general, the intensity based Z-Average and Intensity
values are larger than a diameters obtained from transmission
electron microscopy (TEM) because of a) sixth power relationship
between light scattering intensity and particle diameter, the
larger particles dominate the signal, and b) DLS measures the
hydrodynamic diameter (i.e. diameter of the particle plus ligands,
ions or molecules that are associated with the surface of the
particle) so the particle appear larger to the instrument in
comparison to TEM
[0184] Samples are prepared using 30 .mu.L of liposomal formulation
in 1.5 mL of phosphate buffered saline (pH 7.4) and were
equilibrated to 25.degree. C. prior to analysis. Size measurements
were done in triplicates for each sample.
[0185] Cryo-Transmission Electron Microscopy Analysis of
Doxorubicin Containing Liposomes.
[0186] Copper 400 mesh+carbon film" grids (EMS) were glow
discharged using an EMS100x glow discharge unit. Three microliter
of a sample diluted 15.times. in the provided buffer were applied
on a glow discharged grid and subsequently plunge-frozen in liquid
ethane using a Vitrobot.TM. Mark II (FEI) and then stored in liquid
nitrogen. The grids were imaged using a FEI CM200 field emission
gun transmission electron microscope at an accelerating voltage of
200 kV. The grid was thoughtfully observed and representative
images were acquired at magnification of 15 k.times., 27.5
k.times., 38 k.times., 50 k.times. and 66 k.times. using a TVIPS
F224 2 k.times.2 k detector.
[0187] pH Measurements.
[0188] All analyses were performed using a Mettler Toledo
SevenCompact pH meter with a Mettler Toledo InLab pH
microelectrode.
[0189] Coarse Suspension Preparation.
[0190] Coarse suspension was prepared by dissolving PC, DMPC, FC,
and P188 in 10 mL of DCM at the ratios indicated in Table 6. The
mixture was dried under the stream of nitrogen until viscous film
was formed. The film was further dried in vacuum oven overnight.
Next day dried lipid film was hydrated with 300 mM solution of the
following ammonium salts: ammonium-oxalate, or ammonium-sulfate, or
ammonium-picolinate, or ammonium-phosphate, or ammonium-citrate, or
ammonium-acetate, or ammonium-formate pre-warmed to 65.degree. C.,
and immediately homogenized with a hand-held homogenizer for 2-3
min. Particle size of coarse suspension was determined and always
was in the range of 800-1200 nm. Maleic acid, cysteine, NAC,
ascorbic acid, malonic acid, tartaric acid, fumaric acid, or
succinic acid were first titrated with ammonium hydroxide to pH
4.8-5.0 and then used as hydration media.
[0191] Mf Processing.
[0192] MF processing volume was always 100 ml unless specified
differently. MF processing pressure was always 10 KPSI.
Microfluidization of coarse suspension was performed in recycling
mode (return of the material into the feed reservoir) at controlled
(.ltoreq.65.degree. C.) temperature. Processing time was in 10-16
min range. The target particle size (Z-average) was 60-70 nm.
[0193] Tangential Flow Filtration
[0194] Translucent nanosuspension was harvested from microfluidizer
and subjected to tangential flow filtration (TFF) with 15-20.times.
volumes of PBS, pH 7.4. The purpose of TFF was to replace external
buffers encompassing ammonium-oxalate, or ammonium-sulfate, or
ammonium-phosphate, or ammonium-tartrate, or ammonium-citrate, or
maleic acid, cysteine, NAC, ascorbic acid, malonic acid, tartaric
acid, fumaric acid, or succinic acid titrated with ammonium
hydroxide to pH 4.8-5.0 with PBS, and to majorly remove ammonium
from external buffer and intraliposomal space. Ammonium in external
buffer was measured by using ammonium specific electrode. TFF was
stopped when ammonium concentration in external buffer was
.ltoreq.3 mM.
[0195] Remote Loading of Doxorubicin
[0196] Doxorubicin hydrochloride was dissolved in saline to the
final concentration 6 mg/mL. Saline solution of doxorubicin was
added to the liposomal nanosuspension in PBS, pH 7.4 to the final
concentration 1 mg/mL. The mixture was heated to 70.degree. C.
After 30 min of incubation at 70.degree. C. the mixture was allowed
to cool down to ambient temperature (.about.25.degree. C.) and
subjected to another TFF 5.times. cycle with PBS pH 7.4 containing
6% of sucrose.
[0197] The cold loading of doxorubicin into liposomes was performed
as follows: saline solution of doxorubicin was added to the
liposomal nanosuspension at room temperature to final concentration
1 mg/mL, gently inverted (2-3 times) and incubated at room
temperature for 10 min. After 10 min of incubation at room
temperature the mixture was: a) subjected to another TFF 5.times.
cycle with PBS pH 7.4 containing 6% of Sucrose, and/or b) placed in
2-8.degree. C. refrigerator for 16 hrs, and then was sterile
filtered or subjected to another TFF 5.times. cycle with PBS pH 7.4
containing 6% of Sucrose.
[0198] Following cold loading, liposomal nanosuspension was sterile
filtered into sterile Nalgene flask via 0.22 um filter. Particle
size, pH, HPLC content of doxorubicin, Fi, Ft, free doxorubicin,
and doxorubicin release profile were determined. The sterile
nanosuspension was aseptically dispensed into 2 mL pre-sterilized
vials, stoppered, and sealed. The vials were stored at 2-8.degree.
C.
[0199] In Vitro Cell-Based Cytotoxicity Assay.
[0200] Daudi cells (ATCC, CCL-213). Cells were plated in 96 well
plates at the density 100K cells per well. Doxorubicin
hydrochloride, DOXIL.RTM., or doxorubicin oxalate liposomes were
appropriately diluted in Growth medium (RPMI-1640, 10% FBS) and
added to the cells. After 1 hr of incubation at 37.degree. C.
plates were centrifuged, supernatant was removed via aspiration,
100 .mu.L of fresh media were added into each well, and cells were
cultured for 48 hrs.
[0201] Hela cells (HeLa (ATCC CCL-2). Cells were plated in 96 well
plates at the density 25K cells per well. Doxorubicin
hydrochloride, DOXIL.RTM., or doxorubicin oxalate liposomes were
appropriately diluted in Growth medium (DMEM, 5% FBS, 1%
antibiotic, 1% HEPES) and added to the cells. After 1 hr of
incubation at 37.degree. C. plates were centrifuged, supernatant
was removed via aspiration, 100 .mu.L of fresh media were added
into each well, and cells were cultured for 48 hrs.
[0202] On the day, 3 to assess cell viability, Alamar Blue solution
was prepared in Growth medium (RPMI-1640, 10% FBS) at 1:250
dilution and added directly into cell media. After 6 hrs of
incubation at 37.degree. C. the resulting fluorescence was read on
a plate reader. The fluorescence of cells that have not received
any drug treatment was defined as maximum viability. The
fluorescence of cell free media was defined as complete cell death.
Data analysis was performed by using Prism nonlinear regression
software (GraphPad Software) for the curve-fitting and
determination of IC50 values.
[0203] In Vivo Study
[0204] Human B lymphoma cell line Ramos (RA 1) (ATCC CRL-1596) were
cultured in RPMI-1640 medium containing 20 mM HEPES, 10% Fetal
bovine serum, 2 mM L-glutamine, and 1 mM sodium pyruvate. Cell
density was maintained in 3.times.10.sup.5-1.5.times.10.sup.6
range, and viability in 90-95% range. On the same day prior to
administration into animals cell were counted, centrifuged at 12000
rpm, washed with sterile PBS, centrifuged again at 1200 rpm,
resuspended in sterile PBS to the final cell count 35.times.10-per
mL. Suspension of cells was administered into animals intravenously
via single bolus injection to deliver 5.times.10.sup.6 cells per
mouse.
[0205] Four weeks old immune deficient SCID Beige,
(Line--CB17.Cg-Prkdc.sup.scidLyst.sup.bg-J/Crl) mice were
maintained on Chow diet. At the age of 8 weeks mice were subdivided
in 3 groups. On the day 1, 5.times.10.sup.6 B lymphoma cells were
intravenously injected into each mouse (FIG. 2). On the day 2 one
group received placebo (doxorubicin free liposomes), and another
two received treatment with DOXIL.RTM. (lot #FAZSR00) or
doxorubicin Oxalate liposomes, respectively. On the day 2 and 3 all
treatments were repeated (FIG. 2). Animals were monitored daily and
weighed twice a week.
[0206] Prior to injection DOXIL.RTM. liposomes, or doxorubicin
Oxalate liposomes were diluted appropriately with PBS to final
concentration 0.5 mg/mL. Treatment articles were administered
intravenously in 120 .mu.L via single bolus injection to deliver 60
.mu.g of doxorubicin per mouse or .about.3 mg/kg dose.
[0207] Materials used are shown in Table 4.
TABLE-US-00004 TABLE 4 List of Materials Vendor Reagents MW Catalog
# Manufacturer Phosphatidyl choline (PC) egg lecithin 770
510800-KG-1 Lipoid (LIPOID EPC S)
1,2-dimyristoyl-sn-glycero-3-phosphocholine 678 850345P Avanti
Polar Lipids (DMPC) DSPE-PEG(2000) Amine 2790 880128P Avanti Polar
Lipids 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-
N-[amino(polyethylene glycol)-2000] (ammonium salt) Kolliphor P 188
(Poloxamer P188) 7680-9510 WPCH537B Mutchler P188/Synperonic PE/F68
7680-9510 ETK1229 Croda Free Cholesterol (FC) 387 A11470 Alfa Aesar
doxorubicin hydrochloride 579.98 7000A002113 Sicor, TEVA* API
Division DOXIL .RTM. (Caelyx) N/A FAZSROO TTY Biopharm Company Ltd,
DOXIL .RTM. (Caelyx) N/A L01DB01 Ben Venue, Ohio, USA Irinotecan
hydrochloride trihydrate 677.18 1-4122 LC Labs Irinotecan
hydrochloride 623.14 11406 Sigma-Aldrich Mitoxantrone
dihydrochloride 516.70 14842 Cayman Chemicals Ammonium Sulfate
132.14 A4418 Sigma-Aldrich Oxalic acid 90.03 241172 Sigma-Aldrich
Ammonium oxalate monohydrate 142 09898 Sigma-Aldrich Ammonium
Phosphate monobasic 115.03 216003 Sigma-Aldrich Ammonium Phosphate
dibasic 132.06 215996 Sigma-Aldrich Ammonium Citrate dibasic 226.18
25102 Sigma-Aldrich Ammonium Citrate tribasic 243.22 A1332
Sigma-Aldrich L - (+) Tartaric acid 150.09 251380 Sigma-Aldrich
Cysteine 121.16 C 7352 Sigma-Aldrich Ammonium Acetate 77.08 AX
1222-5 EMD Picolinic acid 123.11 P 42800 Sigma-Aldrich Malonic acid
104.06 M 1296 Sigma-Aldrich Maleic acid 116.07 M0375 Sigma-Aldrich
Fumaric acid 116.07 47910 Sigma-Aldrich Ammonium Formate solution,
10M 63.06 78314 Sigma-Aldrich Succinic acid 118.09 S3674
Sigma-Aldrich L-Ascorbic acid 176.12 A5960 Sigma-Aldrich Butylated
hydroxytoluene (BHT) 220.36 B1196 Spectrum N-acetyl L cysteine
163.19 A7250 Sigma-Aldrich Ammonium Hydroxide 35.04 AX1303-6 EMD
HCl 36.5 HX0603 EMD Millipore Dichloromethane (DCM) 85 P3813
Sigma-Aldrich Phosphate buffered saline NA P5368-10PAK
Sigma-Aldrich Triton X-100 625 9400 OmniPur Low endotoxin Sucrose
342 1.00892.1003 EMD Millipore Lactose 360.31 Tabletose 80 MEGGE
Pharma Mannitol 182.17 Pearlitol 160C Roquette
Ethylenediaminetetraacetic acid (EDTA) 292.25 PN: 0322 Amresco
Sodium Chloride 58 M-11619 Fisher Scientific L-Ascorbic acid
6-palmitate 414.53 A1968 Sigma-Aldrich CoenzymeQ10 863.34 C9538
Sigma-Aldrich Resazurin Sodium Salt (Alamar Blue) 251.17 R7017
Sigma Aldrich Water for Irrigation NA PN: BMGR5007 B. Braun Water
for Injection NA PN: 2B0309 Baxter 2 mL vials N/A RTF8409 Afton
Scientific Pooled normal Human Serum N/A IPLA-SER Innovative
Research Single Donor Human Whole Blood-Na N/A IPLA-WB1 Innovative
Research EDTA anticoagulant Mice Serum Wild Type N/A IGMS-057-SER
Innovative Research *Drug Master File holder.
[0208] Equipment used is shown in Table 5.
TABLE-US-00005 TABLE 5 List of Equipment Instrument Model
Vendor/Manufacturer High shear lab mixer Silverson L5M-A Silverson
Microfluidizer M 110P Microfluidics Zetasizer Nano ZSP Malvern
Microplate Reader SpectraMax Gemini EM Molecular devices Tangential
Flow Filtration Unit Labscale TFF System w/ MD Millipore Pellicon
XL cassette Repeater Pump BAXA Baxter VWR recirculating water bath
1160 A VWR Scientific Lyophilizer VirTis Genesis SQ25EL VirTis
Analytical balance XS 6002S Mettler Toledo pH meter SevenCompact
Mettler Toledo Eppendorf centrifuge 5417 Eppendorf Handheld pH
meter w/ ammonium SP21 VWR specific electrode Beckman Coulter
Centrifuge Allegra 6R Beckman Coulter HPLC Agilent Technologies
1220 Infinity LC Agilent Vacuum Oven 1430 D VWR Scientific
Microscope Olympus BHA Olympus
[0209] Summary of Abbreviations Used: HPLC--high pressure liquid
chromatography; MFD--manufacturing date; DCM--Dichloromethane;
PC--phosphatidylcholine; FC--free cholesterol; P188--Poloxamer 188;
DMPC--1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine;
MF--microfluidizer; W/V--weight to volume; mfg--manufacturing;
ND--not determined; Fi--fluorescence of intact liposomes loaded
with fluorescent drug; Ft--total fluorescence of the drug derived
from the ruptured liposomes; TFF--tangential flow filtration;
W/W--weight to weight ratio; Lipid to Drug Ratio--W/W Ratio of
(PC+DMPC+FC)/doxorubicin, or irinotecan, or mitoxantrone.
Example 2: Doxorubicin Loading at 70.degree. C.: Comparison of
Different Counter Ions at Fixed 50:1 (i.e. 50 to 1 or 50/1)
Lipid/Drug (i.e. Lipid to Drug) Ratio
[0210] Hydration Media Used:
[0211] a) 300 mM solution of the following ammonium salts:
ammonium-oxalate, or ammonium-sulfate, or ammonium-picolinate, or
ammonium-phosphate, or ammonium-citrate, or ammonium-acetate, or
ammonium-formate.
[0212] b) oxalic acid, maleic acid, cysteine, malonic acid,
tartaric acid, fumaric acid, succinic acid, ascorbic acid, or
N-acetyl L cysteine (NAC) were first titrated with ammonium
hydroxide to pH 4.8-5.0 and then used as hydration media.
[0213] Remote loading was carried out at 70.degree. C. with 1 mg/mL
of doxorubicin hydrochloride (e.g. 0.936 mg of doxorubicin free
base per mL). Hydrochloride. Formulation composition is shown in
Table 6. All formulations were prepared at 50:1 lipid/drug ratio
(Table 6). Lipid/Drug ratio represents weight/weight (w/w) ratio of
total lipids to doxorubicin free base in final suspension of
doxorubicin loaded liposomes.
[0214] The data for picolinate, maleate, cysteinate, malonate,
fumarate, formate, succinate, acetate, ascorbic acid, or NAC are
not shown since no doxorubicin loading was observed and liposomal
material precipitated after overnight storage at 2-8.degree. C.
TABLE-US-00006 TABLE 6 Formulation Composition. Amounts of solids
used in formulations, W/W, % Hydration P Doxorubicin Lot # Media PC
DMPC FC 188 Hydrochloride Lipid/Drug 647-2-106 Ammonium- 65.50
16.38 11.46 4.91 1.75 50 Sulfate 647-2-121 A Ammonium- 65.50 16.38
11.46 4.91 1.75 50 Oxalate 647-2-145 B Oxalic 65.50 16.38 11.46
4.91 1.75 50 acid + NH.sub.4OH to pH 4.8-5.0 647-2-144 B Ammonium-
65.50 16.38 11.46 4.91 1.75 50 Phosphate 647-2-151 B Tartaric 65.50
16.38 11.46 4.91 1.75 50 acid + NH.sub.4OH to pH 4.8-5.0 647-2-105
B Ammonium- 65.50 16.38 11.46 4.91 1.75 50 Citrate
[0215] Coarse suspension was prepared and MF processed. After 9-12
min of MF processing the particle size (Z-average) reached
.about.60-75 nm. A sample was collected and sterile filtered into
Nalgene flask. The particle size of filtered nanosuspension was
determined (Table 7).
TABLE-US-00007 TABLE 7 Summary of MF processing and resultant
emulsion parameters. Particle Processing size Pressure, Z avrg, Lot
# Counter Ion MFD KPSI nm 647-2-106 Sulfate 28 Mar. 2016 10 60
647-2-121 A Oxalate 12 Apr. 2016 10 59 647-2-145 B Oxalate 09 May
2016 10 63 647-2-144 B Phosphate 06 May 2016 10 60 647-2-151 B
Tartrate 05 May 2016 10 65 647-2-105 B Citrate 03 May 2016 10
60
[0216] The liposomes were subjected to TFF followed by remote
loading with doxorubicin, and another TFF cycle with PBS sucrose.
Doxorubicin hydrochloride concentration used for remote loading:
1.0 mg/mL (doxorubicin free base concentration: 0.936 mg/mL).
[0217] The particle size of doxorubicin loaded liposomes is
presented in Table 8.
TABLE-US-00008 TABLE 8 Particle size of doxorubicin loaded
liposomes. Lot # Counter Ion Loading Date Particle size (Z avrg,
nm) 647-2-106 Sulfate 31 Mar. 2016 66 647-2-121 A Oxalate 12 Apr.
2016 68 647-2-145 B Oxalate 09 May 2016 72 647-2-144 B Phosphate 06
May 2016 70 647-2-151 B Tartrate 18 May 2016 70 647-2-105 B Citrate
03 May 2016 72
[0218] Determination of doxorubicin in liposomal suspension.
Followed doxorubicin loading liposomal suspension was subjected to
5.times.TFF to majorly remove free (not encapsulated) doxorubicin.
To determine total doxorubicin concentration at T0 (within one week
of MFD) TFF washed liposomes were diluted with methanol or IPA and
subjected to HPLC analysis. Doxorubicin content, percent of
recovery (doxorubicin content in liposomal suspension relative to
doxorubicin free base concentration used for remote loading), and
encapsulation efficiency (%) are presented in the Table 9.
Encapsulation efficiency (%) represents the difference between
doxorubicin recovery (%) and free doxorubicin (%).
TABLE-US-00009 TABLE 9 Total doxorubicin content and Encapsulation
efficiency. Doxorubicin Assay, HPLC Encapsulated free base used
Doxorubicin content doxorubicin, % for loading, (Liposomal
Suspesion) Recovery, [Recovery, %] - Lot # Counter .mu.g/mL
.mu.g/mL % [Free, %] 647-2-106 Ion 936 936 100 100 647-2-121 A
Oxalate 936 841 90 100 647-2-145 B Oxalate 936 792 85 100 647-2-144
B Phosphate 936 795 85 84 647-2-151 B Tartrate 936 814 87 87
647-2-105 B Citrate 936 750 80 79
[0219] The amount of free (not encapsulated) doxorubicin was
determined within one week of manufacturing (Table 10).
TABLE-US-00010 TABLE 10 Free doxorubicin content. Lot # Counter Ion
% of Total 647-2-106 Sulfate 0.02 647-2-121 A Oxalate 0.13
647-2-145 B Oxalate 0.01 647-2-144 B Phosphate 0.46 647-2-151 B
Tartrate 0.03 647-2-105 B Citrate 0.90
[0220] The change of free (not encapsulated) doxorubicin content
during the storage at 2-8.degree. C. is presented in the Table
11.
TABLE-US-00011 TABLE 11 Change of Free doxorubicin content during
the storage at 2-8.degree. C. Lot # Counter Ion Days past T0 % of
Total 647-2-106 Sulfate 0 0.02 62 0.03 647-2-121 A Oxalate 0 0.13
45 0.3 647-2-145 B Oxalate 0 0.01 36 0.19 647-2-144 B Phosphate 0
0.46 26 0.25 647-2-151B Tartrate 0 0.03 26 0.1 647-2-105 B Citrate
0 0.90 34 1.10
[0221] Liposomal doxorubicin release studies were carried out at
25.degree. C. (Table 12) and 37.degree. C. (Table 13). For each
sample doxorubicin release was determined at 2, 4 and 8 hrs time
points.
TABLE-US-00012 TABLE 12 Doxorubicin release rate determined at
25.degree. C. Counter pH 5, Release, % pH 7.4, Release, % Lot # Ion
Pka1 2 hrs 4 hrs 8 hrs 2 hrs 4 hrs 8 hrs 647-2-106 Sulfate -3 1 0 1
0 0 1 647-2-121 A Oxalate 1.27 2 5 7 0 0 0 647-2-144 B Phosphate
1.96 2 3 4 0 1 0 647-2-151 B Tartrate 3.03 0 4 4 0 0 0 647-2-105 B
Citrate 3.13 3 4 6 0 0 0
TABLE-US-00013 TABLE 13 Doxorubicin release rate determined at
37.degree. C. Counter pH 5, Release, % pH 7.4, Release, % Lot # Ion
Pka1 2 hrs 4 hrs 8 hrs 2 hrs 4 hrs 8 hrs DOXIL .RTM. Sulfate -3 0.9
1.1 1.4 0.9 1.1 1.1 647-2-106 Sulfate -3 0.1 2.7 2.4 0.01 0.6 0.1
647-2-121 A Oxalate 1.27 47 55 73 2 2 5 647-2-145 B Oxalate 1.27 41
58 70 1 4 5 647-2-144 B Phosphate 1.96 18 20 22 0 0 1 647-2-151 B
Tartrate 3.03 14 27 39 0 1 1 647-2-105 B Citrate 3.13 14 19 24 0 0
0
[0222] Doxorubicin release rate (at 37.degree. C.) was also
monitored during storage of samples at 2-8.degree. C. conditions
(Table 14).
TABLE-US-00014 TABLE 14 Doxorubicin release rate at 37.degree. C.
Effect of the storage at 2-8.degree. C. Counter Days pH 5, Release,
% pH 7.4, Release, % Lot # Ion past T0 2 hrs 4 hrs 8 hrs 2 hrs 4
hrs 8 hrs 647-2- Sulfate 0 0.1 2.7 2.4 0 0.6 0.1 106 62 1 2 2 0 0 1
647-2- Oxalate 0 47 55 73 2 2 4 121 A 45 41 56 72 4 6 5 647-2-
Oxalate 0 41 58 70 1 4 5 145 B 36 30 50 68 1 4 6 647-2- Phosphate 0
18 20 22 0 0 1 144 B 38 16 17 19 1 1 3 647-2- Tartrate 0 14 27 39 0
1 1 151 B 26 15 24 35 1 1 2 647-2- Citrate 0 14 19 24 0 0 0 105 B
59 22 27 31 0 0 0
[0223] Particle size. It can be seen from the Tables 7 that
microfluidization of different liposomal formulation resulted in
similar particle sizes. Doxorubicin loading resulted in slight
increase of particle size of all formulations (Table 8).
[0224] Efficiency of doxorubicin encapsulation varied from 79% to
100%. The highest encapsulation 100% was observed when sulfate was
used as a counter ion (Table 9).
[0225] Free doxorubicin reflects concentration of not encapsulated
drug determined (within one week after manufacturing (T0), and
during the storage of liposomal material at 2-8.degree. C. It can
be seen from the Table 10 that free doxorubicin content--at T0 was
in the range from 0.02-0.9%. There was no significant change in
concentration of free doxorubicin observed over at least one month
of storage at 2-8.degree. C. (Table 11).
[0226] Liposomal doxorubicin release rate. Drug release studies
were carried out at 25.degree. C. and 37.degree. C.
[0227] At 25.degree. C. all formulations (e.g. with sulfate,
oxalate, phosphate, tartrate, and citrate) demonstrated very low
and similar release rate (Table 12) with .DELTA.pH7.4/5.0 release
differential close to zero.
[0228] At 37.degree. C., when oxalate or tartrate were used as a
counter ions the difference between doxorubicin release at pH7.4
and pH 5.0 (.DELTA.pH7.4/5.0 release differential) was markedly
higher compare to other used counter ions (Table 13 and FIG.
3).
[0229] It is worth mentioning that regardless of whether
ammonium-oxalate salt or oxalic acid (titrated to pH 4.8-5.0 with
NH.sub.4OH) were used to prepare hydration media, the particle size
of empty or doxorubicin loaded liposomes (Tables 7-8), efficiency
of doxorubicin encapsulation (Table 9), and release profile (Tables
13-14) were essentially the same.
[0230] The 37.degree. C. release rate and extent of
.DELTA.pH7.4/5.0 differential observed at T0 (within one week after
MFD) were sustained during the storage at 2-8.degree. C. for at
least .about.two months (Table 14).
[0231] The poor doxorubicin release rates observed for all tested
counter is at 25.degree. C. (Table 12) and dramatic increase of
doxorubicin release at 37.degree. C. observed with oxalate or
tartrate compared to sulfate, phosphate, and citrate (Table 13)
suggests uniqueness of physical state(s) of doxorubicin-oxalate or
-tartrate aggregates at 37.degree. C. that may facilitate their
dissolution, and therefore doxorubicin release. The observed
difference in .DELTA.pH7.4/5.0 release differential determined for
specified counter ions at 25.degree. C. and 37.degree. C. might
also indicates on more profound temperature dependent transition of
the physical state of doxorubicin-oxalate or -tartrate
intraliposomal aggregates compared to -sulfate or -phosphate in
some embodiments.
[0232] In embodiments, doxorubicin forms aggregates when
encapsulated in liposomes in response to a pH gradient and
counter-ions, an observation that has been confirmed by several
research groups [1, 13, 15, 23-25]. The physicochemical properties
of the counter ions (e.g., oxalate, sulfate, phosphate, tartrate,
and citrate) such as size, pKa values, stereochemistry, dipole
moment, polarizability, etc. may interplay to generate different
precipitated structures, and therefore control release of
doxorubicin from the liposomes.
[0233] Andreas Fritze, et all [13] used Cryotransmission electron
microscopy (C-TEM) to visualize doxorubicin loaded liposomes
prepared in 300 mM (NH.sub.4).sub.2HPO.sub.4 solution. As shown in
FIG. 4, entrapped and precipitated doxorubicin forms bundles appear
as linear structures and induce a change in liposomal shape,
resulting in a characteristic "coffee bean"-structure. It was
demonstrated that doxorubicin release into pH 7.4 buffer from
liposomes containing doxorubicin phosphate bundles was <2-3% at
1 hr and at 25.degree. C. [13].
[0234] Xingong Li, et all [15] have examined doxorubicin's (DOX)
physical state in solution and inside EPC/cholesterol liposomes
that were loaded via a transmembrane pH gradient. Using cryogenic
electron microscopy (cryo-EM) they noted that doxorubicin loaded to
200-300 mM internal concentrations in citrate containing liposomes
formed linear, curved, and circular bundles of fibers with no
significant interaction/perturbation of the vesicle membrane [15]
(FIG. 5). It was also demonstrated that doxorubicin release into pH
7.6 buffer from liposomes containing doxorubicin citrate fibers was
relatively slow (.about.4% at 1 hr).
[0235] Doxorubicin aggregates in the presence of sulfate typically
have rigid linear fiber bundles (interfiber spacing is
approximately 27 A.degree.) compared with the doxorubicin-citrate
aggregates in the presence of citrate, which appear mostly linear
or curved (interfiber spacing is approximately 30-35 A.degree.) [1,
15, 23-25] (FIG. 6). These results suggest that the sulfate anion,
being smaller than the citrate anion, may allow a tighter packing
arrangement, resulting in a decreased flexibility of fiber bundles
and therefore lower rate of drug release from liposomes.
[0236] Cryo-TEM analysis of doxorubicin-oxalate-containing
liposomes (lot #647-2-157). The doxorubicin-oxalate-containing
liposomes (lot #647-2-157) were characterized by cryo transmission
electron microscopy (cryo-TEM). Cryo-TEM analysis revealed
well-defined dense liposomal particles of spherical morphology
(FIGS. 7 A, 7B, and 7C) with a well-define bilayer membrane (5-6 nm
thickness, FIG. 7C) and a minute internal density of the liposomes
that is in line with high (50:1) drug to lipid ratio. It is also
worth mentioning that free (not encapsulated) doxorubicin
determined by ultrafiltration method was .about.0.3% (Table
21).
[0237] Even though the particles appear densely packed on the
support, the on-grid spreading of the sample was relatively even
and no particle clustering, nor aggregation was observed. At higher
magnification, the membrane bilayer can be clearly distinguished
(FIGS. 7B and 7C). The vast majority (97%) of the particles was
unilamellar.
[0238] In embodiments, doxorubicin-oxalate aggregates appeared to
have non-crystalline nature (FIGS. 7A-7C) and did not form tightly
packed bundles observed when sulfate or phosphate was used as a
counter ions (FIGS. 4-6). This finding signifies unique physical
state of the intraliposomal doxorubicin-oxalate aggregates compared
to doxorubicin-sulfate and -phosphate aggregates, and is in a good
agreement with observed difference in drug release profiles (FIG.
3, Tables 13-14).
[0239] Overall obtained data demonstrate efficient loading of
doxorubicin and formation of stable liposomal formulations when
sulfate, oxalate, phosphate, tartrate, and citrate were used as
counter ions. Although all formulations similarly released
doxorubicin at 25.degree. C., oxalate and tartrate showed desirable
.DELTA.pH7.4/5.0 release differential when doxorubicin release rate
was determined at 37.degree. C. This data indicate that temperature
dependent physical state transition of doxorubicin-oxalate or
-tartrate aggregates may be more extensive compared to
doxorubicin-sulfate or -phosphate aggregates.
[0240] In embodiments, not to be bound by theory, although pKa
values of the counter-ions determine response to the change of
external pH, pKa may be important not just for liposomal
doxorubicin release on molecular level (when in solution), but
combined with other physical-chemical properties of the
counter-ions (e.g. size, stereochemistry, dipole moment,
polarizability, etc.) are also important in controlling
intraliposomal doxorubicin packaging, physical state of formed
aggregates, and therefore their dissolution rate.
[0241] Thus, the effect of counter ion for optimal pH dependent
drug release was demonstrated, and performed studies strongly
suggest that optimal counter ions are oxalate and tartrate at least
in some embodiments.
[0242] In some alternative embodiments, citrate can be used as a
counter ion.
Example 3: Further Characterization of Doxorubicin-Oxalate
Containing Liposomes. Variable Lipid/Drug Ratios
[0243] Hydration media: 300 mM Ammonium-oxalate.
[0244] Liposomes were prepared at different lipid/drug ratios
(5:1-100:1) and various concentrations of P188 (Table 15). Final
doxorubicin hydrochloride concentrations used for remote loading
were 0.5 or 1.0 mg/mL Remote loading was performed at 70.degree.
C.
TABLE-US-00015 TABLE 15 Formulation composition. Amounts of solids
used in formulations, W/W, % Ratios Doxorubicin Lipid/ Lot # PC
DMPC FC P 188 Hydrochloride Drug 647-1-175 65.50 16.38 11.46 4.91
1.75 50 647-1-174 66.05 16.51 11.56 4.95 0.88 100 647-2-13 63.94
15.98 11.19 7.99 0.85 100 647-2-48 65.50 16.38 11.46 4.91 1.75 50
647-2-121 A 65.50 16.38 11.46 4.91 1.75 50 647-2-157 65.50 16.38
11.46 4.91 1.75 50 647-2-159 B 68.89 17.22 12.06 0.00 1.84 50
647-2-99 A 61.72 15.43 10.80 7.71 4.34 20 647-2-99 B 54.61 13.65
9.56 6.83 15.35 5
[0245] Coarse suspension was prepared and MF processed at 10 KPSI
processing pressure. After 9-15 min of MF processing the particle
size (Z-average) reached .about.60-65 nm. A sample was collected
and sterile filtered into Nalgene flask. The particle size of
filtered nanosuspension was determined (Table 16).
TABLE-US-00016 TABLE 16 Summary of MF processing and resultant
emulsion parameters. Particle size Lot # MFD Z avrg, nm 647-1-175
15 JUL 15 62 647-1-174 15 JUL 15 62 647-2-13 08 OCT 15 63 647-2-48
20 JAN 16 63 647-2-121 A 12 APR 16 60 647-2-157 24 MAY 16 65
647-2-159 B 02 JUN 16 65 647-2-99 A 22 MAR 16 64 647-2-99B 23 MAR
16 61
[0246] The liposomes were subjected to TFF followed by remote
loading with doxorubicin, and another TFF cycle with PBS sucrose.
Doxorubicin hydrochloride concentration used for remote loading:
0.5 or 1.0 mg/mL.
[0247] The particle size of doxorubicin loaded liposomes is
presented in Table 17.
TABLE-US-00017 TABLE 17 Particle size of doxorubicin loaded
liposomes. Particle size Lot # Lipid/Drug Loading Date Z avrg, nm
647-1-175 50 21 JUL 15 66 647-1-174 100 22 JUL 15 66 647-2-13 100
09 OCT 15 70 647-2-48 50 21 JAN 16 73 647-2-121A 50 12 APR 16 68
647-2-157 50 24 MAY 16 73 647-2-159B 50 02 JUN 16 67 647-2-99 A 20
22 MAR 16 81 647-2-99 B 5 23 MAR 16 80
[0248] Particle size stability data are presented in Tables 18.
TABLE-US-00018 TABLE 18 Particle size of doxorubicin loaded
liposomes. Stability at 2-8.degree. C. Stability Days past Particle
size Lot# Lipid/Drug T0 Z avrg, nm 647-1-175 50 0 66 41 67 113 67
647-1-174 100 0 66 42 67 114 68 647-2-13 100 0 70 34 71 114 72
647-2-48 50 0 73 82 74 647-2-121A 50 0 68 45 69 647-2-99 A 20 0 81
7 81 65 83 647-2-99 B 5 0 80 7 80 65 84
[0249] Determination of doxorubicin in liposomal suspension.
Followed doxorubicin loading liposomal suspension was subjected to
5.times.TFF to majorly remove free (not encapsulated) doxorubicin.
To determine total doxorubicin concentration at T0 (within one week
of MFD) TFF washed liposomes were diluted with methanol or IPA and
subjected to HPLC analysis. Doxorubicin content, percent of
recovery (doxorubicin content in liposomal suspension relative to
doxorubicin free base concentration used for remote loading), and
encapsulation efficiency (%) are presented in the Table 19.
Encapsulation efficiency (%) represents the difference between
doxorubicin recovery (%) and free doxorubicin (%).
TABLE-US-00019 TABLE 19 Total doxorubicin content and Encapsulation
efficiency. Assay, HPLC Encapsulated Doxorubicin Doxorubicin
doxorubicin, free base content % used (Liposomal Re- [Recovery,
Lipid/ for loading, Suspesion) covery, %]- Lot# Drug .mu.g/mL
.mu.g/mL % [Free, %] 647-1-175 50 936 839 90 90 647-1-174 100 468
436 93 93 647-2-13 100 468 461 98 98 647-2-48 50 936 899 95 95
647-2-121A 50 936 841 90 90 647-2-157 50 936 861 92 92 647-2-159 B
50 936 920 98 98 647-2-99 A 20 936 526 56 52 647-2-99 B 5 936 384
41 31
[0250] Stability of liposomal doxorubicin was assessed during the
storage at 2-8.degree. C. The percent of recovery during the
storage relative to initial doxorubicin content determined at T0 is
shown in the Table 20.
TABLE-US-00020 TABLE 20 Stability of Encapsulated doxorubicin.
Storage conditions: 2-8.degree. C. Lot# Lipid/Drug Days past T0
Content, .mu.g/m Recovery, % 647-1-175 50 41 861 102 647-1-174 100
42 452 103 647-2-13 100 118 461 100 647-2-48 50 84 812 91
647-2-121A 50 45 799 95
[0251] The amount of free doxorubicin was determined within one
week of manufacturing (Table 21).
TABLE-US-00021 TABLE 21 Free doxorubicin content. Lot# Lipid/Drug %
of Total 647-1-175 50 0.38 647-1-174 100 0.20 647-2-13 100 0.35
647-2-48 50 0.41 647-2-121A 50 0.13 647-2-157 50 0.29 647-2-159 B
50 0.02 647-2-99 A 20 4 647-2-99 B 5 10
[0252] The change of free doxorubicin content during the storage at
2-8.degree. C. is presented in the Table 22.
TABLE-US-00022 TABLE 22 Change of Free doxorubicin content during
the storage at 2-8.degree. C. Lot# Lipid/Drug Days past T0 % of
Total 647-1-175 50 0 0.38 41 0.49 114 0.74 647-1-174 100 0 0.20 41
0.34 114 0.64 647-2-13 100 0 0.35 38 0.5 124 0.5 647-2-48 50 0 0.41
82 0.9 647-2-121A 50 0 0.13 45 0.69 647-2-99 A 20 0 4 7 5 647-2-99
B 5 0 10 7 15
[0253] Liposomal doxorubicin release studies were carried out at
37.degree. C. within one week after manufacturing (Table 23). For
each sample doxorubicin release was determined at 2, 4 and 8 hrs
time points.
TABLE-US-00023 TABLE 23 Doxorubicin release rate determined at TO
(within one week after manufacturing). Lipid/ pH 5, Release, % pH
7.4, Release, % Lot # Drug 2 hrs 4 hrs 8 hrs 2 hrs 4 hrs 8 hrs
647-1-175 50 55 65 81 2 1 5 647-1-174 100 38 57 81 2 3 7 647-2-13
100 ND* ND* ND* ND* ND* ND* 647-2-48 50 50 56 75 0 0 0 647-2-121 A
50 47 55 73 2 2 5 647-2-157 50 43 56 73 0 3 5 647-2-159 B 50 50 65
78 2 7 7 647-2-99 A 20 22 35 53 14 27 35 647-2-99 B 5 27 43 64 12
20 42 ND* -- not done
[0254] Liposomal doxorubicin release rate (at 37.degree. C.) was
also monitored during the further storage of samples at 2-8.degree.
C. conditions (Table 23a).
TABLE-US-00024 TABLE 23a Change of doxorubicin release rate during
the storage at 2-8.degree. C. Lipid/ Days pH 5, Release, % pH 7.4,
Release, % Lot # Drug past T0 2 hrs 4 hrs 8 hrs 2 hrs 4 hrs 8 hrs
647-1-175 50 0 55 65 81 2 1 5 41 66 75 96 4 5 7 114 65 72 84 1 3 3
647-1-174 100 0 38 57 81 2 3 7 41 42 60 75 3 5 10 114 28 45 62 0 2
5 647-2-13 100 0 ND* ND* ND* ND* ND* ND* 38 29 34 54 0 0 3 119 40
42 71 4 7 8 647-2-48 50 0 50 56 75 0 0 0 82 40 54 74 0 0 2
647-2-121 A 50 0 47 55 73 2 2 5 45 41 56 72 4 6 8 647-2-99 A 20 0
22 35 53 14 27 35 647-2-99 B 5 0 27 43 64 12 20 42 ND* -- not
done
[0255] Particle size. It can be seen from the Tables 15 and 16 that
microfluidization of all specified liposomal formulations resulted
in similar particle size. Doxorubicin loading resulted in only
slight increase of particle size of formulations with lipid/drug
ratios from 50:1 to 100:1 (Table 17), whereas marked increase was
observed for formulations with lipid/drug ratios 20:1 and 5:1
(Table 17). Particle size of doxorubicin loaded liposomes with
lipid/drug ratios from 50:1 to 100:1 remains stable for at least
four months (Table 18), whereas particle size of liposomes with
lipid/drug ratios 20:1 and 5:1 was unstable and showed significant
increase.
[0256] Efficiency of doxorubicin encapsulation. Efficiency of
doxorubicin encapsulation into liposomes with lipid/drug ratios
from 50:1 to 100:1 varied from 90 to 98% (Table 19), and there was
no significant change of liposomal doxorubicin content observed
during storage at 2-8.degree. C. (Table 20). In contrast, markedly
lower encapsulation efficiency (31%-52%) was observed for liposomes
with lipid/drug ratios 20:1 and 5:1 (Table 19).
[0257] Free (not encapsulated) doxorubicin. Free doxorubicin
reflects concentration of drug that did not get encapsulated into
liposomes during loading step or leaked from the liposome during
the storage. It can be seen from the Table 21 that free doxorubicin
content in formulations with lipid/drug ratios 50:1 and 100:1 was
in the range from 0.2-0.41%. There was no significant change in
concentration of free doxorubicin observed up to .about.4 months of
storage at 2-8.degree. C. (Table 22).
[0258] Free doxorubicin content determined in formulations with
20:1 and 5:1 lipid/drug ratios was markedly higher (Table 21), and
markedly increased after 7 day of storage at 2-8.degree. C. (Table
22). These data indicate on evident leakage of doxorubicin from the
liposomes made at lower than 50:1 lipid/drug ratios and stored at
2-8.degree. C. and pH 7.4.
[0259] Liposomal doxorubicin release rate. It can be seen from the
Table 23 that doxorubicin release rate at pH 5 was markedly higher
compare to that at pH 7.4 for the formulations with lipid/drug
ratios 50:1 and 100:1 (Table 23 and FIG. 3). The .DELTA.pH7.4/5.0
release differential observed at T0 (within one week of MFD) was
sustained during the storage at 2-8.degree. C. for up to 114 days
(Table 23a).
[0260] In contrast, the formulations with lower lipid/drug ratios
(20:1 and 5:1) demonstrated poor .DELTA.pH7.4/5.0 release
differential (Table 23 and FIG. 8) and marked leakage of the
doxorubicin at pH 7.4 (Tables 22-23a).
[0261] In embodiments, not to be bound by theory, lower lipid/drug
ratios may lead to increased surface tension and compromised lipid
layer integrity that upon dilution could result in increased not pH
dependent leakage of liposomal content into dissolution media due
to concentration gradient, and could offset the pH driven release
of drug. In contrast, higher lipid to drug ratios may result in
formation of the liposomes with lower surface tension and higher
integrity lipid layer(s) that are capable of preventing "off
target" leakage of intraliposomal material into dissolution media,
and release the drug only in response to pH transition.
[0262] Thus, the effect of lipid/drug ratio for both pH dependent
drug release and stable performance of liposomes was demonstrated.
Performed studies suggest that optimal lipid/drug ratios are in the
range from 20:1 to 50:1 in some embodiments. Other ratios that can
be used include 20:1 to 100:1 in some other embodiments.
[0263] It is also worth mentioning that addition of P188 to the
liposomal formulation did not have any significant impact on
particle size (Tables 15-17), efficiency of doxorubicin
encapsulation (Tables 19 and 21), and doxorubicin release profile
(Table 23) compared to liposomal formulation prepared with no P188
(lot #647-2-159 B.). However, P188 was elected for use in liposomal
formulations due to its possible advantageous impact on biological
performance of drug-loaded liposomes [10-11, 16-19].
Example 4: In Vitro Cell-Based Cytotoxicity Assay
[0264] The human lymphoma Daudi B-cell line, commonly used for
evaluating drugs for treatment of B-cell lymphomas [21, 22] was
next used to test the B-cell cytotoxicity of the
doxorubicin-oxalate loaded liposomes vs free doxorubicin, and
DOXIL.RTM.. It can be seen from the Table 24 that three different
lots of doxorubicin-oxalate loaded liposomes demonstrated cell
cytotoxicity similar to free doxorubicin and were .about.50-70 fold
more potent relative to DOXIL.RTM. (Lot #L01DB01), a difference
predictive of increased efficacy in vivo.
[0265] Hela cells, the human cell line derived from cervical cancer
was also used for evaluating drugs cytotoxicity. It can be seen
from the Table 24 that doxorubicin-oxalate loaded liposomes
demonstrated cell cytotoxicity 2-3 fold lower than free doxorubicin
but were 4-6 fold higher more potent relative to DOXIL.RTM. (Table
24).
TABLE-US-00025 TABLE 24 CC.sub.50 (.mu.M) values obtained for
doxorubicin and liposomal doxorubicin formulations. Daudi, Hela,
Lipid/ Storage CC.sub.50, CC.sub.50, Lot# Name Drug T.degree. C.
.mu.M .mu.M 7000AO02113 Doxorubicin N/A N/A 0.4 7 Hydrochloride
L01DB01 DOXIL .RTM. 8 2-8 29 100 (Liposomal Doxorubicin Sulfate)
647-1-175 Liposomal 50 2-8 0.4 26 Doxorubicin Oxalate 647-1-174
Liposomal 100 2-8 0.4 20 Doxorubicin Oxalate 647-2-13 Liposomal 100
2-8 0.2 15 doxorubicin Oxalate
[0266] Obtained cytotoxicity data demonstrated markedly increased
potency of doxorubicin-oxalate containing liposomes compared to
DOXIL.RTM. that is in a good agreement with markedly higher
.DELTA.pH7.4/5.0 release differential of doxorubicin-oxalate
compared to doxorubicin-sulfate containing liposomes.
Example 5: In Vivo Study
[0267] The efficacy of doxorubicin-oxalate loaded liposomes (Lot
#647-2-13) vs DOXIL.RTM. (Lot #FAZSR00) was evaluated in a standard
lymphoma model in Beige,
(Line--CB17.Cg-Prkdc.sup.scidLyst.sup.bg-J/Crl) mice. As
illustrated by the timeline in FIG. 2, B lymphoma cells
(5.times.10.sup.6) were injected intravenously on day 0 and allowed
to disseminate for 24 hours, followed by dosing mice on days 1, 2,
and 3 with drug-free lipid formulation (Placebo), or
doxorubicin-oxalate containing liposomes (3 mg doxorubicin/kg), or
DOXIL.RTM. (3 mg doxorubicin/kg).
[0268] Animals treated with Placebo (doxorubicin free liposomes)
reached a median survival time (MST) in 21 days, whereas the
doxorubicin-oxalate loaded liposomes increased the MTS to 33 days
(FIG. 5). In contrast, DOXIL.RTM. liposomes exhibited a MST of 15
days (FIG. 5). The shorter MST observed with DOXIL.RTM. compared to
Placebo (FIG. 5) treated mice indicate potential DOXIL.RTM.
toxicity, whereas no such toxicity was observed with
doxorubicin-Oxalate containing liposomes. Group of untreated mice
(not injected with any material) showed survival rate identical to
Placebo (not shown in the graph). Approximately 8% maximum group
average weight loss was observed on the day 11 in
doxorubicin-oxalate treated animals with complete recovery on the
day 17, whereas in DOXIL.RTM. treated mice .about.30% weight loss
was observed on day 14 resulting in death of 4 from 8 animals. High
toxicity was also observed in a group of mice treated with free
doxorubicin (50% death rate on day 13), although survivors
demonstrated longest MST that validates the model. The lower
toxicity observed for doxorubicin-oxalate containing liposomes
could be in part due to their faster clearance. The lower toxicity
and higher efficacy of the tested liposomes in accordance with the
disclosure compared to DOXIL.RTM. is a highly desirable and
encouraging outcome. Thus, under the same experimental protocol,
the treatment with doxorubicin-oxalate containing liposomes
demonstrated no obvious toxicity and significant improvement of
survival rates compared to Placebo control, whereas treatment with
DOXIL.RTM. at the same doses and regimen demonstrated noticeable
toxicity and did not result in any significant improvement of MST
compared to Placebo. Better performance of doxorubicin-oxalate
liposomes was in line with the optimized .DELTA.pH release
differential and overall resulted in improved safety and efficacy
compared to DOXIL.RTM. (doxorubicin-sulfate) liposomes.
[0269] Thus, under the same experimental protocol, the treatment
with doxorubicin-oxalate containing liposomes demonstrated no
obvious toxicity and significant improvement of survival rates
compared to Placebo control, whereas treatment with DOXIL.RTM. at
the same doses and regimen demonstrated noticeable toxicity and did
not result in any significant improvement of MST compared to
Placebo (FIG. 9). Better performance of doxorubicin-oxalate
containing liposomes was in line with the optimized
.DELTA.pH7.4/5.0 release differential and overall resulted in
improved safety and efficacy compared to DOXIL.RTM.
(doxorubicin-sulfate) liposomes.
Example 6: Further Characterization of Doxorubicin-Tartrate
Containing Liposomes: Variable Lipid to Drug Ratios
[0270] Preparation and further characterization of
doxorubicin-tartrate containing liposomes. Liposomes were prepared
at different lipid/drug ratios (5:1, which is equally referred to 5
to 1 or 5/1-100:1, which is equally referred to 100 to 1 or 100/1)
(Table 25). Doxorubicin hydrochloride concentrations used for
remote loading were 0.5 or 1.0 mg/mL (i.e. 0.468 and 0.936 mg of
doxorubicin free base per mL). Remote loading was performed with
doxorubicin Hydrochloride at 70.degree. C. Tartaric acid was first
titrated with ammonium hydroxide to pH 4.8-5.0 and then used as
hydration media.
TABLE-US-00026 TABLE 25 Formulation Composition. Amounts of solids
used in formulations, W/W, % Ratios P Doxorubicin Lipid/ Lot # PC
DMPC FC 188 Hydrochloride Drug 647-2-186 B 66.0.5 16.51 11.56 4.95
0.88 100 647-2-151 B 65.50 16.38 11.46 4.91 1.75 50 647-2-178 B
63.68 15.92 11.14 4.78 4.48 20 647-2-178 D 56.15 14.04 9.83 4.21
15.78 5
[0271] Coarse suspension was prepared and MF processed at 10 KPSI
processing pressure. After 9-15 min of MF processing the particle
size (Z-average) reached .about.60-65 nm. A sample was collected
and sterile filtered into Nalgene flask. The particle size of
filtered nanosuspension was determined (Table 25a).
TABLE-US-00027 TABLE 25a Summary of MF processing and resultant
emulsion parameters. Particle size Lot# MFD Z avrg, nm 647-2-186B
18 JUL. 2016 62 647-2-151B 5 MAY 2016 65 647-2-178B 11 LUL 16 61
647-2-178D 11 LUL 16 61
[0272] The liposomes were subjected to TFF followed by remote
loading with doxorubicin, and another TFF cycle with PBS sucrose.
Doxorubicin hydrochloride concentration used for remote loading:
0.5 or 1.0 mg/mL.
[0273] The particle size of doxorubicin loaded liposomes is
presented in Table 25b.
TABLE-US-00028 TABLE 25b Particle size of doxorubicin loaded
liposomes. Lipid/ Particle size Lot# Drug Loading Date Z avrg, nm
647-2-186B 100 19 JUL. 2016 64 647-2-151B 50 18 MAY 2016 68
647-2-178B 20 11 JUL. 2016 67 647-2-178D 5 11 LUL 16 66
[0274] Determination of doxorubicin in liposomal suspension.
Followed doxorubicin loading liposomal suspension was subjected to
5.times.TFF to majorly remove free (not encapsulated) doxorubicin.
To determine total doxorubicin concentration at T0 (within one week
of MFD) TFF washed liposomes were diluted with methanol or IPA and
subjected to HPLC analysis. Doxorubicin content, percent of
recovery (doxorubicin content in liposomal suspension relative to
doxorubicin free base concentration used for remote loading), and
encapsulation efficiency (%) are presented in the Table 25c.
Encapsulation efficiency (%) represents the difference between
doxorubicin recovery (%) and free doxorubicin (%).
TABLE-US-00029 TABLE 25c Total doxorubicin content and
Encapsulation efficiency. Assay, HPLC Doxorubicin Doxorubicin
Encapsulated free base content doxorubicin, used (Liposomal Re- %
[Recovery, Lipid/ for loading, Suspesion) covery, %]- Lot# Drug
.mu.g/mL .mu.g/mL % [Free, %] 647-2-186 B 100 468 354 76 76
647-2-151 B 50 936 814 87 87 647-2-178 B 20 936 897 96 96 647-2-178
D 5 936 413 44 41
[0275] The amount of free (not encapsulated) doxorubicin was
determined within one week of manufacturing (Table 25d).
TABLE-US-00030 TABLE 25d Free doxorubicin content. Lot# Lipid/Drug
% of Total 647-2-186 B 100 0.01 647-2-151 B 50 0.03 647-2-178 B 20
0.01 647-2-178 D 5 3.20
[0276] Liposomal doxorubicin release studies were carried out at
37.degree. C. within one week after manufacturing (Table 25e). For
each sample doxorubicin release was determined at 2, 4 and 8 hrs
time points.
TABLE-US-00031 TABLE 25e Doxorubicin release rate determined at TO
(within one week after manufacturing). Lipid/ pH 5, Release, % pH
7.4, Release, % Lot # Drug 2 hrs 4 hrs 8 hrs 2 hrs 4 hrs 8 hrs
647-2-186 B 100 18 35 56 2 1 0 647-2-151 B 50 14 27 39 0 1 1
647-2-178 B 20 9 15 21 1 2 3 647-2-178 D 5 17 23 27 1 4 5
[0277] Particle size. It can be seen from the Tables 25 and 25a
that microfluidization of all specified liposomal formulations
resulted in similar particle size. Doxorubicin loading resulted in
only slight increase of particle size (Table 25b).
[0278] Efficiency of doxorubicin encapsulation. Efficiency of
doxorubicin encapsulation into liposomes with lipid/drug ratios
from 5:1 to 100:1 varied from 76 to 96% (Table 25c). In contrast,
markedly lower encapsulation efficiency (41%) was observed for
liposomes with lipid/drug ratios 5:1 (Table 25c).
[0279] Free (not encapsulated) doxorubicin. Free doxorubicin
reflects concentration of not encapsulated drug that did not get
encapsulated into liposomes during loading step or leaked from the
liposome during the storage. It can be seen from the Table 25d that
free doxorubicin content in formulations with lipid/drug ratios
20:1 to 100:1 was in the range from 0.01-0.03%, whereas increased
levels of free doxorubicin (3.2%) were observed at 5:1 lipid/drug
ratio. These data are in agreement with results obtained for
Oxalate containing liposomes (Table 21) and indicate on evident
leakage of doxorubicin at neutral pH from the liposomes made at
lower than 20:1 lipid/drug ratios.
[0280] Liposomal doxorubicin release rate. Although doxorubicin
release rate at pH 5 was higher compare to that at pH 7.4 for all
formulations (Table 25e), the extra leakage at neutral pH was
observed for formulations with lipid/drug ratios below 50:1. It is
worth mentioning, however, that doxorubicin leakage at pH 7.4 was
markedly less for Tartrate containing liposomes compared to that of
oxalate containing liposomes (Table 21, 23, FIG. 8, 10).
[0281] Thus, the effect of lipid/drug ratio for both counter ions
oxalate and tartrate was demonstrated. Performed studies suggest
that optimal lipid/drug ratios are in the range from 20:1 to 50:1
in some embodiments. Other ratios that can be used include 20:1 to
100:1 in some other embodiments.
Example 7: Cold Loading of Doxorubicin into Oxalate- and
Tartrate-Containing Liposomes: Variable Lipid/Drug Ratio
[0282] The cold loading of doxorubicin into liposomes was performed
as follows: saline solution of doxorubicin Hydrochloride was added
to the liposomal nanosuspension at room temperature to the final
concentration 0.5 or 1 mg/mL (i.e. 0.468 and 0.936 mg of
doxorubicin free base per mL), gently inverted (2-3 times) and
incubated at room temperature for 10-20 min. After 10-20 min of
incubation at room temperature the mixture was: a) subjected to
another TFF 5.times. cycle with PBS pH 7.4 containing 6% of
Sucrose, and/or b) placed in 2-8.degree. C. refrigerator for 16
hrs, and then subjected to another TFF 5.times. cycle with PBS pH
7.4 containing 6% of Sucrose. There was no notable difference
observed between doxorubicin release profiles of the liposomes
loaded at RT, or RT followed by 2-8.degree. C. overnight incubation
Data for RT followed by 2-8.degree. C. overnight incubation not
shown.
[0283] Formulation composition is shown in the Table 26.
TABLE-US-00032 TABLE 26 Formulation Composition. Amounts of solids
used in formulations, W/W, % Ratios Counter P Doxorubicin Lipid/
Lot # Ion PC DMPC FC 188 Hydrochloride Drug 647-2- Oxalate 66.0.5
16.51 11.56 4.95 0.88 100 181 A 647-2- Oxalate 65.57 16.38 11.46
4.91 1.75 50 163 B 647-2- Oxalate 63.68 15.92 11.14 4.78 4.48 20
185 A 647-2- Oxalate 56.15 14.04 9.83 4.21 15.78 5 185 C 647-2-
Tartrate 66.0.5 16.51 11.56 4.95 0.88 100 186 A 647-2- Tartrate
65.50 16.38 11.46 4.91 1.75 50 170 B 647-2- Tartrate 63.68 15.92
11.14 4.78 4.48 20 178 A 647-2- Tartrate 56.15 14.04 9.83 4.21
15.78 5 178 C
[0284] Coarse suspension was prepared and MF processed at 10 KPSI
processing pressure. After 9-15 min of MF processing the particle
size (Z-average) reached .about.60-66 nm. A sample was collected
and sterile filtered into Nalgene flask. The particle size of
filtered nanosuspension was determined (Table 26a).
TABLE-US-00033 TABLE 26a Summary of MF processing and resultant
emulsion parameters. Lipid/ Particle size Lot# Counter Ion MFD Drug
Z avrg, nm 647-2-181 A Oxalate 24 MAY 2016 100 66 647-2-163 B
Oxalate 24 MAY 2016 50 66 647-2-185 A Oxalate 19 JUL. 2016 20 62
647-2-185 C Oxalate 19 JUL. 2016 5 63 647-2-186 A Tartrate 19 JUL.
2016 100 62 647-2-170 B Tartrate 23 JUN. 2016 50 63 647-2-178 A
Tartrate 11 LUL 16 20 61 647-2-178 C Tartrate 11 LUL 16 5 61
[0285] The liposomes were subjected to TFF followed by remote
loading with doxorubicin, and another TFF cycle with PBS sucrose.
The particle size of doxorubicin loaded liposomes is presented in
Table 26b.
TABLE-US-00034 TABLE 26b Particle size of doxorubicin loaded
liposomes. Counter Loading Particle size Lot# Ion Date Lipid/Drug Z
avrg, nm 647-2-181 A Oxalate 14 JUL. 2016 100 67 647-2-163 B
Oxalate 10 JUN. 2016 50 67 647-2-185 A Oxalate 19 JUL. 2016 20 62
647-2-185 C Oxalate 19 JUL. 2016 5 62 647-2-186 A Tartrate 19 JUL.
2016 100 62 647-2-170 B Tartrate 23 JUN. 2016 50 63 647-2-178 A
Tartrate 11 JUL. 2016 20 60 647-2-178 C Tartrate 11 JUL. 2016 5
65
[0286] Determination of doxorubicin in liposomal suspension.
Followed doxorubicin loading liposomal suspension was subjected to
5.times.TFF to majorly remove free (not encapsulated) doxorubicin.
To determine total doxorubicin concentration at T0 (within one week
of MFD) TFF washed liposomes were diluted with methanol or IPA and
subjected to HPLC analysis. Doxorubicin content, percent of
recovery (doxorubicin content in liposomal suspension relative to
doxorubicin free base concentration used for remote loading), and
encapsulation efficiency (%) are presented in the Table 26c.
Encapsulation efficiency (%) represents the difference between
doxorubicin recovery (%) and free doxorubicin (%).
TABLE-US-00035 TABLE 26c Total doxorubicin content and
Encapsulation efficiency. Doxo- Assay, HPLC rubicin Doxorubicin
free base content Encapsulated used for (Liposomal Doxorubicin, %
Counter Lipid/ loading, Suspension) Recovery [Recovery,%]- Lot# Ion
Drug .mu.g/mL .mu.g/mL % [Free, %] 647-2-181 A Oxalate 100 468 425
91 91 647-2-163 B Oxalate 50 936 909 97 97 647-2-185 A Oxalate 20
936 892 95 95 647-2-185 C Oxalate 5 936 252 27 22 647-2-186 A
Tartrate 100 468 388 83 83 647-2-170 B Tartrate 50 936 850 91 91
647-2-178 A Tartrate 20 936 850 91 91 647-2-178 C Tartrate 5 936
645 69 67
[0287] The amount of free doxorubicin was determined within one
week of manufacturing (Table 26d).
TABLE-US-00036 TABLE 26d Free doxorubicin content. Lot# Counter Ion
Lipid/Drug % of Total 647-2-181 A Oxalate 100 0.07 647-2-163 B
Oxalate 50 0.08 647-2-185 A Oxalate 20 0.01 647-2-185 C Oxalate 5
4.96 647-2-186 A Tartrate 100 0.01 647-2-170 B Tartrate 50 0.04
647-2-178 A Tartrate 20 0.01 647-2-178 C Tartrate 5 2.51
[0288] Liposomal doxorubicin release studies were carried out at
37.degree. C. within one week after manufacturing (Table 26e). For
each sample doxorubicin release was determined at 2, 4 and 8 hrs
time points.
TABLE-US-00037 TABLE 26e Doxorubicin release rate determined at
37.degree. C. Counter Lipid/ pH 5, Release, % pH 7.4, Release, %
Lot# Ion Drug 2 hrs 4 hrs 8 hrs 2 hrs 4 hrs 8 hrs 647-2-181 A
Oxalate 100 93 100 100 0 0 0 647-2-163 B Oxalate 50 92 100 100 0 0
0 647-2-185 A Oxalate 20 51 66 84 3 8 4 647-2-185 C Oxalate 5 40 49
60 3 8 11 647-2-186 A Tartrate 100 56 72 81 0 0 0 647-2-170 B
Tartrate 50 63 89 100 0 0 0 647-2-178 A Tartrate 20 20 32 42 0 1 2
647-2-178 C Tartrate 5 13 18 24 0 2 4
[0289] Particle size. It can be seen from the Tables 26 and 26a
that microfluidization of all specified liposomal formulations
resulted in similar particle size. Doxorubicin loading resulted in
only slight increase of particle size of some formulations.
[0290] Efficiency of doxorubicin encapsulation. Efficiency of
doxorubicin encapsulation into Oxalate containing liposomes with
lipid/drug ratios from 20:1 to 100:1 varied from 91 to 97% (Table
26c). In contrast, markedly lower encapsulation efficiency (22%)
was observed for liposomes with lipid/drug ratio 5:1 (Table
26c).
[0291] When tartrate was used as counter ion efficiency of
doxorubicin encapsulation into liposomes with lipid/drug ratios
from 20:1 to 100:1 varied from 83 to 91% (Table 26c), while lower
encapsulation efficiency (67%) was observed for liposomes with
lipid/drug ratio 5:1 (Table 26c).
[0292] Free (not encapsulated) doxorubicin. Free doxorubicin
reflects concentration of the drug that did not get encapsulated
into liposomes during loading step or leaked from the liposome
during the storage. It can be seen from the Table 26d that free
doxorubicin content in oxalate containing liposomes with lipid/drug
ratios 20:1 and 100:1 was in the range from 0.01-0.08%. Free
doxorubicin content determined in formulations with 5:1 lipid/drug
ratio was 4.96% (Table 26d).
[0293] When tartrate was used as counter ion free doxorubicin
content of liposomes with lipid/drug ratios from 20:1 to 100:1
varied from 0.01 to 0.04% (Table 26d), while lower encapsulation
efficiency and higher free doxorubicin content (2.51%) was observed
for liposomes with lipid/drug ratio 5:1 (Table 26d).
Liposomal Doxorubicin Release Rate.
[0294] Effect of the lipid/drug ratio. It can be seen from the
Table 26e that for both counter ions doxorubicin release at pH 5
was dramatically higher compare to that at pH 7.4 for the
formulations with lipid/drug ratios 50:1 and 100:1 (Table 26e,
FIGS. 11-12). Moreover, doxorubicin leakage at pH 7.4 was fully
suppressed (Table 26e, FIGS. 11-12).
[0295] At pH 5 doxorubicin release from the cold loaded
oxalate-containing liposomes achieved .about.100% at 2 hrs time
point, and was doubled that of liposomes loaded at 70.degree. C.
(FIG. 13).
[0296] At pH 5 doxorubicin release from the cold loaded
tartrate-containing liposomes achieved .about.90-100% at 4 hrs time
point, and was tripled that of liposomes loaded at 70.degree. C.
(FIG. 14).
[0297] Surprisingly, when oxalate and/or tartrate were used as
counter ions, cold loading (e.g. mixing at room temperature)
further improved (maximized) .DELTA.pH 7.4/5.0 release differential
compared to liposomes loaded at 70.degree. C. by markedly
increasing drug release at pH 5 while suppressing its release at pH
7.4 (Table 26e and FIG. 13 and FIG. 14). At acidic pH doxorubicin
release from the cold loaded oxalate-containing liposomes achieved
.about.100% at 2 hrs time point, and was doubled--that of liposomes
loaded at 70.degree. C. (FIG. 13). Doxorubicin release from the
cold loaded tartrate-containing liposomes achieved .about.90% at 4
hrs (FIG. 14), and was triple that of the liposomes loaded at
70.degree. C.
[0298] Interestingly, doxorubicin-citrate containing liposomes
demonstrated low release rate at pH 5, although .DELTA.pH 7.4/5.0
release differential was acceptable due to extremely low release at
pH 7.4 (Table 13 and FIG. 3).
[0299] In contrast, the formulations with lipid/drug ratios below
5:1 demonstrated poor .DELTA.pH7.4/5.0 release differential due to
lower release at pH 5.0 and leakage of doxorubicin at pH 7.4 (Table
26e and FIGS. 11-12). It is worth mentioning, however, that
tartrate containing liposomes at lower than 50:1 lipid/drug ratios
demonstrated stronger suppression of doxorubicin leakage at neutral
pH compared to oxalate liposomes (Table 26e and FIGS. 11-12).
[0300] Thus, in some embodiments, lipid to drug ratio for both
counter ions oxalate and tartrate may play a role. Performed
studies suggest that in some embodiments, optimal lipid to drug
ratios can be in the range from 20:1 to 50:1 at least in some
embodiments. Other ratios that can be used include 10:1 to 100:1 in
some other embodiments in some other embodiments.
[0301] It has been also demonstrated that in some embodiments, cold
loading of doxorubicin maximized .DELTA.pH7.4/5.0 release
differential achieved with oxalate and tartrate as counter ions and
50:1 and 100:1 lipid/drug ratios. Other temperatures that can be
used for remote doxorubicin loading in oxalate and tartrate include
2-8.degree. C. to 70.degree. C. in some other embodiments.
Example 8.1: Cold Loading of Lyophilized and Reconstituted
Doxorubicin into Oxalate and/or Tartrate Containing Liposomes
[0302] Lyophilization. Doxorubicin-hydrochloride was dissolved to
the final concentration of 6 mg/mL in sterile water for injection
containing 6% sucrose, or 4% mannitol, or 1% lactose, or 3%
lactose, sterile filtered, aseptically filled in 2 mL vials (1 mL
fill volume), and lyophilized in VirTis Genesis SQ25EL lyophilizer.
Vials containing doxorubicin solution were transferred to a
pre-frozen to -40.degree. C. lyophilizer and allowed to freeze
overnight (16) hrs. After 16 hrs the vacuum was turned on and the
temperature was increased to -32.degree. C. at a temperature rump
up rate 1.degree. C./4 min. After 24 hrs of lyophilization at
32.degree. C. the temperature was further increased to 20.degree.
C. at a temperature ramp up rate 1.degree. C./20 min. Vials were
stoppered under vacuum.
[0303] Remote Loading: Lyophilized material was reconstituted in
sterile water for injection to the final concentration 6 mg/mL and
0.4 mL of reconstituted material (.about.2.24 mg of doxorubicin
free base) were added to 2 mL of the liposomal nanosuspension at
room temperature to the final concentration 0.936 mg/mL of
doxorubicin free base, gently inverted (2-3 times) and incubated at
room temperature for 10 min.
[0304] Formulation composition is shown in the Table 27.
TABLE-US-00038 TABLE 27 Formulation composition. Amounts of solids
used in formulations, W/W, % Ratios Doxorubicin Lipid/ Lot# Counter
Ion PC DMPC FC P 188 Hydrochloride Drug 647-2-196 Oxalate 65.44
16.36 11.45 4.91 1.84 50 647-2-198 Tartrate 65.44 16.36 11.45 4.91
1.84 50
[0305] Coarse suspension was prepared and MF processed at 10 KPSI
processing pressure. After 9-15 min of MF processing the particle
size (Z-average) reached .about.60-70 nm. A sample was collected
and sterile filtered into Nalgene flask. The particle size of
filtered nanosuspension was determined (Table 27a).
TABLE-US-00039 TABLE 27a Summary of MF processing and resultant
emulsion parameters. Counter Lipid/Drug Particle size Lot# Ion MFD
Ratio Z avrg, nm 647-2-196 Oxalate 5 AUG. 2016 50 64 647-2-198
Tartrate 9 AUG. 2016 50 65
[0306] The liposomes were subjected to TFF followed by remote
loading with doxorubicin at RT for 10 min. The particle size of
doxorubicin loaded liposomes is presented in Table 27b.
TABLE-US-00040 TABLE 27b Particle size of doxorubicin loaded
liposomes. Cryoprotectant Particle used for size Counter
doxorubicin Lipid/ Z avrg, Lot# Ion lyophilization MFD Drug nm
647-2-196 A Oxalate Mannitol, 4% 12 AUG. 2016 50 64 647-2-196 B
Oxalate Lactose, 1% 12 AUG. 2016 50 64 647-2-196 C Oxalate Lactose,
3% 12 AUG. 2016 50 64 647-2-198 A Tartrate Mannitol, 4% 12 AUG.
2016 50 65 647-2-198 B Tartrate Lactose, 1% 12 AUG. 2016 50 65
647-2-198 C Tartrate Lactose, 3% 12 AUG. 2016 50 65
[0307] The amount of free (not encapsulated) doxorubicin was
determined within one week of manufacturing (Table 27c).
TABLE-US-00041 TABLE 27c Free doxorubicin content. Cryoprotectant
used for doxolubicin Lot# Counter Ion lyophilization % of Total
647-2-196 A Oxalate Mannitol, 4% 0.23 647-2-196 B Oxalate Lactose,
1% 0.24 647-2-196 C Oxalate Lactose, 3% 0.24 647-2-198 A Tartrate
Mannitol, 4% 0.14 647-2-198 B Tartrate Lactose, 1% 0.18 647-2-198 C
Tartrate Lactose, 3% 0.17
[0308] Determination of doxorubicin in liposomal suspension. No
second TFF was performed followed the doxorubicin loading step. To
determine total doxorubicin concentration at T0 (within one week of
MFD) doxorubicin loaded liposomes were diluted with methanol or IPA
and subjected to HPLC analysis. Doxorubicin content, percent of
recovery (doxorubicin content in liposomal suspension relative to
doxorubicin free base concentration used for remote loading), and
encapsulation efficiency (%) are presented in the Table 27d.
Encapsulation efficiency (%) represents the difference between
doxorubicin recovery (%) and free doxorubicin (%).
TABLE-US-00042 TABLE 27d Total doxorubicin content and
Encapsulation efficiency. Doxo- Assay, HPLC rubicin Doxorubicin
Encapsu- free base content ulated used for (Liposomal doxo- Counter
loading, Suspesion) Recovery rubicin, Lot# Ion Cryoprotectant
.mu.g/mL .mu.g/mL % % 647-2-196 A Oxalate Mannitol, 4% 936 936 100
100 647-2-196 B Oxalate Lactose, 1% 936 936 100 100 647-2-196 C
Oxalate Lactose, 3% 936 934 97 97 647-2-198 A Tartrate Mannitol, 4%
936 934 97 97 647-2-198 B Tartrate Lactose, 1% 936 936 100 100
647-2-198 C Tartrate Lactose, 3% 936 936 100 100
[0309] Liposomal doxorubicin release studies were carried out at
37.degree. C. within one week after manufacturing (Table 27e). For
each sample doxorubicin release was determined at 2, 4 and 8 hrs
time points.
TABLE-US-00043 TABLE 27e Doxorubicin release rate determined at
37.degree. C. Cryoprotectant used for Counter doxolubicin pH 5,
Release, % pH 7.4, Release, % Lot# Ion lyophilization 2 hrs 4 hrs 8
hrs 2 hrs 4 hrs 8 hrs 647-2-196 A Oxalate Mannitol, 4% 92 98 100 0
0 0 647-2-196 B Oxalate Lactose, 1% 88 100 100 0 0 0 647-2-196 C
Oxalate Lactose, 3% 97 100 100 0 0 0 647-2-198 A Tartrate Mannitol,
4% 33 54 74 0 0 0 647-2-198 B Tartrate Lactose, 1% 28 50 70 0 0 0
647-2-198 C Tartrate Lactose, 3% 31 53 72 0 0 0
[0310] Lyophilized doxorubicin product. Lyophilized doxorubicin
water solution containing Mannitol or Lactose resulted in readily
(i.e. instantly) reconstitutable doxorubicin product. In contrast,
lyophilization in presence of 6% sucrose yielded not readily
reconstitutable material. Therefore, lyophilized doxorubicin
product containing 6% sucrose was not considered for further
development and was not used in loading experiments.
[0311] Particle size. It can be seen from the Tables 27 and 27a
that microfluidization of liposomal formulations resulted in
similar particle size. Doxorubicin loading did not affect the
particle size of the liposomes independent of counter ion and
cryoprotectant (Table 27b).
[0312] Free (not encapsulated) doxorubicin. Free doxorubicin
reflects concentration of the drug that did not get encapsulated
into liposomes during loading step or leaked from the liposome
during the storage. It can be seen from the Table 27c that free
doxorubicin content in Oxalate containing liposomes was
.about.0.24%. When Tartrate was used as counter ion free
doxorubicin content in the liposomal suspension was in the range
from 0.14 to 0.18% (Table 27d).
[0313] Efficiency of doxorubicin encapsulation. Efficiency of
doxorubicin encapsulation was from 97 to 100% (considering the
levels of free doxorubicin) independently of counter ion and
cryoprotectant (Table 27d).
[0314] Liposomal doxorubicin release rate. Cold (room temperature)
loading of lyophilized and reconstituted doxorubicin into oxalate
and/or tartrate containing liposomes resulted in rapid (within 10
min) encapsulation of the doxorubicin. Liposomal doxorubicin
product demonstrated exceptional .DELTA.pH7.4/5.0 release
differential with high release of doxorubicin at pH 5, while
doxorubicin release at pH 7.4 was fully suppressed (Table 27e).
[0315] Thus, successful lyophilization of the doxorubicin that
results in readily reconstitutable at RT in water for injection
lyophilized product, unique ability of our novel liposomes to
rapidly encapsulate reconstituted doxorubicin product at RT, and
provide exceptional .DELTA.pH7.4/5.0 release differential were
demonstrated.
[0316] Overall, stability of liposomal doxorubicin products depends
on both the stability of the liposomes and the stability of the
drug product inside the liposomes. Lyophilized and readily
reconstitutable at RT doxorubicin, and the capability of oxalate-
or tartrate-containing liposomes to rapidly load doxorubicin at RT
address these potential stability issues, while also providing
superior release profiles. This finding leads to particular product
presentation format consisting of two vials: a vial with
lyophilized doxorubicin and a vial with liposomal vehicle
suspension. Mixing (via simple inversion) the reconstituted content
of two vials at room temperature will yield the final ready-for-use
product within minutes.
[0317] Product Stability. The both suspension of doxorubicin free
oxalate- or tartrate-containing liposomes (vehicle) and lyophilized
doxorubicin were stable when stored at 2-8.degree. C. amb RH for at
least 6 months. Six month time point represents last stability
testing performed for the formulations described in Section 8.1.
The vehicle was loaded with lyophilized and reconstituted
doxorubicin and stability testing at included: HPLC assay of
lyophilized and reconstituted doxorubicin material (98.+-.2,%),
efficiency of doxorubicin encapsulation into liposomes (98.+-.2,%),
determination of free (not encapsulated) doxorubicin (0.1-1%),
particle size (Zavrg=63-68 nm), pH (7.2-7.4), and .DELTA.pH7.4/5.0
doxorubicin release differential was close to 100%. Moreover, older
batch (647-1-190) of oxalate-containing liposomes (vehicle) stored
at 2-8.degree. C. amb RH showed acceptable stability for at least
18 months with no notable changes observed in above described
parameters. Development of lyophilized liposomal vehicle is also
considered.
Example 8.2: Comparison of Different Counter Ions at Fixed 50:1
Lipid/Drug Ratio: Cold Loading
[0318] Hydration Media Used:
[0319] a) 300 mM solution of the following ammonium salts:
ammonium-oxalate, or ammonium-sulfate, or ammonium-phosphate, or
ammonium-citrate.
[0320] b) tartaric acid, ascorbic acid, or N-acetyl L cysteine
(NAC) were first titrated with ammonium hydroxide to pH 4.8-5.0 and
then used as hydration media.
[0321] Formulation composition is shown in Table 27. All
formulations were prepared at 50:1 fixed lipid/drug ratio (Table
28)
[0322] The data for NAC are not shown since no doxorubicin loading
was observed and liposomal material precipitated after overnight
storage at 2-8.degree. C.
TABLE-US-00044 TABLE 28 Formulation Composition. Amounts of solids
used in formulations, W/W, % Counter Doxorubicin Lipid/ Lot# Ion PC
DMPC FC P 188 Hydrochloride Drug 647-2-169 B Sulfate 65.50 16.38
11.46 4.91 1.75 50 647-2-163 B Oxalate 65.50 16.38 11.46 4.91 1.75
50 647-2-169 C Phosphate 65.50 16.38 11.46 4.91 1.75 50 647-2-170 B
Tartrate 65.50 16.38 11.46 4.91 1.75 50 647-2-169 D Citrate 65.50
16.38 11.46 4.91 1.75 50 647-2-164 B Ascorbate 65.50 16.38 11.46
4.91 1.75 50
[0323] Coarse suspension was prepared and MF processed. After 9-12
min of MF processing the particle size (Z-average) reached
.about.60-75 nm. A sample was collected and sterile filtered into
Nalgene flask. Then liposomes were subjected to TFF followed by
remote loading with doxorubicin, and another TFF cycle with PBS
sucrose. Particle size of the Microfluidized liposomal material was
similar to that shown herein.
[0324] Doxorubicin hydrochloride concentration used for remote
loading: 1.0 mg/mL (doxorubicin free base concentration: 0.936
mg/mL).
[0325] The cold loading of doxorubicin into liposomes was performed
as follows: Saline solution of doxorubicin Hydrochloride (6 mg/mL)
was added to the liposomal nanosuspension at room temperature to
the final concentration 1 mg/mL (i.e. 0.936 mg of doxorubicin free
base per mL), gently inverted (2-3 times) and incubated at room
temperature for 10-20 min. After 10-20 min of incubation at room
temperature the mixture was: a) subjected to another TFF 5.times.
cycle with PBS pH 7.4 containing 6% of Sucrose, and/or b) placed in
2-8.degree. C. refrigerator for 16 hrs, and then subjected to
another TFF 5.times. cycle with PBS pH 7.4 containing 6% of
Sucrose. There was no notable difference observed between
doxorubicin release profiles of the liposomes loaded at RT, or RT
followed by 2-8.degree. C. overnight incubation. Data for RT
followed by 2-8.degree. C. overnight incubation are not shown.
[0326] The particle size of doxorubicin loaded liposomes is
presented in Table 28a.
TABLE-US-00045 TABLE 28a Particle size of doxorubicin loaded
liposomes. Counter Loading Particle size Lot# Ion Date Z avrg, nm
647-2-169 B Sulfate 17 JUN. 16 62 647-2-163 B Oxalate 10 JUN. 16 67
647-2-169 C Phosphate 17 JUN. 16 62 647-2-170 B Tartrate 23 JUN. 16
63 647-2-169 D Citrate 17 JUN. 16 61 647-2-164 B Ascorbate 15 JUN.
16 67
[0327] Determination of doxorubicin in liposomal suspension.
Followed doxorubicin loading liposomal suspension was subjected to
5.times.TFF to majorly remove free (not encapsulated) doxorubicin.
To determine total doxorubicin concentration at T0 (within one week
of MFD) TFF washed liposomes were diluted with methanol or IPA and
subjected to HPLC analysis. Doxorubicin content, percent of
recovery (doxorubicin content in liposomal suspension relative to
doxorubicin free base concentration used for remote loading), and
encapsulation efficiency (%) are presented in the Table 28b.
Encapsulation efficiency (%) represents the difference between
doxorubicin recovery (%) and free doxorubicin (%).
TABLE-US-00046 TABLE 28b Total doxorubicin content and
Encapsulation efficiency. Doxorubicin Assay, HPLC Encapsulated free
base used Doxorubicin content doxorubicin, % Counter for loading,
(Liposomal Suspesion) Recovery, [Recovery,%] - Lot# Ion .mu.g/mL
.mu.g/mL % [Free,%] 647-2-169 B Sulfate 936 828 88 88 647-2-163 B
Oxalate 936 909 97 97 647-2-169 C Phosphate 936 725 78 78 647-2-170
B Tartrate 936 850 91 91 647-2-169 D Citrate 936 853 91 91
647-2-164 B Ascorbate 936 822 88 86
[0328] The amount of free doxorubicin was determined within one
week of manufacturing (Table 28c).
TABLE-US-00047 TABLE 28c Free doxorubicin content. Lot# Counter Ion
% of Total 647-2-169 B Sulfate 0.01 647-2-163 B Oxalate 0.08
647-2-169 C Phosphate 0.01 647-2-170 B Tartrate 0.04 647-2-169 D
Citrate 0.01 647-2-164 B Ascorbate 1.85
[0329] Liposomal doxorubicin release studies were carried out at
37.degree. C. (Table 28d). For each sample doxorubicin release was
determined at 2, 4 and 8 hrs time points.
TABLE-US-00048 TABLE 28d Doxorubicin release rate determined at
37.degree. C. Counter pH 5, Release, % pH 7 4, Release, % Lot# Ion
Pka1 2 hrs 4 hrs 8 hrs 2 hrs 4 hrs 8 hrs 647-2-169 B Sulfate -3 2 1
2 0 0 0 647-2-163 B Oxalate 1.27 92 100 100 0 0 2 647-2-169 C
Phosphate 1.96 2 2 2 0 0 0 647-2-170 B Tartrate 3.03 63 89 100 0 0
0 647-2-169 D Citrate 3.13 19 33 44 0 0 0 647-2-164 B Ascorbate
4.17 97 97 100 58 66 69
[0330] Particle size. Microfluidization of different liposomal
formulation resulted in similar particle sizes closely resembling
to that shown herein. Cold doxorubicin loading resulted in slightly
less increase of particle size (except ascorbate-containing
liposomes) compared to the liposomes loaded with doxorubicin at
70.degree. C. (Table 28a and Table 8).
[0331] Efficiency of doxorubicin encapsulation. Efficiency of
doxorubicin encapsulation for the most of the formulas varied from
86 to 97%, except phosphate containing liposomes that showed 78% of
doxorubicin recovery (Table 27b). The most efficient encapsulation
was observed when oxalate was used as a counter ion, and least
efficient with phosphate (Table 28b).
[0332] Free (not encapsulated) doxorubicin. Free doxorubicin
reflects concentration of not encapsulated drug determined at T0
(within one week after manufacturing). It can be seen from the
Table 27c that free doxorubicin content for all formulations (but
Ascorbate) was in the range from 0.01-0.08%. Doxorubicin-ascorbate
containing liposomes demonstrated markedly higher leakage of free
doxorubicin (Table 28c).
[0333] Liposomal doxorubicin release rate. Drug release studies
were carried out at 37.degree. C.
[0334] Cold loading of doxorubicin resulted in some improvement of
.DELTA.pH7.4/5.0 release differential for citrate (Table 28d, FIG.
15), marked improvement of .DELTA.pH7.4/5.0 release differential
for oxalate, and major improvement for tartrate (Table 28d, FIG.
15) compared to the liposomes loaded with doxorubicin at 70.degree.
C. (Table 13, FIG. 3).
[0335] Doxorubicin-sulfate and -phosphate containing liposomes
retained poor .DELTA.pH 7.4/5.0 release differential (Table 28d,
FIG. 15) due to low release at both pH. Although
doxorubicin-ascorbate containing liposomes demonstrated high
doxorubicin release at pH 5, the .DELTA.pH 7.4/5.0 release
differential was very poor due to high release/leakage of the
doxorubicin at pH 7.4 (Table 28d, FIG. 15) that defeats the purpose
of doxorubicin encapsulation, and will compromise product stability
and in vivo performance.
[0336] Thus, when oxalate or tartrate were used as a counter ions
the difference between doxorubicin release at pH 5 and pH 7.4 was
markedly higher compare to other used counter ions regardless of
the loading conditions (Table 13, FIG. 3 and Table 28d, FIG. 15).
It is worth mentioning, however, that cold loading further improved
.DELTA.pH 7.4/5.0 release differential for doxorubicin-oxalate and
-tartrate containing liposomes relative to same liposomal
formulations that were loaded at 70.degree. C. The observed
difference suggests uniqueness of physical state(s) of
doxorubicin-oxalate or -tartrate aggregates at 37.degree. C. that
may facilitate their dissolution in response to the temperature and
pH.
Example 8.3: Cold Loading of Doxorubicin into Oxalate or Tartrate,
or Citrate Containing Liposomes
[0337] This example provides (i) further characterization regarding
pH dependent doxorubicin release at pH 7.4, 6.7, 6.0, and 5.0 and
(ii) effect of lipid/drug and phospholipid/free cholesterol (PL/FC)
ratios on pH dependent doxorubicin release. Comparison was made
with MYOCET-like formulation ("MYOCET", doxorubicin-citrate) and
DOXIL.RTM. (doxorubicin-sulfate).
[0338] Oxalate-, tartrate-, and citrate-containing liposomes
(vehicle) were prepared and loaded with doxorubicin hydrochloride
as described in Methods and section 8.1 to the final concentration
of 1 mg/mL. The various lipid/drug and phospholipid/free
cholesterol (PL/FC) ratios were achieved by adjusting relative
amounts of phospholipid and/or free cholesterol (Table 28e) and/or
through respective dilutions of the liposomes before loading with
doxorubicin. In the Tables 28e-28h "lipid/drug" represents
weight/weight (w/w) ratio of total lipids to doxorubicin free base
in final suspension of doxorubicin loaded liposomes. "PL/FC"
represents mol/mol ratio of phospholipids (PL) to free cholesterol
(FC) in final suspension of doxorubicin loaded liposomes.
[0339] Commercial DOXIL.RTM. and MYOCET-like ("MYOCET") liposomes
were used as comparators. MYOCET-like ("MYOCET") liposomes were
prepared by hydrating lipid film containing 6.9 g of PC and 2.84 g
of FC (55/45 molar ratio) with 100 mL of 0.3M citric Acid pH 4.0 at
65.degree. C. Microfluidization and TFF were performed as described
in Methods and section 8.1. The resultant liposomes were sterile
filtered. 1.9 mL aliquot was taken and loaded with 50 mg of
doxorubicin in total 25 mL of loading media at 70.degree. C.
according to the protocol described in MYOCET package insert.
Detailed formulation composition is presented in the Table 28e.
TABLE-US-00049 TABLE 28e Formulation Composition. Amounts of solids
used in formulations, W/W, % Doxo- PL/ rubicin Lipid/ FC, Counter
Hydro- Drug mol/ Lot# Ion PC DMPC FC P 188 chloride w/w mol
761-1-36 Oxalate 65.50 16.38 11.46 4.91 1.75 50 3.68 761-1-55
Oxalate 57.91 14.48 21.72 4.34 1.54 58 1.72 761-1-63 Oxalate 54.01
13.50 27.00 4.05 1.44 63 1.29 761-1-63-23 Oxalate 52.61 13.13 26.31
3.94 4.00 23 1.29 761-1-63-16 Oxalate 51.70 12.93 25.85 3.87 5.65
16 1.29 761-1-64 Oxalate 47.58 11.90 35.69 3.57 1.27 68 0.86
761-1-37 Tartrate 65.50 16.38 11.46 4.91 1.75 50 3.68 761-1-38
Citrate 65.50 16.38 11.46 4.91 1.75 50 3.68 761-1-69 Citrate 54.99
N/A 22.63 N/A 22.38 3.5 1.22 "MYOCET" Doxo- DSPE-PEG HSPC FC P188
rubicin DOXIL .RTM. Sulfate 17.76 53.34 17.76 N/A 11.14 8.0 1.62
PL-Phospholipid, FC-Free Cholesterol.
[0340] Doxorubicin release testing was carried out at 20.times.
(Table 28f) and 50.times. (Table 28g) dilutions in the dissolution
media.
TABLE-US-00050 TABLE 28f Doxorubicin release rate determined after
8 hrs of incubation in dissolution medias pH 7.4, 6.7, 6.0, and 5.0
at 37.degree. C. Dilution in the dissolution media = 20X. PL/
Lipid/ FC, Counter Drug mol/ Doxorubicin Release, % Lot# Ion w/w
mol pH 7.4 pH 6.7 pH 6.0 pH 5.0 761-1-36 Oxalate 50 3.68 0 35 88
100 761-1-55 Oxalate 58 1.72 0 35 64 100 761-1-63 Oxalate 63 1.29 0
25 64 100 761-1-63-23 Oxalate 23 1.29 0 15 50 80 761-1-63-16
Oxalate 16 1.29 0 10 40 70 761-1-64 Oxalate 68 0.86 0 5 54 100
761-1-37 Tartrate 50 3.68 0 7 40 81 761-1-38 Citrate 50 3.68 0 3 12
44 761-1-69 Citrate 3.5 1.22 0 0 5 15 "MYOCET" DOXIL .RTM. Sulfate
8.0 1.62 0 0 2 3
TABLE-US-00051 TABLE 28g Doxorubicin release rate determined after
8 hrs of incubation in dissolution medias pH 7.4, 6.7, 6.0, and 5.0
at 37.degree. C. Dilution in the dissolution media = 50X. Lipid/
Counter Drug PL/FC, Doxorubicin Release, % Lot# Ion w/w mol/mol pH
7.4 pH 6.7 pH 6.0 pH 5.0 761-1-36 Oxalate 50 3.68 0 40 93 100
761-1-55 Oxalate 58 1.72 0 39 95 100 761-1-63 Oxalate 63 1.29 0 36
95 100 761-1-63-23 Oxalate 23 1.29 4 30 80 100 761-1-63-16 Oxalate
16 1.29 6 25 70 100 761-1-64 Oxalate 68 0.86 0 19 77 100 761-1-37
Tartrate 50 3.68 0 14 60 100 761-1-38 Citrate 50 3.68 0 7 25 68
761-1-69 Citrate 3.5 1.22 0 5 13 23 "MYOCET" DOXIL .RTM. Sulfate
8.0 1.62 0 0 3 6
[0341] Effect of counter ions. It can be seen from the Tables 28f,
28g, and FIGS. 16-17 that doxorubicin-Oxalate containing liposomes
demonstrated the highest .DELTA.pH (7.4/6.7/6.0/5.0) doxorubicin
release differential at both 20.times. and 50.times. dilutions when
compared with doxorubicin-tartrate and doxorubicin-citrate
containing liposomes made with the same (50/1) lipid/drug ratio. It
is worth mentioning that pH dependent doxorubicin release from
doxorubicin-oxalate containing liposomes (Table 28f, 28g, FIGS.
16-17) and in slightly lesser extent of doxorubicin-tartrate
liposomes is in line with physiological pH gradient that occurs in
vivo (FIG. 1) indicating pH targeting capability of our liposomal
delivery system. Markedly lower .DELTA.pH (7.4/6.7/6.0/5.0)
doxorubicin release differential was observed for the "MYOCET" at
both 20.times. and 50.times. dilutions (Table 28f, 28g, FIGS.
16-17). .DELTA.pH (7.4/6.7/6.0/5.0) doxorubicin release
differential of DOXIL.RTM. was close to zero (Table 28f, 28g, FIGS.
16-17).
[0342] Effect of Lipid/Drug ratio. The dependence of .DELTA.pH
(7.4/6.7/6.0/5.0) doxorubicin release differential on the
lipid/drug ratio can be clearly seen when .DELTA.pH release
differential of doxorubicin-Oxalate liposomes (50/1 lipid/drug
ratio) compared to that of doxorubicin-oxalate liposomes made at
23/1 and/or 16/1 lipid/drug ratio (Table 28f and FIG. 18; 20.times.
dilution of liposomes in the dissolution media). Similar dependence
but in a lesser extent was observed at 50.times. dilution of
liposomal material in the dissolution media (Table 28g and FIG.
19). It is worth mentioning that liposomes made at low (23/1 and
16/1) lipid/drug ratios showed marked dependence on dilution factor
(i.e. 20.times. or 50.times.), whereas liposomes with higher (50/1)
lipid/drug ratio demonstrated essentially similar drug release at
both conditions (Table 28f, 28g, and FIGS. 16-19). Some leakage of
doxorubicin from the liposomes prepared at low (23/1 and 16/1)
lipid to drug ratio was observed at 50.times. dilution (Table 28g
and FIG. 19).
[0343] Marked dependence of .DELTA.pH (7.4/6.7/6.0/5.0) doxorubicin
release differential on the lipid/drug ratio can be seen from
comparison of doxorubicin-citrate liposomes made at 50/1 and 3.5/1
lipid/drug ratios (Table 28f, 28g, and FIGS. 16-17) at both
20.times. and 50 dilutions.
[0344] Thus, obtained data demonstrate that in some embodiments,
higher lipid/drug ratios achieve higher .DELTA.pH 7.4/6.7, 7.4/6.0,
and 7.4/5.0 doxorubicin release differentials. In some embodiments,
at lower lipid/drug ratios doxorubicin-oxalate, or -tartrate, or
possibly citrate aggregates may be forced to form denser fibril
like structures that would negatively impact dissolution of
aggregates. These data also indicate that in some embodiments,
higher lipid/drug ratios accommodate/support the pH sensitive,
possibly disorganized, physical state of doxorubicin aggregates.
The leakage of doxorubicin at neutral pH observed for lower
lipid/drug ratio formulations could be due to the lower lipid
content resulting in increased surface tension and compromised
integrity of the lipid bilayer of doxorubicin loaded liposomes at
least in some embodiments.
[0345] Effect of PL/FC (Phospholipid/Free Cholesterol) ratio. Free
cholesterol content can affect unilamellar liposomes lipid bilayer
rigidity that may potentially translate in serum/blood stability of
the liposomes [52, 53], and may also lay a path to further
development of lyophilized product. To test whether or not
increased FC content will negatively affect .DELTA.pH
(7.4/6.7/6.0/5.0) doxorubicin release differential the experiments
on effect of FC content, and therefore PL/FC ratio on .DELTA.pH
release differential were carried out. It can be seen in the Table
28f and FIG. 18 (20.times. dilution in dissolution media) that
increase of FC content and respective decrease of PL/FC ratio from
3.68 to 0.86 in the doxorubicin-oxalate liposomes resulted in
notable decrease of drug release at pH 6.7, while release at pH 6.0
and 5.0 was not significantly affected.
[0346] No notable effect of FC on .DELTA.pH (7.4/6.7/6.0/5.0)
doxorubicin release differential was observed when release
experiments were performed at 50.times. dilution (Table 28g and
FIG. 19) using liposomes with 3.68 to 1.29 PL/FC ratios. It is
worth mentioning that PL/FC ratio <1.0 resulted in notable
changes of doxorubicin release at both 20.times. and 50.times.
dilutions (Table 28f and 28g).
[0347] Thus, overall data demonstrate that in some embodiments,
.DELTA.pH (7.4/6.7/6.0/5.0) doxorubicin release differential of
doxorubicin-oxalate liposomes can highly tolerate decrease of PL/FC
molar ratio from 3.68 to 1.29 when release experiments are
performed at 50.times. dilution. At 20.times. dilution the decrease
of PL/FC molar ratio from 3.68 to 1.29 mostly affected .DELTA.pH
(7.4/6.7) release differential while no significant changes of
doxorubicin release rate were observed at pH 6.0 and 5.0. In
contrast, decrease of PL/FC molar ratio of doxorubicin-citrate
liposomes from 3.68 to 1.22 resulted in decrease of .DELTA.pH
release differential across of entire pH range (Table 28f, 28g, and
FIGS. 16-17).
Example 8.4: Cold Loading of Doxorubicin into Oxalate or Tartrate,
or Citrate Containing Liposomes
[0348] This examples provides (i) further characterization
regarding the stability of doxorubicin-oxalate,
doxorubicin-tartrate and (ii) doxorubicin-citrate containing
liposomes in human serum. Effect of lipid/drug and
phospholipid/free cholesterol (PL/FC) ratios is demonstrated.
[0349] Oxalate-, tartrate-, and citrate-containing liposomes
(vehicle) were prepared and loaded with doxorubicin hydrochloride
as described in Methods and section 8.1 to the final concentration
of 1 mg/mL. The various lipid/drug and phospholipid/free
cholesterol (PL/FC) ratios were achieved by manipulating of
relative amounts of phospholipid and/or free cholesterol (Table
28e), and/or through appropriate dilution of the liposomes before
loading with doxorubicin.
[0350] Commercial DOXIL.RTM. and MYOCET-like ("MYOCET") liposomes
were used as comparators. MYOCET-like ("MYOCET") liposomes were
prepared by hydrating lipid film containing 6.9 g of PC and 2.84 g
of FC (55/45 molar ratio) with 100 mL of 0.3M Citric Acid pH 4.0 at
65.degree. C. Microfluidization and TFF were performed as described
in Methods and section 8.1. The resultant liposomes were sterile
filtered. 1.9 mL aliquot was taken and loaded with 50 mg of
doxorubicin in total 25 mL of loading media at 70.degree. C.
according to the protocol described in MYOCET package insert.
Detailed formulation composition is presented in the Table 28e.
[0351] Serum stability studies were conducted at 50.times. dilution
(simulates administration of 60 mg/m.sup.2 or 110 mg/70 kg human
dose of doxorubicin) of the liposomal material in human serum by
adding of 50 uL of the doxorubicin loaded liposomes to 2.45 mL
(50.times. dilution) of the human serum. T0 samples were analyzed
immediately and other samples were incubated at 37.degree. C. for
2, 4, and 8 hrs. At each time point fluorescence of intact
liposomes (Fi) and total fluorescence of liposomes ruptured with
Triton X-100 (Ft) was measured. The data are presented in the Table
28h.
TABLE-US-00052 TABLE 28h Doxorubicin release determined after 2, 4,
and 8 hrs of incubation of the doxorubicin loaded liposomes in
human serum at 37.degree. C. and 50X dilution. PL/ Lipid/ FC,
Doxorubicin Counter Drug mol/ Release, % Lot# Ion w/w mol 2 hrs 4
hrs 8 hrs 761-1-36 Oxalate 50 3.68 45 50 52 761-1-55 Oxalate 58
1.72 42 45 52 761-1-63 Oxalate 63 1.29 25 27 33 761-1-63-23 Oxalate
23 1.29 46 65 77 761-1-63-16 Oxalate 16 1.29 60 92 100 761-1-64
Oxalate 68 0.86 12 14 20 761-1-37 Tartrate 50 3.68 48 49 53
761-1-38 Citrate 50 3.68 40 43 46 761-1-69 Citrate 3.5 1.22 100 100
100 "MYOCET" DOXIL .RTM. Sulfate 8.0 1.62 4 6 8
[0352] Effect of counter ions. Similar stability in human serum was
observed for doxorubicin-Oxalate, -Tartrate, and -Citrate liposomes
made at the same (50/1) lipid/drug ratio (Table 28h). This data
indicate that counter ions do not determine stability of the tested
articles in human serum. It is worth mentioning that marked
stability of DOXIL.RTM. liposomes in serum is achieved via using
pegylated lipids [2-5, 7-8].
[0353] Effect of lipid/drug ratio. It can be seen in the Table 28h
and FIG. 20 that serum stability of the doxorubicin-oxalate
liposomes decreases with decrease of the lipid/drug ratio.
Stability of doxorubicin-oxalate liposomes made at .gtoreq.50/1
lipid/drug ratios is markedly higher compared to
doxorubicin-oxalate liposomes made with 23/1 and 16/1 lipid/drug
ratios. Same trend was observed upon comparison of
doxorubicin-citrate liposomes made at 50/1 and 3.5/1 lipid/drug
ratios (Table 28h and FIG. 20). These data clearly demonstrate that
lipid/drug ratio may contribute to serum stability of the
liposomes. The effect of lipid/drug ratio on serum stability of the
liposomes as well as its effect on .DELTA.pH (7.4/6.7/6.0/5.0)
doxorubicin release differential demonstrates advantage of using
higher lipid/drug ratios for optimal performance of liposomal
delivery system in some embodiments.
[0354] Effect of PL/FC (phospholipid/free cholesterol) ratio.
Further increase of serum stability of doxorubicin-oxalate
liposomes was observed with increase of FC content and subsequent
decrease of PL/FC ratio (Table 28h and FIG. 21). PL/FC ratio 0.86
demonstrated the highest protection of the liposomes in human
serum, although notable negative impact of 0.86 PL/FC ratio on
.DELTA.pH (7.4/6.7/6.0/5.0) doxorubicin release differential was
observed (Table 28f and 28g). The obtained data suggest that the
optimal PL/FC ratio for doxorubicin-oxalate liposomes would lie
between 3.68 and 1 (Table 28f-28h, and FIGS. 20-21). It is worth
mentioning however, that doxorubicin-oxalate liposomes with near
optimal PL/FC ratio (1.29) but lower than 50/1 lipid/drug ratio
(i.e. 23/1 and 16/1) demonstrated lower serum stability (Table 28h
and FIG. 20). These data demonstrate contribution of both
lipid/drug and PL/FC ratio to liposomal serum stability. Similar
relationships between lipid/drug and PL/FC ratios were observed for
doxorubicin-citrate liposomes ((Table 28h, and FIG. 20). It is also
worth mentioning that human blood as well as mouse serum showed
less deleterious effect on the liposomes compared to human serum
(data not shown).
[0355] Obtained data clearly demonstrate impact of lipid/drug ratio
and PL/FC ratio on both serum stability of the liposomes and
.DELTA.pH (7.4/6.7/6.0/5.0) doxorubicin release differential
emphasizing beneficial effect of higher lipid/drug ratios and lower
than 3.68 but higher than 1.0 PL/FC ratios in some embodiments.
[0356] Overall, obtained data demonstrate that doxorubicin-oxalate
liposomes exhibit markedly higher .DELTA.pH (7.4/6.7/6.0/5.0)
doxorubicin release differential compared to "MYOCET" and
DOXIL.RTM. (Table 28e-28g and FIGS. 16-17). The serum stability of
doxorubicin-oxalate liposomes is markedly higher than that of
"MYOCET" but lower then DOXIL.RTM., although lowering of PL/FC
ratio while keeping the lipid/drug ratio .gtoreq.50/1 further
improves serum stability of doxorubicin-oxalate liposomes and makes
it somewhat comparable to DOXIL.RTM. (Table 28e, 28h, and FIG. 21).
Several physical and chemical properties of liposomes, such as
size, lipid composition, charge, and surface coatings, are known to
determine their interaction with plasma proteins that influence the
clearance pharmacokinetics of the vesicles [53]. Therefore, if in
vivo pharmacokinetics of the liposomal delivery system in
accordance with the disclosure herewith (i.e. area under the plasma
concentration-time curve, elimination half-life . . . etc) is
reflective of serum performance (i.e. between MYOCET and
DOXIL.RTM.) that would be highly desirable outcome.
Example 9: Cold Loading of Doxorubicin into Fixed (50:1) Lipid/Drug
Ratio Liposomes: Addition of Ascorbic Acid (AA), or
N-Acetylcysteine (NAC), Ascorbyl Palmitate (AP), Ubiquinone
(CoQ10), or Ethylenediaminetetraacetic Acid (EDTA) to
Ammonium-Oxalate and/or -Tartrate Hydration Media
[0357] Hydration medias used: 300 mM solution of ammonium-oxalate;
tartaric acid was first titrated with ammonium hydroxide to pH
4.8-5.0 and then used as hydration media.
[0358] Liposomes were prepared as described in the methods section,
except that NAC, or AA, or AP, or CoQ10, or EDTA were added to 300
mM Ammonium-Oxalate hydration media to the final concentrations 100
mM (NAC), or 36 and 100 mM (AA), 2 mM or 20 mM (AP), 1 mM (CoQ10),
2 mM or 20 mM (EDTA).
[0359] The cold loading of doxorubicin into liposomes was performed
as follows: saline solution of doxorubicin hydrochloride (6 mg/mL)
was added to the liposomal nanosuspension at room temperature to
the final concentration 1 mg/mL (i.e. 0.936 mg of doxorubicin free
base per mL), gently inverted (2-3 times) and incubated at room
temperature for 10-20 min. After 10-20 min of incubation at room
temperature the mixture was: a) subjected to another TFF 5.times.
cycle with PBS pH 7.4 containing 6% of sucrose, and/or b) placed in
2-8.degree. C. refrigerator for 16 hrs, and then subjected to
another TFF 5.times. cycle with PBS pH 7.4 containing 6% of
sucrose. There was no notable difference observed between
doxorubicin release profiles of the liposomes loaded at RT, or RT
followed by 2-8.degree. C. overnight incubation. Data for RT
followed by 2-8.degree. C. overnight incubation are not shown.
[0360] Formulation composition is shown in Table 29.
TABLE-US-00053 TABLE 29 Formulation composition. Amounts of solids
used in formulations, W/W, % Doxo- rubicin Ratios Hydration Hydro-
Lipid/ Lot# Media PC DMPC FC P 188 chloride Drug 647-2-163 B
Ammonium Oxalate 65.50 16.38 11.46 4.91 1.75 50 647-2-170 B
Ammonium Tartrate 65.50 16.38 11.46 4.91 1.75 50 647-2-153 C
Ammonium Oxalate 65.50 16.38 11.46 4.91 1.75 50 Ascorbic acid (36
mM) 647-2-161 B Ammonium Oxalate 65.50 16.38 11.46 4.91 1.75 50
Ascorbic acid (100 mM) 647-2-165 B Ammonium Oxalate 65.50 16.38
11.46 4.91 1.75 50 NAC (100 mM) 647-2-172 B Ammonium Oxalate 65.50
16.38 11.46 4.91 1.75 50 Ascorbyl Palmitate (2 mM) 647-2-173 B
Ammonium Oxalate 65.50 16.38 11.46 4.91 1.75 50 Ascorbyl Palmitate
(20 mM) 647-2-175 B Ammonium Oxalate 65.50 16.38 11.46 4.91 1.75 50
Ubiquinone (1 mM) 647-2-190 B Ammonium Oxalate 65.50 16.38 11.46
4.91 1.75 50 EDTA (2 mM) 647-2-192 B Ammonium Oxalate 65.50 16.38
11.46 4.91 1.75 50 EDTA (20 mM) 647-2-193 B Ammonium Tartrate 65.50
16.38 11.46 4.91 1.75 50 EDTA (2 mM) 647-2-194 B Ammonium Tartrate
65.50 16.38 11.46 4.91 1.75 50 EDTA (20 mM)
[0361] Coarse suspension was prepared and MF processed at 10 KPSI
processing pressure. After 9-15 min of MF processing the particle
size (Z-average) reached .about.60-75 nm. A sample was collected
and sterile filtered into Nalgene flask. The particle size of
filtered nanosuspension was determined (Table 29a).
TABLE-US-00054 Table 29a. Summary of MF processing and resultant
emulsion parameters. Particle size Lot# MFD Z avrg, nm 647-2-163 B
24 MAY 16 66 647-2-170 B 23 JUN. 16 63 647-2-153 C 19 MAY 16 62
647-2-161 B 03 JUN. 16 62 647-2-165 B 16 JUN. 16 65 647-2-172 B 05
JUL. 16 63 647-2-173 B 06 JUL. 16 67 647-2-175 B 07 JUL. 16 62
647-2-190 B 28 JUL. 16 62 647-2-192 B 29 JUL. 16 62 647-2-193 B 01
AUG. 16 62 647-2-194 B 02 AUG. 16 62
[0362] The liposomes were subjected to TFF followed by remote
loading with doxorubicin, and another TFF cycle with PBS
sucrose.
[0363] The particle size of doxorubicin loaded liposomes is
presented in Table 29b.
TABLE-US-00055 TABLE 29b Particle size of doxorubicin loaded
liposomes. Particle size Lot# Loading Date Z avrg, nm 647-2-163 B
10 JUN. 16 67 647-2-170 B 23 JUN. 16 63 647-2-153 C 03 JUN. 16 63
647-2-161 B 03 JUN. 16 66 647-2-165 B 16 JUN. 16 69 647-2-172 B 05
JUL. 16 64 647-2-173 B 06 JUL. 16 67 647-2-175 B 07 JUL. 16 62
647-2-190 B 28 JUL. 16 62 647-2-192 B 29 JUL. 16 62 647-2-193 B 01
AUG. 16 62 647-2-194 B 02 AUG. 16 62
[0364] The amount of free (not encapsulated) doxorubicin was
determined within one week of manufacturing (Table 29c).
TABLE-US-00056 TABLE 29c Free doxorubicin content. Lot# % of Total
647-2-163 B 0.08 647-2-170 B 0.04 647-2-153 C 0.07 647-2-161 B 0.03
647-2-165 B 0.01 647-2-172 B 0.08 647-2-173 B 0.09 647-2-175 B 0.16
647-2-190 B 0.1 647-2-192 B 0.07 647-2-193 B 0.02 647-2-194 B
0.01
[0365] Determination of doxorubicin in liposomal suspension.
Followed doxorubicin loading liposomal suspension was subjected to
5.times.TFF to majorly remove free (not encapsulated) doxorubicin.
To determine total doxorubicin concentration at T0 (within one week
of MFD) TFF washed liposomes were diluted with methanol or IPA and
subjected to HPLC analysis. Doxorubicin content, percent of
recovery (doxorubicin content in liposomal suspension relative to
doxorubicin free base concentration used for remote loading), and
encapsulation efficiency (%) are presented in the Table 29d.
Encapsulation efficiency (%) represents the difference between
doxorubicin recovery (%) and free doxorubicin (%).
TABLE-US-00057 TABLE 29d Total doxorubicin content and
Encapsulation efficiency. Assay, HPLC Doxorubicin Doxorubicin
Encapsulated free base content used (Liposomal doxorubicin, % for
loading, Suspesion) Recovery, [Recovery,%] - Lot# .mu.g/mL .mu.g/mL
% [Free,%] 647-2-163 B 936 909 97 97 647-2-170 B 936 850 91 91
647-2-153 C 936 750 80 80 647-2-161 B 936 745 80 80 647-2-165 B 936
910 97 97 647-2-172 B 936 936 100 100 647-2-173 B 936 936 100 100
647-2-175 B 936 912 97 97 647-2-190 B 936 936 100 100 647-2-192 B
936 926 99 99 647-2-193 B 936 926 99 99 647-2-194 B 936 926 99
99
[0366] Liposomal doxorubicin release studies were carried out at
37.degree. C. within one week after manufacturing (Table 29e). For
each sample doxorubicin release was determined at 2, 4 and 8 hrs
time points.
TABLE-US-00058 TABLE 29e Doxorubicin release rate determined at
37.degree. C. pH 5, pH 7.4, Release, % Release, % Lot# 2 hrs 4 hrs
8 hrs 2 hrs 4 hrs 8 hrs 647-2-163 B 92 100 100 0 0 2 647-2-170 B 63
89 100 0 0 0 647-2-153 C 90 97 100 0 3 3 647-2-161B 92 100 100 0 1
4 647-2-165 B 82 90 100 1 5 13 647-2-172 B 80 87 94 0 0 0 647-2-173
B 69 71 75 0 0 0 647-2-175 B 80 94 94 0 0 0 647-2-190 B 77 88 94 0
0 0 647-2-192 B 6 9 18 0 0 0 647-2-193 B 33 50 64 0 0 0 647-2-194 B
3 5 12 0 0 0
[0367] Efficiency of doxorubicin encapsulation in presence or
absence of ascorbic acid or NAC was in the range 80-97% (Table
29d). Addition of ascorbic acid or NAC to ammonium-oxalate
hydration media did not have significant impact on the particle
size of the liposomes.+-.doxorubicin (Tables 289 and 29b), free
(not encapsulated or leaked) doxorubicin (Tables 29c), and more
importantly doxorubicin release profile (Table 29e) compared to
liposomes formed with oxalate or tartrate alone (Tables 29a-e).
[0368] Addition of 2 mM or 20 mM of EDTA, 2 mM or 20 mM of AP, or 1
mM of CoQ10 did not have any significant effect on particles size
of empty or doxorubicin loaded liposomes (Tables 29a and 29b), free
doxorubicin (29c), and encapsulation efficiency (Table 29d).
[0369] Complementing oxalate with 2 mM of AP (AP/Oxalate--1:150) or
1 mM of CoQ10 (CoQ10/Oxalate--1:300) did not affect the doxorubicin
release profile (Table 29e). Although some decrease of doxorubicin
release was observed upon addition of 20 mM AP (AP/Oxalate--1:15),
the .DELTA.pH7.4/5.0 release differential was sufficiently high
(Table 29e).
[0370] Complementing oxalate with 2 mM of EDTA (Oxalate/EDTA at
1:150 ratio) did not affect the doxorubicin release profile (Table
28e). Although notable decrease of doxorubicin release was observed
when 2 mM EDTA was added to 300 mM of tartrate
(tartrate/EDTA--1:150) hydration media (Table 29e), the
.DELTA.pH7.4/5.0 release differential was sufficiently high. In
contrast, very low doxorubicin release was observed when 20 mM of
EDTA were added to 300 mM of either oxalate or tartrate hydration
media (Table 29e).
[0371] These findings enable use of ascorbic acid, and/or NAC,
and/or ascorbyl palmitate, and/or CoQ10, and/or EDTA in combination
with oxalate or tartrate (preferred counter ions in some
embodiments) or citrate to alleviate oxidative stress during
processing and may result in more stable product with a longer
shelf life. Moreover, ascorbic acid can exert cardioprotective
effect to prevent or alleviate doxorubicin induced cardiac toxicity
[28-29, and 33]. In embodiments, this may result from unrestrained,
drug induced, cardiac reactive oxygen metabolism [28-29].
Investigations have shown that electron transfer after treatment
with doxorubicin is markedly enhanced in the heart, and leads to a
substantial increase in superoxide anion and hydrogen peroxide
formation in mitochondria and sarcoplasmic reticulum, two major
sites of cardiac damage from doxorubicin [28, 30-31]. Vitamin C
(ascorbic acid) is an antioxidant vitamin that has been shown to
antagonize the effects of reactive oxygen species-generating
antineoplastic drugs [29, 32-33]. It has been also demonstrated
that treatment of experimental animals with pharmacologic dosages
of the NAC selectively rescues the heart from the toxicity of
doxorubicin [28, 29, 34]. Chelating agents such as EDTA can reduce
generation of reactive oxygen species (ROS) by chelating transition
metal ions, therefore decrease damage to the cardiomyocyte membrane
and the risk of doxorubicin-related cardiomyopathy [36, 37].
Besides EDTA can increase the stability of liposomal formulations
by inhibiting metal catalyzed lipid oxidation.
[0372] Thus, complementing oxalate or tartrate (preferred counter
ion in some embodiments) or citrate with ascorbic acid
(AA/oxalate--1:8 or 1:3), and/or NAC (NAC/oxalate--1:3), and/or AP
(AP/oxalate--1:150 or 1:15), and/or CoQ10 (CoQ10/Oxalate--1:300),
and/or EDTA (EDTA/oxalate 1:150) did not have considerable negative
effect on .DELTA.pH7.4/5.0 doxorubicin release differential.
Therefore this powerful combination of optimized .DELTA.pH7.4/5.0
release differential and antioxidant(s)/chelators maybe efficacious
for the cancer patients and advantageous for alleviation of
cardiotoxic effect of free doxorubicin. Other ratios of ascorbic
acid or NAC to oxalate or tartrate or citrate that can be used
include 1:10 to 1:1. Other ratios of AP to oxalate or tartrate or
citrate that can be used include 1:300 to 1:10. Other ratios of
CoQ10 to oxalate or tartrate or citrate that can be used include
1:300 to 1:10. Other ratios of EDTA to oxalate or tartrate or
citrate that can be used include 1:300 to 1:5.
[0373] The ratios of a chelator (e.g. ascorbic acid (AA), or
N-Acetylcysteine (NAC), ascorbyl palmitate (AP), ubiquinone
(CoQ10), or ethylenediaminetetraacetic acid (EDTA)) to a counter
ion (e.g. oxalate, tartrate or citrate), i.e. a chelator/counter
ion ration can be about 1:1 to about 1:10,000.
[0374] Thus, in embodiments, the ratio of AA/oxalate can be about
1:1, about 1:2, about 1:3, about 1:5, about 1:8, about 1:10, about
1:15, about 1:20, about 1:50, about 1:100, about 1:200, about
1:300, about 1:500, about 1:1000, about 1:2000, about 1:5000, about
1:10,000 or more. In embodiments, the ratio of NAC/oxalate can be
about 1:1, about 1:2, about 1:3, about 1:5, about 1:8, about 1:10,
about 1:15, about 1:20, about 1:50, about 1:100, about 1:200, about
1:300, about 1:500, about 1:1000, about 1:2000, about 1:5000, about
1:10,000 or more. In embodiments, the ratio of AP/oxalate can be
about 1:1, about 1:2, about 1:3, about 1:5, about 1:8, about 1:10,
about 1:15, about 1:20, about 1:50, about 1:100, about 1:200, about
1:300, about 1:500, about 1:1000, about 1:2000, about 1:5000, about
1:10,000 or more. In embodiments, the ratio of CoQ10/oxalate can be
about 1:1, about 1:2, about 1:3, about 1:5, about 1:8, about 1:10,
about 1:15, about 1:20, about 1:50, about 1:100, about 1:200, about
1:300, about 1:500, about 1:1000, about 1:2000, about 1:5000, about
1:10,000 or more. In embodiments, the ratio of EDTA/oxalate can be
about 1:1, about 1:2, about 1:3, about 1:5, about 1:8, about 1:10,
about 1:15, about 1:20, about 1:50, about 1:100, about 1:200, about
1:300, about 1:500, about 1:1000, about 1:2000, about 1:5000, about
1:10,000 or more.
[0375] In embodiments, the ratio of AA/tartrate can be about 1:1,
about 1:2, about 1:3, about 1:5, about 1:8, about 1:10, about 1:15,
about 1:20, about 1:50, about 1:100, about 1:200, about 1:300,
about 1:500, about 1:1000, about 1:2000, about 1:5000, about
1:10,000 or more. In embodiments, the ratio of NAC/tartrate can be
about 1:1, about 1:2, about 1:3, about 1:5, about 1:8, about 1:10,
about 1:15, about 1:20, about 1:50, about 1:100, about 1:200, about
1:300, about 1:500, about 1:1000, about 1:2000, about 1:5000, about
1:10,000 or more. In embodiments, the ratio of AP/tartrate can be
about 1:1, about 1:2, about 1:3, about 1:5, about 1:8, about 1:10,
about 1:15, about 1:20, about 1:50, about 1:100, about 1:200, about
1:300, about 1:500, about 1:1000, about 1:2000, about 1:5000, about
1:10,000 or more. In embodiments, the ratio of CoQ10/tartrate can
be about 1:1, about 1:2, about 1:3, about 1:5, about 1:8, about
1:10, about 1:15, about 1:20, about 1:50, about 1:100, about 1:200,
about 1:300, about 1:500, about 1:1000, about 1:2000, about 1:5000,
about 1:10,000 or more. In embodiments, the ratio of EDTA/tartrate
can be about 1:1, about 1:2, about 1:3, about 1:5, about 1:8, about
1:10, about 1:15, about 1:20, about 1:50, about 1:100, about 1:200,
about 1:300, about 1:500, about 1:1000, about 1:2000, about 1:5000,
about 1:10,000 or more.
[0376] In embodiments, the ratio of AA/citrate can be about 1:1,
about 1:2, about 1:3, about 1:5, about 1:8, about 1:10, about 1:15,
about 1:20, about 1:50, about 1:100, about 1:200, about 1:300,
about 1:500, about 1:1000, about 1:2000, about 1:5000, about
1:10,000 or more. In embodiments, the ratio of NAC/citrate can be
about 1:1, about 1:2, about 1:3, about 1:5, about 1:8, about 1:10,
about 1:15, about 1:20, about 1:50, about 1:100, about 1:200, about
1:300, about 1:500, about 1:1000, about 1:2000, about 1:5000, about
1:10,000 or more. In embodiments, the ratio of AP/citrate can be
about 1:1, about 1:2, about 1:3, about 1:5, about 1:8, about 1:10,
about 1:15, about 1:20, about 1:50, about 1:100, about 1:200, about
1:300, about 1:500, about 1:1000, about 1:2000, about 1:5000, about
1:10,000 or more. In embodiments, the ratio of CoQ10/citrate can be
about 1:1, about 1:2, about 1:3, about 1:5, about 1:8, about 1:10,
about 1:15, about 1:20, about 1:50, about 1:100, about 1:200, about
1:300, about 1:500, about 1:1000, about 1:2000, about 1:5000, about
1:10,000 or more. In embodiments, the ratio of EDTA/citrate can be
about 1:1, about 1:2, about 1:3, about 1:5, about 1:8, about 1:10,
about 1:15, about 1:20, about 1:50, about 1:100, about 1:200, about
1:300, about 1:500, about 1:1000, about 1:2000, about 1:5000, about
1:10,000 or more.
[0377] Other counter ions and/or antioxidants, and/or chelators
that can be advantageous and/or used in combination with oxalate
and/or tartrate include citrate, and/or phytate and/or glutathione,
and/or vitamin e, and/or dexrazoxane, and/or deferoxamine.
[0378] Overall, obtained data demonstrated the effect of:
[0379] a) proper counter ions such as oxalate and tartrate that
determine physical state of intraliposomal doxorubicin aggregates
and optimal .DELTA.pH7.4/5.0 and .DELTA.pH7.4/6.7/6.0/5.0 release
differential;
[0380] b) lipid/drug ratio (preferred range is 20:-50:1 in some
embodiments) for selective response to pH change;
[0381] c) doxorubicin loading temperature (advantage of cold
loading for maximizing .DELTA.pH7.4/5.0 and
.DELTA.pH7.4/6.7/6.0/5.0 release differential);
[0382] for optimal pH dependent drug release profile and stable
performance of doxorubicin loaded liposomes.
[0383] Moreover, complementing of preferred counter ion(s) with
other counter ions, and/or antioxidants, and/or chelators may be
beneficial for the final product stability and its biological
performance.
Example 10: Irinotecan
[0384] Methods:
[0385] Fluorometry
[0386] All analyses were performed using a Molecular Devices
SpectraMax Gemini EM Fluorescence Plate Reader. SoftMax Pro
software controlled the device and was used for analysis and
reporting of values.
[0387] Standard stock solution of Irinotecan hydrochloride was
prepared in a 6% sucrose solution in sterile water for injection
(e.g., 6.0 mg Irinotecan hydrochloride powder in 1 mL water
containing 6% of Sucrose). Calibration standards were prepared by
diluting the stock solution in phosphate buffered saline, pH 7.4
and 5.0 to bracket the target concentration for analysis. The plate
reader temperature was set to 25.degree. C., and excitation and
emission wavelengths were set at 370 nm and 470 nm, respectively.
The linear response range was determined to be 0.5-4 .mu.g/mL of
Irinotecan hydrochloride. To remain in the linear response range,
the Irinotecan hydrochloride calibration standards and samples were
diluted accordingly.
[0388] To determine fluorescence of total Irinotecan in liposomal
formulation (Ft), the liposomes were ruptured by addition of Triton
X-100 to the final concentration 1%, mixed by inversion, and
incubated for 5-10 min prior to quantification.
[0389] To determine fluorescence of intraliposomal Irinotecan (Fi)
the liposomal formulation was subjected to fluorometric analysis
without pretreatment with Triton X-100.
[0390] Quantification of Irinotecan Release from Liposomal
Formulations
[0391] The method which employs a fluorescence dequenching
technique and relays it to fluorescence (liposomes ruptured with
Triton X-100) has been used for determination of Irinotecan
release. This approach is based on the fact that fluorescence of
Irinotecan quenched upon encapsulation into liposomes and markedly
increases upon Irinotecan release from liposomes. Therefore,
increase of fluorescence of intact liposomes (Fi) during the
incubation of sample in dissolution media represents release of
Irinotecan into the media. The difference between Fi values at
different time points and T0 relayed to Ft (fluorescence of
ruptured liposomes), and represents percent of released drug.
[0392] The study was carried out for the following time points: T0,
T2 hrs, T4 hrs, and T8 hrs. Individual samples were diluted in 2
separate diluents/dissolution medias; PBS pH 7.4 and PBS pH 5 by a
factor of 20 times (e.g. 100 .mu.L of sample+1.9 mL of diluent) to
simulate intravenous injection into mouse. For T0 time point
determination, liposomal formulations were diluted in PBS pH 7.4
and pH 5 buffers at .about.25.degree. C. The fluorescence of intact
liposomes (Fi) and total fluorescence of liposomes ruptured with
Triton X-100 (Ft) were measured immediately (within 10 min). The
plate reader temperature was set to 25.degree. C. and excitation
and emission wavelengths were set at 370 nm and 470 nm,
respectively.
[0393] Other liposomal samples were diluted 20.times. in PBS pH 7.4
and pH 5 buffers pre-warmed to 37.degree. C. (to simulate in vivo
temperature) and incubated for 2, 4, and 8 hrs at 37.degree. C. At
each time point fluorescence of intact liposomes (Fi) and total
fluorescence of liposomes ruptured with Triton X-100 (Ft) was
measured. The percent of drug release was quantified as
[(Fi_n-Fi_t0)/Ft_avrg)]*100%, where Fi_n-Fi measured at 2, 4, or 8
hrs, Fi_t0-Fi measured at T0, and Ft_avrg-average of Ft values
determined for T0 time point.
[0394] It is worth mentioning that total fluorescence (Ft)
increased significantly over 8 hrs of incubation of the liposomes
at pH 7.4. This observation was in agreement with reported
conversion of Irinotecan to carboxylate form in neutral medium
[26-27].
[0395] Particle Size Determination
[0396] All analyses were performed using a Malvern Zetasizer Nano
ZS with 4 mW He--Ne laser operating at a wavelength of 633 nm and a
detection angle of 173.degree.. Zetasizer software controlled the
device and was used for analysis and reporting of values.
[0397] The intensity-averaged particle diameters (Z-average) were
calculated from the cumulants analysis as defined in ISO 13321
(International Organization for Standardization 1996).
[0398] Samples are prepared using 30 .mu.L of liposomal formulation
in 1.5 mL of phosphate buffered saline (pH 7.4) and were
equilibrated to 25.degree. C. prior to analysis. Size measurements
were done in triplicates for each sample.
[0399] pH Measurements
[0400] All analyses were performed using a Mettler Toledo
SevenCompact pH meter with a Mettler Toledo InLab pH
microelectrode.
[0401] Preparation of Liposomes: Coarse Suspension Preparation.
[0402] Coarse suspension was prepared by dissolving PC, DMPC, FC,
and P188 in 10 mL of DCM at the ratios indicated in Table 29. The
mixture was dried under the stream of Nitrogen until viscous film
was formed. The film was further dried in vacuum oven overnight.
Next day dried lipid film was hydrated with 300 mM
Ammonium-Oxalate, or Ammonium-Sulfate, or Ammonium-Phosphate, or
Ammonium-Citrate buffer pre-warmed to 65.degree. C., and
immediately homogenized with a hand-held homogenizer for 2-3 min.
Tartaric acid was first titrated to pH 4.8-5.0 with NH.sub.4OH and
then used as hydration media. Particle size of coarse suspension
was determined and always was in the range of 800-1200 nm.
[0403] MF Processing
[0404] MF processing volume was always 100 ml unless specified
differently. MF processing pressure was always 10 KPSI.
Microfluidization of coarse suspension was performed in recycling
mode (return of the material into the feed reservoir) at controlled
(.ltoreq.65.degree. C.) temperature. Processing time was in 9-12
min range. The target particle size was 60-70 nm (Z-average).
[0405] Tangential Flow Filtration
[0406] Translucent nanosuspension was harvested from Microfluidizer
and subjected to tangential flow filtration (TFF) with 15-20.times.
volumes of PBS, pH 7.4. The purpose of TFF was to replace
ammonium-oxalate, or ammonium-sulfate, or ammonium-phosphate,
ammonium-citrate or ammonium-tartrate external, buffer by PBS and
to majorly remove ammonium from intraliposomal space. Ammonium in
external buffer was measured by using ammonium specific electrode.
TFF was stopped when ammonium concentration in external buffer was
.ltoreq.3 mM.
[0407] Remote Loading of Irinotecan
[0408] Irinotecan hydrochloride was dissolved in sterile water for
injection containing 6% sucrose to the final concentration 6 mg/mL.
Solution of irinotecan hydrochloride was added to the liposomal
nanosuspension at room temperature to the final concentration 1
mg/mL (e.g. 0.94 mg of irinotecan free base per mL), and incubated
at room temperature for 10-30 min with or without overnight
incubation at 2-8.degree. C., and then subjected to another TFF
5.times. cycle with PBS pH 7.4 containing 6% of sucrose. The
purpose of this TFF cycle was to wash out free (not encapsulated)
Irinotecan. There was no notable difference observed between
Irinotecan release profiles of the liposomes loaded at RT, or RT
followed by 2-8.degree. C. overnight incubation. Data for RT
followed by 2-8.degree. C. overnight incubation not shown.
[0409] Then liposomal nanosuspension was sterile filtered into
sterile Nalgene flask via 0.22 um filter. Particle size, pH, Fi,
Ft, and Irinotecan release profile were determined. The sterile
nanosuspension was aseptically dispensed into 2 mL pre-sterilized
vials, stoppered, and sealed. The vials were stored at 2-8.degree.
C.
Example 11: Cold Loading of Irinotecan into Fixed (50:1) Lipid/Drug
Ratio Liposomes
[0410] The counter ions that demonstrated ability to facilitate
loading of doxorubicin into 50:1 lipid/drug ratio liposomes were
used in this example.
[0411] Hydration media used: 300 mM ammonium-sulfate,
ammonium-oxalate, ammonium-phosphate, ammonium-citrate.
[0412] Tartaric acid, was first titrated with ammonium hydroxide to
pH 5 and then used as hydration media.
[0413] Cold remote loading was carried out with 1 mg/mL (i.e. 0.94
mg of irinotecan free base per mL) of irinotecan hydrochloride.
Formulation composition is shown in Table 30. All formulations were
prepared at 50:1 fixed lipid/drug ratio (Table 30).
TABLE-US-00059 TABLE 30 Formulation Composition. Amounts of solids
used in formulations, W/W, % Irinotecan Lipid/ Lot# Hydration Media
PC DMPC FC P 188 Hydrochloride Drug 647-2-142 A Ammonium-Sulfate
65.50 16.38 11.46 4.91 1.75 50 647-2-91 Ammonium-Oxalate 65.50
16.38 11.46 4.91 1.75 50 647-2-142 B Ammonium-Phosphate 65.50 16.38
11.46 4.91 1.75 50 647-2-161 A Ammonium-Tartrate 65.50 16.38 11.46
4.91 1.75 50 647-2-142 C Ammonium-Citrate 65.50 16.38 11.46 4.91
1.75 50
[0414] Coarse suspension was prepared and MF processed at 10 KPSI
processing pressure. After 9-12 min of MF processing the particle
size (Z-average) reached .about.60-65 nm. A sample was collected
and sterile filtered into Nalgene flask. The particle size of
filtered nanosuspension was determined (Table 31).
TABLE-US-00060 TABLE 31 Summary of MF processing and resultant
emulsion parameters. Particle size Lot# Counter Ion MFD Z avrg, nm
647-2-142 A Sulfate 28 MAR. 16 60 647-2-91 Oxalate 11 MAR. 16 65
647-2-142 B Phosphate 28 APR. 16 64 647-2-161 A Tartrate 17 MAY 16
65 647-2-142 C Citrate 03 MAY 16 62
[0415] The liposomes were subjected to TFF followed by remote
loading with Irinotecan, and another TFF cycle of Irinotecan loaded
liposomes with PBS containing 6% sucrose. Irinotecan hydrochloride
concentration used for remote loading: 1.0 mg/mL (Irinotecan free
base concentration: 0.94 mg/mL).
[0416] The particle size of Irinotecan loaded liposomes is
presented in Table 32.
TABLE-US-00061 TABLE 32 Particle size of irinotecan loaded
liposomes. Particle size Lot# Counter Ion MFD Z avrg, nm 647-2-142
A Sulfate 28 MAR. 16 64 647-2-91 Oxalate 11 MAR. 16 65 647-2-142 B
Phosphate 28 APR. 16 65 647-2-161 A Tartrate 17 MAY 16 65 647-2-142
C Citrate 03 MAY 16 63
[0417] Followed irinotecan loading liposomal suspension was
subjected to 5.times.TFF to majorly remove free (not encapsulated)
irinotecan. To determine liposomal irinotecan concentration at T0
(within one week of MFD) TFF washed liposomes were ruptured by
addition of Triton X-100 to the final concentration 1%, mixed by
inversion, and incubated for 5-10 min prior to quantification via
fluorometric analysis. Irinotecan content and percent of recovery
(Irinotecan content in liposomal suspension relative to irinotecan
free base concentration used for remote loading) are presented in
the Table 33.
TABLE-US-00062 TABLE 33 Liposomal irinotecan content. Assay, HPLC
Irinotecan Irinotecan content free base used (Liposomal Counter for
loading, Suspesion) Recovery, Lot# Ion .mu.g/mL .mu.g/mL %
647-2-142 A Sulfate 0.94 870 100 647-2-91 Oxalate 0.94 850 98
647-2-142 B Phosphate 0.94 850 98 647-2-161 A Tartrate 0.94 869 100
647-2-142 C Citrate 0.94 800 93
[0418] Liposomal irinotecan release studies were carried out at
37.degree. C. within one week after manufacturing (Table 34). For
each sample irinotecan release was determined at 2, 4 and 8 hrs
time points.
TABLE-US-00063 TABLE 34 Irinotecan release rate determined at TO
(within one week after MFD). pH 5, Release, % Lot# Counter Ion Pka
1 2 hrs 4 hrs 8 hrs 647-2-142 A Sulfate -3 0 3 3 647-2-91 Oxalate
1.27 95 95 95 647-2-142 B Phosphate 1.96 9 11 7 647-2-161 A
Tartrate 3.03 76 93 100 647-2-142 C Citrate 3.13 25 67 70
[0419] Particle size. It can be seen from the Tables 31 that
microfluidization of different liposomal formulation resulted in
similar particle sizes. Irinotecan loading did not result in any
significant increase of particle size for any of prepared
formulations (Tables 31-32).
[0420] Liposomal Irinotecan Content. The 100% recovery was observed
when sulfate was used as a counter ion (Table 33). Interestingly,
similar trend was observed for doxorubicin containing liposomes
(Table 9). The .about.100% recovery of liposomal Irinotecan after
5.times.TFF with PBS (pH 7.4) strongly suggest no leakage of
encapsulated irinotecan.
[0421] Liposomal Irinotecan release rate. Drug release studies were
carried out within one week after MFD. Since Irinotecan is highly
unstable at neutral pH and rapidly converts to carboxylate form in
neutral medium [26-27], the release experiments were carried out at
pH 5 only. It can be seen from the Table 34 that when oxalate or
tartrate were used as a counter ions, irinotecan release rate at pH
5 reached .about.100% and plateaued at 2-4 hrs time points. When
citrate was used as a counter ion the release at acidic pH was more
modest and reached 70% at 4-8 hrs. Formulations with sulfate and
phosphate--showed very low release at pH 5 (Table 34 and FIG. 22).
Overall, the obtained data on liposomal Irinotecan content and its
release at pH 5 indicate high .DELTA.pH7.4/5.0 release differential
achieved when oxalate, or tartrate, or citrate were used as a
counter ions.
[0422] Interestingly, that release of either doxorubicin or
irinotecan from sulfate, phosphate, or citrate containing liposomes
showed dependence on pKa1 value of corresponding counter ion (FIGS.
15 and 16). However, release of either doxorubicin or irinotecan
from oxalate or tartrate (preferred counter ions in some
embodiments) containing liposomes did not line up with pKa1 values
of tested counter ions (FIGS. 15 and 16). These data strongly
suggest contribution of unique physical state of
doxorubicin-oxalate and/or -tartrate as well as irinotecan-oxalate
and/or -tartrate intraliposomal aggregates into doxorubicin or
irinotecan release profiles, and are in agreement with c_TEM
analysis of doxorubicin-oxalate containing liposomes.
[0423] Overall, obtained data on irinotecan liposomes are in a good
agreement with results obtained for doxorubicin and support the
effect of oxalate and tartrate counter ions at preferred lipid/drug
ratio. Thus, in some embodiments, oxalate or tartrate counter ion
can be used for delivery of irinotecan. The data also suggest the
use of citrate as a counter ion for irinotecan in some
embodiments.
Example 12: Mitoxantrone
[0424] Methods.
[0425] Mitoxantrone Release from Liposomal Formulations
[0426] The study was carried out at 37.degree. C. for the following
time points: T0 and T24 hrs. Individual samples were diluted in 2
separate diluents/dissolution medias; PBS pH 7.4 and PBS pH 5 by a
factor of 20 times (e.g. 100 .mu.L of sample+1.9 mL of diluent) to
simulate intravenous injection into mouse. For T0 time point,
liposomal formulations were diluted in PBS pH 7.4 and pH 5 buffers
at .about.25.degree. C.
[0427] Other liposomal samples were diluted 20.times. in PBS pH 7.4
and pH 5 buffers pre-warmed to 37.degree. C. (to simulate in vivo
temperature) and incubated for 0 and T24 hrs at 37.degree. C. At
each time point the release of mitoxantrone from the liposomes was
assessed by visual observation: PBS/saline solution of mitoxantrone
has intense blue color and it turns purple upon encapsulation into
liposomes. Release of mitoxantrone from the liposomes results in
changing the color from purple to blue.
[0428] Particle Size Determination
[0429] All analyses were performed using a Malvern Zetasizer Nano
ZS with 4 mW He--Ne laser operating at a wavelength of 633 nm and a
detection angle of 173.degree.. Zetasizer software controlled the
device and was used for analysis and reporting of values.
[0430] The intensity-averaged particle diameters (Z-average) were
calculated from the cumulants analysis as defined in ISO 13321
(International Organization for Standardization 1996).
[0431] Samples are prepared using 30 .mu.L of liposomal formulation
in 1.5 mL of phosphate buffered saline (pH 7.4) and were
equilibrated to 25.degree. C. prior to analysis. Size measurements
were done in triplicates for each sample.
[0432] pH Measurements
[0433] All analyses were performed using a Mettler Toledo
SevenCompact pH meter with a Mettler Toledo InLab pH
microelectrode.
[0434] Coarse Suspension Preparation.
[0435] Coarse suspension was prepared by dissolving PC, DMPC, FC,
and P188 in 10 mL of DCM at the ratios indicated in Table 34. The
mixture was dried under the stream of Nitrogen until viscous film
was formed. The film was further dried in vacuum oven overnight.
Next day dried lipid film was hydrated with 300 mM ammonium-oxalate
buffer pre-warmed to 65.degree. C., and immediately homogenized
with a hand-held homogenizer for 2-3 min. Particle size of coarse
suspension was determined and always was in the range of 800-1200
nm.
[0436] MF Processing
[0437] MF processing volume was always 100 ml unless specified
differently. MF processing pressure was always 10 KPSI.
Microfluidization of coarse suspension was performed in recycling
mode (return of the material into the feed reservoir) at controlled
(.ltoreq.65.degree. C.) temperature. Processing time was in 9-12
min range. The target particle size was 60-70 nm (Z-average).
[0438] Tangential Flow Filtration
[0439] Translucent nanosuspension was harvested from Microfluidizer
and subjected to tangential flow filtration (TFF) with 15-20.times.
volumes of PBS, pH 7.4. The purpose of TFF was to replace
ammonium-oxalate, external buffer by PBS and to primarily remove
ammonium from intraliposomal and external space. Ammonium in
external buffer was measured by using ammonium specific electrode.
TFF was stopped when ammonium concentration in external buffer was
.ltoreq.3 mM.
[0440] Remote Loading of Mitoxantrone
[0441] Mitoxantrone hydrochloride was dissolved in saline to the
final concentration 6 mg/mL. Saline solution of mitoxantrone
hydrochloride was added to the liposomal nanosuspension in PBS, pH
7.4 to the final concentration 1 mg/mL, incubated at room
temperature for 30 minutes, and placed at 2-8.degree. C. After 16
hrs of incubation at 2-8.degree. C. the mixture was subjected to
another TFF 5.times. cycle with PBS pH 7.4 containing 6% of
Sucrose. The purpose of this TFF cycle was to remove free (not
encapsulated) mitoxantrone.
[0442] Then liposomal nanosuspension was sterile filtered into
sterile Nalgene flask via 0.22 um filter. Particle size and pH were
determined. The sterile nanosuspension was aseptically dispensed
into 2 mL pre-sterilized vials, stoppered, and sealed. The vials
were stored at 2-8.degree. C.
Example 13: Cold Loading of Mitoxantrone into Fixed (50:1)
Lipid/Drug Ratio Liposomes
[0443] Oxalate demonstrated ability to facilitate loading of
doxorubicin and irinotecan into 50:1 lipid/drug ratio liposomes.
Therefore, Oxalate was used in this example (Table 35).
TABLE-US-00064 TABLE 35 Formulation Composition. Amounts of solids
used in formulations, W/W, % Mitoxantrone Lipid/ Lot# Hydration
Media PC DMPC FC P 188 Dihydrochloride Drug 647-2-97 B
Ammonium-Oxalate 65.50 16.38 11.46 4.91 1.75 50
[0444] Coarse suspension was prepared and MF processed at 10 KPSI
processing pressure. After 9-12 min of MF processing the particle
size (Z-average) reached .about.60-70 nm. A sample was collected
and sterile filtered into Nalgene flask. The particle size of
filtered nanosuspension was determined (Table 36).
TABLE-US-00065 TABLE 36 Summary of MF processing and resultant
emulsion parameters. Processing Particle Pressure, size Lot#
Counter Ion MFD KPSI Z avrg, nm 647-2-97B Oxalate 11 MAR. 16 10
65
[0445] The liposomes were subjected to TFF followed by remote
loading with mitoxantrone, and another TFF cycle with PBS sucrose.
Mitoxantrone hydrochloride concentration used for remote loading:
1.0 mg/mL
[0446] The particle size of mitoxantrone loaded liposomes is
presented in Table 37.
TABLE-US-00066 TABLE 37 Particle size Mitoxantrone loaded
liposomes. Loading Particle size Lot# Counter Ion Date Z avrg, nm
647-2-97 B Oxalate 15 MAR. 16 67
[0447] Liposomal mitoxantrone release studies were carried out
within one week after manufacturing. At each time point (0 and 24
hrs) the release of mitoxantrone from the liposomes was assessed by
visual observation. It is worth mentioning that mitoxantrone
release from the liposomes was markedly slower compare to
doxorubicin and irinotecan.
[0448] Since mitoxantrone release study continued for 24 hrs, the
particle size of incubated samples was determined at T0 and T24 to
prove that integrity of the liposomes was not compromised. It can
be seen from the Table 37 that there was no dramatic change of the
particle size or aggregation observed after 24 hrs of incubation of
20 fold diluted sample at 37.degree. C.
TABLE-US-00067 TABLE 38 Particle size measurements during release
study. Particle size Lot# Time point pH Z avrg, nm 647-2-97 B 0 hrs
5 63 647-2-97 B 24 hrs 70 647-2-97 B 0 hrs 7 63 647-2-97 B 24 hrs
69
[0449] Discussion
[0450] Particle size. It can be seen from the Tables 36 and 37 that
microfluidization and loading of mitoxantrone resulted in similar
particle sizes comparable to doxorubicin (Tables 16 and 17) and
irinotecan (Tables 31 and 32).
[0451] Liposomal Mitoxantrone release. Drug release studies were
carried out within one week after MFD. It is worth mentioning that
PBS/saline solution of mitoxantrone has intense blue color and it
turns purple upon encapsulation into liposomes. It can be seen from
FIG. 23A that at T0 the color of mitoxantrone remains purple at
both pH 7.4 and 5 that is indicative of encapsulated Mitoxantrone.
When oxalate was used as counter ion, the color of the sample
turned blue after 24 hrs at pH 5.0, whereas it remained purple at
pH 7.4 (FIG. 23B). These data indicate release of mitoxantrone from
the liposomes at pH 5.0 and absence of release at pH 7.4.
[0452] Thus, overall obtained data demonstrate the effect of
oxalate and tartrate counter ions, lipid/drug ratio, and drug
loading conditions for preferential physical state of the
intraliposomal doxorubicin aggregates that result in optimal
.DELTA.pH7.4/5.0 release differential at least in some embodiments.
All these factors may contribute to efficient pH targeted delivery
of the weak bases chemotherapeutic agents to the tumor sites.
[0453] Summary of Experimental Findings
[0454] In embodiments, identification of proper counter ions
oxalate and tartrate (e.g. preferred counter ions in some
embodiments) that can form intraliposomal pharmaceutical salts
aggregates with weak bases, proper lipid/drug ratio (e.g. preferred
range 20:1 to 65:1 in some embodiments), and PL/FC ratio (e.g.
preferred range 1/1 to 4/1 in some embodiments) to maximize drug
release differential between neutral and acidic pH and to increase
liposomal stability in serum/blood for optimal pH targeted delivery
of weak bases chemotherapeutic agents (for various molecular
targets) to tumor site. The marked difference between in vitro
characteristics of oxalate or tartrate containing liposomes, and
other tested counter ions strongly suggests uniqueness of physical
state(s) of intraliposomal doxorubicin-oxalate or -tartrate
aggregates that evidently facilitates their dissolution in response
to the temperature and pH.
[0455] Only 5 (sulfate, oxalate, phosphate, tartrate, and citrate)
from 15 tested counter ions facilitated remote loading of
doxorubicin and formation of stable doxorubicin-salt containing
liposomes. Other 10 tested counter ions resulted in no loading of
doxorubicin into the liposomes and caused precipitation of the
liposomal material during overnight storage at 2-8.degree. C.
[0456] In embodiments, it was surprising finding that only oxalate
and tartrate yielded doxorubicin-containing liposomes with
desirable pH dependent drug release profile (.DELTA.pH 7.4/5.0 or
.DELTA.pH 7.4/6.7/6.0/5.0 release differential) at 37.degree. C.
(body temperature). Therefore, oxalate and tartrate were selected
as preferred counter-ions in some embodiments.
[0457] Increased cytotoxicity of doxorubicin-oxalate-containing
liposomes compared to DOXIL.RTM. (doxorubicin-sulfate) was observed
with two cancerous cell lines (Daudi and Hela cells)
[0458] Improved safety and efficacy of
doxorubicin-oxalate-containing liposomes compared to DOXIL.RTM.
(doxorubicin-sulfate) was observed in mouse B lymphoma model;
[0459] In some embodiments, it was surprising finding that oxalate
and tartrate containing liposomes can efficiently and rapidly
encapsulate doxorubicin at room temperature. It was also surprising
finding that in some embodiments, room temperature (RT) loading of
doxorubicin into oxalate and tartrate containing liposomes further
improved (maximized) .DELTA.pH 7.4/5.0 release differential
compared to oxalate- and/or tartrate-containing liposomes loaded at
70.degree. C. Other temperatures that can be used for remote
doxorubicin loading in oxalate containing liposomes include
2-8.degree. C. to 70.degree. C., with or without overnight
incubation at 2-8.degree. C.
[0460] In some embodiments, citrate was another counter ion that
showed notable improvement of .DELTA.pH 7.4/5.0 release
differential upon cold loading of doxorubicin. In contrast, room
temperature loading did not result in improvement of doxorubicin
release profile when sulfate and/or phosphate were used as counter
ions. Other temperatures that can be used for remote doxorubicin
loading in tartrate and citrate containing liposomes include
2-8.degree. C. to 70.degree. C. with or without overnight
incubation at 2-8.degree. C.
[0461] In embodiments, successful lyophilization of the doxorubicin
in presence of lactose and/or mannitol resulted in lyophilized
material that was readily reconstitutable in water for injection at
room temperature to the final concentration up to 6 mg/mL. In
embodiments, mixing of lyophilized and reconstituted doxorubicin
material with the novel oxalate and tartrate (preferred counter
ions in some embodiments) containing liposomes resulted in
efficient and rapid encapsulation of the doxorubicin at room
temperature. The resultant product demonstrated exceptional
.DELTA.pH 7.4/5.0 or .DELTA.pH 7.4/6.7/6.0/5.0 release differential
in some embodiments. In embodiments, this finding leads to
particular product presentation format having or consisting of two
vials: a vial with lyophilized doxorubicin and a vial with
liposomal vehicle suspension. Mixing (via simple inversion) the
reconstituted content of two vials at room temperature will yield
the final ready-for-use product within minutes--a very convenient
formulation to prepare at the bedside. Development of lyophilized
liposomal vehicle is also considered.
[0462] In embodiments, the effect of lipid/drug ratio for achieving
maximum .DELTA.pH 7.4/5.0 or .DELTA.pH 7.4/6.7/6.0/5.0 release
differential and optimal serum/blood stability was demonstrated for
both preferred counter ions oxalate and tartrate. In embodiments,
it was also surprising finding that liposomes exhibited high
.DELTA.pH 7.4/5.0 or .DELTA.pH 7.4/6.7/6.0/5.0 release differential
and increased serum/blood stability when lipid to drug ratio was
above 20 (preferred range is 20:1-65:1). In embodiments, when
lipid/drug ratio was .ltoreq.20, doxorubicin loading was poor and
leakage of the doxorubicin from liposomes was evident. Other ratios
that can be used include 20:1 to 100:1. Performed studies suggest
that in some embodiments, optimal lipid/drug ratios for achieving
maximum pH release differential are in the range from 20:1 to 65:1.
Other ratios that can be used include 10:1 to 100:1. In
embodiments, the effect of specific range of PL/FC ratios (e.g. 1/1
to 4/1) for optimal serum/blood stability and .DELTA.pH
7.4/6.7/6.0/5.0 release differential was demonstrated for preferred
counter ion oxalate.
[0463] Applicability of specified counter ions and optimized
Lipid/Drug ratio to achieving pH discriminative drug release
profile was demonstrated for -3 structurally different weak bases
cancer therapeutic agents: doxorubicin, irinotecan, and
mitoxantrone. Other ratios that can be used include 20:1 to
100:1.
[0464] Cryo transmission electron microscopy (cryo-TEM) led to
important finding that doxorubicin-oxalate aggregates appeared to
have non-crystalline nature and did not form tightly packed bundles
observed when sulfate, phosphate, or citrate were used as a counter
ions. This finding signifies unique physical state of the
intraliposomal doxorubicin-oxalate aggregates compared to
doxorubicin-sulfate, phosphate, and citrate aggregates, and is in a
good agreement with observed difference in drug release
profiles.
[0465] In embodiments, the poor .DELTA.pH 7.4/5.0 release
differential was observed at 25.degree. C. for all tested counter
ions, while at 37.degree. C. dramatic increase of .DELTA.pH7.4/5.0
release differential was observed with oxalate or tartrate, but not
with sulfate, phosphate, or citrate. The observed difference in
.DELTA.pH 7.4/5.0 release differential determined at 25.degree. C.
and 37.degree. C. might also indicates on more profound temperature
dependent transition of the physical state of doxorubicin-oxalate
or -tartrate intraliposomal aggregates compared to -sulfate,
-phosphate, or -citrate at least in some embodiments.
[0466] In embodiments, addition of P188 to the liposomal
formulation did not have any significant impact on particle size,
efficiency of doxorubicin encapsulation, and doxorubicin release
profile compared to liposomal formulation prepared with no P188.
However, P188 was used in liposomal formulations due to its
possible advantageous impact on biological performance of
drug-loaded liposomes [10-11, 16-19] in some embodiments.
[0467] In embodiments, complementing preferred counter ions oxalate
and/or tartrate with ascorbic acid (e.g. AA/oxalate--1:8 or 1:3),
and/or NAC (e.g. NAC/oxalate--1:3), and/or ascorbyl palmitate (e.g.
AP/oxalate--1:150 or 1:15), and/or CoQ10 (e.g.
CoQ10/oxalate--1:300), and/or EDTA (e.g. EDTA/oxalate 1:150) did
not show considerable negative effect on doxorubicin release
profile compared to liposomes containing oxalate or tartrate alone.
In embodiments, this finding enables use of ascorbic acid and/or
NAC, and/or ascorbyl palmitate, and/or CoQ10, and/or EDTA in
combination with oxalate or tartrate. In embodiments, citrate can
be used with any above-listed chelators in any ratio. In
embodiments, addition of antioxidants and/or chelators can
alleviate oxidative stress during liposomes processing, and
therefore may be beneficial for the final product stability.
Moreover, in embodiments, the combination of optimized
.DELTA.pH7.4/5.0 release differential and antioxidant(s) and/or
chelators may be advantageous for the final product biological
performance. It has been shown that ascorbic acid, NAC, and EDTA
exert cardioprotective effect alleviating doxorubicin induced
cardiac toxicity [28-29, 33, 36-37]. In embodiments, other ratios
of PA to oxalate or tartrate that can be used include 1:300 to
1:10. Other ratios of CoQ10 to oxalate or tartrate that can be used
include 1:300 to 1:10. Other ratios of EDTA to oxalate or tartrate
that can be used include 1:300 to 1:5. Other counter ions and/or
antioxidants, and/or chelators that can be advantageous and/or used
in combination with oxalate and/or tartrate include citrate, and/or
phytate, and/or glutathione, and/or vitamin e, and/or dexrazoxane,
and/or deferoxamine.
[0468] In embodiments, it was surprising finding that structurally
different chemotherapeutic agents irinotecan and mitoxantrone
(Table 2) demonstrated similar to doxorubicin pH discriminative
release profile when oxalate and/or tartrate were used as a counter
ions at 50:1 lipid/drug ratio (other ratios that can be used
include 20:1 to 100:1). It is worth mentioning that doxorubicin,
irinotecan, and mitoxantrone are weak bases (Table 2). Thus, in
embodiments, basicity of chemotherapeutic agents, proper counter
ions, optimal lipid/drug ratio, and loading conditions may
contribute to pH discriminative drug release and efficient delivery
of weak bases chemotherapeutic agents (Table 3) to the tumor.
[0469] In embodiments, release of either doxorubicin or irinotecan
from sulfate, phosphate, or citrate containing liposomes showed
dependence on pKa1 value of corresponding counter ion. However,
release of either doxorubicin or irinotecan from oxalate or
tartrate (preferred counter ions in some embodiments) containing
liposomes did not line up with pKa1 values of tested counter ions.
These data may suggest contribution of unique physical state of
doxorubicin-oxalate and -tartrate intraliposomal aggregates into
doxorubicin release profile, and are in agreement with c_TEM
analysis of doxorubicin-oxalate containing liposomes.
Embodiments
[0470] Embodiment 1. A pharmaceutical composition comprising a
liposome, the liposome encompassing a weakly basic anticancer
compound and an acid or salt thereof, wherein the acid is oxalic
acid or tartaric acid.
[0471] Embodiment 2. The pharmaceutical composition of Embodiment
1, wherein the weakly basic anticancer compound is doxorubicin,
irinotecan, mitoxantrone or a combination thereof.
[0472] Embodiment 3. The pharmaceutical composition of Embodiment 1
or 2, wherein the liposome comprises a poloxamer.
[0473] Embodiment 4. The pharmaceutical composition of Embodiment
3, wherein the poloxamer is poloxamer 188.
[0474] Embodiment 5. The pharmaceutical composition of any one of
Embodiments 1-3, wherein the liposome comprises a plurality of
lipid compounds and the weight ratio of the plurality of lipids to
the weakly basic anticancer agent is at least 20/1.
[0475] Embodiment 6. The pharmaceutical composition of any one of
Embodiments 1-3, wherein the liposome comprises a plurality of
lipid compounds and the weight ratio of the plurality of lipids to
the weakly basic anticancer agent is about 20/1 to about 100/1.
[0476] Embodiment 7. The pharmaceutical composition of any one of
Embodiments 1-3, wherein the liposome comprises a plurality of
lipid compounds and the weight ratio of the plurality of lipids to
the weakly basic anticancer agent is 20/1 to about 50/1.
[0477] Embodiment 8. The pharmaceutical composition of any one of
Embodiments 1-7, wherein the weakly basic anticancer compound is
substantially released from the liposome only at acidic pH.
[0478] Embodiment 9. The pharmaceutical composition of Embodiment
8, wherein at least 40% of the weakly basic anticancer compound is
released from the liposome at pH 5 under standard assay conditions
and wherein less than 5% of the weakly basic anticancer compound is
released from the liposome at pH 7.4 under standard assay
conditions.
[0479] Embodiment 10. The pharmaceutical composition of Embodiment
8, wherein at least 80% of the weakly basic anticancer compound is
released from the liposome at pH 5 under standard assay conditions
and wherein less than 5% of the weakly basic anticancer compound is
released from the liposome at pH 7.4 under standard assay
conditions.
[0480] Embodiment 11. The pharmaceutical composition of Embodiment
9, wherein the standard assay conditions comprise 20.times.
dilution in PBS buffer pH 7.4 or pH 5 and incubation at 25.degree.
C. or 37.degree. C. for 2, 4, or 8 hours.
[0481] Embodiment 12. The pharmaceutical composition of any one of
Embodiments 1-11, wherein the liposome is substantially
spherical.
[0482] Embodiment 13. The pharmaceutical composition of any one of
Embodiments 1-12, wherein the pharmaceutical composition comprises
a plurality of the liposome having mean longest dimension of about
60-80 nm determined by the intensity-averaged particle diameters
(Z-average) measured by Dynamic Light Scattering.
[0483] Embodiment 14. The pharmaceutical composition of any one of
Embodiments 1-12, wherein the pharmaceutical composition comprises
a plurality of the liposome having the mean longest dimension of
about 10-30 nm determined by the number-based particle diameters
measured by Dynamic Light Scattering.
[0484] Embodiment 15. The pharmaceutical composition of any one of
Embodiments 1-12, wherein the pharmaceutical composition comprises
a plurality of the liposome having a mean longest dimension from
10-30 nm determined by Cryo-Transmission Electron Microscopy.
[0485] Embodiment 16. The pharmaceutical composition of any of
Embodiments 1-14, wherein the liposome comprises about 500-1000
.mu.g/mL of the weakly basic anticancer compound and an acid or
salt thereof.
[0486] Embodiment 17. The pharmaceutical composition of any of
Embodiments 1-14, wherein the liposome comprises about 700-850
.mu.g/mL of the weakly basic anticancer compound and an acid or
salt thereof.
[0487] Embodiment 18. The pharmaceutical composition of Embodiment
1-17, wherein the liposome comprises a plurality of the weakly
basic anticancer compound forming a disorganized non-crystalline
aggregate.
[0488] Embodiment 19. The pharmaceutical composition of any of
Embodiments 1-18, wherein the liposome comprises a plurality of the
weakly basic anticancer compound and retains greater than 90% of
the plurality of weakly basic anticancer compound after 40 days
when stored at 2-8.degree. C. under standard storage
conditions.
[0489] Embodiment 20. The pharmaceutical composition of Embodiment
1-2 or 4-19, wherein the liposome does not comprise a cholesterol
or a poloxamer 188.
[0490] Embodiment 21. The pharmaceutical composition of Embodiment
1, wherein the liposome does not comprise an acidic organic
compound other than oxalic acid, tartaric acid, or salts
thereof.
[0491] Embodiment 22. The pharmaceutical composition of any one of
Embodiments 1-21, wherein the drug loaded liposome is formed by
loading the weakly basic anticancer compound into an unloaded
liposome containing an encapsulated acid or salt thereof, followed
by incubation at a room temperature.
[0492] Embodiment 23. A method for preparing a liposome
encompassing a weakly basic anticancer compound and an acid or salt
thereof, wherein the acid is oxalic acid or tartaric acid, the
method comprising mixing a solution of the weakly basic anticancer
compound with a suspension comprising the liposomes containing an
encapsulated acid or salt thereof and incubating the solution of
the weakly basic anticancer compound with the suspension comprising
the liposomes containing an encapsulated acid or salt thereof.
[0493] Embodiment 24. The method of Embodiment 23, wherein about
85-100% of the weakly basic anticancer compound used in mixing with
a suspension comprising the liposomes containing an encapsulated
acid or salt thereof is retained within the liposomes.
[0494] Embodiment 25. The method of Embodiment 23, wherein about
95-100% of the weakly basic anticancer compound used in mixing with
a suspension comprising the liposomes containing an encapsulated
acid or salt thereof is retained within the liposomes.
[0495] Embodiment 26. The method of any of Embodiments 23-26,
wherein the incubating step occurs at room temperature.
[0496] Embodiment 27. The method of Embodiment 25, wherein the
incubating step is about 10-30 minutes.
[0497] Embodiment 28. The method of Embodiment 25, wherein the
incubating step is about 5-25 minutes.
[0498] Embodiment 29. A kit comprising a first vial comprising a
weakly basic anticancer compound, and a second vial with a
suspension comprising the liposomes containing an encapsulated acid
or salt thereof.
[0499] Embodiment 30. The kit of Embodiment 29, wherein the weakly
basic anticancer compound of the first vial is a lyophilized weakly
basic anticancer.
[0500] Embodiment 31. The kit of Embodiment 29, wherein the
liposome suspension of the second vial is an aqueous suspension of
liposomes containing an encapsulated acid or salt thereof.
[0501] Embodiment 32. A method of using the kit of any one of
Embodiments 28-31, comprising mixing the contents of the first vial
with the contents of the second vial.
[0502] Embodiment 33. The method of Embodiment 32, wherein the
mixing is at room temperature.
[0503] Embodiment 34. The method of any of Embodiments 23-25,
wherein the incubating step occurs at Room temperature followed by
incubation at 2-8.degree. C.
[0504] Embodiment 35. The method of Embodiment 34, wherein the
incubating step at RT is about 10-30 minutes.
[0505] Embodiment 36. The method of any of Embodiments 34, wherein
the incubating step at 2-8.degree. C. is about 60-960 minutes.
[0506] Embodiment 37. The method of any of Embodiments 23-35,
wherein the incubating step occurs at 70.degree. C.
[0507] Embodiment 38. The method of Embodiment 37, wherein the
incubating step is about 10-30 minutes.
[0508] Embodiment 39. A method for preparing a liposome
encompassing a weakly basic anticancer compound and an acid or salt
thereof, wherein the acid is citric acid, the method comprising
mixing a solution of the weakly basic anticancer compound with a
suspension comprising the liposomes containing an encapsulated acid
or salt thereof and incubating the solution of the weakly basic
anticancer compound with the suspension comprising the liposomes
containing an encapsulated acid or salt thereof.
[0509] Embodiment 40. A pharmaceutical composition comprising a
liposome, the liposome encompassing a weakly basic anticancer
compound and an acid or salt thereof, wherein the acid is citric
acid and wherein the liposome comprises a plurality of lipid
compounds and the weight ratio of the plurality of lipids to the
weakly basic anticancer agent is at least 20/1.
[0510] Embodiment 41. A pharmaceutical composition comprising a
liposome, the liposome encompassing a weakly basic anticancer
compound and an acid or salt thereof, wherein the acid is oxalic
acid or tartaric acid.
[0511] Embodiment 42. The pharmaceutical composition of Embodiment
41, wherein the weakly basic anticancer compound is doxorubicin,
irinotecan, mitoxantrone or a combination thereof.
[0512] Embodiment 43. The pharmaceutical composition of Embodiment
41, wherein the liposome comprises a poloxamer.
[0513] Embodiment 44. The pharmaceutical composition of Embodiment
43, wherein the poloxamer is poloxamer 188.
[0514] Embodiment 45. The pharmaceutical composition of Embodiment
41, wherein the liposome comprises a plurality of lipids and the
weight ratio of the lipids to the weakly basic anticancer compound
and an acid or salt thereof is at least 10/1.
[0515] Embodiment 46. The pharmaceutical composition of Embodiment
41, wherein the liposome comprises a plurality of lipids and the
weight ratio of the lipids to the weakly basic anticancer compound
and an acid or salt thereof is about 10/1 to about 100/1.
[0516] Embodiment 47. The pharmaceutical composition of Embodiment
41, wherein the liposome comprises a plurality of lipids and the
weight ratio of the lipids to the weakly basic anticancer compound
and an acid or salt thereof is 20/1 to about 50/1.
[0517] Embodiment 48. The pharmaceutical composition of Embodiment
41, wherein the liposome comprises a plurality of free
cholesterols.
[0518] Embodiment 49. The pharmaceutical composition of Embodiment
41, wherein the liposome comprises a plurality of
phospholipids.
[0519] Embodiment 50. The pharmaceutical composition of Embodiment
48, wherein said pharmaceutical composition comprises phospholipids
and a molar ratio of the phospholipids to the free cholesterols is
at least 0.5/1.
[0520] Embodiment 51. The pharmaceutical composition of Embodiment
48, wherein said pharmaceutical composition comprises phospholipids
and a molar ratio of the phospholipids to the free cholesterols is
at least 0.5/1 to about 4/1.
[0521] Embodiment 52. The pharmaceutical composition of Embodiment
48, wherein said pharmaceutical composition comprises phospholipids
and a molar ratio of the phospholipids to the free cholesterols is
in the range of about 0.86/1 to about 3.68/1.
[0522] Embodiment 53. The pharmaceutical composition of Embodiment
41, wherein the weakly basic anticancer compound is substantially
released from the liposome at pH<7.4.
[0523] Embodiment 54. The pharmaceutical composition of Embodiment
41, wherein at least 40% of the weakly basic anticancer compound is
released from the liposome at pH 5 under standard assay
conditions.
[0524] Embodiment 55. The pharmaceutical composition of Embodiment
41, wherein less than 5% of the weakly basic anticancer compound is
released from the liposome at pH 7.4 under standard assay
conditions.
[0525] Embodiment 56. The pharmaceutical composition of Embodiment
41, wherein at least 80% of the weakly basic anticancer compound is
released from the liposome at pH 5 under standard assay
conditions.
[0526] Embodiment 57. The pharmaceutical composition of Embodiment
41, wherein at least 10% of the weakly basic anticancer compound is
released from the liposome at about pH 6.0 under standard assay
conditions.
[0527] Embodiment 58. The pharmaceutical composition of Embodiment
41, wherein at least 50% of the weakly basic anticancer compound is
released from the liposome at pH 6.0 under standard assay
conditions.
[0528] Embodiment 59. The pharmaceutical composition of Embodiment
41, wherein at least 7% of the weakly basic anticancer compound is
released from the liposome at pH 6.7 under standard assay
conditions.
[0529] Embodiment 60. The pharmaceutical composition of Embodiment
41, wherein at least 30% of the weakly basic anticancer compound is
released from the liposome at pH 6.7 under standard assay
conditions.
[0530] Embodiment 61. The pharmaceutical composition of Embodiment
41, wherein the liposome comprises a plurality of weakly basic
anticancer compounds and retains greater than 35-50% of the
plurality of weakly basic anticancer compound for up to about 8 hrs
of incubation in serum or blood when tested under standard assay
conditions.
[0531] Embodiment 62. The pharmaceutical composition of any one of
Embodiments 54-61, wherein the standard assay conditions comprise
20.times. or 50.times. dilution of the liposomes in PBS buffer.
[0532] Embodiment 63. The pharmaceutical composition of any one of
Embodiments 54-61, wherein the standard assay conditions comprise
incubation at pH 7.4, pH 6.7, pH 6.0 or pH 5.0.
[0533] Embodiment 64. The pharmaceutical composition of any one of
Embodiments 54-61, wherein the standard assay conditions comprise
incubation at about 25.degree. C. or about 37.degree. C.
[0534] Embodiment 65. The pharmaceutical composition of any one of
Embodiments 14-21, wherein the standard assay conditions comprise
incubation for about 2, about 4 or about 8 hours.
[0535] Embodiment 66. The pharmaceutical composition of any one of
Embodiments 54-61, wherein the standard assay conditions comprise
50.times. dilution of the liposomes in serum or blood and
incubation at 37.degree. C. for 2, 4, or 8 hours at a physiological
pH (pH 7.4).
[0536] Embodiment 67. The pharmaceutical composition of Embodiment
41, wherein the liposome is substantially spherical.
[0537] Embodiment 68. The pharmaceutical composition of Embodiment
41, wherein the pharmaceutical composition comprises a plurality of
the liposome having a mean longest dimension of about 60-80 nm
determined by the intensity-averaged particle diameters (Z-average)
measured by Dynamic Light Scattering.
[0538] Embodiment 69. The pharmaceutical composition of Embodiment
41, wherein the pharmaceutical composition comprises a plurality of
the liposome having a mean longest dimension of about 10-30 nm
determined by the number-based particle diameters measured by
Dynamic Light Scattering.
[0539] Embodiment 70. The pharmaceutical composition of Embodiment
41, wherein the pharmaceutical composition comprises a plurality of
the liposome having a mean longest dimension from 10-30 nm
determined by Cryo-Transmission Electron Microscopy.
[0540] Embodiment 71. The pharmaceutical composition of Embodiment
41, wherein the liposome comprises about 500-1000 .mu.g/mL of the
weakly basic anticancer compound and an acid or salt thereof.
[0541] Embodiment 72. The pharmaceutical composition of Embodiment
41, wherein the liposome comprises about 700-850 .mu.g/mL of the
weakly basic anticancer compound and an acid or salt thereof.
[0542] Embodiment 73. The pharmaceutical composition of Embodiment
41, wherein the liposome comprises a plurality of the weakly basic
anticancer compound forming a disorganized non-crystalline
aggregate.
[0543] Embodiment 74. The pharmaceutical composition of Embodiment
41, wherein the liposome comprises a plurality of the weakly basic
anticancer compound and retains greater than 90% of the plurality
of weakly basic anticancer compound after 40 days when stored at
2-8.degree. C. under standard storage conditions.
[0544] Embodiment 75. The pharmaceutical composition of Embodiment
41, wherein the liposome does not comprise a cholesterol or a
poloxamer 188.
[0545] Embodiment 76. The pharmaceutical composition of Embodiment
41, wherein the liposome does not comprise an acidic organic
compound other than oxalic acid, tartaric acid, or salts
thereof.
[0546] Embodiment 77. The pharmaceutical composition of Embodiment
41, wherein the liposome does not comprise the weakly basic
anticancer compound and an acid or salt thereof other than oxalic
acid, tartaric acid or salts thereof.
[0547] Embodiment 78. The pharmaceutical composition of Embodiment
41, wherein the liposome encompassing the weakly basic anticancer
compound and an acid or salt thereof is formed by loading the
weakly basic anticancer compound into an unloaded liposome
containing an encapsulated acid or salt thereof, followed by
incubation at a room temperature.
[0548] Embodiment 79. The pharmaceutical composition of Embodiment
38, wherein the unloaded liposome after 180 and/or 540 days of
storage at 2-8.degree. C. under standard storage conditions retains
greater than 90% of the weakly basic anticancer compound and an
acid or salt thereof upon loading.
[0549] Embodiment 80. The pharmaceutical composition of Embodiment
39, wherein about 40-80% of the loaded weakly basic anticancer
compound and an acid or salt thereof is released from the liposome
at pH 5.0 under standard assay conditions.
[0550] Embodiment 81. The pharmaceutical composition of Embodiment
39, wherein about 20-60% of the loaded weakly basic anticancer
compound and an acid or salt thereof is released from the liposome
at pH 6.0 under standard assay conditions.
[0551] Embodiment 82. The pharmaceutical composition of Embodiment
39, wherein about 7-30% of the loaded weakly basic anticancer
compound and an acid or salt thereof is released from the liposome
at pH 6.7 under standard assay conditions.
[0552] Embodiment 83. The pharmaceutical composition of Embodiment
39, wherein less than 5% of the weakly basic anticancer compound
and an acid or salt thereof is released from the liposome at pH 7.4
under standard assay conditions.
[0553] Embodiment 84. The pharmaceutical composition of claim 41
further comprising a compound selected from the group consisting of
ascorbic acid (AA), or N-Acetylcysteine (NAC), ascorbyl palmitate
(AP), ubiquinone (CoQ10), and ethylenediaminetetraacetic acid
(EDTA).
[0554] Embodiment 85. A method for preparing a liposome
encompassing a weakly basic anticancer compound and an acid or salt
thereof, wherein the acid is oxalic acid or tartaric acid, the
method comprising mixing a solution comprising a weakly basic
anticancer compound with a suspension comprising a plurality of
liposomes, wherein each of the liposomes comprise an oxalic acid or
tartaric acid or salts thereof.
[0555] Embodiment 86. The method of Embodiment 85, wherein further
comprising incubating the solution and the suspension.
[0556] Embodiment 87. The method of Embodiment 85, wherein about
85-100% of the weakly basic anticancer compound is incorporated
within the plurality of liposomes subsequent to said mixing.
[0557] Embodiment 88. The method of Embodiment 85, wherein about
95-100% of the weakly basic anticancer compound is incorporated
within the plurality of liposomes subsequent to said mixing.
[0558] Embodiment 89. The method of Embodiment 86, wherein the
incubating step occurs at room temperature (RT).
[0559] Embodiment 90. The method of Embodiment 89, wherein the
incubating step is about 10-30 minutes.
[0560] Embodiment 91. The method of Embodiment 89, wherein the
incubating step is about 5-25 minutes.
[0561] Embodiment 92. The method of Embodiment 85, wherein the
incubating step occurs at room temperature (RT) followed by
incubation at 2-8.degree. C.
[0562] Embodiment 93. The method of Embodiment 92, wherein the
incubating step at RT is about 10-30 minutes.
[0563] Embodiment 94. The method of Embodiment 92, wherein the
incubating step at 2-8.degree. C. is about 60-960 minutes.
[0564] Embodiment 95. The method of Embodiment 86, wherein the
incubating step occurs at 70.degree. C.
[0565] Embodiment 96. The method of Embodiment 95, wherein the
incubating step is about 10-30 minutes.
[0566] Embodiment 97. A kit comprising a first container comprising
a weakly basic anticancer compound, and a second container
comprising a suspension, said suspension comprising a plurality of
liposomes, wherein each of said plurality of liposome comprise an
acid or salt of said weakly basic anticancer compound, wherein the
acid is oxalic acid or tartaric acid.
[0567] Embodiment 98. The kit of Embodiment 97, wherein the weakly
basic anticancer compound is a lyophilized weakly basic anticancer
compound.
[0568] Embodiment 99. The kit of Embodiment 97, wherein the
suspension is an aqueous suspension.
[0569] Embodiment 100. A method of using the kit of Embodiment 97,
comprising mixing the contents of the first container with the
contents of the second container.
[0570] Embodiment 101. The method of Embodiment 100, wherein the
mixing is at room temperature.
[0571] Embodiment 102. A method for preparing a liposome comprising
a weakly basic anticancer compound and an acid or salt thereof,
wherein the acid is citric acid, the method comprising mixing a
solution comprising the weakly basic anticancer compound with a
suspension comprising a plurality of liposomes, wherein each of aid
plurality of liposome comprise an acid or salt thereof.
[0572] Embodiment 103. The method of Embodiment 102 further
comprising incubating the solution with the suspension.
[0573] Embodiment 104. The method of Embodiment 102, wherein the
incubating step occurs at room temperature (RT).
[0574] Embodiment 105. The method of Embodiment 104, wherein the
incubating step is about 10-30 minutes.
[0575] Embodiment 106. The method of Embodiment 104, wherein the
incubating step is about 5-25 minutes.
[0576] Embodiment 107. The method of Embodiment 103, wherein the
incubating step occurs at room temperature (RT) followed by
incubation at 2-8.degree. C.
[0577] Embodiment 108. The method of Embodiment 107, wherein the
incubating step at RT is about 10-30 minutes.
[0578] Embodiment 109. The method of Embodiment 107, wherein the
incubating step at 2-8.degree. C. is about 60-960 minutes.
[0579] Embodiment 110. The method of Embodiment 103, wherein the
incubating step occurs at 70.degree. C.
[0580] Embodiment 111. The method of Embodiment 110, wherein the
incubating step is about 10-30 minutes.
[0581] Embodiment 112. A pharmaceutical composition comprising a
liposome, the liposome comprising a weakly basic anticancer
compound and an acid or salt thereof, wherein the acid is citric
acid.
[0582] Embodiment 113. The pharmaceutical composition of Embodiment
112, wherein the liposome comprises a plurality of lipids and the
weight ratio of the plurality of lipids to the weakly basic
anticancer compound and an acid or salt thereof is at least 10 to
1.
[0583] Embodiment 114. The pharmaceutical composition of Embodiment
112, wherein the liposome comprises a plurality of free
cholesterols.
[0584] Embodiment 115. The pharmaceutical composition of Embodiment
114, wherein the liposome comprises phospholipids, wherein the
molar ratio of the phospholipid to the free cholesterols is at
least 1 to 1.
[0585] Embodiment 116. The pharmaceutical composition of Embodiment
112 further comprising a compound selected from the group
consisting of ascorbic acid (AA), or N-Acetylcysteine (NAC),
ascorbyl palmitate (AP), ubiquinone (CoQ10), and
ethylenediaminetetraacetic acid (EDTA).
[0586] Embodiment 117. A method of treating a cancer in a subject,
the method comprising: administering an effective amount of the
pharmaceutical composition of Embodiment 40 to the subject in need
of the treatment.
[0587] Embodiment 118. The method of Embodiment 117, wherein the
weakly basic anticancer compound is doxorubicin, irinotecan,
mitoxantrone or a combination thereof.
[0588] Embodiment 119. The method of Embodiment 117, wherein the
liposome comprises a poloxamer.
[0589] Embodiment 120. The method of Embodiment 117, wherein the
poloxamer is poloxamer 188.
[0590] Embodiment 121. The method of Embodiment 117, wherein the
liposome comprises lipids and the weight ratio of the lipids to the
weakly basic anticancer compound is at least 10 to 1.
[0591] Embodiment 122. The method of Embodiment 117, wherein the
liposome comprises a lipids and the weight ratio of the lipids to
the weakly basic anticancer compound is about 10 to 1 to about 100
to 1.
[0592] Embodiment 123. The method of Embodiment 117, wherein the
liposome comprises a plurality of lipids and the weight ratio of
the lipids to the weakly basic anticancer compound is about 20 to 1
to about 50 to 1.
[0593] Embodiment 124. The method of Embodiment 117, wherein the
liposome comprises a plurality of free cholesterols.
[0594] Embodiment 125. The method of Embodiment 117, wherein the
liposome comprises a plurality of phospholipids.
[0595] Embodiment 126. The method of Embodiment 125, wherein said
liposome comprises phospholipids and a molar ratio of the
phospholipids to the free cholesterols is at least 0.5 to 1.
[0596] Embodiment 127. The method of Embodiment 125, wherein said
liposome comprises phospholipids and a molar ratio of the
phospholipids to the free cholesterols is at least 0.5 to 1 to
about 4 to 1.
[0597] Embodiment 128. The method of Embodiment 125, wherein said
liposome comprises phospholipids and a molar ratio of the
phospholipids to the free cholesterols is in the range of about
0.86 to 1 to about 3.68 to 1.
[0598] Embodiment 129. The method of Embodiment 117, wherein the
weakly basic anticancer compound is substantially released from the
liposome at pH<7.4.
[0599] Embodiment 130. The method of Embodiment 117, wherein at
least 40% of the weakly basic anticancer compound is released from
the liposome at pH 5 under standard assay conditions.
[0600] Embodiment 131. The method of Embodiment 117, wherein less
than 5% of the weakly basic anticancer compound is released from
the liposome at pH 7.4 under standard assay conditions.
[0601] Embodiment 132. The method of Embodiment 117, wherein at
least 80% of the weakly basic anticancer compound is released from
the liposome at pH 5 under standard assay conditions.
[0602] Embodiment 132. The method of Embodiment 117, wherein at
least 10% of the weakly basic anticancer compound is released from
the liposome at about pH 6.0 under standard assay conditions.
[0603] Embodiment 134. The method of Embodiment 117, wherein at
least 50% of the weakly basic anticancer compound is released from
the liposome at pH 6.0 under standard assay conditions.
[0604] Embodiment 135. The method of Embodiment 117, wherein at
least 7% of the weakly basic anticancer compound is released from
the liposome at pH 6.7 under standard assay conditions.
[0605] Embodiment 136. The method of Embodiment 117, wherein at
least 30% of the weakly basic anticancer compound is released from
the liposome at pH 6.7 under standard assay conditions.
[0606] Embodiment 137. The method of Embodiment 117, wherein the
liposome comprises a plurality of weakly basic anticancer compounds
and retains greater than 35-50% of the plurality of weakly basic
anticancer compound for up to about 8 hrs of incubation in serum or
blood when tested under standard assay conditions.
[0607] Embodiment 138. The method of any one of Embodiments
130-137, wherein the standard assay conditions comprise 20.times.
or 50.times. dilution of the liposomes in PBS buffer.
[0608] Embodiment 139. The method of any one of Embodiments
130-137, wherein the standard assay conditions comprise incubation
at pH 7.4, pH 6.7, pH 6.0 or pH 5.0.
[0609] Embodiment 140. The method of any one of Embodiments
130-137, wherein the standard assay conditions comprise incubation
at about 25.degree. C. or about 37.degree. C.
[0610] Embodiment 141. The method of any one of Embodiments
130-137, wherein the standard assay conditions comprise incubation
for about 2, about 4 or about 8 hours.
[0611] Embodiment 142. The method of any one of Embodiments
130-137, wherein the standard assay conditions comprise 50.times.
dilution of the liposomes in serum or blood and incubation at
37.degree. C. for 2, 4, or 8 hours at a physiological pH (pH
7.4).
[0612] Embodiment 143. The method of Embodiment 117, wherein the
liposome is substantially spherical.
[0613] Embodiment 144. The method of Embodiment 117, wherein the
liposome comprises about 500-1000 .mu.g/mL of the weakly basic
anticancer compound and an acid or salt thereof.
[0614] Embodiment 145. The method of Embodiment 117, wherein the
liposome comprises about 700-850 .mu.g/mL of the weakly basic
anticancer compound and an acid or salt thereof.
[0615] Embodiment 146. The method of Embodiment 117, wherein the
liposome comprises a plurality of the weakly basic anticancer
compound forming a disorganized non-crystalline aggregate.
[0616] Embodiment 147. The method of Embodiment 117, wherein the
liposome does not comprise a cholesterol or a poloxamer 188.
[0617] Embodiment 148. The method of Embodiment 117, wherein the
liposome does not comprise an acidic organic compound other than
oxalic acid, tartaric acid, or salts thereof.
[0618] Embodiment 149. The method of Embodiment 117, wherein the
liposome does not comprise the weakly basic anticancer compound and
an acid or salt thereof other than oxalic acid, tartaric acid or
salts thereof.
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