U.S. patent application number 17/041094 was filed with the patent office on 2021-04-22 for poly (alkyl cyanoacrylate) nanoparticles for use in treatment of cancer.
The applicant listed for this patent is Oslo Universitetssykehus HF, Sintef TTO AS. Invention is credited to Kjersti Flatmark, Heidi Johnsen, Gunhild M.ae butted.landsmo, Yrr Morch, Kirsten Sandvig, Ruth Schmid, Tore Skotland, Per Stenstad, Einar Sulheim.
Application Number | 20210113482 17/041094 |
Document ID | / |
Family ID | 1000005359399 |
Filed Date | 2021-04-22 |
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United States Patent
Application |
20210113482 |
Kind Code |
A1 |
Morch; Yrr ; et al. |
April 22, 2021 |
Poly (Alkyl Cyanoacrylate) Nanoparticles for Use in Treatment of
Cancer
Abstract
The invention is related to the field nanoparticles and medical
use. In particular, it relates to cabazitaxel (CBZ) as active
ingredient encapsulated into poly (alkyl cyanoacrylate)
nanoparticles for use in treatment of cancer.
Inventors: |
Morch; Yrr; (Trondheim,
NO) ; Schmid; Ruth; (Tiller, NO) ; Sulheim;
Einar; (Trondheim, NO) ; Stenstad; Per;
(Trondheim, NO) ; Johnsen; Heidi; (Trondheim,
NO) ; Sandvig; Kirsten; (Nittedal, NO) ; M.ae
butted.landsmo; Gunhild; (Oslo, NO) ; Skotland;
Tore; (Nittedal, NO) ; Flatmark; Kjersti;
(Oslo, NO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sintef TTO AS
Oslo Universitetssykehus HF |
Trondheim
Oslo |
|
NO
NO |
|
|
Family ID: |
1000005359399 |
Appl. No.: |
17/041094 |
Filed: |
March 27, 2019 |
PCT Filed: |
March 27, 2019 |
PCT NO: |
PCT/EP2019/057678 |
371 Date: |
September 24, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/5138 20130101;
A61K 9/0019 20130101; A61K 9/5192 20130101; B82Y 5/00 20130101;
A61P 35/04 20180101; A61K 31/337 20130101 |
International
Class: |
A61K 9/51 20060101
A61K009/51; A61K 31/337 20060101 A61K031/337; A61P 35/04 20060101
A61P035/04 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 27, 2018 |
NO |
20180429 |
Claims
1. A drug delivery system comprising PEGylated poly (alkyl
cyanoacrylate) (PACA) nanoparticles (NPs) loaded with cabazitaxel
(CBZ), or a pharmaceutically acceptable salt thereof, for use in
treatment of cancer, wherein the CBZ comprises 1-50 wt % of the NP,
provided that the drug delivery system does not comprise
NP-stabilized microbubbles (MBs).
2. The drug delivery system according to claim 1, wherein the PACA
NPs are produced according to a miniemulsion anionic polymerization
process.
3. The drug delivery system according to any of claim 1, wherein
the NPs further are surface modified by a targeting moiety.
4. The drug delivery system according to claim 1, wherein the PACA
NP is below 800 nm.
5. A method of administering the drug delivery system according to
claim 1, wherein the drug delivery system is administered
parenterally.
6. The method according to claim 5, wherein the drug delivery
system is administered into the blood.
7. The drug delivery system according to claim 1, further
comprising pharmaceutically acceptable excipients.
8. The method according to claim 5, wherein the cancer is a tumor
in a vascular phase.
9. The drug delivery system according to claim 1, wherein the CBZ
comprises 5-20 wt % of the NPs.
10. The drug delivery system according to claim 1, wherein the PACA
NP is in a range of 1-800 nm.
11. The drug delivery system according to claim 1, wherein the PACA
NP is in a range of 10-500 nm.
12. The drug delivery system according to claim 1, wherein the PACA
NP is in a range of 70-150 nm.
13. The drug delivery system according to claim 1, wherein the drug
delivery system is in a form for parenteral administration.
Description
FIELD OF INVENTION
[0001] The invention is related to the field nanoparticles and
medical use. In particular, it relates to an active ingredient
encapsulated into poly (alkyl cyanoacrylate) nanoparticles for use
in treatment of cancer.
BACKGROUND
[0002] The use of nanotechnology in medicine offers many exciting
possibilities with potential in a number of medicinal applications
envisaged. In particular, nanomedicine is expected to lead to big
improvements in the treatment of complex diseases. Two areas in
which the use of nanoparticles has begun to demonstrate particular
value are drug delivery and molecular imaging.
[0003] Poly(alkyl cyanoacrylate) (PACA) was first developed and
approved as surgical glue. PACA nanoparticles (NPs) have later
demonstrated promising abilities as a drug carrier, being
biodegradable and allowing high drug loading capacity.
[0004] WO2014191502 A1 discloses a one-step polymerization process
for preparing stealth NPs of PACA homopolymer or copolymer
comprising anionic polymerization of an oil-in-water miniemulsion.
As disclosed, by utilizing a miniemulsion in combination with a
particular class of polyalkylene glycol derivatives, it is possible
to covalently attach targeting moieties to polyalkylene glycols,
thereby enabling the simultaneous introduction of a targeting group
and formation of a stealth corona. It is described that the
miniemulsion may contain active agents, and a list of therapeutic
agents are disclosed. However, none of the examples include
encapsulation of any of these agents, and neither in vitro nor in
vivo data are disclosed.
[0005] Although new, targeted treatment options and immunotherapy
are being developed, chemotherapy is still the main therapeutic
option for patients with advanced cancer. However, the therapeutic
effect is not sufficient for certain cancer types and the treatment
also results in severe side effects. Several products of
drug-loaded NPs have reached the market, and many new product
candidates are in clinical trials. These aspects, including the
challenges and opportunities of using nanoparticles in cancer drug
delivery, have been discussed in multiple reviews and commentaries
including Shi et al (2017) and Torchilin (2014). In addition to
improving efficacy by benefiting from the enhanced permeability and
retention (EPR) effect (Matsumura et Maeda, 1986), NP encapsulated
drug delivery may demonstrate reduced toxicity. The main advantage
of the drug-loaded NPs in the market is that they give less adverse
effects than free drug, while the therapeutic efficacy is rather
similar, as described in Parahbakar et al 2013.
[0006] In Snipstad et al (2017), it is disclosed medical use of
PEGylated PEBCA NPs in combination with microbubbles (MBs) and
ultrasound. The drug delivery system as described consists of
microbubbles stabilized by polymeric nanoparticles (NPMBs), which
enables ultrasound-mediated drug delivery. The NPs are synthesized
by miniemulsion polymerization. It is disclosed NPs containing
cabazitaxel (CBZ), and in vitro toxicity of these NPs in
triple-negative human breast adenocarcinoma cells, MDA-MB-231. The
in vivo data of the drug delivery system disclosed in Snipstad et
al (2017) described the therapeutic effect achieved by
NP-stabilized MBs on localized, solid tumors, and how an improved
effect is achieved by applying focused ultrasound.
[0007] Breast cancer is the most common non-cutaneous malignancy in
women and second only to lung carcinoma in cancer mortality. There
are several types of breast cancer, and in the last decade it has
been possible to preform molecular classification of breast cancer
based on gene expression profiles. Analyses of human breast tumors
have revealed remarkably robust molecular subtypes with distinctive
gene signatures and clinical outcome (Toft and Cryns, Mol
Endocrinol. 2011 February; 25(2): 199-211.). Basal-like breast
cancer (BLBC) is a particularly aggressive molecular subtype
defined by a robust cluster of genes expressed by epithelial cells
in the basal or outer layer of the adult mammary gland. BLBC is a
major clinical challenge because these tumors are prevalent in
young women, often relapsing rapidly. Additionally, most (but not
all) basal-like tumors lack expression of steroid hormone receptors
(estrogen receptor and progesterone receptor) and human epidermal
growth factor receptor 2, limiting targeted therapeutic options for
these predominantly triple-negative breast cancers. As described in
Engebraaten et al (2013), for basal-like tumors no targeted
therapies are available, and the patients would therefore benefit
from improved chemotherapy regimens.
[0008] The male counterpart to breast cancer is prostate cancer. It
is the most common cancer among men, and develops in the prostate,
a gland in the male reproductive system. Most prostate cancers are
slow growing; however, some grow relatively quickly. The cancer
cells may spread from the prostate to other area of the body,
particularly the bones and lymph nodes. Prostate cancer can often
be treated successfully if found in the early stages.
[0009] Taxanes are important chemotherapeutic agents with proven
efficacy in human cancers. Taxanes include paclitaxel, docetaxel,
cabazitaxel and their pharmaceutically acceptable salts. Paclitaxel
was originally derived from the Pacific yew tree. Docetaxel is a
semi-synthetic analogue of paclitaxel. CBZ, which has been
characterized by Vrignaud et al (2013), is a relatively novel
semi-synthetic taxane derivative. CBZ has a potent cytostatic
effect by microtubule stabilization but its use has been limited
due to its toxicity. CBZ has been included in several clinical
trials investigating efficacy against several types of cancer. It
has been approved by the US Food and Drug Administration (FDA) for
treatment of refractory prostate cancer as a second line drug after
docetaxel chemotherapy. Taxanes present difficulties in formulation
as medicines because they are poorly soluble in water.
[0010] It is therefore desirable, and hence an object of the
present invention, to develop a new drug delivery system which is
capable of effectively delivering a therapeutic agent to a specific
location. In particular, a drug delivery system which demonstrate
efficacy in addition to fewer adverse side effects would be
desirable.
[0011] It is further desired if the new drug delivery system is
capable of delivering hydrophobic and/or poorly soluble therapeutic
agents.
[0012] Ultimately, it is desired if the drug delivery system is
suitable for treatment of tumors where no targeted therapies are
available.
SUMMARY OF INVENTION
[0013] In a first aspect of the present invention, it is provided
herein a drug delivery system comprising PEGylated poly (alkyl
cyanoacrylate) (PACA) nanoparticles (NPs) loaded with cabazitaxel
(CBZ), or a pharmaceutically acceptable salt thereof, for use in
treatment of cancer, provided that the drug delivery system does
not comprise NP-stabilized microbubbles (MBs).
[0014] In one embodiment of this aspect, the drug delivery system
does not comprise NPs that stabilize the MBs or NPs that are used
to stabilize gas-filled MBs. In another embodiment, the drug
delivery system does not comprise NPs that are associated with the
MBs. In yet another embodiment, the drug delivery system does not
comprise gas-filled MBs. In a further embodiment, the drug delivery
system does not comprise MBs.
[0015] In a further embodiment, the PACA NPs are produced according
to a miniemulsion anionic polymerization process.
[0016] In another embodiment, the NPs are further surface modified
by a targeting moiety.
[0017] According to different embodiments of the first aspect, the
PACA NP is below 800 nm, such as in a range selected from 1-800 nm
or 10-500 nm or 70-150 nm.
[0018] In yet other embodiments, the CBZ comprises 1-90 wt % of the
NP, preferentially 5-50 wt % of the NP, more preferentially 5-20 wt
% or most preferentially 5-15 wt % of the NP. In a particular
embodiment, CBZ comprises from 6-13 wt % of the NP, more
particularly about 6, 7, 8, 9, 10, 11, 12 or 13 wt % of the NP.
[0019] In other embodiments of the first aspect, the drug delivery
system is administered parenterally and may further comprising
pharmaceutically acceptable excipients.
[0020] In yet another embodiment of the first aspect, the cancer is
a tumor in a vascular phase.
[0021] In a further embodiment, the tumor belongs to a type of
cancer selected from the group consisting of prostate cancer,
breast cancer, glioma, lung cancer, adrenocortical carcinoma,
testicular cancer, urothelium transitional cell carcinoma and
ovarian cancer. In yet another embodiment, the drug delivery system
is for use in prophylactic treatment of cancer to prevent
metastasis through the lymph node.
[0022] In yet a further embodiment, the drug delivery system is for
use as an immune modulator and/or as a vehicle to enhance the
therapeutic effect of encapsulated drugs.
[0023] In a second aspect of the invention, it is also provided a
method for treating cancer comprising administering a drug delivery
system according to the first aspect to a patient in need
thereof.
[0024] In a third aspect, it is provided a composition or solution
comprising the drug delivery system according to the first aspect
of the invention. The composition or solution may be a
pharmaceutical formulation comprising pharmaceutically acceptable
excipients and diluents.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1. Size distribution of the batches used in the
MAS98.12 efficacy study. The size distributions for PEBCA-CBZ (the
batch with size z-average of 215 nm in Table 1) is shown in dark
blue and for PEBCA (without drug; the batch with z-average of 156
nm in Table 1) is shown in light blue. The size distribution of
non-encapsulated CBZ, solubilized in a polysorbate 80 solution, is
shown in red. Intensity (%) on the y-axis means percent intensity
of total scattering.
[0026] FIG. 2. Treatment efficacy and toxicity studied in mice
bearing MAS98.12 patient derived xenograft (PDX) breast tumor
models. Tumor growth inhibition (A) and body weight change (B)
after treatment. PEBCA-CBZ and CBZ were injected 2.times.15 mg
CBZ/kg body weight at day 33 and 36, indicated by the red arrows.
Empty PEBCA NPs (same dose as PEBCA-CBZ) and saline were used as
negative controls (mean.+-.SEM; n=8-9 tumors/5-6 mice). Tumor size
is relative to the size measured at time of randomization.
Non-palpable tumors at day 57 after implantation are indicated as
complete remissions. The statistical p-value of Welch t-test of
areas under the curves is indicated (p=0.02).
[0027] FIG. 3. Biodistribution of PEBCA particles containing the
fluorescent dye NR668 measured in MAS98.12 tumor-bearing mice.
Whole body images were obtained with IVIS 1, 4, 24 and 96 h after
intravenous administration of the NPs; color scale on the right
indicates radian efficiency.times.10.sup.9(A). Ex vivo fluorescence
images of isolated organs were obtained 24 h after injection (B).
Quantification of fluorescence intensity as relative radiant
efficiency per region of interest pixel data of tissues collected
24 h after injection (C). Mean values obtained for 2 animals; error
bars show estimated SD values. PEBCA-CBZ: PEBCA containing CBZ and
NR668; PEBCA: PEBCA containing NR668, but not CBZ. LNs: lymph
nodes.
[0028] FIG. 4. CBZ concentrations in plasma and organs measured
with mass spectrometry after administration of 15 mg CBZ/kg. Plasma
concentration measured as function of time (A). CBZ concentrations
in tumors and organs measured after 24 h (B) and 96 h (C). LNs:
lymph nodes. Data shown are mean values.+-.SD (n=3). Note that
logarithmic scales are used on all y-axes.
[0029] FIG. 5. Macrophage infiltration in treated MAS98.12 tumors.
The macrophage infiltration was measured in the MAS98.12 tumors 96
h after injection of saline (control), PEBCA NPs (without drug),
non-encapsulated CBZ, and PEBCA-CBZ. (A): The total population of
infiltrated macrophages was quantified using an antibody to CD68.
(B): The population of anti-tumorigenic (pro-inflammatory)
macrophages was quantified using an antibody to iNOS. (C): The
population of pro-tumorigenic (anti-inflammatory) macrophages was
quantified with an antibody to CD206. Data shown as mean.+-.SEM
(n=3 for control samples; n=4 for PEBCA and n=5 for CBZ and
PEBCA-CBZ). Asterisks indicate statistical significance obtained by
unpaired parametric t-test, where p<0.0005 is marked with ***
and p<0.0001 is marked with ****.
[0030] FIG. 6. Treatment efficacy in the MAS98.12 PDX model. Each
bar represents the mean area under the curve (AUC) of individual
MAS98.12 tumors shown in FIG. 2. Data shown are mean.+-.SEM (n=9 or
10 tumors in each group). The statistical p-values have been
calculated using Welch's unequal variance t-test comparing the
indicated groups.
[0031] FIG. 7. Treatment efficacy in mice bearing MDA-MB-231
tumors. Tumor growth inhibition was measured following two
injections of PEBCA-CBZ, non-encapsulated CBZ, PEBCA NPs without
drug and saline. The red arrows indicate days for injections.
PEBCA-CBZ and CBZ were injected at 2.times.15 mg CBZ/kg; PEBCA
particles not containing drug were injected 2.times.175 mg (similar
to the amount of NPs in the PEBCA-CBZ group); 2.times.0.1 ml per 10
g body weight of saline were injected as a control.
[0032] FIG. 8. Ex vivo fluorescence images of isolated organs
obtained after injection of PEBCA particles containing NR668.
Images are shown for organs taken 1 h, 4 h and 96 h after injection
of the particles (images taken 24 h after injection are shown in
FIG. 3). LNs: lymph nodes.
[0033] FIG. 9. Immunohistochemical staining of macrophage
infiltration in treated MAS98.12 tumors. The macrophage
infiltration was measured in the tumors 96 h after injection of
saline (control), PEBCA NPs (without drug), non-encapsulated CBZ,
and PEBCA-CBZ (left to right columns). Immunohistochemical staining
with (A) anti-CD68, (B) anti-iNOS, and (C) anti-CD206. Scale bar:
100 .mu.m.
[0034] FIG. 10. In vitro toxicity measured as cell viability and
cell proliferation in three breast cancer cell lines. PEBCA-CBZ
with 100 nM CBZ contains 4.5 .mu.g/ml PEBCA materials; equivalent
amount of empty PEBCA NPs were given for comparison. Left column:
Cell viability measured with the MTT assay after incubation for 72
h. Right column: Cell proliferation measured as [.sup.3H]thymidine
incorporation after incubation for 24 h. The following cell lines
were used: (A): MDA-MB-231, (B): MDA-MB-468, and (C): MCF-7. Data
shown as mean.+-.SD; n=3.
[0035] FIG. 11: Treatment effects of a prostate carcinoma tumor
model (PC3) with CBZ formulated as Jevtana.RTM., CBZ in PEBCA NPs
and control. Plots of mean tumor size in the three groups. Arrows
show treatment days, error bars show standard deviation and ***
indicates p>0.001, t-test.
DEFINITIONS
[0036] The term `nanoparticle, (NP)` is used herein to describe
particles or capsules with linear dimensions less than 800 nm.
[0037] The term "PEGylation" is used herein to describe the process
of both covalent and non-covalent attachment or amalgamation of
polyethylene glycol (PEG) polymer chains to nanoparticles, which is
then described as PEGylated (pegylated). As will be known to the
skilled person, the association of PEG to the NP surface can "mask"
the NP from the host's immune system by creating a water corona
around the NP. This can reduce the immunogenicity and antigenicity
of the NP, and prolong its circulatory time by reducing renal
clearance. Depending on the density of PEG on the surface, the PEG
is classified as being in a brush or mushroom conformation. The
PEGylation can be performed either during or after synthesis of the
NPs, by either a covalent or noncovalent bond, resulting in varying
properties of the PEGylation.
[0038] The term "targeting moiety" is used herein to describe any
molecule that can be bound to the surface of the NP and result in
selective binding to specific cells or biological surfaces.
[0039] The term "passive targeting" is used herein to describe the
accumulation and/or retention of nanoparticles in inflamed and
malignant tissue that occurs due to leaky blood vessels and
impaired lymphatic drainage. Passive targeting is independent of
targeting moieties on the surface of NPs.
[0040] The term "active targeting" is used herein to describe the
accumulation and/or retention of the nanoparticle on specific cells
or biological surfaces due to the specific interaction between the
targeting moiety and the cell surface or the biological
surface.
[0041] The term "enhanced permeability and retention (EPR)" effect
is used herein to describe the phenomenon where molecules of
certain sizes (typically liposomes, nanoparticles, and
macromolecular drugs) tend to accumulate in tumor tissue much more
than they do in normal tissues. The NPs as described herein are
typically of a size from about 1-800 nm, such as about 10-500,
preferably about 70-150 nm. Accordingly, the EPR effect will allow
the NPs as described herein to selectively extravasate and
accumulate in tumors.
[0042] The terms "parenteral administration" and "administered
parenterally" are art recognized terms, and include modes of
administration other than enteral and topical administration, such
as injections, and include without limitation intravenous,
intramuscular, intrapleural, intravascular, intrapericardial,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, transtracheal, subcutaneous,
subcuticular, intraarticular, subcapsular, subarachnoid,
intraspinal and intrastemal injection and infusion.
[0043] The term "pharmaceutically acceptable" as used herein
denotes that the system or composition is suitable for
administration to a subject, including a human patient, to achieve
the treatments described herein, without unduly deleterious side
effects in light of the severity of the disease and necessity of
the treatment.
[0044] The terms "therapy", "treat," "treating," and "treatment"
are used synonymously to refer to any action providing a benefit to
a patient at risk for or afflicted with a disease, including
improvement in the condition through lessening, inhibition,
suppression or elimination of at least one symptom, delay in
progression of the disease, prevention, delay in or inhibition of
the likelihood of the onset of the disease, etc.
[0045] The terms "microbubble associated with nanoparticles" or
"nanoparticles associated with microbubbles" are used herein to
describe in what way nanoparticles can interact with the
microbubble interface. The term "associated with" as used in
connection with this include association by any type of chemical
bonding, such as covalent bonding, non-covalent bonding, hydrogen
bonding, ionic bonding or any other surface-surface
interactions.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The present invention describes a drug delivery system
comprising PEGylated poly (alkyl cyanoacrylate) (PACA)
nanoparticles (NPs) loaded with cabazitaxel (CBZ) for use in
treatment of cancer, provided that the drug delivery system does
not comprise microbubbles (MBs).
[0047] The effect of PACA NPs loaded with the cytotoxic drug CBZ is
demonstrated in several in vitro and in vivo studies. As disclosed
herein, the studies include demonstration of effects in three
breast cancer cell lines, one basal-like patient-derived xenograft
model grown in the mammary fat pad of immunodeficient mice and one
prostate carcinoma tumor model. It is demonstrated that
NP-encapsulated CBZ has similar or even better efficacy than
similar concentrations of non-encapsulated drug. As demonstrated in
the basal-like patient-derived xenograft, the results show complete
remission of 6 out of 8 tumors. The different drug concentrations
obtained with NP-encapsulated versus non-encapsulated CBZ was
investigated with mass spectrometry analyses of CBZ, which was
performed using blood and selected tissue samples. The results show
that the nanoparticle-encapsulated drug has a longer circulation
time in blood and a higher content in tumor tissue. The tissue
biodistribution, which is obtained after 24 h using mass
spectrometry analyses, correlates well with biodistribution data
obtained using IVIS.RTM. Spectrum in vivo imaging of nanoparticles
labeled with the fluorescent substance NR668. This is a clear
indication that these data also are representative for the
nanoparticle distribution. Furthermore, immunohistochemistry was
used to estimate infiltration of macrophages into the tumor tissue
following injection of NP-encapsulated CBZ and injection of
non-encapsulated CBZ. It was also demonstrated a higher content of
anti-tumorigenic versus pro-tumorigenic macrophages in tumors
treated with the NPs. Without being bound by theory, this may
further contribute to the improved effect obtained with the
NP-encapsulated drug. In summary, encapsulation of CBZ in PACA NPs
is a promising alternative to the clinically available formulation
of the drug.
[0048] As will be understood by a person skilled in the art, the
invention as disclosed herein is different form the drug delivery
system as described in Snipstad et al (2017). As described herein,
the drug delivery system of the invention does not comprise
NP-stabilized MBs, as is described by Snipstad et al, 2017. In
different embodiments, the drug delivery system according to the
invention does not comprise NPs that stabilize the MBs nor NPs that
are used to stabilize gas-filled MBs. Accordingly, the drug
delivery system of the invention is not dependent on ultrasound to
achieve treatments effects, in contrast to the delivery system
described in Snipstad et al 2017, which is ultrasound-mediated.
[0049] Accordingly, one embodiment of the invention as provided
herein is a drug delivery system comprising PEGylated PACA NPs
loaded with CBZ, or a pharmaceutically acceptable salt thereof, for
use in treatment of cancer, provided that the drug delivery system
is not mediated by an acoustic field, such as ultrasound or focused
ultrasound.
[0050] In a further embodiment of the invention, the drug delivery
system does not comprise NPs that are associated with the MB. It is
also disclosed a drug delivery system that does not comprise
gas-filled MBs. In yet a further embodiment, the drug delivery
system does not comprise MBs.
[0051] Degradation rate of PACA NPs can be controlled by the choice
of the alkyl chain of the cyanoacrylate monomer, as demonstrated by
Sulheim et al. (2016). It has also been demonstrated, using a panel
of cell lines, that the cytotoxicity is dependent on the monomers
used, i.e. n-butyl-, 2-ethyl-butyl-, or octyl cyanoacrylate (BCA,
EBCA and OCA, respectively), see Sulheim et al (2017).
[0052] In different embodiments of the invention, the alkyl chain
of the cyanoacrylate monomer is a linear or branched C4-C10 alkyl
chain. In preferred embodiments the monomer used is selected from
the group consisting of n-butyl-(BCA), 2-ethyl butyl (EBCA),
polyisohexyl (IHCA) and octyl cyanoacrylate (OCA). Accordingly, in
different embodiments, the drug delivery system comprises NPs
selected from the group consisting of PBCA, PEBCA, PIHCA and
POCA.
[0053] As described herein, the NPs are PEGylated, i.e. coated with
a hydrophilic polymer such as polyethylene glycol (PEG).
[0054] The rationale of PEGylation in drug delivery is to obtain
increased circulation time after parenteral administration, such as
intravenous (i.v.) injection. This is well demonstrated and is
known to the skilled person. Aslund et al. (2017) has studied the
quantitative and qualitative effects of different types of
PEG-comprising molecules on PACA nanoparticles. For example, it has
been demonstrated that longer PEG on the surface will increase
blood circulation time and diffusion of PACA NPs in collagen
gels.
[0055] Accordingly, by varying the type of PEG one can achieve NPs
with different surface modifications. This will influence the zeta
potential, the protein adsorption, diffusion, cellular interaction
and blood circulation half-life of the NPs described herein.
[0056] In different embodiments of the invention, the NPs are
PEGylated with PEG-comprising molecules selected from the group
consisting of Jeffamine, Brij, Kolliphor, Pluronic or combinations
thereof.
[0057] According to an embodiment, the NPs are PEGylated with the
PEG-comprising molecules selected from Pluronic and Kolliphor.
[0058] According to another embodiment, the NPs are PEGylated with
the PEG-comprising molecules selected from Brij and Kolliphor.
[0059] In an embodiment of the invention, the PACA NPs is produced
by a miniemulsion anionic polymerization process, in particular a
one-step process as described in WO2014/191502, both with or
without targeting moieties.
[0060] By using NPs that is further surface modified with targeting
moieties, for example by using NPs prepared by miniemulsion anionic
polymerization technique with polyalkylene glycols that is
covalently attached to a targeting moiety, one can enable active
targeting and potentially enhanced retention at specific locations,
such as in tumors or diseased tissue. Also, this can facilitate
uptake in cancer cells that is dependent upon specific
ligand-receptor interactions.
[0061] The targeting moiety may be any suitable moiety that causes
the NPs to bind specifically at targeted locations.
[0062] Preferably, the targeting moiety has a molecular weight in
the range 100 to 200 000 Da, more preferably 200 to 50000 Da, even
more preferably 300 to 15000 Da.
[0063] It should be appreciated that a single targeting moiety or a
mixture of different targeting moieties may be used.
[0064] Example targeting moieties are selected from the group
consisting of an amino acid, protein, peptide, antibody, antibody
fragment, saccharide, carbohydrate, glycan, cytokine, chemokine,
nucleotide, lectin, lipid, receptor, steroid, neurotransmitter,
cell surface marker, cancer antigen, glycoprotein antigen, aptamer
or mixtures thereof. Particularly preferred targeting moieties
include linear and cyclic peptides. In one embodiment, the
targeting moiety does not belong to the group consisting of amino
acids and lipids
[0065] It is previously known that the size of nanoparticles
influences the targeting effects of the nanoparticles when they are
administrated systemically into the blood, as they accumulate in
the areas around tumors with leaky vasculature. This is known as
`enhanced permeability and retention` (EPR) effect in tumor tissue.
The EPR effect is as a type of targeting, commonly referred to as
"passive targeting".
[0066] Traditionally, tumor targeting approaches are classified
into `passive targeting` and `active targeting`.
[0067] The EPR effect will be known to the skilled person as a form
of passive targeting. The introduction of targeting moieties on the
surface of the NP will be known to the skilled person as a type of
active targeting.
[0068] Angiogenesis is a biological process by which new
capillaries are formed. It is essential in many physiological
conditions, such as embryo development, ovulation and wound repair,
and pathological conditions, such as arthritis, diabetic
retinopathy, and tumors.
[0069] Tumors can grow to a size of approximately 1-2 mm.sup.3
before their metabolic demands are restricted due to the diffusion
limit of oxygen and nutrients. In order to grow beyond this size,
the tumor switches to an angiogenic phenotype and initiates the
formation of neovasculature from surrounding blood vessels.
Accordingly, tumors are endowed with angiogenic capability and
their growth, invasion and metastasis are angiogenesis-dependent.
Apart from some exemptions, in most cases, neoplastic cell
populations will form a clinically observable tumor only after
angiogenic capability has been acquired and a vascular network
sufficient to sustain their growth is produced. Furthermore, new
blood vessels provide them with a gateway through which they enter
the circulation and metastasize to distant sites. Tumor
angiogenesis is essentially mediated by angiogenic molecules
elaborated by tumor cells.
[0070] Without being bound by theory, the phenomenon of the EPR
effect is caused by the tumors ability to stimulate the production
of blood vessels in order for tumor cells to proliferate. Vascular
endothelial growth factor (VEGF) and other growth factors, known to
the skilled person, are involved in cancer angiogenesis. Tumor cell
aggregates as small as 150-200 .mu.m, will start to become
dependent on blood supply carried out by neovasculature for their
nutritional and oxygen supply. However, these newly formed tumor
vessels are usually abnormal in form and architecture. They have
poorly aligned defective endothelial cells with fenestrations,
lacking a smooth muscle layer, or innervation with a wider lumen,
and impaired functional receptors for angiotensin II. Furthermore,
tumor tissues usually lack effective lymphatic drainage. All of
these factors lead to abnormal molecular and fluid transport
dynamics, especially suitable for NPs as disclosed herein.
[0071] Accordingly, the EPR effect will results in passive
accumulation of NPs in tumors due to the hyperpermeability of the
vasculature and the lack of lymphatic drainage. This is contrary to
what is seen in normal tissue where the NPs are constrained to the
blood vessels. This makes the NPs as described herein attractive
for tumor targeting. To increase the fraction of NPs reaching the
tumor, the systemic circulation time of the NPs are increased. By
extending the circulation time, an enhanced number of NPs will be
able to accumulate in the tumors, as it increases the probability
for the NPs to diffuse through openings in the blood vessels.
[0072] Accordingly, in preferred embodiments of the invention, the
drug delivery system is for treatment of cancer, such as tumors,
including but not limited to tumors of the colon, lung, breast,
cervix, bladder, prostate and pancreas.
[0073] Further, it has been demonstrated that the NPs according to
the invention partly accumulate in the lymph nodes. This may be
utilized to treat metastasizing cancer cells in lymph nodes.
Accordingly, in one embodiment of the invention, the drug delivery
system is for treating metastasizing cancer cells in lymph nodes.
In yet another embodiment, the drug delivery system is for
prophylactic treatment of cancer by preventing metastasis through
the lymph nodes. In a further embodiment, the drug delivery system
is for treatment of tumors and for prophylactic treatment of
metastasis.
[0074] In another embodiment of the invention, the tumor has
vasculature that is hyperpermeable and/or lack lymphatic
drainage.
[0075] Tumor growth consists of an avascular and a subsequent
vascular phase. In one embodiment of the invention, the tumor is in
a vascular phase.
[0076] The NPs used in the examples contain the cytotoxic drug
cabazitaxel (CBZ). CBZ is a semi-synthetic taxane derivative that
inhibits microtubule disassembly. CBZ has a very low water
solubility, which complicates the administration of the free,
non-encapsulated drug.
[0077] However, as demonstrated in the examples, due to excellent
compatibility and solubility of CBZ in alkyl cyanoacrylate
monomers, high concentrations of the drug can be dissolved in alkyl
cyanoacrylate monomer solution and thus become encapsulated in
PACAs.
[0078] According to different embodiments of the invention the
loading capacity of CBZ in NPs can be 1-90 wt % of the NP,
preferentially 5-50 wt % of the NP. In particularly preferred
embodiments, the loading capacity of CBZ is from 5-15 wt % of the
NP, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 wt % of the
NP.
[0079] Accordingly, the drug delivery system according to the
invention has a high loading capacity, which is shown to influence
the treatment effects of the invention.
[0080] As CBZ is insoluble in water, the conventional formulation
is CBZ solubilized in a polysorbate 80 solution. As used herein,
non-encapsulated or free CBZ refers to the conventional
formulation.
[0081] CBZ has been included in several clinical trials that study
the effects on different types of cancer including several types of
prostate cancer, adrenocortical carcinoma, testicular cancer,
urothelium transitional cell carcinoma and ovarian cancer. The
inventors have also demonstrated therapeutic effects of CBZ in
glioma and lung cancer.
[0082] In the clinical studies, it has been demonstrated that the
efficacy of CBZ is accompanied by serious side effects and toxic
deaths. The toxicity rates observed in clinical trials has been
assumed to pose an obstacle to use and management of CBZ, a drug
that, on the other hand, has demonstrated great activity. In the
transition from clinical trial to clinical practice, it has been
speculated that CBZ will not be used much because of the risk of
side effects, as well as high cost and discomfort derived from the
administration regimes and the lack of patient compliance with the
administration regimes previously proposed for CBZ-treatments.
Thus, limiting the administration regimes, for example from
tree-weekly to weekly has been proposed in treatment of for example
prostate cancer, to improve hematologic tolerance along with a
better therapeutic range to be able to increase the dose intensity
and activity without increasing the associated toxicity.
[0083] Accordingly, the advantage that drug-loaded NPs give less
adverse effects than free drug makes the drug delivery system as
described by the inventors highly relevant for CBZ. Encapsulating
CBZ in NPs offers a more sustained release profile of the drug,
which can ameliorate parts of the toxicity and allows for
administration of higher doses. The reduction of adverse effects
allows for administration of increased doses of drugs. Accordingly,
encapsulation of drug in the NPs will further improve the treatment
effects. Accordingly, the inventors propose the idea that the drug
delivery system as described herein will enhance treatments effects
and/or reduce side effects when used in treatment of cancer.
[0084] In different embodiments, the invention provides a drug
delivery system comprising PEGylated PACA NPs loaded with CBZ, or a
pharmaceutically acceptable salt thereof, for use in treatment
cancer, wherein the tumors belong to a type of cancer selected from
the group consisting of prostate cancer, breast cancer, glioma,
lung cancer, adrenocortical carcinoma, testicular cancer,
urothelium transitional cell carcinoma and ovarian cancer.
[0085] In one particular embodiment of the invention, the tumor
belongs to a type of prostate cancer, such as prostate carcinoma,
hormone refractory prostate cancer, prostatic neoplasms or bone
metastatic prostate cancer.
[0086] In another particular embodiment of the invention, the tumor
belongs to a type of breast cancer, including luminal-like and
basal-like breast cancer.
[0087] Breast cancer can be classified into major subgroups based
on the gene expression pattern. Tumors belonging to the most
aggressive subtypes are commonly treated with antracyclin and
taxane-based chemotherapy regimens. In the examples, the growth
inhibitory effect of CBZ encapsulated into PEBCA NPs is
demonstrated in breast cancer models in vitro and in vivo, and in a
prostate carcinoma tumor model in vivo. Three breast cancer cell
lines representing the two main types of breast cancer, the luminal
and basal-like subgroups was used. One of the cell lines is also
injected and grown in the mammary fat pad of immunodeficient mice.
Additionally, one patient-derived xenograft (PDX) previously
demonstrated to be highly representative for aggressive basal like
breast cancer is included in the examples.
[0088] One finding presented here is the surprising demonstration
of improved therapeutic effect of PEBCA-CBZ compared to the
non-encapsulated CBZ demonstrated in the basal-like PDX model.
Analysis with mass spectrometry indicated that this could be due to
the increased delivery of CBZ to the tumor following from
encapsulation of the drug in NPs. Also, immunohistochemical
staining revealed a lower content of pro-tumorigenic macrophages in
the PEBCA-CBZ treated tumors than in tumors treated with
non-encapsulated CBZ. Without being bound by theory, this could
contribute to the improved efficacy. To elucidate the difference in
efficacy of
[0089] PEBCA-CBZ and non-encapsulated CBZ, the examples demonstrate
in vivo biodistribution of particles loaded with the lipophilic and
near-infrared fluorescent substance NR668. Quantitative mass
spectrometry analyses were used to describe the biodistribution of
CBZ in tissues and the kinetics of CBZ in blood plasma after
injection of both PEBCA-CBZ and non-encapsulated CBZ.
[0090] The example with prostate carcinoma demonstrates that the
CBZ encapsulated into PEBCA NPs had similar growth inhibition as
the clinically approved formulation. With the enhanced retention of
drug loaded NPs in cancer cells compared to normal tissue, the drug
delivery system of the present invention allows for administration
of higher dosage with lower adverse effects.
[0091] Delivery of drug to lymph nodes has been discussed for
treatment of metastasizing tumors. Since local lymph nodes are a
significant metastatic site in many cancer types, accumulation of
PEBCA-CBZ to lymphatic tissue may contribute additionally to both
therapeutic and prophylactic treatment.
[0092] In summary, the PEBCA-CBZ NPs demonstrate promising results
for treatment of prostate cancer and breast cancer, supporting the
therapeutic effect of a drug delivery system for use in treatment
of tumors as disclosed herein.
[0093] Furthermore, the observation that the PACA NPs seems to
enhance intratumoral presence of anti-tumorigenic macrophages might
be of more general value, and support that the drug delivery system
of the invention can be used as an immune modulator and/or as a
vehicle able to enhance the therapeutic effect of encapsulated
drugs.
[0094] According to an embodiment of the invention, the drug
delivery system is provided in a composition to be administered
systemically, such as parenterally.
[0095] A last aspect of the invention includes a method of treating
cancer comprising administering a drug delivery system according to
the first aspect of the invention to a patient in need thereof.
EXAMPLES
Example 1
Materials and Methods
[0096] Synthesis and characterization of nanoparticles. PEGylated
PEBCA NPs were synthesized by miniemulsion polymerization. An oil
phase consisting of 2.5 g 2-ethylbutyl cyanoacrylate (monomer,
Cuantum Medical Cosmetics, Spain) containing 0.2% (w/w) butylated
hydroxytoluene (Fluka, Switzerland) and 2% (w/w) Miglyol 812
(Cremer, USA) was prepared. Fluorescent particles for optical
imaging were prepared by adding NR668 (modified Nile Red), custom
synthesis, 0.2% (w/w) to the oil phase. Particles containing
cytostatic drug for treatment were prepared by adding CBZ (10%
(w/w), Biochempartner Co. Ltd., China, product item number
BCP02404) to the oil phase.
[0097] An aqueous phase consisting of 0.1 M HCl (20 ml) containing
Pluronic F68 (2 mM, Sigma, USA) and Kolliphor HS15 (6 mM, Sigma,
Germany) was added to the oil phase and immediately sonicated for 3
min on ice (6.times.30 sec intervals, 60% amplitude, Branson
Ultrasonics digital sonifier 450, USA). The solution was rotated
(15 rpm, SB3 rotator, Stuart, UK) at room temperature overnight
before adjusting the pH to 5 using 1 M NaOH. The polymerization was
continued for 5 h at room temperature on rotation. The dispersion
was dialyzed (Spectra/Por dialysis membrane MWCO 100,000 Da,
Spectrum Labs, USA) against 1mM HCl to remove unreacted PEG. The
size, polydispersity index (PDI) and the zeta potential of the NPs
were measured by dynamic light scattering and laser Doppler
Micro-electrophoresis using a Zetasizer Nano ZS (Malvern
Instruments, UK). To calculate the amount of encapsulated drug, the
drug was extracted from the particles by dissolving them in acetone
(1:10), and quantified by liquid chromatography coupled to mass
spectrometry (LC-MS/MS) as described below.
[0098] CBZ quantification by LC-MS/MS. CBZ, as the pure chemical or
part of NPs, was quantified by LC-MS/MS, using an Agilent 1290 HPLC
system coupled to an Agilent 6490 triple quadrupole mass
spectrometer. The HPLC column was an Ascentis Express C8,
75.times.2.1 mm, 2.7 .mu.m particles size with a 5.times.2.1 mm
guard column of the same material (Sigma), run at 40.degree. C.
Eluent A was 25 mM formic acid in water and eluent B was 100%
methanol, and flow rate was 0.5 ml/min. The mobile phase gradient
was isocratic at 55% B for 1.5 min, then from 55% to 80% B over 1
min, followed by 1 min washout time and subsequently column
re-equilibration. Injection volume was 5.00 .mu.l. MS detection was
in positive ESI mode (Agilent Jetstream) quantified in multiple
reaction monitoring (MRM) mode using the transition m/z
858.3.fwdarw.577.2. The parent ion was chosen to be the Na adduct
as this gave the best sensitivity. Similarly, the hexadeuterated
internal standard was detected on the 864.4.fwdarw.583.2
transition. Both analytes were run at 380 V fragmentor and 20 V
collision energy.
[0099] Reference standards were used for accurate quantification.
The unlabeled CBZ standard was the same as used for synthesis (see
above) at >98% purity. Hexadeuterated CBZ internal standard was
purchased from Toronto Research Chemicals (Toronto, Canada;
catalogue number C046502 at 99.6% isotopic purity). Standards were
dissolved in acetone and were used to build an unlabeled standard
series spanning at least five concentration points.
[0100] The limit of quantification (LOQ) was calculated from six
replicate quantifications of the lowest concentration point in the
standard curves (0.1 ng/ml), specifically as the average plus six
standard deviations; this amounted to an LOQ of 0.19 ng/ml
(signal/noise ratio >20). Accuracy based on the same standard
sample set was 8.8% and precision was 18.0%.
[0101] Processing of tissue samples before LC-MS/MS analyses. In
order to process the tissue samples such that their CBZ content
could be quantified, we developed a protocol for enzymatic
digestion of tissue followed by extraction and quantification of
CBZ using the LC-MS/MS method described above. The enzyme buffer
consisted of Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher
Scientific, USA, 41965039) with 1% (v/v) penicillin-streptomycin
stock solution (Sigma-Aldrich, P0781) to a final concentration of
100 U/ml penicillin and 100 .mu.g/ml streptomycin, 0.125 mg/ml
papain (Merck, F275644), 2.5 mg/ml trypsin (Sigma-Aldrich, T7409),
0.8 mg/ml collagenase (Sigma-Aldrich, C7926), 0.69 mg/ml
hyaluronidase (Sigma-Aldrich, H3506) and 1% (v/v) Triton X-100
(Sigma-Aldrich, T-8787). To determine the biodistribution of CBZ,
frozen organs (liver, spleen, lymph nodes, kidneys, tumors) were
thawed and freshly prepared enzyme buffer was added at 1 ml per 50
mg tissue; the entire organs were digested. The samples were heated
to 37.degree. C. for 72 h with vortexing once a day, until the
tissue was completely dissolved. The tissue digests, as well as the
plasma samples from the animals, were diluted 10.times. in acetone
before centrifugation; this has the dual effect of both
precipitating proteins and other macromolecules, thus cleaning up
the sample, and making sure all CBZ is solubilized. Internal
standard (hexadeuterated CBZ) dissolved in acetone was added to a
final concentration of 10 ng/ml during the acetone dilution to
correct for possible matrix effects.
[0102] Cell lines. Three breast cancer cell lines were used in this
study. The MDA-MB-231 (triple negative; Claudin low), was cultured
in RPMI 1640; the MDA-MB-468 (triple negative; basal) and the MCF-7
(luminal A) cell lines were cultured in DMEM. All medium was
fortified with 10% (w/v) fetal calf serum albumin (Sigma) and 100
units/ml penicillin/streptomycin (PenStrep.RTM., Sigma). All cell
lines were obtained from ATCC and were routinely tested for
mycoplasma. Cells growing in 24- or 96-well plates were incubated
with serial dilutions of PEBCA-CBZ, CBZ (non-encapsulated CBZ)
dissolved in Tween-80 (Fluka)), and PEBCA without CBZ for 24, 48 or
72 h at 37.degree. C. in an atmosphere of 5% CO.sub.2. The toxicity
was assessed either by the commonly used MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell
viability assay, by measuring cell proliferation based on
[.sup.3H]thymidine incorporation, by measuring protein synthesis by
incorporation of [.sup.3H]leucine and by measuring ATP levels using
CellTiter Glo.RTM..
[0103] MTT cell viability assay. The cells were incubated for 24,
48 and 72 h with the different NPs/substances. The cell medium was
then aspirated and exchanged with 100 .mu.l of medium containing a
final concentration of 250 .mu.g MTT/ml. The incubation was
continued for 3 h at 37.degree. C. for formation of the
formazan-particles, which were dissolved in DMSO with 1% (v/v)
NH.sub.4Cl. The absorbance was read in a plate reader (Biosys Ltd,
Essex, UK) at 570 nm, and background from absorbance at 650 nm was
subtracted.
[0104] Cell proliferation measured by [.sup.3H]thymidine
incorporation. Incorporation of [.sup.3H]thymidine into DNA was
used to estimate cell proliferation. The cells were incubated for
24 h with the different NPs/substances. The cell medium was then
aspirated and substituted with serum free cell medium containing
[.sup.3H]thymidine (3 .mu.g/ml; 75 .mu.Ci/ml). The incubation was
continued for 30 min at 37.degree. C. The medium was removed and 5%
(w/v) trichloroacetic acid (TCA) was added. After 5 min the cells
were washed twice with TCA and solubilized with 200 .mu.l of 0.1 M
KOH, before mixing with 3 ml scintillation fluid (Perkin Elmer,
USA). The radioactivity was counted for 1 min in a scintillation
counter (Tri-Carb 2100TR, Packard Bioscience, USA).
[0105] Protein synthesis measured by [.sup.3H]leucine
incorporation. To determine the impact of PEBCA-CBZ and CBZ on
protein synthesis, the cells were incubated with these substances
for 24 h. The cell medium was then aspirated, the cells washed once
with leucine-free HEPES medium (28 mM HEPES,
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) in MEM) and
further incubated with leucine-free HEPES medium containing
[.sup.3H]leucine (2 .mu.Ci/ml) for 30 min at 37.degree. C. The
medium was removed and 5% (w/v) TCA was added to precipitate
proteins. After 5 min the cells were washed again with 5% (w/v) TCA
and solubilized with 200 .mu.l of 0.1 M KOH, before mixing with 3
ml scintillation fluid and counting the radioactivity as described
above.
[0106] Cell viability estimated by measuring ATP. Viability of the
cells was tested measuring the ATP levels by using the
CellTiter-Glo.RTM. (Promega, Wis., USA) assay, as described by the
supplier. Cells were incubated with PEBCA-CBZ or CBZ for 72 h,
thereafter one half of the volume was removed, replaced with an
equal volume of the ATP reagent and gently mixed. After incubation
for 10 min the cell lysate was transferred to a light-protected
96-well plate and luminescence measured in a plate reader (Biosys
Ltd, Essex, UK).
[0107] Treatment efficacy evaluation in nude mice. All animal
experiments were approved and performed according to the Norwegian
Animal Research Authority (Permit number 15-136041) and were
conducted according to the regulations of the Federation of
European Laboratory Animal Science Association (FELASA). The mice
were kept under pathogen-free conditions, at constant temperature
(21.5.+-.0.5.degree. C.) and humidity (55.+-.5%); 15 air changes/h
and a 12 h light/dark cycle. They had access to distilled water ad
libitum, which was supplemented with 17-.beta.-estradiol at a
concentration of 4 mg/l. All mice used in this study were female
athymic nude foxn1.sup.nu mice (age 5-6 weeks and body weights of
18-20 g), locally bred at the Department of Comparative Medicine,
Oslo University Hospital, Norway.
[0108] The ortothopically growing basal-like xenograft model
MAS98.12 has been established in house and was used as previously
described in Lindholm et al (2012). When using the MDA-MB-231 cell
line, 1.5 million cells were injected into the mammary fat pad and
growing tumors were used for sequential implantation. As for the
MAS98.12 model, 1-2 mm.sup.3 pieces of healthy tumor tissue were
implanted bilaterally into the mammary fat pad of female athymic
mice. After the tumors reached approximately 5 mm in diameter, the
mice were randomly assigned to the different treatment groups (the
average volume of each group was 49-57 mm.sup.3).
[0109] The PEBCA-CBZ NPs were given twice (day 1 and day 4 after
randomization) as i.v. tail vein injections with a dose of CBZ 15
mg/kg and NPs 175 mg/kg. A comparable amount of empty PEBCA NPs
were given as control. Non-encapsulated CBZ was prepared as a stock
solution in Polysorbate 80 (40 mg/ml) and further diluted with 13%
(v/v) ethanol to a working solution of 10 mg/ml CBZ. The injection
solution was prepared directly before the administration by
dilution of the working solution with 0.9% (w/v) NaCl. The same
amount of ethanol (1.2-1.4% (v/v)) was used as vehicle control;
injection volumes were in the range 200-290 .mu.l. From the first
day of treatment, tumor diameter and body weight were measured
twice weekly. Mice were monitored daily for health status and were
killed by cervical dislocation if they became moribund or if tumor
reached 1500 mm.sup.3. The tumor was measured by calipers and the
tumor volume was calculated according to the formula
0.5.times.length.times.width and related to the mean tumor volume
at start of treatment.
[0110] In vivo imaging. PEBCA NPs labeled with the lipophilic and
fluorescent dye NR668 were used to study the biodistribution in
MA98.12 bearing mice using an IVIS.RTM. Spectrum in vivo imaging
system (Perkin Elmer). Mice were intravenously injected the same
dose PEBCA-CBZ or PEBCA without drug as in the efficacy study. The
batch containing CBZ has somewhat larger particles than the batch
not containing CBZ (Table 51). The excitation/emission wavelength
pair of 535/640 nm was found to give the best signal-to-noise ratio
and was thus used for imaging of the NPs. Whole body images were
obtained 1, 4, 24 and 96 h after injection; the animals were then
sacrificed by cervical dislocation and organs were harvested. The
organs were imaged ex vivo with the IVIS scanner using the same
settings as above. Relative signal intensity in the organs was
calculated, using Living Image software (Perkin Elmer), as radiant
efficiency (Emission light [photons/sec/cm.sup.2/str]/Excitation
light [.mu.W/cm.sup.2].times.10.sup.9) per pixel of the region of
interest, which was drawn around the respective organ. Fluorescent
measurements of the PEBCA NPs used for in vivo imaging showed that
the particles without CBZ had a fluorescent intensity of 1.17 times
that of the PEBCA NPs containing CBZ and the data shown in FIG. 3C
are corrected for this difference.
[0111] Biodistribution and pharmacokinetics in blood. Blood and
tissue samples were obtained following a single i.v. injection of
PEBCA-CBZ and non-encapsulated CBZ (15 mg/kg) into the tail vein of
mice bearing the MAS98.12 tumor (n=3). Empty particles (PEBCA) and
saline were used as negative controls. The blood samples were taken
either per tail vein puncture (approximately after 2 min and
thereafter at 1, 4 and 24 h after injection) or by terminal cardiac
puncture (96 h after injection) in Vacutainer tubes containing EDTA
(BD Biosciences, San Jose, Calif., USA) and kept on ice. The 0-24 h
samples were collected consecutively from the same animals (n=3)
while samples after 96 h were obtained from separate mice (n=3).
The blood was centrifuged for 15 min at 4.degree. C. and
3400.times.g, and the supernatant plasma was collected and stored
at -80.degree. C. until LC-MS/MS analysis. The animals were killed
after 24 or 96 h and tissue samples (tumors, livers, spleens, lymph
nodes and kidneys) were harvested. The organs were gently washed
with saline and then snap frozen in liquid nitrogen and stored at
-80.degree. C. until further processing and LC-MS/MS analyses as
described above. Statistical analyses were performed using the
t-test.
[0112] Immunohistochemistry. Tumors from MAS98.12 bearing mice were
collected 96 h after a single injection of the same substances as
were injected in the MAS98.12 efficacy study. The tumors were
preserved in 4% (v/v) formalin and then paraffinized and sliced to
prepare consecutive slides (3 .mu.m thick). The deparaffinization
agent Neo-clear and mounting agent Neo-mount were obtained from VWR
(Radnor, Pa., USA). Heat induced epitope retrieval was performed by
placing deparaffinized slides with 10 mM sodium citrate buffer (pH
6.0) in water bath for 20 min at 100.degree. C. Endogenous
peroxidase activity was blocked by incubating slides with 3% (v/v)
hydrogen peroxide in Tris-buffered saline (TBS; 50 mM Tris-Cl, 150
mM NaCl, pH 7.6). Sections were washed and blocking of non-specific
binding was performed with 3% (w/v) bovine serum albumin (Roche
diagnostics GmbH, Mannheim, Germany) in TBS for 30 min. Sections
were then incubated for 60 min with the primary antibodies. Three
different antibodies, i.e. anti-CD68 (1 mg/ml; ab125212, Abcam,
Cambridge, UK), anti-CD206 (0.1 .mu.g/ml; ab64693, Abcam,
Cambridge, UK), and anti-iNOS (0.5 .mu.g/ml; ab15323, Abcam,
Cambridge, UK) were used to detect different population of
macrophages. CD68 is a commonly used marker for the whole
macrophage population, iNOS (inducible nitric oxide synthase) is a
marker for M1 macrophages (anti-tumourigenic and pro-inflammatory
macrophages), and CD206 is a marker for M2 macrophages
(pro-tumourigenic and anti-inflammatory macrophages). TBS was used
for washing the slides between steps. Detection of primary
antibodies was performed using MACH 3 rabbit HRP-polymer detection
kit according to the manufacturer's protocol (Biocare Medical,
Concord, Calif., USA). Signals were developed by incubation with
the Chromogen solutions provided with the Betazoid DAB Chromogen
kit (Biocare Medical). For counter staining a haematoxylin and 37
mM ammonium hydroxide containing solution (Sigma-Aldrich, St.
Louis, Mo., USA) were used.
[0113] Stained tissue sections were scanned (NanoZoomer HT,
Hamamatsu Photonics, Hamamatsu, Japan) using a 40.times. objective.
The extent of CD68, iNOS and CD206 was automatically scored using
the ImmunoPath software (Room4 Ltd., Crowborough, UK). The tumor
areas were marked manually, excluding necrotic areas as well as
blood vessels to avoid unspecific or false positive staining. The
annotated areas were then broken down to smaller frames per image
of interest for efficient processing. The annotated representative
areas were analyzed further by computerized image analysis. The
image analysis protocols were set up on randomly selected images
from different tumors to educate the software to differentiate
between haematoxylin stained blue negative staining and brown
positive staining. The output of the analysis provides the number
of the positive pixel fraction and the negative pixel fraction of
total annotated area.
[0114] Statistical analyses. To calculate the significance in the
efficacy studies, the area under the curve was calculated for each
tumor and the mean values of the groups were compared using the
Welch unequal variance. To determine whether there is a significant
difference between the frequencies of the tumor regression in the
different treatment groups, we used the Fischer's exact test. If
not stated otherwise, an unpaired two-sided Student t-test without
Welch correction was used. The statistical analyses were performed
using either GraphPad Prism (version 7.00 for Windows, GraphPad
Software, La Jolla, Calif., US) or Microsoft Excel.
Results
[0115] Characterization of PEBCA particles. The particle size,
polydispersity index (PDI) and zeta potential for the batches used
were in the range of 148-227 nm (z-average), 0.04-0.19 and
-(0.6-2.4) mV, respectively. The drug content in the final
particles was 6.0-8.6% (w/w), giving 2.0-3.4 mg CBZ/ml in the NP
stock solutions (Table S1). The size of PEBCA NPs increased when
adding the drug CBZ or the fluorescent label NR668 (Table S1). The
size distribution curves for the two batches used for efficacy
studies in the MAS98.12 tumor model and that of CBZ (forming
clusters in solution) are shown in FIG. 1.
[0116] PEBCA-CBZ inhibits tumor growth more efficiently than free
CBZ in the MAS98.12 mice model. To test the efficacy of the PEBCA
NPs with incorporated CBZ in a PDX mode, MAS98.12 tumors were
implanted into the mammary fat pad of nude mice and treated with
the drug-loaded particles (PEBCA-CBZ), empty particles (PEBCA),
non-encapsulated (free) CBZ and saline as control (FIG. 2A). The
injected dose was 2.times.15 mg CBZ/kg body weight, which
corresponds to a particle dose of 2.times.175 mg/kg (FIG. 2A).
Tumor growth was not affected by empty PEBCA NPs. CBZ treatment
markedly inhibited tumor growth, and the effect was more pronounced
with PEBCA-CBZ treatment, which induced reduction in tumor size
(FIG. 2A). In the PEBCA-CBZ treated group 6 out of 8 tumors went
into complete remission while this was the case in only 2 out of 9
CBZ-treated tumors and none in the negative control groups
(Fisher's exact test (1-sided): p=0.04; comparing PEBCA-CBZ with
CBZ).
[0117] To further evaluate differences in tumor growth between the
four groups, we calculated the area under the curve (AUC) for each
individual tumor and compared the mean values of each treatment
group. The efficacy of PEBCA-CBZ is significantly better than
treatment with non-encapsulated CBZ (p=0.02; Figure S1). The
toxicity of the different treatments measured as body weight
relative to the weight at treatment start is shown in FIG. 2B. The
two treatments with CBZ (non-encapsulated or encapsulated as
PEBCA-CBZ) caused a decline in body weight by approximately 15%,
but one week after the last injection the toxicity was reversed.
This tolerable body weight loss and the recovery time were
comparable for PEBCA-CBZ and CBZ. Administration of empty PEBCA NPs
did not cause any toxic effect as estimated from the body
weights.
[0118] The efficacy of PEBCA-CBZ and non-encapsulated CBZ was also
studied in MDA-MB-231 tumor-bearing mice. In this model we did not
detect a significant difference between the two CBZ formulations,
but a delay in tumor growth was observed (Figure S2). When compared
to MAS98.12, the CBZ (free and encapsulated drug) was less
effective in the MDA-MB-231 tumors. It was not possible to improve
the efficacy by increasing the dose of non-encapsulated drug, due
to toxicity of the free formulation of the drug.
[0119] In vivo biodistribution of PEBCA particles. The
biodistribution of the PEBCA NPs in MAS98.12 bearing mice was
studied by fluorescence imaging up to 96 h after injection of
particles containing the fluorescent dye NR668. The mice were
imaged using the IVIS.RTM. Spectrum scanner after 1, 4, 24 and 96 h
(FIG. 3A), and then sacrificed such that organs could be harvested
and visualized ex vivo. The images of all organs harvested 24 h
after injections are shown in FIG. 3B, and the mean radiant
efficiency relative to the pixel size of the region of interest per
organ is plotted in FIG. 3C. The images of organs obtained at 1, 4
and 96 h are shown in Figure S3. Injection of the free NR668 dye
did not give any detectable fluorescence with the wavelengths used
(data not shown), thus indicating that the detected signals are
from PEBCA-bound NR668. The images shown in FIG. 3 and Figure S3
demonstrate a rapid uptake in all tissues shown; 24 h after
injection the strongest signals were observed in liver, spleen and
lymph nodes, although fluorescence also was easily detectable in
tumors, kidneys, hearts and lungs (FIG. 3C).
[0120] Pharmacokinetics and biodistribution of encapsulated and
non-encapsulated CBZ. PEBCA-CBZ and non-encapsulated CBZ (15 mg/kg)
were intravenously injected into the tail vein of mice bearing the
MAS98.12 tumor. Blood samples were taken approximately 2 min after
injection, and 1, 4, 24 and 96 h after injection, and the plasma
samples were analyzed for CBZ using an LC-MS/MS method. The CBZ
concentrations at almost all time points were at least 10-fold
higher in mice receiving PEBCA-CBZ compared to mice receiving free
CBZ (FIG. 4A). The plasma concentration/time curves for both
PEBCA-CBZ and CBZ indicate an initial distribution phase, followed
by a terminal elimination phase. The low number of data points did
not allow determination of distribution or elimination half-life
through non-linear regression. Interpolation based on the two first
data points (up to 1 hr) indicate distribution half-lives in the
range of approximately 50 min and 30 min for PEBCA-CBZ and CBZ,
respectively. Similarly, calculation of the elimination half-lives
based upon the mean values of the two last time points (24 and 96
h) indicates half-lives of this phase of about 60 h for both
compounds. The higher plasma concentration observed after
administration of PEBCA-CBZ compared to CBZ suggests a lower
distribution volume and lower total clearance for PEBCA-CBZ
compared to CBZ. This is consistent with the NP formulation being
less able to escape the vascular compartment, and elimination of
NPs primarily through the reticuloendothelial system.
[0121] The CBZ concentrations were measured in tumor, liver,
spleen, lymph nodes, and kidney following a single injection of
PEBCA-CBZ and CBZ (15 mg/kg). The results obtained with samples
taken 24 and 96 h after injections are shown in FIGS. 4B and C,
respectively. The highest amount of drug per mg tissue was obtained
in spleen. However, when assuming that the mass of liver is
approximate 13 times that of spleen in mice.sup.15 the data in FIG.
4 indicates that the liver/spleen ratio of PEBCA-CBZ is 2.1 times
24 h after injection and 4.4 times 96 h after injection
demonstrating that liver is taking up the largest part of these
NPs. The amounts of CBZ in the tumor samples measured as ng CBZ/mg
tissue were 20% (24 h) and 1.4% (96 h) of that in liver after
injection of PEBCA-CBZ.
[0122] The concentration of CBZ was significantly higher (t-test;
p-value<0.01) in all tissues analyzed 24 h following injection
of particle bound (PEBCA-CBZ) as compared to non-encapsulated drug
(CBZ), with the largest differences observed in liver and spleen
which contained the highest amounts of PEBCA-CBZ. Following
injection of non-encapsulated CBZ, the highest concentration of CBZ
was found in the tumor, although this level was only about 1/3 of
that obtained in the PEBCA-CBZ group. In the samples obtained 96 h
after injection, CBZ could be detected (i.e. being above the LOQ of
the LC-MS/MS method) in all tissues analyzed after injection of
PEBCA-CBZ, but only in the tumor tissue following injection of free
CBZ (FIG. 4C). At this time point the concentration of CBZ in
tumors following injection of free CBZ was approximately 50% of
that obtained following injection of PEBCA-CBZ, but due to high
variance in the PEBCA-CBZ samples this difference was not
statistically significant (p=0.18). When comparing the CBZ
concentrations in the tissue samples obtained 24 and 96 h after
injection of PEBCA-CBZ, there is a decrease in tumor, spleen and
kidney, and an increase in liver and lymph nodes from 24 to 96 h.
The only significant difference is the decrease in the MAS98.12
tumor tissue (p=0.02). Also, the CBZ concentration after injection
of non-encapsulated CBZ was significantly lower in tumors after 96
h when compared to 24 h (p<0.001). Plasma and tissue samples
were also analyzed for CBZ following injection of PEBCA NPs which
did not contain CBZ; all these samples (similar to those samples
analyzed after injection of PEBCA-CBZ) were below the LOQ of the
analytical method.
[0123] Macrophage infiltration in treated MAS98.12 tumors. The
infiltration of macrophages into the MAS98.12 tumors during
treatment was estimated by immunohistochemistry. The extent of
total population of infiltrating macrophages was quantified using
an antibody against CD68 and automatic quantification of scanned
slides. An increased level of tumor infiltrating macrophages was
observed in mice injected PEBCA-CBZ or PEBCA without drug compared
to mice receiving free CBZ or saline, but the differences did not
reach statistical significance (FIG. 5A). Also the marker of
pro-inflammatory M1 macrophages (iNOS) demonstrated increased
macrophage infiltration into tumors of mice receiving PEBCA-CBZ or
PEBCA (FIG. 5B). However, the anti-inflammatory M2 macrophages
subset, measured as CD206 positive cells, demonstrated increased
infiltration compared to the saline control only in tumors
receiving PEBCA (FIG. 5C). Furthermore, the tumors of mice
receiving PEBCA-CBZ showed significant lower levels of this
pro-tumorigenic macrophage population than tumors receiving PEBCA
alone (p<0.001; FIG. 5C).
[0124] In vitro cell studies. We tested cellular toxicity of
PEBCA-CBZ, non-encapsulated CBZ and PEBCA without drug in three
cell lines, i.e. MDA-MB-231, MDA-MB-468 and MSF-7 using two
different test systems, i.e. measuring cell proliferation by
incorporation of [.sup.3H]thymidine after 24 h and cell viability
after 72 h using the MTT assay. PEBCA-CBZ and CBZ were
significantly more toxic than PEBCA without drug for all cell lines
(range 130-350 fold), but there was no difference in the toxic
effect of PEBCA-CBZ and CBZ in any of these test systems in any
cell line (FIG. 10). It was also apparent that a small fraction
(10-20%) of the cells in all cell lines tested survived even very
high CBZ concentrations when using the MTT assay after incubation
for 72 h. The toxicity of PEBCA-CBZ and non-encapsulated CBZ were
also tested in all three cell lines using two other test systems.
The effect on protein synthesis (incorporation of [.sup.3H]leucine)
was measured following 24 h of incubation and the ATP levels
(CellTiter-Glo.RTM.) were measured after 72 h. The results obtained
with these test systems (data not shown) were very similar to those
shown in Figure S5. The MTT assay was also performed on MDA-MB-231
cells after 24 and 48 h of incubation; the data obtained were
similar to those shown after 72 h of incubation (FIG. 10), although
the toxic effect was smaller after these shorter incubation times
(data not shown).
Discussion
[0125] The main finding of the present study was the remarkably
good therapeutic effect observed with PEBCA NPs containing CBZ in
the basal-like PDX mice model, where complete remission was
obtained in 6 out of 8 tumors following two injections of 15 mg
CBZ/kg (FIG. 2A). Several possible explanations for the
advantageous effect of PEBCA-CBZ versus non-encapsulated CBZ can be
envisioned, and the obtained data point at least to the following
factors: The longer circulation in blood of the NP-CBZ and higher
concentration of the drug in the tumor (FIG. 4) and the higher
ratio of anti-iNOS labeled macrophages to anti-CD206 labeled
macrophages 16-17 in the treated tumors (FIG. 5).
[0126] The amount of CBZ was quantified in blood plasma and in
several tissues using an LC-MS/MS method. The plasma data clearly
show CBZ to be circulating for a longer time when incorporated in
the NPs, and the CBZ concentration was at least 10-fold higher at
nearly all time points in mice receiving PEBCA-CBZ compared to
those receiving free CBZ (FIG. 4A). When evaluating such data, it
is important to remember that the CBZ incorporated in NPs does not
have a therapeutic effect before being released from the NPs. Thus,
biodegradation of the NPs is necessary to obtain a good therapeutic
effect, as exemplified in the MAS98.12 model.
[0127] The in vivo fluorescence imaging data obtained with PEBCA
NPs labeled with NR668 demonstrates accumulation of the
fluorescence in the same tissues as those where CBZ was found to be
present. As expected these data show that most of the NPs end up in
the liver. The liver/spleen ratio of the fluorescence per pixel
measured 24 h after injection was estimated to be 2.9 for NPs
without CBZ and 2.4 for NPs with CBZ, whereas the ratio of total
CBZ content in liver to spleen was calculated to 2.1 based on the
MS analyses. Although one should be careful in interpreting the
quantitative data from the fluorescent imaging (based upon per
pixel measurements) these biodistribution data obtained with two
different methods showed similar results. The observation herein
that injection of free NR668 did not give any measurable in vivo
signal, the fact that NR668 did not leak from NPs with a similar
composition (as demonstrated in Snipstad et al. 2017), and the
rapid elimination of non-encapsulated CBZ from blood indicate that
most of CBZ and NR668 are enclosed within the NPs 24 h after
injection. Therefore, the biodistribution of these low molecular
substances seem to represent well the distribution of the PEBCA NPs
at this time point. In the study disclosed in Snipstad et al. 2017,
with somewhat similar NPs, but using another PEGylation and
fluorescent marker, a liver/spleen ratio of 5.3 was reported 6 h
after injection. In this study, the mean fluorescent signal
obtained in the tumors 24 h after injection was 12% of that in
liver for the NPs not containing CBZ and 3% for the NPs containing
CBZ. The IVIS data (FIGS. 3A, B) indicate higher fluorescence
following injection of PEBCA NPs without drug than for NPs
containing CBZ. Perhaps the somewhat larger size of the NPs with
drug (Table S1) contributes to this difference.
[0128] Macrophages are the most abundant immune cells in mammary
tumors. Tumor associated macrophages (TAMs) were originally thought
to exert anti-tumor activities, but increasing clinical and
experimental evidence show that TAMs also may promote tumor
progression and influence anticancer drug responses. The
pro-tumorigenic macrophages are known as alternatively activated
and referred to as anti-inflammatory (M2-type), whereas the
classically activated pro-inflammatory (M1-type) macrophages
exhibit anti-tumorigenic properties. Plasticity is a hallmark of
the macrophage population and dynamic changes in their phenotype
define the different subtypes. Markers of M1 and M2 are commonly
used to recognize the main phenotypes or functions, but a set of
markers is recommended for a more comprehensive characterization of
the whole population. In the present examples, the well accepted
nitric oxide synthase (iNOS) have been used for detection of the M1
phenotype and mannose receptor (CD206) to define the M2-type.
[0129] The TAMs are influenced by the context, e.g. by factors in
the microenvironment or externally added anticancer drugs.
Interestingly, the effect of docetaxel has been shown to partly
depend upon depletion of M2 macrophages and expansion of M1
macrophages in models of breast cancer. In contrast, no response in
the macrophage populations upon treatment with free CBZ, another
taxane, was observed, despite efficient growth retardation.
However, treatment with PEBCA encapsulated CBZ, resulted in
significant improved anti-tumor efficacy, and complete remission in
75% of the tumors. Even though the number of tumors used for
immunohistochemical quantification is small, the data suggest two
possible mechanisms that may explain this good effect. First, a
trend towards elevated inflammation in tumors upon treatment with
PEBCA NPs (with or without drug) was observed. This may imply a
role of PEBCA NPs in homing anti-tumorigenic M1 macrophages into
the tumors and thus further support the effect of CBZ. Secondly, we
also showed that PEBCA-CBZ treatment significantly reduced CD206
expression in tumors compared to treatment with NPs without drug,
which may point towards depletion of pro-tumorigenic
macrophages.
[0130] Recently it was published that M2 macrophages show a
vigorous endocytic uptake by macropinocytosis, whereas this uptake
was virtually inactive in the M1 macrophages. Furthermore, another
study showed that the M2 macrophages use endocytosis to degrade
collagen and promote tumor growth in solid tumors. Without being
bound by theory, the decrease in M2 macrophages observed after
treatment with PEBCA-CBZ and the strong effect on the tumor growth
is related to the macropinocytic uptake of PEBCA-CBZ and subsequent
killing of these M2 macrophages. Thus, the inherent properties of
M1 and M2 macrophages and selective toxic effect of drug containing
particles on M2 macrophages may increase the efficacy of the
treatment. It has earlier been published that driving TAMs toward
M1 polarization (increasing the ratio of M1/M2 macrophages) has
shown promising therapeutic effects in mice cancer models.
[0131] The effect of PEBCA-CBZ and non-encapsulated CBZ was also
examined in orthotopically growing MDA-MB-231 tumors without
showing the same efficacy as in the MAS98.12 PDX model. Even though
the data demonstrate a therapeutic effect of PEBCA-CBZ in
MDA-MB-231 tumors compared to empty PEBCAs, the effect is not equal
to what is seen with non-encapsulated CBZ.
[0132] Since the outcome of these NPs are based on the EPR effect,
the tumor vasculature is a critical factor for the accumulation of
NPs in tumor. It is shown earlier that the basal-like MAS98.12
tumor has a higher vascularization than the luminal-like MAS98.06
tumor. Although angiogenesis also has been visualized in MDA-MB-231
tumors in mice, the difference in vascularization of the MAS98.12
and MDA-MB-231 tumors is less well characterized. However, in two
previously published studies, the blood volume constituted 2.4% of
the tumor volume 5 weeks after inoculation of MDA-MB-231 and 5.9%
at 5 weeks after transplantation of MAS98.12, suggesting more
efficient vascularization in the latter model. Accordingly, the
degree of vascularization may influence the efficacy.
[0133] Both the fluorescence in vivo imaging data and the
quantification of CBZ in tissue samples clearly demonstrate
accumulation of the PEBCA NPs in lymph nodes (FIGS. 3B, C and FIG.
4B). Accumulation of drugs or imaging agents in lymph nodes was
recently reviewed, suggesting a benefit of injecting very small NPs
for the best accumulation. A study reported accumulation into lymph
nodes of particles similar to those used in the present study, i.e.
poly(butylcyanoacrylate) modified with Pluronic F127 and loaded
with vincristine. As these NPs were shown to rapidly release
vincristine and vincristine also accumulated in lymph nodes
following injection of the non-encapsulated drug, it is difficult
to evaluate to which extent these NPs actually accumulated in the
lymph nodes. Delivery of drug to lymph nodes has been discussed for
treatment of metastasizing tumors. Since local lymph nodes are the
first site of locoregional breast cancer metastasis, and also a
significant metastatic site in the aggressive triple negative basal
like breast cancer, accumulation of PEBCA-CBZ to lymphatic tissue
may contribute additionally to such treatment.
[0134] In summary, the PEBCA-CBZ NPs demonstrate promising results
for treatment of breast cancer, supporting the therapeutic effect
of a drug delivery system for use in treatment of solid tumors as
disclosed herein.
[0135] Furthermore, the observation that the PACA NPs seems to
enhance intratumoral presence of anti-tumorigenic macrophages might
be of more general value, and support that the drug delivery system
of the invention can be used as an immune modulator and/or as a
vehicle able to enhance the therapeutic effect of encapsulated
drugs.
TABLE-US-00001 TABLE 1 Description of size, PDI, zetapotential, NP
and drug content of the batches used in this study. Size z-avg.
Size number- Zeta-potential CBZ content in CBZ content in NP
content in Study NP description (nm) avg. (nm) PDI (mV) NPs (% w/w)
stock sol. (mg/ml) stock sol. (mg/ml) MAS.98.12 PEBCA-NR668 156 86
0.19 -2.2 -- -- 39 MAS.98.12 PEBCA-NR668- 215 161 0.17 -2.4 8.6 2.1
24 CBZ MDA-MB-231 PEBCA 148 118 0.09 -0.6 -- -- 74 and
biodistribution MDA-MB-231 PEBCA-CBZ 214 196 0.07 -1.1 7.0 3.4 49
and biodistribution IVIS imaging PEBCA-NR668 172 152 0.04 -0.8 --
-- 57 IVIS imaging PEBCA-NR668- 227 186 0.15 -1.1 6.0 3.4 56
CBZ
Example 2
Treatment of a Prostate Carcinoma Tumor model with CBZ formulated
as Jevtana.RTM., or Encapsulated into PEBCA NPs
Methods:
[0136] Jevtana was formulated as described in the treatment study
on breast cancer. Briefly, CBZ was dissolved to 40 mg/ml in Tween,
80, then diluted 1:4 in 13% EtOH. CBZ-loaded PEBCA NPs was prepared
and characterized as described in the breast cancer treatment
study. Briefly, CBZ was added to the monomer phase at 10% w/v and
the pegylated PEBCA NPs were made in a one step miniemulsion
polymerization. The NPs were characterized for size, size
distribution and zeta-potential with DLS.
[0137] Human PC3 prostate adenocarcinoma cells were grown in DMEM
with 10% FBS and 1% Pencillin/streptavidin. 3 million cells in 50
.mu.l cell medium were injected subcutaneously on the hind leg of
the mouse. When the tumor reached a volume of 200 mm.sup.3,
treatment was started and the continued with one treatment weekly
for three weeks. The animals were randomly distributed into three
groups receiving: 1; control--no treatment, 2; 10 mg/kg CBZ-PEBCA,
3; 10 mg/kg Jevtana. The drugs were administered through a catheter
in the tail vein. The tumors were measured twice weekly using a
caliper and the animals were euthanized when tumors reached 1000
mm.sup.3.
Results:
[0138] The NPs were characterized with dynamic light scattering,
and had a diameter of 180 nm, PDI of 0.18 and zeta-potential of
-1.7 mV. CBZ-encapsulation was measured with mass spectrometry and
was found to be 8.5% w/w of the NP mass.
[0139] It was found that CBZ had a similar growth inhibition when
formulated either as the clinical approved formulation or in PEBCA
NPs (FIG. 1). Both treatments were significantly different
(p<0.001, t-test) from the untreated control 14 days after the
first treatment.
[0140] In FIG. 11, mean tumor size in the three groups are plotted.
Arrows show treatment days, error bars show standard deviation and
*** indicates p>0.001, t-test. As can be seen from the figure,
the cytostatic effects of CBZ formulated in PEBCA NPs was similar
to the effects of the clinical formulation of the drug in the
prostate cancer model PC3.
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