U.S. patent application number 15/307642 was filed with the patent office on 2017-08-31 for agent, product and use.
This patent application is currently assigned to FUNDACION PEDRO BARRIE DE LA MAZA, CONDE DE FENOSA. The applicant listed for this patent is BIOMERIX CORPORATION, FUNDACION PEDRO BARRIE DE LA MAZA, CONDE DE FENOSA, FUNDACION RAMON DOMINGUEZ, SERGAS, UNIVERSITY OF SANTIAGO DE COMPOSTELA. Invention is credited to Alexandre DE LA FUENTE GONZALEZ, Lawrence Patrick LAVELLE, Jr., Rafael LOPEZ, Miguel Abal POSADA.
Application Number | 20170246258 15/307642 |
Document ID | / |
Family ID | 53199939 |
Filed Date | 2017-08-31 |
United States Patent
Application |
20170246258 |
Kind Code |
A2 |
DE LA FUENTE GONZALEZ; Alexandre ;
et al. |
August 31, 2017 |
AGENT, PRODUCT AND USE
Abstract
The present invention relates to a composition for modulating
tumor cell dissemination, in particular metastatic cancer cells. In
particular, the invention relates to an agent for modulating
metastatic tumor cell dissemination for use in the treatment and/or
prevention of a metastatic cancer wherein the agent an
extracellular matrix (ECM) protein carried on a polycarbonate
polyurethane matrix. The invention also relates to a product,
comprising an agent for modulating metastatic tumor cell
dissemination, and to a method of treatment or prevention of
cancer.
Inventors: |
DE LA FUENTE GONZALEZ;
Alexandre; (Santiago de Compostela, ES) ; LOPEZ;
Rafael; (Santiago de Compostela, ES) ; POSADA; Miguel
Abal; (Santiago de Compostela, ES) ; LAVELLE, Jr.;
Lawrence Patrick; (Colonia, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FUNDACION PEDRO BARRIE DE LA MAZA, CONDE DE FENOSA
SERGAS
UNIVERSITY OF SANTIAGO DE COMPOSTELA
FUNDACION RAMON DOMINGUEZ
BIOMERIX CORPORATION |
La Coruna
La Coruna
La Coruna
La Coruna
Somerset |
NJ |
ES
ES
ES
ES
US |
|
|
Assignee: |
FUNDACION PEDRO BARRIE DE LA MAZA,
CONDE DE FENOSA
Canton Grande, 9
NJ
SERGAS
La Coruna
UNIVERSITY OF SANTIAGO DE COMPOSTELA
La Coruna
FUNDACION RAMON DOMINGUEZ
Santiago de Compostela
BIOMERIX CORPORATION
Somerset
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20170049860 A1 |
February 23, 2017 |
|
|
Family ID: |
53199939 |
Appl. No.: |
15/307642 |
Filed: |
April 30, 2015 |
PCT Filed: |
April 30, 2015 |
PCT NO: |
PCT/EP2015/059602 PCKC 00 |
371 Date: |
October 28, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/19 20130101; B01J
20/262 20130101; B01J 20/3212 20130101; A61K 9/0019 20130101; A61K
9/0024 20130101; A61K 38/39 20130101; A61K 45/06 20130101; B01J
20/3274 20130101; A61K 47/34 20130101; A61K 38/014 20130101; B01J
20/3021 20130101; B01J 20/3225 20130101; A61P 35/04 20180101; A61P
35/00 20180101 |
International
Class: |
A61K 38/39 20060101
A61K038/39; A61K 9/19 20060101 A61K009/19; A61K 9/00 20060101
A61K009/00; A61K 47/34 20060101 A61K047/34; A61K 45/06 20060101
A61K045/06 |
Claims
1. An agent for modulating metastatic tumor cell dissemination
comprising an extracellular matrix (ECM) protein or adhesion
molecule and a polycarbonate polyurethane matrix.
2-3. (canceled)
4. The agent of claim 1, wherein the extracellular matrix (ECM)
protein or adhesion molecule is selected from the group consisting
of collagen, fibronectin, elastin and fibrillin.
5-9. (canceled)
10. The agent of claim 1, wherein the extracellular matrix protein
or adhesion molecule comprises covalently cross-linked particles of
the molecule.
11. (canceled)
12. The agent of claim 1, wherein the polycarbonate polyurethane
matrix is porous, is a 3D scaffold, or is porous and a 3D
scaffold.
13. The agent of claim 1, further comprising one or more
chemotherapeutic agents.
14-18. (canceled)
19. The agent of claim 1, wherein the agent attracts metastatic
tumor cells.
20. The agent of claim 19, wherein once the tumor cells have been
attracted or captured by the agent they are removed or
inactivated.
21. A method of attracting or capturing tumor cells in a subject
comprising administering to the subject an agent for modulating
tumor cell dissemination comprising an extracellular matrix (ECM)
protein or adhesion molecule and a polycarbonate polyurethane
matrix.
22. (canceled)
23. The method of claim 21 wherein the subject has a cancer
selected from the group comprising breast cancer, colorectal
cancer, pancreatic cancer, kidney cancer, prostate cancer,
urothelial cancer, oesophageal cancer, head and neck cancer,
hepatocellular cancer, mesothelioma, Kaposi's sarcoma, ovarian
cancer, soft tissue sarcoma, glioma, melanoma, small-cell and
non-small-cell lung cancer, endometrial cancer, basal cell
carcinoma, transitional cell carcinoma of the urothelial tract,
cervical cancer, endometrial cancer, gastric cancer, bladder
cancer, uterine sarcoma, multiple myeloma, soft tissue and bone
sarcoma, cholangiocarcinoma and a cancer disseminated
thereform.
24.-37. (canceled)
38. The method of claim 21 wherein the subject has a cancer of the
peritoneal cavity or a cancer disseminated into the peritoneal
cavity.
39. The method of claim 21, wherein the subject has a peritoneal
cancer and the agent is implanted in the abdomen of a subject.
40. The method of claim 21, further comprising the step of removing
or inactivating the attracted or captured tumor cells.
41. The method of claim 21, wherein the polycarbonate polyurethane
matrix is cross-linked with urea.
42. A method of manufacturing an agent for modulating metastatic
tumor cell dissemination, the method comprising the steps of:
preparing a suspended solution of an ECM protein; coating a
polycarbonate polyurethane matrix by saturation within the solution
of the ECM protein; and lyophilization of the the ECM protein
within the polycarbonate polyurethane matrix to form the agent for
modulating metastatic tumor cell dissemination.
43. The method according to claim 42, wherein the ECM protein is
cryogenically ground to a smaller particle size prior to
coating.
44. The method according to claim 42, wherein the ECM protein is
ground to an average particle size of between about 10 and 20
microns.
45. The method according to claim 42, wherein the solution of ECM
protein is a solution of ECM protein and deionised water, and
wherein the amount of ECM protein in solution is between about 30
and about 80 mg ECM protein/g water.
46. The method according to claim 42, wherein the polycarbonate
polyurethane matrix is saturated by repeated mechanical
compressions under the surface of the ECM protein solution
fluid.
47. The method according to claim 42, wherein drying is via a
lyophilisation process that utilizes sublimation under vacuum after
the material has been frozen.
48. The method according to claim 42, further comprising
crosslinking the ECM protein.
49. The method according to claim 42, wherein the lyophilized ECM
protein is collagen.
50. The agent of claim 1, wherein the extracellular matrix (ECM)
protein or adhesion molecule is collagen.
51. The agent of claim 50, wherein the collagen is fibrillar Type I
bovine collagen.
52. The agent of claim 50, wherein the total collagen loading
within the polycarbonate polyurethane matrix is between about 0.01
and about 0.2 mg collagen/mm.sup.3 polycarbonate polyurethane
matrix.
53. The agent of claim 50, wherein the total collagen loading
within the polycarbonate polyurethane matrix is at least 0.03 mg
collagen/mm.sup.3 polycarbonate polyurethane matrix.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a 35 U.S.C. .sctn.371 National Phase
Entry Application of International Application No.
PCT/EP2015/059602 filed Apr. 30, 2015, which designated the U.S.,
and which claims benefit of EP Application No. 14382160.1 filed
Apr. 30, 2014, and which claims benefit of GB Application No.
1501474.9 filed Jan. 29, 2015, the contents of each of which are
incorporated herein by reference in their entireties.
[0002] The present invention relates to an agent for modulating the
dissemination of cancer cells, in particular metastatic cancer
cells, and to the use of the agent in the treatment or prevention
of cancer. The composition may capture and/or attract cancer cells,
in particular metastatic cancer cells. The invention also relates
to a method of treatment or prevention of cancer.
[0003] The process of metastasis is associated with more than 90%
of cancer-related deaths and represents the main challenge in
oncology. While primary disease is reasonably accessible to surgery
and/or radiotherapy and presents an acceptable response to
chemotherapy leading to a good prognosis; metastatic dissemination
is associated with a contraindication to surgery and radiotherapy
and especially resistance to chemotherapy, and offers a much worse
prognosis.
[0004] In recent years, the process of metastasis has been
characterized as a stepwise process where aggressive tumor cells
acquire the abilities to invade the surrounding stroma and tissues,
to intravasate and survive in the blood flow, and to extravasate
and generate a micrometastasis at distant organs. In general, there
are two main ways of tumor cell dissemination from the primary
lesion: systemic dissemination of metastatic tumor cells through
the blood and lymphatic vessels, and loco-regional dissemination by
release or migration/invasion of metastatic tumor cells into the
surroundings. Tumor cells which disseminate from the primary tumor
into the bloodstream, are known as circulating tumor cells (CTC),
and are the main cause of metastasis. For dissemination through
blood and lymphatic vessels, the consensus is that tumor cells must
acquire an aggressive phenotype allowing migration and invasion of
the surrounding stroma (epithelial to mesenchymal transition);
activate neoangiogenesis by attracting endothelial cells and
creating new blood vessels that provide the tumor not only with
nutrients but also generating routes for dissemination; then tumor
cells invade and incorporate into the new blood vessels
(intravasation) and disseminate to those sites in the organism
where they will attach and exit the blood vessels (extravasation);
finally, these metastatic tumor cells will be able to establish a
niche and generate a micrometastasis that will evolve into a
metastatic lesion. The whole process is extremely inefficient but
dramatically lethal. Alternatively, dissemination may occur through
cellular migration and invasion of the surrounding stroma and
organs, or like in ovarian cancer where tumor cells are exposed and
released to the peritoneal cavity, by incorporation of metastatic
cells into the ascitic fluid and implantation in the peritoneum and
organs accessible in the cavity.
[0005] The molecular and cellular bases that determine the process
of metastasis suggest an intense dialogue of the primary tumor with
the environment (Sleeman, J P et al., Semin Cancer Biol. 2012 June;
22(3):174-86). Tissue specific metastasis (Nguyen et al., Nat Rev
Cancer. 2009; 9(4):274-84) and pre-metastatic niches (Psaila &
Lyden, Nat Rev Cancer. 2009; 9(4):285-93) are concepts that are
beginning to illustrate an active role of carcinomas in the
determination of the most adequate sites to colonize: signals
emitted from the tumor and from the environment may govern the
remodeling of targeted tissues for a favored reception of tumor
cells disseminated from primary lesions.
[0006] An aim of the present invention is to interfere with the
communication between tumor cells, and in particular metastatic
tumor cells, and the host, to allow the pattern of metastatic
dissemination to be modulated. The invention may operate, in
certain embodiments, by physically trapping such cells and/or by
providing a preferential site for homing of such cells.
[0007] According to a first aspect, the invention provides an agent
for modulating tumor cell dissemination comprising an extracellular
matrix protein (ECM) or adhesion molecule and a reticulated
elastomeric matrix for use in the treatment and/or prevention of a
cancer.
[0008] The invention also provides the use of an agent for
modulating tumor cell dissemination comprising an extracellular
matrix protein (ECM) or adhesion molecule and a reticulated
elastomeric matrix in the treatment and/or prevention of
cancer.
[0009] The invention also further provides the use of an agent for
modulating tumor cell dissemination comprising an extracellular
matrix protein (ECM) or adhesion molecule and a reticulated
elastomeric matrix in the preparation of a medicament or a medical
device for the treatment and/or prevention of cancer. The medical
device may have a non-pharmacological mode of action.
[0010] Preferably the agent for modulating tumor cell dissemination
is for modulating metastatic tumor cell dissemination. Preferably
the agent is for use in the treatment and/or prevention of a
metastatic cancer.
[0011] The agent for modulating tumor cell dissemination may act to
interfere with the natural process of tumor cell dissemination,
preferably to modulate the behaviour of such cells such that they
are attracted to or captured at a particular site, preferably at
the location of the agent for modulating tumor cell
dissemination.
[0012] The agent for modulating tumor cell dissemination may act as
a chemoattractant for tumor cells, in particular for metastatic
tumor cells. The metastatic tumor cells may be circulating tumor
cells, disseminated tumor cells or any cell disseminated from a
primary tumor.
[0013] The agent for modulating tumor cell dissemination may be
intended to capture or trap tumor cells, and in particular
metastatic tumor cells, such as circulating tumor cells,
disseminated tumor cells or any cell disseminated from a primary
tumor. The agent may directly mediate capture of the tumor cells,
for example by adhering to the tumor cells, or may have an indirect
effect which improves adhesion of the tumor cells at specific sites
in the host.
[0014] The reticulated elastomeric matrix may be a polycarbonate
polyurethane matrix, the polymer of the matrix may be cross linked;
the polymer may also contain urea within the molecular
structure.
[0015] An agent for use in the invention may further be capable of
physically capturing tumor cells and trapping them, the agent may
be an adhesive material to which tumor cells adhere.
[0016] The agent may capture/trap the cells by providing a favoured
substrate for the metastatic cells to attach and anchor to. This
substrate may be a solid 2D or 3D polymer surface, or a chemically
modified surface, or a patterned surface, or a gel, or a hydrogel,
etc, where the cell can create adhesive structures such as focal
adhesions, tight junctions, anchoring junctions, GAP junctions,
etc.
[0017] The agent may comprise a 2D or a 3D porous structure. In one
embodiment the capture agent may be a 3D porous tissue scaffold
type material. The agent may be a 3D porous mesh structure.
[0018] The 2D or 3D surface or structure may be provided by the
reticulated elastomeric matrix, such as a polycarbonate
polyurethane matrix, preferably with one or more ECM proteins
contained or carried therein. The polycarbonate polyurethane
structure may be cross-linked with urea.
[0019] The agent may comprise a polycarbonate polyurethane matrix
with urea segments and additional crosslinking, such as the
Biomerix.TM. 3D Scaffolds from Sigma Aldrich, USA.
[0020] The one or more ECM proteins may be physically and/or
chemically contained or carried in the reticulated elastomeric
matrix, such as a polycarbonate polyurethane matrix. The ECM
proteins may be permanently contained within the matrix or they may
be released in a controlled or uncontrolled manner.
[0021] The agent of the invention may comprise a 2D or a 3D
scaffold of a reticulated elastomeric matrix, such as a
polycarbonate polyurethane matrix, which is decorated with or has
embedded therein ECM proteins to improve the attachment of tumor
cells, and metastatic tumor cells in particular, to the surface.
The ECM proteins may mediate cell-cell adhesion or cell-substrate
adhesion. In addition, the agent may capture/trap the metastatic
cells by remodeling the site of implantation of the invention, by
means of remodeling the cellular architecture of the site of
implantation, or by remodeling the extracellular matrix, by
remodeling the site through a foreign body reaction (Anderson et
al., Semin Immunol 2008) or an inflammatory reaction.
[0022] The agent for modulating tumor cell dissemination may
alternatively or additionally comprise a chemoattractant for tumor
cells, and in particular metastatic tumor cells, such as
circulating tumor cells, disseminated tumor cells or any cell
disseminated from a primary tumor.
[0023] In some embodiments the agent for modulating tumor cell
dissemination may therefore act as both a capture agent and a
chemoattractant for tumor cells.
[0024] Useful chemoattractants may be any agent capable of
attracting tumor cells, and in particular metastatic tumor cells,
such as circulating tumor cells, disseminated tumor cells or any
cell disseminated from a primary tumor. The tumor cells may be
attracted directly or indirectly through the attraction of an
intermediate cell (i.e. immune cell or stem cell). For example, the
implantation of an agent of the invention may generate an
inflammatory reaction that provides an additional chemotactic
effect for metastatic cancer cells. Furthermore, the tumor cells
that are attracted by the agent of the invention may themselves
provide an additional chemotactic effect for other tumor cells, and
in particular metastatic tumor cells.
[0025] The agent for modulating tumor cell dissemination may
further comprise vesicles derived from cells, including exosomes.
Exosomes are cell-derived microvesicles that are present in many
and perhaps all biological fluids, including blood, urine, and
ascitic fluid. They typically have a diameter of between 30 and 100
nm. They are released by many cells types during normal
physiological processes; however tumors appear to aberrantly
secrete large quantities of exosomes. Exosomes for use in the
invention may be obtained from a bodily fluid, such as blood or
urine, or obtained from many different cell types in an organism.
The bodily fluid from which exosomes are purified may be from a
healthy donor. Exosomes for use in the invention may be secreted by
cancer cells, such as ovarian cancer cells; or alternatively, or in
addition, the exosomes may be secreted by non cancer cells, such as
mesenchymal stem cells. It may be preferable to use exosomes from
non cancer cells
[0026] Alternatively, or additionally, the agent for modulating
tumor cell dissemination may further comprise ascitic fluid from a
subject with ovarian cancer. The ascitic fluid may comprise
exosomes. The capture agent or chemoattractant may be exosomes
obtained from the ascitic fluid of a subject with ovarian
cancer.
[0027] Alternatively, or additionally, the agent for modulating
tumor cell dissemination may further comprise mesenchymal stem
cells themselves, or indeed another form of stem cells, but
preferably not human embryonic stem cells. Mesenchymal stem cells
of adipose, umbilical cord or bone marrow origin may be used as a
chemoattractant.
[0028] Alternatively or additionally, the agent for modulating
tumor cell dissemination may further comprise a cell adhesion
molecule, such as a selectin, a member of the immunoglobulin (Ig)
superfamily, an integrin or a cadherin. The cell adhesion molecule
may be found associated with exosomes such as CD9 and/or CD81.
[0029] Alternatively, or additionally, the agent for modulating
tumor cell dissemination may further comprise one or more
chemokines and/or one or more growth factors, for example one or
more of SDF1, 90K, osteopontin, EGF, TGFb1, FGF, and IGF. In one
embodiment the chemoattractant comprises a combination of EGF,
TGFb1 and FGF.
[0030] The agent for modulating tumor cell dissemination preferably
includes one or more adhesion molecule or extracellular matrix
component selected from the list comprising cell adhesion
molecules, calcium-independent IgSF, CAM, N-CAM (Myelin protein
zero), ICAM (1, 5), VCAM-1, PE-CAM, L1-CAM, Nectin (PVRL1, PVRL2,
PVRL3), integrins, LFA-1 (CD11a+CD18), integrin alphaXbeta2
(CD11c+CD18), macrophage-1 antigen (CD11b+CD18), VLA-4
(CD49d+CD29), glycoprotein IIb/IIIa (ITGA2B+ITGB3),
Calcium-dependent cadherins, Classical CDH1, CDH2, CDH3, desmosomal
Desmoglein (DSG1, DSG2, DSG3, DSG4), desmocollin (DSC1, DSC2,
DSC3), protocadherin, PCDH1, PCDH15, unconventional/ungrouped
T-cadherin, CDH4, CDH5, CDH6, CDH8, CDH11, CDH12, CDH15, CDH16,
CDH17, CDH9, CDH10, selectins, E-selectin, L-selectin, P-selectin,
other lymphocyte homing receptors, CD44, L-selectin, integrin
(VLA-4, LFA-1), carcinoembryonic antigen, CD22, CD24, CD44, CD146,
CD164, proteins and glycosaminoglycans as components of the
extracellular matrix (ECM): heparan sulfate, chondroitin sulfates,
keratan sulfates, hyaluronic acid, collagens, elastins, fibrillin,
fibronectins and laminins.
[0031] The agent may comprise a component of the extracellular
matrix (ECM), including one or more of heparan sulfate, chondroitin
sulfates, keratan sulfates, hyaluronic acid, collagens, elastins,
fibrillin, fibronectins and laminins. The agent may comprise
collagen and/or fibronectin.
[0032] If included, a further chemoattractant may remain attached
to or within the matrix of the agent of the invention, or may be
released or leached from the matrix to create a gradient of
chemoattractant around the matrix.
[0033] The agent is biocompatible, such that if placed in a human
or non-human animal it does not cause an unacceptable immune
response. In some embodiments the agent may be associated with a
limited immune response at the site of placement, for example an
inflammatory immune response or foreign body reaction.
[0034] The agent may comprise a porous matrix as described above.
The matrix may be a 2D or a 3D porous structure. In one embodiment
the matrix may be a 3D porous tissue scaffold type material. The
matrix may be a 3D porous mesh structure.
[0035] Where the agent includes a chemoattractant, which may or may
not be the one or more ECM proteins, the agent may create a
gradient of the chemoattractant, essentially by releasing the
chemoattractant over time. The chemoattractant may be released in a
controlled or an uncontrolled manner Release of the chemoattractant
may be active or passive, or both. The chemoattractant may be the
one or more ECM proteins.
[0036] Preferably the agent, in use, retains at least 10%, 20% 30%,
40%, 50% or more of the chemoattractant and or ECM proteins for at
least 12 hours, at least 24 hours, at least 48 hours, at least 3
days, at least 4 days, at least 5 days, at least 6 days, at least 1
week, at least 2 weeks, at least 3 weeks, at least 4 weeks or
longer. Preferably the agent is quite stable and no significant ECM
protein is released, preferably over 80% of the ECM protein is
retained in or on the matrix for at least a week when in use. In
another embodiment, over 80% of the ECM protein is retained in or
on the matrix for at least a month when in use. In another
embodiment, over 80% of the ECM protein is retained in or on the
matrix for at least 3 months or 6 months when in use. In another
embodiment, over 80% of the ECM protein is retained in or on the
matrix for at least 12 months when in use.
[0037] The agent, in use, may release at least 10%, 20% 30%, 40%,
50% or more of the chemoattractant and or ECM proteins over at
least 12 hours, at least 24 hours, at least 48 hours, at least 3
days, at least 4 days, at least 5 days, at least 6 days, at least 1
week, at least 2 weeks, at least 3 weeks, at least 4 weeks or
longer.
[0038] The agent, in use, may create a chemoattractant gradient for
at least 12 hours, at least 24 hours, at least 48 hours, at least 3
days, at least 4 days, at least 5 days, at least 6 days, at least 1
week, at least 2 weeks, at least 3 weeks, at least 4 weeks or
longer.
[0039] Preferably the agent of the invention is able to release
sufficient chemoattractant for modulating tumor cell dissemination
to generate a gradient of chemoattractant effective for
loco-regional dissemination and/or for systemic dissemination in a
subject for a period sufficient to avoid metastatic
dissemination.
[0040] The agent of the invention may contain, for example, from
about 10% to about 98% by weight, preferably about 80%, preferably
at least about 20%, 25%, 30%, 35%, 40%, 45%, 50% or more by weight
of the ECM protein.
[0041] The agent of the invention may contain between 0.1 nanograms
and 10 mg of an agent for modulating tumor cell dissemination, such
as a capture agent and/or chemoattractant. Preferably between 0.1
nanograms and 1 mg, or 0.1 nanograms and 100 micrograms of the
agent for modulating tumor cell dissemination, such as a capture
agent and/or chemoattractant. Where the capture agent is collagen
the collagen may be present at between about 0.1 .mu.g to 1 mg,
e.g. between about 25 .mu.g and 500 .mu.g, e.g. 250 .mu.g.
[0042] The agent of the invention may further comprise a
chemotherapeutic agent, such as a cytostatic agent. Wherein a
cytostatic agent is a pharmacologically active compound capable of
inhibiting or suppressing cellular growth and multiplication.
Depending on the mechanism of action and on the dose of the
compound, it may also represent a cytotoxic agent. In particular,
the cytostatic agent may be a compound that is capable of killing,
or inhibiting the growth of, tumor cells, preferably metastatic
tumor cells, such as circulating tumor cells, disseminated tumor
cells or any cell disseminated from a primary tumor.
[0043] The cytostatic agent may be selected, for example, from:
[0044] a. anthracyclines and analogs thereof, such as daunomycin,
doxorubicin, idarubicin, epirubicin, valrubicin, aclacinomycin, and
mitoxantrone; [0045] b. antimetabolites, such as gemcitabine,
cytosine arabinoside, cytarabine, vidarabine, thioguanine,
pentostatin, cladribine, methotrexate, floxuridine, fluorouracil
and other fluorinated pyrimidines, purines, or nucleosides; [0046]
c. alkylating agents, such as nitrogen mustards, including
cyclophosphamide, melphalan, chlorambucil, ifosfamide;
nitrosoureas, including carmustine, lomustine, and streptozocin;
alkyl sulfonates, including busulfan; thiotepa; platinum compounds,
including cisplatin, carboplatin, oxaliplatin, nedaplatin,
satraplatin, and triplatin tetranitrate; procarbazine; and
altretamine; [0047] d. plant alkaloids and terpenoids, such as
vinca alkaloids, including vincristine, vinblastine, vinorelbine,
and vindesine; taxanes, including taxol, paclitaxel, docetaxel; and
podophyllotoxin; [0048] e. topoisomerase inhibitors, such as
amsacrine, etoposide, etoposide phosphate, teniposide and other
derivatives of epipodophyllotoxins; irinotecan, topotecan and other
camptothecins; and [0049] f. other antineoplastics, such as
dactinomycin, bleomycin, mitomycin, etoposide, bleomycin, and
plicamycin.
[0050] The agent for modulating tumor cell dissemination may be
used alone or in combination with other active agents, for example
in combination with one or more cytostatic agents.
[0051] The agent for modulating tumor cell dissemination of the
invention may be placed at a site of use by surgery. Similarly,
after use the agent may be removed by surgery.
[0052] The agent for modulating tumor cell dissemination of the
invention may be intended for use with many types of cancer,
including, but not limited to, breast cancer, colorectal cancer,
pancreatic cancer, kidney cancer, prostate cancer, urothelial
cancer, oesophageal cancer, head and neck cancer, hepatocellular
cancer, mesothelioma, Kaposi's sarcoma, ovarian cancer, soft tissue
sarcoma, glioma, melanoma, small-cell and non-small-cell lung
cancer, endometrial cancer, basal cell carcinoma, transitional cell
carcinoma of the urothelial tract, cervical cancer, endometrial
cancer, gastric cancer, bladder cancer, uterine sarcoma, multiple
myeloma, soft tissue and bone sarcoma, cholangiocarcinoma and
cancers disseminated therefrom.
[0053] In particular, the agent for modulating tumor cell
dissemination of the invention may be intended for use with cancers
of the peritoneal cavity, such as, stomach, gall bladder, liver,
small intestine, GIST, esophagus, abdominal sarcoma, soft tissue
sarcoma, mesothelioma, ovarian, pancreatic, colon, rectal, uterine,
cervical, kidney cancer and cancers disseminated therefrom. In a
preferred embodiment the cancer is ovarian cancer or a cancer
disseminating therefrom. Where the cancer is ovarian cancer or a
cancer disseminating therefrom, the product of the invention may be
implanted in the abdominal wall of the subject. Alternatively the
cancer may be colon cancer. The cancer may be pancreatic
cancer.
[0054] The present invention may be intended for use in the
prevention of cancer metastases, in particular for the prevention
of peritoneal metastases.
[0055] The agent of the invention may provide a favoured and
preferred site for the attachment or implantation of metastatic
tumor cells, for example in the peritoneal cavity if that is where
the agent is placed. In an embodiment, the agent has a
non-pharmacological mode of action when placed in the peritoneal
cavity that is further facilitated by the trascoelomic flow present
in the peritoneal cavity: that is, cells turn around in the
peritoneal cavity and are gradually trapped within the agent of the
invention where the agent is acting as a medical device. The agent
of the invention may therefore not need to act as a chemoattractant
itself, and may simply act to trap metastatic tumor cells.
[0056] According to another aspect, the invention provides the use
of an agent of the invention that traps or captures metastatic
cancer cells in the preparation of a medicament for the treatment
or prevention of cancer.
[0057] According to another aspect, the invention provides a
medical device comprising an agent of the invention that traps or
captures metastatic cancer cells. The device may be used for the
treatment or prevention of cancer.
[0058] Preferably the treatment or prevention of cancer comprises
the attraction and/or trapping of tumor cells, and in particular
metastatic tumor cells, such as circulating tumor cells,
disseminated tumor cells or any cell disseminated from a primary
tumor.
[0059] Preferably the attracted cells are held or trapped by the
action of the agent for modulating tumor cell dissemination, and
any chemoattractant present, thus localizing them to a particular
location and allowing them to be treated.
[0060] The agent for modulating tumor cell dissemination preferably
comprises an ECM protein contained in, or attached to, a matrix as
described herein. In some embodiments the matrix itself may be an
agent for modulating tumor cell dissemination capable of attracting
and/or trapping tumor cells, and the provision of a further capture
agent and/or chemoattractant is optional. In some embodiments the
agent for modulating tumor cell dissemination comprises a 3D
polymer scaffold, or hydrogel. Such polymers have been found to
have adhesive properties and to be capable of trapping tumor cells,
e.g. by providing a niche to which such cells can adhere, and/or by
providing a preferential site for homing of such cells.
[0061] In some embodiments, the agent for modulating tumor cell
dissemination comprises a cross-linked, polycarbonate
polyurethane-urea matrix (3D-Kube Biomerix scaffold) decorated with
an ECM protein, such as collagen and/or fibronectin.
[0062] Preferably the agent for modulating tumor cell dissemination
is administered to a non-vital organ. Thus the tumor cells will be
attracted to and retained in this tissue and may then be removed by
surgery. Such a location may allow any chemoattractant present to
be accessible from everywhere in the body; for example, it would
allow the agent of the invention to become vascularized and to
reach the blood circulation.
[0063] Alternatively the agent for modulating tumor cell
dissemination may be administered into the fat of a subject.
[0064] In a yet further embodiment the agent for modulating tumor
cell dissemination may be administered into the peritoneum of a
subject to attract and/or capture metastatic cells disseminating in
the peritoneal cavity. Similarly, the pleura may be a good place to
locate the agent for modulating tumor cell dissemination when
treating lung carcinomas and mesiotheliomas or tumor cells
disseminating into the pleura.
[0065] The agent for modulating tumor cell dissemination may be
administered by direct injection into the fat of a human or
non-human animal. For example, for the attraction of peritoneal
metastatic tumor cells the agent for modulating tumor cell
dissemination may be injected into the peritoneum or surrounding
tissue including fat tissue, for example, the gonadal fat.
Alternatively, the agent of the invention may be administered by
surgery.
[0066] Preferably, once in situ, the agent for modulating tumor
cell dissemination causes tumor cells, and in particular metastatic
tumor cells, such as circulating tumor cells, disseminated tumor
cells or any cell disseminated from a primary tumor, to be
attracted to it, and to congregate or be "trapped". In a preferred
embodiment the attracted cells are held or trapped by the action of
the agent for modulating tumor cell dissemination until the cells
are treated. Preferably at least 5%, 10%, 20%. 30%, 40% , 50%, 60%,
70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more of the attracted
cells are captured by the product of the invention.
[0067] The attracted or trapped cells may then be treated. The
attracted or trapped cells may be treated by physically removing
them, for example by surgery, or by treating them to destroy or
inactivate the cells, for example by chemotherapy or radiotherapy.
If the agent includes a cytostatic or cytotoxic agent, or the agent
for modulating tumor cell dissemination is administered with a
cytostatic or cytotoxic agent, then this may act to eradicate or
prevent the replication of the attracted cells.
[0068] The tumor cells to be treated may be one or more of any of
the cancers described above, in particular, the tumor cells may be
derived from/disseminated from a peritoneal cancer, such as ovarian
cancer.
[0069] Preferably the agent of the invention provides an adhesive
surface for metastatic cells, and this provides a preferred site of
implantation in competition with the natural sites for
implantation. The agent may act as an artificial pre-metastatic
niche.
[0070] According to another aspect the invention provides a method
of attracting tumor cells, and in particular metastatic tumor
cells, such as circulating tumor cells, disseminated tumor cells or
any cell disseminated from a primary tumor, in a subject comprising
administering to the subject an agent for modulating tumor cell
dissemination, and in particular metastatic tumor cells, such as
circulating tumor cells, disseminated tumor cells or any cell
disseminated from a primary tumor. The agent for modulating tumor
cell dissemination may comprise an ECM protein contained within or
attached to a matrix as described with reference to any aspect of
the invention. Preferably the attracted cells are retained or
trapped by the action of the agent for modulating tumor cell
dissemination. Once trapped by the agent for modulating tumor cell
dissemination the cancer cells themselves may act as a capture
agent and/or chemoattractant for other cancer cells.
[0071] According to a still further aspect, the invention provides
a method of treating or preventing cancer, in particular a
metastatic cancer, comprising administering to a subject in need
thereof, an agent for modulating tumor cell dissemination, and in
particular metastatic tumor cell dissemination, such as circulating
tumor cells, disseminated tumor cells or any cell disseminated from
a primary tumor. The agent for modulating tumor cell dissemination
may comprise an ECM protein contained within or attached to a
matrix as described herein. Preferably the subject in need of
treatment has already been diagnosed with a primary cancer, both
metastatic or not metastatic. Preferably tumor cells are retained
or trapped by the action of the agent for modulating tumor cell
dissemination. Preferably the method further comprises the step of
treating the trapped cells.
[0072] The agent may comprise a polycarbonate polyurethane scaffold
with urea segments and additional crosslinking.
[0073] The attracted or trapped cells may be treated by physically
removing them, for example by surgery, or by treating them to
destroy or inactivate the cells, for example by chemotherapy or
radiotherapy. If the agent includes a cytostatic or cytotoxic agent
then this may act to eradicate or prevent the replication of the
attracted cells. The method may comprise the step of surgically
removing the attracted cells, and/or the step of administering
chemotherapy and/or radiotherapy to treat the attracted or trapped
cells.
[0074] The method of the present invention may be used in
combination with current clinical scenarios, including in
combination with one or more of surgery, radiotherapy and
chemotherapy.
[0075] The cancer may be any cancer, in particular a peritoneal
cancer, such as ovarian cancer.
[0076] According to a still further aspect the invention provides a
medical device or implantable device for use in preventing or
treating cancer, preferably metastatic cancer, in a subject,
wherein the device comprises an agent for modulating tumor cell
dissemination as described herein.
[0077] According to another aspect of the invention, there is
provided a method of manufacturing an agent for modulating
metastatic tumor cell dissemination, the method comprising the
steps of: [0078] preparing a suspended solution of an ECM protein;
[0079] coating the polycarbonate polyurethane matrix by saturation
within the solution of the ECM protein; and [0080] lyophilization
of the the ECM protein within the polycarbonate polyurethane matrix
to form the agent for modulating metastatic tumor cell
dissemination.
[0081] The skilled man will appreciate that preferred features of
any one embodiment and/or aspect and/or claim of the invention may
be applied to all other embodiments and/or aspects and/or claims of
the invention.
DETAILED DESCRIPTION
[0082] Polycarbonate Polyurethane Scaffold
[0083] An agent of the invention may comprise a reticulated
elastomeric matrix which comprises a network of cells which forms a
three-dimensional spatial structure. The cells communicate and
connect to each other via the open-celled pores contained within
the cells or within the walls of the cells. This network results in
a matrix with a unique morphology, composed of continuous
interconnected and intercommunicating cells and pores creating a
continuous void. The reticulated elastomeric matrix permits
in-growth and proliferation of cells and tissue into the implant.
Preferably, the reticulated elastomeric matrix is biodurable, is
resiliently compressible and preferably comprises polycarbonate
polyurethane or polycarbonate polyurethane urea. Suitable matrices
include, without limitation, those described in U.S. Pat. Nos.
7,803,395 and 8,337,487; the disclosures of which are hereby
incorporated by reference.
[0084] Certain embodiments of the invention comprise reticulated
biodurable elastomer products, which are also compressible and
exhibit resilience in their recovery, that have a diversity of
applications and can be employed, by way of example, in biological
implantation, especially into humans, for long-term implants that
can stay permanently in the body or can be removed from the body
after a certain period of time. It would be desirable to form
implantable devices suitable for use as scaffolds, tissue
engineering scaffolds, cellular growth scaffolds or other
comparable substrates, to support in-vivo cell capture, growth, or
propagation.
[0085] In another embodiment, the implantable devices suitable for
use as scaffolds, tissue engineering scaffolds, cellular growth
scaffolds or other comparable substrates, to support in-vivo cell
capture and propagation. In one embodiment, the reticulated
elastomeric matrix of the invention facilitates cell capture and
propagation by providing a surface for cellular attachment,
migration, proliferation and/or deposition of new tissues,
extra-cellular matrix, epithelial tissue connective tissue, areolar
tissue, dense regular and irregular tissue, reticular tissue,
adipose tissue, cartilage and bone tissue, skeletal, smooth and
cardiac muscle tissue, fibrovascular tissue, or any combination
thereof. Without being bound by any particular theory, the
reticulated implantable devices having a high void content and an
unfettered access to the inter-connected and inter-communicating
high void content is thought to allow the implantable device to
become at least partially ingrown and/or proliferated, in some
cases substantially ingrown and proliferated, in some cases
completely ingrown and proliferated, with cells and create a
preferential site for the capture of disseminated or circulating
tumor cells. In another embodiment, owing to the biointegrative
three dimensional inter-connected and inter-communicating structure
characteristics of the reticulated matrix of the implantable
devices of the invention, the agent of the invnetion has the
advantage of potentially better and faster dissemination or
circulating tumor cells as compared to natural sites of
metastasis.
[0086] In another embodiment, reticulated biodurable elastomer
products can be satisfactorily implanted or otherwise exposed to
living tissue and fluids for extended periods of time, for example,
at least 29 days. In one embodiment, the implantable device is
biodurable for at least 2 months. In another embodiment, the
implantable device is biodurable for at least 6 months. In another
embodiment, the implantable device is biodurable for at least 12
months. In another embodiment, the implantable device is biodurable
for at least 24 months. In another embodiment, the implantable
device is biodurable for at least 5 years. In another embodiment,
the implantable device is biodurable for longer than 5 years.
[0087] The reticulated biodurable elastomeric products used in the
agent of the invention may be described as having a
"macrostructure" and a "microstructure", which terms are used
herein in the general senses described in the following
paragraphs.
[0088] The "macrostructure" refers to the overall physical
characteristics of an article or object formed of the biodurable
elastomeric product of the invention, such as: the outer periphery
as described by the geometric limits of the article or object,
ignoring the pores or voids; the "macrostructural surface area"
which references the outermost surface areas as though any pores
thereon were filled, ignoring the surface areas within the pores;
the "macrostructural volume" or simply the "volume" occupied by the
article or object which is the volume bounded by the
macrostructural, or simply "macro", surface area; and the "bulk
density" which is the weight per unit volume of the article or
object itself as distinct from the density of the structural
material.
[0089] The "microstructure" refers to the features of the interior
structure of the biodurable elastomeric material from which the
inventive products are constituted such as cell and pore
dimensions; pore surface area, being the total area of the material
surfaces in the pores; and the configuration of the struts and
intersections that constitute the solid structure of certain
embodiments of the inventive elastomeric product. Described
generally, the microstructure of the porous biodurable elastomeric
matrix having a distinct shape or an extended, continuous entity,
comprises a solid phase formed of a suitable biodurable elastomeric
material and interspersed there within, or defined thereby, a
continuous interconnected void phase, the latter being a principle
feature of a reticulated structure and comprises of cells and
pores.
[0090] The individual cells forming the reticulated elastomeric
matrix are characterized by their average cell diameter or, for
non-spherical cells, by their largest transverse dimension. The
reticulated elastomeric matrix comprises a network of cells that
form a three-dimensional spatial structure or void phase which is
interconnected via the open pores therein. In one embodiment, the
cells form a 3-dimensional superstructure. The pores provide
connectivity between the individual cells, or between clusters or
groups of pores which form a cell. The cells of the elastomeric
matrix are formed from clusters or groups of pores, which would
form the walls of a cell except that the cell walls of most of the
pores are absent or substantially absent owing to reticulation. In
particular, a small number of pores may have a cell wall of
structural material also called a "window" or "window pane" such as
cell wall. Such cell walls are undesirable to the extent that they
obstruct the passage of fluid and/or propagation and proliferation
of tissues through pores. Such cell walls that obstruct the passage
of fluid and/or propagation and proliferation of tissues through
pores may, in one embodiment, be removed in a suitable process
step, such as reticulation that can be thermal, explosive or
chemical reticulation.
[0091] In one embodiment the microstructure of elastomeric matrix
is constructed to permit or encourage cellular adhesion to the
surfaces of matrix and cellular proliferation into pores of void
phase, when elastomeric matrix resides in suitable in-vivo
locations for a period of time. In another embodiment, such
cellular ingrowth and proliferation can occur or be encouraged not
just into exterior layers of pores, but into the deepest interior
of and throughout elastomeric matrix. This is possible owing to the
presence of interconnected and inter-communicating cells and pores
and voids, all of which are accessible for cellular ingrowth and
proliferation. Thus, in this embodiment, the space occupied by
elastomeric matrix becomes entirely filled by the cellular ingrowth
and proliferation except for the space occupied by the elastomeric
solid phase.
[0092] The void phase may comprise as little as 5% by volume of
elastomeric matrix referring to the volume provided by the
interstitial spaces of elastomeric matrix. In another embodiment,
void phase may comprise as little as 25% by volume of elastomeric
matrix. In another embodiment, void phase may comprise as little as
50% by volume of elastomeric matrix. In another embodiment, void
phase may comprise as little as 75% by volume of elastomeric
matrix. In another embodiment, void phase may comprise as least 90%
by volume of elastomeric matrix. In another embodiment, void phase
may comprise at least 95% by volume of elastomeric matrix.
[0093] In another embodiment the average diameter or other largest
transverse dimension of pores is not greater than about 800 .mu.m.
In another embodiment the average diameter or other largest
transverse dimension of pores is not greater than about 600 .mu.m.
In another embodiment, the average diameter or other largest
transverse dimension of pores is not greater than about 500 .mu.m.
In another embodiment, the average diameter or other largest
transverse dimension of pores is not greater than about 400 .mu.m.
In another embodiment, the average diameter or other largest
transverse dimension of pores is not greater than about 385 .mu.m.
In another embodiment, the average diameter or other largest
transverse dimension of pores is not greater than about 200 .mu.m.
In another embodiment, the average diameter or other largest
transverse dimension of pores is not greater than about 100 .mu.m.
In another embodiment, the average diameter or other largest
transverse dimension of pores is not greater than about 20
.mu.m.
[0094] In one embodiment to encourage cellular ingrowth and
proliferation and to provide adequate fluid permeability, the
average diameter or other largest transverse dimension of the cells
of elastomeric matrix is at least about 50 .mu.m. In another
embodiment, the average diameter or other largest transverse
dimension of it cells is at least about 200 .mu.m. In another
embodiment, the average diameter or other largest transverse
dimension of it cells is at least about 350 .mu.m. In another
embodiment, the average diameter or other largest transverse
dimension of it cells is at least about 500 .mu.m. In another
embodiment, the average diameter or other largest transverse
dimension of it cells is at least about 700 .mu.m. In another
embodiment, the average diameter or other largest transverse
dimension of it cells is at least about 900 .mu.m. In another
embodiment, the average diameter or other largest transverse
dimension of it cells is at least about 1500 .mu.m. In another
embodiment, the average diameter or other largest transverse
dimension of it cells is at least about 1800 .mu.m.
[0095] In one embodiment, the elastomeric matrix may have a
permeability of greater than 250 Darcy. Alternatively, the
elastomeric matrix may have a permeability of greater than 200
Darcy. The elastomeric matrix may have a permeability of less than
500 Darcy. Alternatively, the elastomeric matrix may have a
permeability of less than 400 Darcy. Alternatively, the elastomeric
matrix may have a permeability of less than 300 Darcy. In one
embodiment, the elastomeric matrix may have a permeability of
between about 200 Darcy and about 500 Darcy.
[0096] In one embodiment, the elastomeric matrix may have a density
of between about 3.5 and about 3.9 lb/ft.sup.3. In another
embodiment, the elastomeric matrix may have a density of between
about 2 and about 10 lb/ft.sup.3.
[0097] The structure, morphology and properties of the elastomeric
matrices of this invention can be engineered or tailored over a
wide range of performance by varying the starting materials and/or
the processing and/or the post processing conditions for different
functional or therapeutic uses. In another embodiment, the
structure, morphology and properties of the device comprising
elastomeric matrices and at least one functional element such as a
coating can be engineered or tailored over a wide range of
performance by varying the starting materials and/or the processing
and/or the post processing conditions.
[0098] In one embodiment, the inventive reticulated biodurable
elastomeric matrix is synthetic polymers, especially, elastomeric
polymers that are resistant to biological degradation, for example,
polycarbonate polyurethane-urea, polycarbonate polyurea-urethane,
polycarbonate polyurethane. Such elastomers are generally
hydrophobic but, pursuant to the invention, may be treated to have
surfaces that are less hydrophobic or somewhat hydrophilic. In
another embodiment, such elastomers may be produced with surfaces
that are less hydrophobic or somewhat hydrophilic. In another
embodiment, such elastomers may be produced with surfaces that are
significantly or largely hydrophobic.
[0099] In further embodiments, the invention provides a porous
biodurable elastomer and a process for polymerizing, cross-linking
and foaming the same which can be used to produce a biodurable
reticulated elastomeric matrix as described herein. In another
embodiment, reticulation follows.
[0100] More particularly, in another embodiment, the invention
provides a biodurable elastomeric polyurethane matrix which
comprises synthesizing the matrix from a polycarbonate polyol
component and an aromatic diisocyanates such as p-phenylene
diisocyanate, 4,4'-diphenylmethane diisocyanate ("4,4'-MDI"),
2,4'-diphenylmethane diisocyanate ("2,4'-MDI") or mixture thereof.
The biodurable elastomeric polyurethane matrix is made by
polymerization, cross-linking and foaming, thereby forming pores,
followed by reticulation of the foam to provide a reticulated
product. Reticulation generally refers to a process for at least
partially removing cell walls, not merely rupturing or tearing them
by a crushing process. Moreover, crushing undesirable creates
debris that must be removed by further processing. In another
embodiment, the reticulation process substantially fully removes at
least a portion of the cell walls. Reticulation may be effected,
for example, by at least partially dissolving away cell walls,
known variously as "solvent reticulation" or "chemical
reticulation"; or by at least partially melting, burning and/or
exploding out cell walls, known variously as "combustion
reticulation", "thermal reticulation" or "explosive reticulation".
The product is designated as a polycarbonate polyurethane or
polycarbonate polyurethane-urea, being a polymer comprising
urethane groups formed from, e.g., the hydroxyl groups of the
polycarbonate polyol component and the isocyanate groups of the
isocyanate component. In this embodiment, the process employs
controlled chemistry to provide a reticulated elastomer product
with good biodurability characteristics. Pursuant to the invention,
the polymerization is conducted to provide a foam product employing
chemistry that avoids biologically undesirable or nocuous
constituents therein.
[0101] In one embodiment, the invention provides a process for
preparing a flexible polyurethane biodurable matrix capable of
being reticulated based on polycarbonate polyol component and
isocyanate component starting materials. In another embodiment, the
foam is substantially free of isocyanurate linkages. In another
embodiment, the foam has no isocyanurate linkages. In another
embodiment, the foam is substantially free of biuret linkages. In
another embodiment, the foam has no biuret linkages. In another
embodiment, the foam is substantially free of allophanate linkages.
In another embodiment, the foam has no allophanate linkages. In
another embodiment, the foam is substantially free of isocyanurate
and biuret linkages. In another embodiment, the foam has no
isocyanurate and biuret linkages. In another embodiment, the foam
is substantially free of isocyanurate and allophanate linkages. In
another embodiment, the foam has no isocyanurate and allophanate
linkages. In another embodiment, the foam is substantially free of
allophanate and biuret linkages. In another embodiment, the foam
has no allophanate and biuret linkages. In another embodiment, the
foam is substantially free of allophanate, biuret and isocyanurate
linkages. In another embodiment, the foam has no allophanate,
biuret and isocyanurate linkages.
[0102] Collagen Coating of an Elastomeric Matrix
[0103] In one embodiment, an elastomeric matrix may have what are
referred to herein as "endopore" features as part of its
microstructure, i.e., features of elastomeric matrix that are
located "within the pores". In one embodiment, the internal
surfaces of pores may be "endoporously coated", i.e., coated or
treated to impart to those surfaces a degree of a desired
characteristic, e.g., hydrophilicity or cell attachment. In one
embodiment, the internal surfaces of struts may be "endoporously
coated", i.e., coated or treated to impart to those surfaces a
degree of a desired characteristic, e.g., hydrophilicity or cell
attachment. In one embodiment, the internal void space or the space
between the cells may be "endoporously coated", i.e., coated or
treated to impart to those surfaces a degree of a desired
characteristic, e.g., hydrophilicity or cell attachment. The
coating or treating medium can have additional capacity to
transport or bond to active ingredients or cells that can then be
preferentially delivered to pores. In one embodiment, this coating
medium or treatment can be used facilitate attachment, growth and
proliferation of cells to the interior pore surfaces. In one
embodiment, this coating medium or treatment can be used facilitate
growth and proliferation of cells to the interior pore surfaces. In
one embodiment, this coating medium or treatment can be used
facilitate covalent bonding of materials to the interior pore
surfaces. In another embodiment, the coating comprises a
biodegradable polymer, a natural polymer, a cellular ingrowth
promoter or an inorganic component.
[0104] Furthermore, one or more coatings may be applied
endoporously by contacting with a biocompatible synthetic polymer,
biocompatible synthetic resorbable polymer, natural polymer or
cellular ingrowth promoter either in a liquid coating media or in a
melt state under conditions suitable to allow the formation of a
biocompatible coating, a biocompatible film coating or a
biocompatible lyophilized coating. The liquid coating media can be
a solution or a slurry or a mixture thereof. In one embodiment, the
polymers or the cellular ingrowth promoter used for such coatings
are film-forming biocompatible polymers or materials that
preferably should adhere to the solid phase. In another embodiment,
the polymers or the cellular ingrowth promoter used for such
coatings are lyophilizable biocompatible polymers that preferably
should adhere to the solid phase. In another embodiment, the
bonding strength is such that the film coating or the lyophilized
coating does not crack or dislodge during handling or deployment or
during placement in the body of reticulated elastomeric matrix.
[0105] In one embodiment, the coating is not continuous across the
entire external surface of the elastomeric matrix. In another, the
coating is not continuous across the entire external surface of the
elastomeric matrix such that the permeability and thus the cellular
infiltration into the interior surfaces of the elastomeric matrix
is not affected. In another, the coating is not continuous across
the entire external surface of the elastomeric matrix such that the
permeability is moderately affected but still permits cellular
infiltration into the elastomeric matrix. It is further thought
that as the coating degrades, permeability is restored in cases
where they may have been affected, and that circulating cells can
invade and infiltrate the reticulated elastomeric matrix in an
unfettered fashion.
[0106] Suitable biocompatible polymers include bioresorbable
aliphatic polyesters include but not limited to polymers and
copolymers of lactide (which includes lactic acid d-, l- and meso
lactide), .epsilon.-caprolactone, glycolide (including glycolic
acid), hydroxybutyrate, hydroxyvalerate, para-dioxanone,
trimethylene carbonate (and its alkyl derivatives) or a mixture
thereof. Suitable biocompatible polymers include hydrophilic
polymers and include but not limited to polyethylene glycol,
polyvinyl alcohol, polyvinyl acetate or mixture thereof.
[0107] In a further embodiment of the invention, described in more
detail below, some or all of the pores or the void phase of
elastomeric matrix are coated or at least partially filled with a
cellular ingrowth promoter. In another embodiment, the promoter can
be foamed. In another embodiment, the promoter can be lyophilized.
In another embodiment, the promoter can be present as a film. The
promoter can be a biodegradable material to promote cellular
invasion of elastomeric matrix in-vivo. Promoters include naturally
occurring materials that can be enzymatically degraded in the human
body or are hydrolytically unstable in the human body, such as
collagen, fibrin, fibrinogen, elastin, hyaluronic acid and
absorbable biocompatible polysaccharides, such as chitosan, starch,
fatty acids (and esters thereof), glucoso-glycans and hyaluronic
acid. In some embodiments, the pore surface of elastomeric matrix
is coated or impregnated, as described in the previous section but
substituting the promoter for the biocompatible polymer or adding
the promoter to the biocompatible polymer, to encourage cellular
ingrowth and proliferation. In a preferred embodiment, the coating
is comprised of collagen.
[0108] Prior to coating, the collagen may be infiltrated into the
void phase of the elastomeric matrix or into the pores of an
elastomeric matrix in form of an aqueous collagen slurry, an
aqueous collagen suspension or an aqueous collagen solution or a
mixture thereof. The collagen may be Type I, II or III or a mixture
thereof. In one embodiment, the collagen type comprises at least
70% collagen I. In one embodiment, the collagen type comprises at
least 80% collagen I. In one embodiment, the collagen type
comprises at least 90% collagen I. The collagen can be derived from
a variety human or animal sources, including porcine, bovine,
equine, and other animal sources suitable for human use, or may be
from a recombinant source. In one embodiment, the collagen can be
derived from bovine tendon which is free of bovine spongiform
encephalopathy. In one embodiment the collagen comprises or
consists of fibrillar Type I bovine collagen. The collagen may be
partially denatured, substantially denatured moderately denatured,
or slightly denatured. In another embodiment, the collagen can be
not denatured.
[0109] The concentration of collagen in the collagen slurry,
collagen suspension or an aqueous collagen solution may range from
about 0.05% to about 4.0% by weight. In another embodiment, the
concentration of collagen in the collagen slurry, collagen
suspension or an aqueous collagen solution range from about 0.1% to
about 2.0% by weight. In another embodiment, the concentration of
collagen in the collagen slurry, collagen suspension or an aqueous
collagen solution range from about 0.2% to about 1.0% by weight.
Alternatively, the concentration of collagen in the collagen
slurry, collagen suspension or an aqueous collagen solution may
range from about 1% to about 10% by weight. In another embodiment,
the concentration of collagen in the collagen slurry, collagen
suspension or an aqueous collagen solution range from about 3% to
about 8% by weight. In another embodiment, the concentration of
collagen in the collagen slurry, collagen suspension or an aqueous
collagen solution range from about 4% to about 5% by weight.
[0110] In one embodiment, the collagen coating can be obtained by
dipping the elastomeric matrix into a collagen slurry or collagen
suspension and drying it under heat and/or vacuum to form a film
coating. In one embodiment, the collagen coating can be obtained by
dipping the elastomeric matrix into a collagen solution and drying
it under heat and/or vacuum to form a film coating. In one
embodiment, the collagen coating can be obtained by dipping the
elastomeric matrix into a mixture of collagen slurry and solution
and drying it under heat and/or vacuum to form a film coating.
[0111] In one embodiment, the collagen coating can be obtained by
dipping the elastomeric matrix into a collagen slurry or collagen
suspension and drying it under lyophilization conditions to form a
lyophilized coating. In one embodiment, the collagen coating can be
obtained by dipping the elastomeric matrix into a collagen solution
and drying lyophilization conditions to form a lyophilized coating.
In one embodiment, the collagen coating can be obtained by dipping
the elastomeric matrix into a mixture of collagen suspension and
solution and drying it under lyophilization conditions to form a
lyophilized coating.
[0112] Optionally, the film or lyophilized collagen coating can be
crosslinked to control the rate of in-vivo enzymatic degradation of
the collagen coating and/or to control the ability of the collagen
coating to bond to elastomeric matrix. The collagen can be
crosslinked by methods known to those in the art, e.g., by heating
in an evacuated chamber, by heating in a substantially
moisture-free inert gas atmosphere, by bringing the collagen into
contact with formaldehyde vapor, or by the use of glutaraldehyde.
The cross-linking may comprise covalent cross-linking In one
embodiment, the film or lyophilized collagen coating can be
crosslinked by bringing the collagen in contact with
carboxyl-reactive chemical groups including carbodiimide compounds
such as EDC (1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide
hydrochloride, and DCC (N',N'-dicyclohexyl carbodiimide).
[0113] Collagen pickup weights range from as low as 0.5
.mu.g/mm.sup.3 up to about 100 .mu.g/mm.sup.3. In one embodiment,
collagen dosing levels range from about 5 .mu.g/mm.sup.3 up to
about 10 .mu.g/mm.sup.3.
[0114] In one embodiment, the total ECM protein, such as collagen,
loading within the scaffold is between about 0.01 and about 0.2 mg
ECM protein/mm.sup.3 scaffold. Alternatively, the total ECM
protein, such as collagen, loading within the scaffold is between
about 0.01 and about 0.1 mg ECM protein/mm.sup.3 scaffold.
Alternatively, the total ECM protein, such as collagen, loading
within the scaffold is between about 0.02 and about 0.08 mg ECM
protein/mm.sup.3 scaffold. Alternatively, the total ECM protein,
such as collagen, loading within the scaffold is between about 0.02
and about 0.05 mg ECM protein/mm.sup.3 scaffold. In one embodiment,
the total ECM protein, such as collagen, loading within the
scaffold is about 0.04 mg ECM protein/mm.sup.3.
[0115] In order to provide sufficient collagen coating on the
scaffold, the collagen may be ground to a smaller particle size,
for example by milling. The milling may comprise cryogenically
grinding. In one embodiment, the collagen may be an average
particle size of about 5 and about 100 .mu.m. The collagen may be
an average particle size of about 10 and about 20 .mu.m.
Alternatively, the collagen may have an average particle size of
less than 50 .mu.m. In another embodiment, the collagen may have an
average particle size of less than 20 .mu.m.
[0116] Design Configurations for Agents/Devices of the
Invention
[0117] Agents or devices of the invention comprising a reticulated
elastomeric matrix with a collagen coating can be readily
fabricated in any desired size and shape. Such agents/devices are
referred to herein with respect to the design configurations as
implants. Suitable designs include, without limitation, those
described in U.S. application Ser. No. 12/699,012 (U.S. Publication
2010/0318108 A1), the disclosures of which are hereby incorporated
by reference. It is a benefit of the invention that the shape and
configuration of elastomeric matrix may vary widely and can readily
be adapted to desired anatomical morphologies.
[0118] The minimum dimension of the implant may be as little as 0.5
mm and the maximum dimension as much as 500 mm or even greater. In
certain embodiments, the implant may be in any two-dimensional or
three-dimensional shape. Exemplary embodiments of a two-dimensional
shape may include regular and irregular shapes, such as, for
example, triangular, rectangular, circular, oval, elliptical,
trapezoidal, pentagonal, hexagonal and irregular configurations,
including one that corresponds to the shape of the defect, and
other shapes. Exemplary embodiments of a three-dimensional shape
may include, plugs, cylinders, tubular structures, stent-like
structures, and other configurations, including one that
corresponds to the contours of the defect, and other
configurations. The device may have a major axis having a length
between about 2 cm to about 50 cm. The device may be in a square
shape with a side having a length between about 2 cm to about 50
cm. In a preferred embodiment, it is contemplated that the implant
would have a shape of a curved sheet with an oval configuration
that optimally contours to the peritoneal cavity. In another
embodiment, the implant would have a shape of a flat sheet with an
oval configuration. The transverse or cross-sectional dimension of
a curved or flat mesh configuration may range from as little as 0.5
mm to up to 10 mm. The length and width dimensions of the implant
may range from as little as 10 mm to up to 500 mm. Optimally, such
implants are dimensioned to allow for delivery using laparoscopic
delivery methods by rolling, folding, or compressing such
implant.
[0119] In another embodiment, the implant would have an elongated
shape, such as the shapes of cylinders, rods, tubes or elongated
prismatic forms, or a folded, coiled, helical or other more compact
configuration. In an alternative embodiment, the implant of the
invention may have a spherical, cubical, tetrahedral, toroidal,
cup-like, or other form having no dimension substantially elongated
when compared to any other dimension and with a diameter or other
maximum dimension of from about 0.5 mm to about 500 mm.
[0120] For metastatic cell capture applications, it is an advantage
of the invention that the implant can be effectively employed
without any need to closely conform to the configuration of the
application site, which may often be complex and difficult to
model. Thus, in one embodiment, the implants have significantly
different and simpler configurations which conformally fit the
target site. Without being bound by any particular theory, the
resilience and recoverable behavior that leads to such a conformal
fit results in the formation of a tight boundary between the walls
of the implantable device and the defect with substantially no
clearance, thereby providing an interface conducive to the capture
of metastatic tumor cells.
[0121] Furthermore, in one embodiment, the implantable device of
the present invention, or implantable devices if more than one is
used, should not completely fill the application site even when
fully expanded in situ. In one embodiment, the fully expanded
implantable device(s) of the present invention are smaller in a
dimension than the application site and provide sufficient space
within the application site to ensure vascularization, tumor cell
capture, and proliferation, and for passage of blood to the
implantable device. In another embodiment, the fully expanded
implantable device(s) of the present invention are substantially
the same in a dimension as the application site. In another
embodiment, the fully expanded implantable device(s) of the present
invention are larger in a dimension than the application site. In
another embodiment, the fully expanded implantable device(s) of the
present invention are smaller in volume than the application site.
In another embodiment, the fully expanded implantable device(s) of
the present invention are substantially the same volume as
application site. In another embodiment, the fully expanded
implantable device(s) of the present invention are larger in volume
than the application site.
[0122] In embodiments of the invention, an optional anti-adhesion
coating can be added. The coating can consist of biodegradable or
biodurable polymeric materials. One embodiment of the invention
incorporates a thin layer, coating or film of either a permanent
polymer or biodegradable polymer used to reduce the potential for
biological adhesions. In a preferred embodiment, a biodegradable or
bioabsorbable coating is made from copolymers of caprolactone with
lactic acid, glycolic acid, acid d-, l- and meso lactide and
para-dioxanone. Compositions considered favorable for anti-adhesion
properties include copolymers of caprolactone with lactic acid in
the ratio of 40/60, 30/70 or 20/80 polycaprolactone to polylactic
acid. This anti-adhesion film may be incorporated with the
reticulated elastomeric matrix using various processing techniques
known in the art including adhesive bonding, melt processing,
compression molding, suturing, and other techniques.
[0123] Other embodiments involve implants for in-vivo delivery via
catheter, endoscope, arthroscope, laparoscope, cystoscope, syringe
or through non-endoscopic open procedures or other suitable
delivery-device. In one embodiment, elastomeric matrices of the
invention have sufficient resilience to allow substantial recovery,
e.g., to at least about 30% of the size of the relaxed
configuration in at least one dimension, after being compressed for
implantation in the human body, In another embodiment, elastomeric
matrices of the invention have sufficient resilience to allow
recovery to at least about 60% of the size of the relaxed
configuration in at least one dimension after being compressed for
implantation in the human body. In another embodiment, elastomeric
matrices of the invention have sufficient resilience to allow
recovery to at least about 90% of the size of the relaxed
configuration in at least one dimension after being compressed for
implantation in the human body. In another embodiment, elastomeric
matrices of the invention have sufficient resilience to allow
recovery to at least about 95% of the size of the relaxed
configuration in at least one dimension after being compressed for
implantation in the human body.
[0124] Following delivery of the implant in-vivo, the device may be
secured to the target area by any means. In one embodiment, the
implant may be sutured to the target area. In another embodiment,
the implant may be stapled in place. Other methods for fixation of
the device include sutureless techniques such as fixation of the
device with a glue (e.g., human fibrin glue).
[0125] There is provided a method of manufacturing an agent for
modulating metastatic tumor cell dissemination, the method
comprising the steps of: [0126] preparing a suspended solution of
an ECM protein; [0127] coating the polycarbonate polyurethane
matrix by saturation within the solution of the ECM protein; and
[0128] lyophilization of the the ECM protein within the
polycarbonate polyurethane matrix to form the agent for modulating
metastatic tumor cell dissemination.
[0129] The ECM protein may be ground to (or provided in) a smaller
particle size prior to coating. The grinding may be cryogenically
grinding. The ECM protein may be ground to an average particle size
of between about 1 and 100 microns. In another embodiment, the ECM
protein may be ground to an average particle size of between about
5 and 50 microns. Alternatively, the ECM protein may be ground to
an average particle size of between about 20 and 30 microns. The
ECM protein may be ground to an average particle size of less than
100 microns. The ECM protein may be ground to an average particle
size of less than 50 microns. The ECM protein may be ground to an
average particle size of less than 20 microns.
[0130] Advantageously, the smaller average particle size
facilitates the coating of a higher amount of ECM protein, such as
collagen, relative to larger particle sizes. In particular large
particles like fibrillary collagen can clog the matrix pores and
reducing the particle size alleviates this problem. Cryogenically
grinding the ECM protein can help to prevent denaturation.
[0131] The solution of ECM protein may be a solution of ECM protein
and deionised water. The amount of ECM protein in solution may be
between about 30 and about 80 mg ECM protein/g water.
Alternatively, the amount of ECM protein in solution may be between
about 35 and about 60 mg ECM protein/g water. The amount of ECM
protein in solution may be between about 40 and about 50 mg ECM
protein/g water.
[0132] The concentration of collagen in solution may range from
about 0.05% to about 4.0% by weight. In another embodiment, the
concentration of collagen in the collagen solution may range from
about 0.1% to about 2.0% by weight. In another embodiment, the
concentration of collagen in the collagen solution may range from
about 0.2% to about 1.0% by weight. Alternatively, the
concentration of collagen in the collagen solution may range from
about 1% to about 10% by weight. In another embodiment, the
concentration of collagen in the collagen solution may range from
about 3% to about 8% by weight. In another embodiment, the
concentration of collagen in the collagen solution may range from
about 4% to about 5% by weight.
[0133] The polycarbonate polyurethane matrix may be saturated by
repeated mechanical compressions under the surface of the ECM
protein solution fluid.
[0134] The drying may be via a lyophilisation process that utilizes
sublimation under vacuum after the material has been frozen, for
example at less than -20.degree. C. or less than -40.degree. C.
[0135] The method may further comprise crosslinking the ECM
protein, such as covalently crosslinking. Crosslinking may be
provided by saturating the ECM protein in a solution of a molecule
capable of covalently crosslinking the ECM protein, for example
molecules comprising carboxyl-reactive chemical groups, such as
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC). The
lyophilisation process may be repeated after crosslinking.
[0136] The lyophilized ECM protein of the method may comprise or
consist of collagen. The collagen may comprise or consist of Type I
bovine collagen.
[0137] The present invention will be further described in more
detail, by way of example only, with reference to the following
figures in which:
[0138] FIG. 1--shows an agent of the invention comprising a
reticulated scaffold coated with 6.36 .mu.g collagen/mm.sup.3
scaffold and imaged by electron microscopy to show the reticulated
scaffold from Biomerix (left panel; bar 500 .mu.m), and the
collagen fibers decorating the surface of the polymeric scaffold
(middle panel at bar 25 .mu.m; right panel at bar 10 .mu.m).
[0139] FIG. 2--shows the polycarbonate polyurethane scaffold and
the open cell intercommunicating network present through the volume
of the material. 35.times. magnification.
[0140] FIG. 3--shows the lyophilized collagen network (0.0400
mg/mm.sup.3 scaffold) distributed within the poycarbonate polymeric
scaffold at 35.times. magnification.
[0141] FIG. 4--shows the lyophilized collagen network distributed
within the poycarbonate polymeric scaffold at 150.times.
magnification.
[0142] FIG. 5--shows the permeability of the composite material
versus the total coat weight of collagen on the clinical size
device.
[0143] FIG. 6A--shows attachment of fluorescent-labelled SKOV3
cells to the fibers of an agent of the invention, referred to
herein as the M-Trap device, decorated with collagen (M-Trap)
compared to the fibers of Biomerix scaffold without collagen
(Scaffold). Attachment of the cells was facilitated by the orbital
circulation of the tumor cells.
[0144] FIG. 6B--shows the capture of SKOV3 cells to an agent of the
invention, wherein the agent comprises a 3D scaffold in the
presence of collagen (M-Trap). The results show that cell capture
was enhanced by the collagen coating compared to adhesion to the 3D
scaffold without collagen (Scaffold). This enhancement was
demonstrated in both a dose dependent manner with 25 and 250 .mu.g
collagen, and in a time-dependent manner for 24, 48 and 72 hours at
37.degree. C. (p<0.001).
[0145] FIG. 7A--shows an increased capture of SKOV3 cells when
cells were exposed to an enhanced adhesive surface.
[0146] FIG. 7B--shows the effect of exposure of
fluorescent-labelled SKOV3 cells to the Biomerix scaffold coated
with collagen (M-Trap) or to the Biomerix scaffold alone
(Scaffold), in gradually increased 3D containers.
[0147] FIG. 7C--shows the effect of exposure of
fluorescent-labelled SKOV3 cells to the Biomerix scaffold coated
with collagen (M-Trap) located at the center (Scaffold 1) or at the
exterior side (Scaffold 3) of the container, or at an intermediate
location between them (Scaffold 2).
[0148] FIG. 7D--shows the reticulated scaffold coated with collagen
(left panel D) and captured fluorescent SKOV3 cells within the
scaffold (right panel D).
[0149] FIG. 8--shows adhesion assays to demonstrate the release of
collagen from scaffolds following incubation in PBS for 0 hours, 48
hours, 5 days and 7 days.
[0150] FIG. 9--shows the efficiency of M-trap for the capture of
non-tumor cell types.
[0151] FIG. 10--shows short-term adhesion assays of SKOV3 cells
labelled with calcein seeded in wells of a polystyrene plate with 5
.mu.g/.mu.l collagen. The coated surfaces were exposed overnight at
37.degree. C. to 7 nM Paclitaxel as IC50 and to 3.5 nM Paclitaxel
as IC50/2, to 10 .mu.M Carboplatin as IC50 and to 5 .mu.M
Carboplatin as IC50/2, and to the combination of both
Paclitaxel+Carboplatin at their respective IC50 (0.7 nM Paclitaxel,
1 .mu.M Carboplatin) and IC50/2 (0.35 nM Paclitaxel; 0.5 .mu.M
Carboplatin) prior to seeding the cells.
[0152] FIG. 11--shows the quantification of M-trap Tumor cell
capture in a time and collagen concentration-dependent manner.
[0153] FIG. 12--shows short term adhesion assays of SKOV3 cells
treated overnight at 37.degree. C. with 7 nM Paclitaxel as IC50 and
3.5 nM Paclitaxel as IC50/2, 10 .mu.M Carboplatin as IC50 and 5
.mu.M Carboplatin as IC50/2, and the combination of both
Paclitaxel+Carboplatin at their respective IC50 (0.7 nM Paclitaxel,
1 .mu.M Carboplatin) and IC50/2 (0.35 nM Paclitaxel; 0.5 .mu.M
Carboplatin).
[0154] FIG. 13--shows the tumor cell saturation capacity of M-trap
in an orbital adhesion assay.
[0155] FIG. 14A--shows peritoneal dissemination of SKOV3 cells
stably expressing the luciferase reporter gene, following
intraperitoneal injection in a mouse model of ovarian cancer.
[0156] FIG. 14B--shows the location of the device when surgically
implanted at the inner wall of the peritoneum opposite to the
natural sites of metastaisi, one week before SKOV3 cell
injection.
[0157] FIG. 15A--shows the complete remodelling of the peritoneal
pattern of metastasis by M-Trap device, this representative image
showing a complete capture of tumor cells within M-Trap device and
a complete eradication of metastasis at natural sites.
[0158] FIG. 15B--quantification of the amount of tumor cells at
natural sites and at M-Trap device with increased amounts of
collagen. This quantification also demonstrates that the main
capture action of the device is provided by the scaffold, the
collagen coating auxiliary improving the capture efficiency.
[0159] FIG. 16A--shows the incomplete efficacy of a pharmacological
mode of action technology (pluronic+EGF) to capture tumor cells
disseminating into the peritoneal cavity. Controlled release of EGF
as chemoattractant was not as efficient as M-Trap device to
completely capture metastatic cells.
[0160] FIG. 16B--shows the incomplete efficacy of another
pharmacological mode of action technology (PLGA+EGF) to capture
tumor cells disseminating into the peritoneal cavity. Controlled
release of EGF as chemoattractant was not as efficient as M-Trap
device to completely capture metastatic cells.
[0161] FIG. 17--shows the efficiacy of the M-trap device which
captured all metastatic cells at each time point, one, three and
six months post-implantation in an in vivo model of metastatic
ovarian peritoneal dissemination.
[0162] FIG. 18A--schematic description of the four arms included in
the preclinical trial demonstrating M-Trap benefit in survival in
the mice model of ovarian peritoneal metastasis.
[0163] FIG. 18B--in vivo follow-up of the pattern of peritoneal
dissemination for each of the four arms included in the preclinical
trial. Biolumiscence imaging of tumor cell implants in the
peritoneal cavity three months after SKOV3 cells and at sacrifice
shows an effective focalization of the disease in the presence of
M-Trap device, implanted both pre- and post-injection, in
comparison to the massive peritoneal dissemination shown in the
control arm. Finally, removal of M-Trap device upon capture
completely eradicates the peritoneal disease.
[0164] FIG. 18C--Kaplan-Meyer survival curve demonstrates the
benefit in survival by the presence of M-Trap device. Focalization
of the disease both before (M-Trap group) and after (Post-injection
group) natural metastasis formation, resulted in an improved
survival. Removal of M-Trap upon capture (Re-operated group)
further impacted in survival (p<0.0001).
[0165] FIG. 18D--Histological examination of organs and mesothelium
at sacrifice confirmed the reduced peritoneal extension of the
disease in the presence of M-Trap device. Representative images of
affected organs for each group included in the preclinical study
are shown.
[0166] FIG. 19A--shows representative images demonstrating that the
efficacy of M-Trap device to completely capture tumor cells
disseminating in the peritoneal cavity is not impaired by the
presence of IC50 concentrations of standard chemotherapy in ovarian
cancer (carboplatin-paclitaxel).
[0167] FIG. 19B--shows the quantification of tumor cell survival to
standard peritoneal chemotherapy in the presence or not of M-Trap
device.
[0168] FIG. 20--shows representative images of the pattern of
peritoneal metastasis in the presence or not of M-Trap devices, for
different clinically relevant ovarian cancer cell lines (serous
TOV112; endometroid OV90; and primary cancer cells isolated from
ascitic fluid of ovarian cancer patients). Histograms show
quantification of the amount of different ovarian tumor cells at
natural sites and captured by M-Trap device, further demonstrating
the universality of M-Trap technology.
[0169] FIG. 21A--shows a comparative in-vivo assay in which
subcutaneous SKOV3 cell tumors were generated in mice under three
different conditions (PBS, Matrigel, M-trap) with quantification of
the bioluminescence at 2 weeks and 4 weeks to assess tumor growth
and proliferation.
[0170] FIG. 21B--shows how M-trap does not contribute to tumor
growth upon cell capture in a murine subcutaneous tumor model.
[0171] FIG. 22A--shows Increasing concentrations of fibronectin
decorating the scaffold were able to capture SKOV3 cells in the in
vitro dynamic orbital assay mimicking transcoelomic peritoneal
flow, in a dose dependent manner.
[0172] FIGS. 22B and 22C--show fibronectin coating of Biomerix
scaffold resulted in a complete remodeled pattern of peritoneal
implants in the in vivo model of ovarian dissemination, with almost
all metastatic tumor cells being captured within the device.
EXAMPLES
Trap Device
[0173] Trap devices of the invention, comprising an agent for
modulating metastatic tumor cell dissemination for use in the
treatment and/or prevention of a metastatic cancer wherein the
agent for modulating metastatic tumor cell dissemination is an
extracellular matrix (ECM) protein carried on a reticulated
elastomeric matrix, preferably a polycarbonate polyurethane urea
matrix, are manufactured by coating the matrix with the ECM
protein. Methods of production of the matrix are known to the
skilled person and are described, for example in issued U.S. Pat.
No. 7,803,395 and U.S. Pat. No. 8,337,487 (the contents of which
are incorporated in their entirety, by reference).
[0174] The polycarbonate polyurethane urea matrix of the Examples
(sometimes referred to as the Biomerix scaffold) is a
non-resorbable, reticulated, cross-linked, polycarbonate
polyurethane-urea matrix (Biomerix, Fremont, Calif., USA) offering
a fully interconnected, highly permeable, macroporous morphology
with over 90-95% void content The material specifications are
provided in Table 1. The scaffold was coated with 250 .mu.g
collagen and imaged by electron microscopy. The results are shown
in FIG. 1.
TABLE-US-00001 TABLE 1 Biomerix HF3 Formulation, Material
Specifications Property Requirement Permeability >250 Darcy
Average cell size <385 .mu.m Density 3.5-3.9 lb/ft.sup.3
Compressive strength 1.0-1.8 psi Tensile strength parallel
.gtoreq.50 psi Elongation parallel >180% Tensile strength
perpendicular .gtoreq.36 psi Elongation perpendicular >180%
[0175] The Biomerix scaffold permits in-growth and proliferation of
host cells and tissue into the volume of the polymeric scaffold.
The polymeric scaffold can be characterized as an open and
interconnected network of polycarbonate polyurethane which forms a
three-dimensional spatial structure with a high void volume and
surface area. The material can be further characterized as having
an elastomeric nature that enables it to be compressible, resilient
and demonstrating good recovery properties after compression or
manipulation. The reticulated elastomeric matrix is comprised of a
biodurable and biocompatible polymer that will not degrade or
change in properties after implantation within the body for the
lifetime of the device. FIG. 2 shows the polycarbonate scaffold and
the open cell intercommunicating network that is present through
the volume of the material.
[0176] Methods to coat the scaffold with collagen are described
above.
[0177] Collagen coated polycarbonate polyurethane urea matrices are
also referred to herein as M-traps.
[0178] The collagen component of the M-trap device is comprised of
a fibrillar Type I bovine collagen that is lyophilized onto the
reticulated elastomeric polycarbonate polyurethane scaffold via a
manufacturing process that ensures that the collagen network
permeates through the entirety of the polymeric scaffold. The
bovine fibrillar collagen is sourced from Maquet/Datascope. The
collagen is crosslinked after lypholization to improve the
durability of the collagen such that it will remain intact and
effective for the intended life of the device. The lyophilized
collagen network has a high permeability and surface area similar
to the polymeric scaffold and as such it does not restrict
in-growth and proliferation of host cells and tissue into the
volume. The collagen within the polymeric scaffold acts as an
attractant to the disseminating tumor cells within the cavity.
FIGS. 3 and 4 show the lyophilized collagen network distributed
within the polycarbonate polymeric scaffold at different
magnifications.
[0179] M-trap Device Configuration and Optimized Collagen Coating
Concentration
[0180] The clinical M-Trap device was configured as a flat sheet of
the composite material with a thickness of 5 mm and an oval shape
with a major axis of 50 mm and a minor axis of 15 mm. The total
collagen loading within the scaffold for the clinical devices was
approximately 0.04 mg collagen/mm.sup.3 for a total delivered
amount of collagen of approximately 120 mg of type I bovine
collagen per device. A range of sizes of the M-Trap device can also
be provided, where the equivalent collagen amounts may be scaled up
or down appropriately.
[0181] Initial Collagen Concentration Optimization Experiments
[0182] Initial conceptual development of the M-trap device utilized
a soluble form of rat tail collagen as the cancer cell attractant
at a dose level of 6.36 .mu.g/mm.sup.3 of scaffold. The initial
development work on the clinical device investigated whether it was
possible to increase the dose level of collagen as the attractant
for additional cell capture. Utilizing a soluble collagen material
was found to limit the amount of collagen that could be applied to
the scaffold as the saturated solution concentration of collagen
was a limiting factor. To address this issue, fibrillar bovine type
I collagen was utilized as this biomaterial has multiple regulatory
clearances within medical devices. Coating of the scaffold with a
fibrillar material was found to be initially ineffective since the
length of the fibrils were greater than the openings within the
polymeric scaffold and the collagen was not able to be uniformly
distributed through the interior of the scaffold. The collagen was
cryogenically ground within an oscillatory ball mill to reduce the
mean particle size to approximately 10-20 .mu.m. Cryogenic grinding
was chosen over standard ball milling of the collagen to ensure
that the proteins of the collagen were not denatured.
[0183] Utilizing the cryogenically ground microparticles of the
bovine collagen, solutions of various concentrations were produced
and coated on the scaffold by a film coating and also by a
lypholization processes. The lyophilized process had an
advantageous morphology at the microscopic level of additional
surface area for cellular adhesion within the scaffold and was
determined to be the preferred method of combining the scaffold and
collagen. To determine the maximum amount of collagen that could be
placed within the scaffold and still maintain device functionality,
experiments were conducting looking at the permeability of the
resulting composite material versus the total coat weight of
collagen on the clinical size device. The results of these
experiments are presented in FIG. 5. It was determined that a high
dose level of 0.0400 mg/mm.sup.3 and a low dose level of 0.0067
mg/mm.sup.3 would be investigated further within the preclinical
models. The preclinical size of the M-Trap device is 6 mm.times.3
mm.times.2 mm. Preclinical testing demonstrated that the optimized
collagen concentration for M-trap is the high dose level of
collagen of 0.0400 mg/mm.sup.3.
[0184] M-Trap Manufacturing Process
[0185] A uniform collagen coating within the polymeric scaffold was
achieved by saturation of a suspension of the cryogenically ground
collagen with an approximate particulate size of 10-20 microns, and
deionized water within the scaffold. The primary control of the
amount of collagen left behind on the surface of the scaffold is
the initial concentration of the collagen within the suspension and
subsequent complete saturation of the sponges prior to the drying
process. To determine the amount of collagen within the solution
needed, the amount of suspension that can be held within the
scaffold must first be understood. The polymeric scaffold is a
hydrophobic polycarbonate polyurethane porous polymer that will not
readily adsorb water onto the surface of the material. However, it
will readily absorb and hold water within the fine, open structure
of the material once saturated due to surface tension. Based on
multiple experiments, the total solution contained within the
scaffold at saturation is 0.00086 g/mm.sup.3 Scaffold.
[0186] Based on the optimized (high dose) amount of collagen
desired, a solution concentration of 46.5 mg Collagen/g H.sub.2O
was made and maintained under constant stirring prior to coating
the scaffolds. To accomplish the saturation of the scaffold, the
material was repeatedly mechanically compressed under the surface
of the fluid to remove any entrained air and filled with
suspension. Saturated scaffolds were placed onto a porous substrate
after being coated so that a flat liquid boundary layer is not
created at the surface of the scaffold prior to drying. The water
was removed from the solution within the scaffold via a
lypholization process that utilizes sublimation under vacuum after
the material has been frozen to -45.degree. C.
[0187] To enable the collagen within the scaffold to have a greater
efficacy over time in-vivo, the collagen within the scaffold was
crosslinked by saturating the lyophilized composite scaffolds in a
100 mM solution of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDAC) and repeating the lyophilization process an additional
time.
[0188] M-Trap Technology Acts as a Preferential Niche for
Implantation and Efficiently Captures Peritoneal Metastatic
Cells.
[0189] To analyze the mode of action of M-Trap devices, with
collagen fibers at the surface of the non-degradable 3D scaffold,
an in vitro assay was developed aiming to mimic the natural flow of
peritoneal fluid within the abdominal cavity directed by gravity to
its most dependent sites and providing a route for the
transcoelomic dissemination of detached tumour cells (Tan et al.,
2006). For this, the capture of 250,000 calcein-labelled SKOV3
cells re-suspended in a 2 ml volume to the M-Trap device located in
a P6 cell culture plate (3.5 cm diameter) subjected to an orbital
movement of 90 rpm was evaluated. Attachment of
fluorescent-labelled SKOV3 cells to the fibers of the M-Trap device
decorated with collagen (M-Trap) compared to the fibers of Biomerix
scaffold without collagen (Scaffold) was further facilitated by the
orbital circulation of tumor cells (FIG. 6, panel A), close to the
clinical scenario of metastatic dissemination within a peritoneal
cavity. Under these dynamic conditions, the capture of SKOV3 cells
to the 3D scaffold in the presence of collagen (M-Trap) was
enhanced compared to adhesion to the 3D scaffold without collagen
(Scaffold) both in a dose dependent manner with 25 and 250
.quadrature.g collagen, and in a time-dependent manner for 24, 48
and 72 hours at 37.degree. C. (p<0.001; FIG. 6 panel B), further
indicative of the specificity of SKOV3 cells attachment to M-Trap
due to the adhesive ability of collagen as a capture agent. These
in vitro results demonstrate that an M-Trap device composed of the
Biomerix scaffold (polycarbonate polyurethane cross linked with
urea) coated with collagen might be acting through a
non-pharmacological mode of action by providing a favored surface
for the adhesion of tumor cells that are orbital circulating within
a 3D container; translated to the clinics, M-Trap device may be
competing with the natural sites of peritoneal implantation and
supporting a preferential niche for the attachment and capture of
metastatic tumor cells.
[0190] To further confirm the non-pharmacological mode of action of
the M-Trap device composed by the Biomerix scaffold coated with
collagen, the dynamic capture of calcein-labelled SKOV3 cells when
exposed to an increased surface of M-Trap device during 24 hours at
37.degree. C. was evaluated. For this, the ability of one or two
units of the Biomerix scaffold without collagen (Scaffold) or with
collagen (M-Trap), to capture SKOV3 cells in the dynamic assay
mimicking peritoneal dissemination was compared. As shown in FIG. 7
panel A, an increased capture of SKOV3 cells was evidenced when
cells were exposed to an enhanced adhesive surface. Likewise, when
fluorescent-labelled SKOV3 cells were exposed to the Biomerix
scaffold coated with collaged (M-Trap) or just to the scaffold
(Scaffold), in gradually increased 3D containers (P6 corresponding
to a 3.5 cm diameter cell culture plate; P100 corresponding to a
8.5 cm diameter cell culture plate; and P150 corresponding to a
13.5 cm diameter cell culture plate), no significant differences in
the efficiency of SKOV3 cells capture (FIG. 7, panel B) was
observed, further indicative of a non-pharmacological mode of
action of the M-Trap technology of the invention. Finally, when the
fluorescent-labeled SKOV3 cells were exposed to the Biomerix
scaffold coated with collagen (M-Trap) located at the center
(Scaffold 1) or at the exterior side (Scaffold 3) of the container,
or at an intermediate location between them (Scaffold 2), an
increased ability to capture SKOV3 cells was observed corresponding
to the heterogeneous distribution of SKOV3 cells in the solution
subjected to an orbital movement during 24 hours (FIG. 7, panel C).
The capture of SKOV3 cells within the M-Trap composed of the
Biomerix scaffold coated with collagen was further confirmed by
fluorescent microscopy. Images show the reticulated scaffold coated
with collagen (FIG. 7, left panel D) and captured fluorescent SKOV3
cells within the scaffold (FIG. 7, right panel D). In addition to
reinforcing the non-pharmacological mode of action of M-Trap
composed of the Biomerix reticulated scaffold (polycarbonate
polyurethane urea matrix) coated with collagen, these results also
demonstrate that the translation of M-Trap device into the clinics
is not limited by the scaling of M-Trap dimensions to the
peritoneal cavity, and must be accompanied by a re-dimension of
M-Trap to an optimal size that might balance the maximal surface of
the device with a minimal impact at the peritoneum (i.e. avoiding
intestinal adherences).
[0191] The stability of the M-Trap device composed of the Biomerix
scaffold coated with collagen was evaluated through a release
experiment combined with a short term adhesion assay. Briefly, we
incubated the M-Trap device in 100 .mu.l of PBS for 0 hours, 48
hours, 5 days and 7 days. At the indicated times, the supernatant
was recovered with the potential collagen traces released from the
scaffolds. A short-term adhesion assay as described was then
performed with both the scaffolds (M-Trap) and their corresponding
supernatants (SN). As can be observed (FIG. 8), no difference could
be found among scaffolds or among supernatants along incubated
times, as well as between supernatants and control basal adhesion
in PBS conditions. These results are indicative that no release of
collagen occurred during incubation of M-Trap devices and
demonstrating the stability of M-Trap technology at least for the
indicated times.
[0192] M-Trap Technology Efficiently Captures Additional Cell Types
with Adhesive Abilities Disseminating in the Peritoneal Cavity.
[0193] The universality of M-Trap technology to capture additional
types of cells with adhesive abilities disseminating in the
peritoneal cavity may beneficially impact on the efficacy of M-Trap
by generating a more clinically relevant niche to compete with the
natural sites of implantation of tumor cells disseminating in the
peritoneum. The efficiency of the polycarbonate polyurethane
scaffold with collagen coated M-Trap prototype to capture different
types of cells was evaluated in the dynamic in-vitro capture assay
with the M-Trap prototype placed in a P6 well plate and
fluorescent-labelled cells added in suspension and incubated under
orbital movement for 24 hours. Quantification of the percentage of
cells captured by M-Trap relative to the cells in suspension at the
end of the experiment as shown in FIG. 9. Cell types evaluated
include the ovarian cancer cell line SKOV3, HUVEC endothelial
cells, JURKAT lymphocytes, fibroblasts and mesenchymal stem cells
of adipose, bone marrow (BM MSC) and umbilical cord (UC MSC)
origin. These cell types are representative of the cell types
present in the peritoneal cavity which could be interacting with
the implanted M-Trap device. As can be observed, the efficiency of
cell capture correlates with the capacity of these cells to attach
to solid surfaces, with tumor cells, fibroblasts, MSC and
endothelial cells being efficiently captured as they adhere rapidly
to adhesive surfaces. In contrast, lymphocytes do not efficiently
adhere to solid surfaces, and in fact, they grow in suspension.
These results reinforce the non-pharmacological mode of action of
M-Trap, without any active selection of cells but a passive
adhesive affect for the capture of cells disseminating into the
peritoneal cavity.
[0194] Impact of Chemotherapy on the Efficacy of M-Trap Device
[0195] The adhesion of SKOV3 cells in the presence or not of the
standard therapy used in ovarian cancer (Paclitaxel+Carboplatin)
was evaluated.
[0196] The impact of chemotherapy on the adhesive properties of a
polymeric surface coated with collagen and the impact on the
adhesive properties of tumor cells, exposed to the IC50
concentration of both drugs individually and in combination, were
evaluated separately. For the impact of chemotherapy on the
adhesive properties of the material, the bottom of a polystyrene
well plate was coated with 5 .mu.g/.mu.l collagen during overnight
at 37.degree. C. The coated surface was exposed overnight at
37.degree. C. to 7 nM Paclitaxel as IC50 and to 3.5 nM Paclitaxel
as IC50/2, to 10 .mu.M Carboplatin as IC50 and to 5 .mu.M
Carboplatin as IC50/2, and to the combination of both
Paclitaxel+Carboplatin at their respective IC50 (0.7 nM Paclitaxel,
1 .mu.M Carboplatin) and IC50/2 (0.35 nM Paclitaxel; 0.5 .mu.M
Carboplatin). Finally a short-term adhesion assay was performed
with 50.times.10.sup.4 SKOV3 cells labeled with calcein seeded in
the different treated well plates for 1 hour before washing and
quantification of adhered cells with a luminometer. As shown in
FIG. 10, no significant differences in SKOV3 cell adhesion were
observed when the polymeric surface coated with collagen was
exposed to the different chemotherapy conditions.
[0197] With reference to FIG. 12, the impact of chemotherapy on the
adhesive ability of SKOV3 cells was also evaluated. For this, SKOV3
cells were treated overnight at 37.degree. C. with 7 nM Paclitaxel
as IC50 and 3.5 nM Paclitaxel as IC50/2, 10 .mu.M Carboplatin as
IC50 and 5 .mu.M Carboplatin as IC50/2, and the combination of both
Paclitaxel+Carboplatin at their respective IC50 (0.7 nM Paclitaxel,
1 .mu.M Carboplatin) and IC50/2 (0.35 nM Paclitaxel; 0.5 .mu.M
Carboplatin). A short-term adhesion assay was then performed to
un-treated collagen coated well plates as described, and a slightly
diminished capacity of SKOV3 cells treated with the combination of
both drugs was observed, although this was not statistically
significant.
[0198] From these results it can be concluded that chemotherapy
should not impact the material and the adhesive properties of
M-Trap technology. An effect of chemotherapy on the capacity of
tumor cells to adhere might be expected, although this effect
should impact similarly on the ability of tumor cells to adhere to
the peritoneal wall and generate metastasis.
[0199] Quantification of M-Trap Tumor Cell Capture in a Time and
Collagen Concentration-Dependent Manner
[0200] An in vitro study determined the mode-of-action of M-Trap by
evaluating the additive contribution of each element of the M-Trap
device (namely, the polyurethane scaffold and the Type I collagen
coating) to the tumor cell capture efficacy of the device in an
in-vitro system. Tumor cell capture efficacy was assessed in an
orbital adhesion assay which mimics peritoneal dissemination in
ovarian cancer. M-Trap devices were immobilized in cell culture
dishes. SKOV3 cells labeled with the fluorescent marker calcein
were added to the plate and placed on an orbital shaker at 90 rpm
for durations of 24, 48 and 72 hours at 37.degree. C. in 5% CO2.
After incubation, SKOV3 cells captured by M-Trap devices were
quantified in a luminometer.
[0201] The experimental groups used were as follows: [0202] Empty
Group: Bare M-Trap scaffolds (polycarbonate polyurethane scaffold,
no collagen coating). [0203] M-Trap Low-Dose Group: M-Trap devices
specially manufactured with a minimal collagen coating. [0204]
M-Trap High-Dose Group: M-Trap devices with the targeted collagen
coating level designed for clinical use.
[0205] As shown in FIG. 11, the principal capture action was
provided by the bare scaffold with an ancillary improved adhesive
efficacy as the concentration of collagen was increased.
Additionally, the linear increase in the capture efficacy as a
function of incubation time further confirmed the
non-pharmacological mode of action of the device.
[0206] In-Vitro Evaluation of M-Trap Tumor Cell Capture
Capacity
[0207] The tumor cell saturation capacity of M-Trap in an orbital
adhesion assay was evaluated. Increasing numbers of ovarian cancer
cells (SKOV3) labeled with calcein were added to the plates and
allowed to be captured by the device for 24 hours before
quantification in a luminometer. The capacity of the device to
capture six different quantities of ovarian cancer cells (1
million, 5 million, 10 million, 15 million, 20 million and 25
million) was quantified. Study results are summarized in FIG. 13.
This study demonstrated that the tumor cell saturation capacity of
a single M-Trap device (preclinical size) is approximately 10
million cells. Scaling the preclinical device size to the clinical
size of the device, the expected saturation capacity of M-Trap in
patients would be up to 1,000.times.10.sup.6 metastatic cells.
Since two M-Trap devices will be implanted in patients in locations
where tumor cells typically disseminate, the saturation capacity of
M-Trap in clinical use is up to 2,000.times.10.sup.6 metastatic
cells.
[0208] Mouse Model of Ovarian Cancer Peritoneal Dissemination
[0209] The non-pharmacological mode-of-action of M-Trap was
demonstrated by the evaluation of the additive contribution of each
element of the M-Trap device (namely, the polyurethane scaffold and
the Type I collagen coating) to the tumor cell capture efficacy of
the device in an in-vivo model. Tumor cell capture efficacy was
assessed in a murine model of ovarian cancer peritoneal
dissemination (SCID mouse) at the one-week timepoint. In this
model, 1.times.10.sup.6 SKOV3 ovarian cancer cells stably
expressing the luciferase reporter gene were intraperitoneally
injected. One week after injection, mice were sacrificed and the
pattern of metastasis was analyzed by bioluminescence to determine
the pattern of natural metastasis in this model system. Testing
demonstrated that the pancreas and gonadal fat pad are the natural
sites for SKOV3 cells implantation (FIG. 14A). Alternatively, to
assess the impact of M-Trap, the device was surgically implanted at
the inner wall of the peritoneum opposite to the natural sites of
metastasis, one week before SKOV3 cell injection (FIG. 14B).
[0210] A total of 32 mice were used to evaluate the mode-of-action
and efficacy of M-Trap in this model. A description of the
experimental groups is as follows: [0211] Control Group (n=8): One
million luciferase-expressing SKOV3 cells are injected
intraperitoneally. One week after tumor cell injection, the mice
are sacrificed and the normal pattern of tumor cell dissemination
was evaluated by bioluminescence. [0212] Empty Group (n=8): Bare
M-Trap scaffolds (polycarbonate polyurethane scaffold, no collagen
coating) were surgically implanted in the inner peritoneal wall of
mice. One week later, one million luciferase-expressing SKOV3 cells
were injected intraperitoneally. One week after tumor cell
injection, the animals were sacrificed and the pattern of tumor
cell dissemination was evaluated. [0213] M-Trap Low-Dose Group
(n=8): M-Trap devices specially manufactured with a minimal
collagen coating were surgically implanted in the inner peritoneal
wall of mice. One week later, one million luciferase-expressing
SKOV3 cells were injected intraperitoneally. One week after tumor
cell injection, the animals were sacrificed and the pattern of
tumor cell dissemination was evaluated. [0214] M-Trap High-Dose
Group (n=8): M-Trap devices with the targeted collagen coating
level designed for clinical use were surgically implanted in the
inner peritoneal wall of mice. One week later, one million
luciferase-expressing SKOV3 cells were injected intraperitoneally.
One week after tumor cell injection, the animals were sacrificed
and the pattern of tumor cell dissemination was evaluated.
[0215] As shown in FIG. 15A, results demonstrated that the pattern
of dissemination of metastatic ovarian tumor cells in the presence
of M-Trap was completely remodeled, with the eradication of the
natural foci of metastasis and the focalization of metastasis in a
unique location within the M-Trap device. Moreover, quantification
of the bioluminescence signal confirmed the non-pharmacological
mode of action with the bare scaffold acting as the principal
capture agent, with 65% of tumoral cells captured by the Empty
scaffold (FIG. 15B). In the M-Trap Low Dose group, approximately
80% of tumoral cells were captured by M-Trap, demonstrating an
improved adhesive capacity. Finally, in the M-Trap High Dose group
(clinical design), 100% of tumoral cells injected were captured by
M-Trap, illustrating that the optimal ancillary adhesive capacity
was achieved by the clinical design.
[0216] In Vivo Efficacy of Two Comparative Devices Composed of a
Biodegradable Scaffold Containing the Epidermal Growth Factor (EGF)
as Bioactive Protein
[0217] With reference to FIG. 16, the efficacy of two comparative
devices composed of a biodegradable scaffold containing the
Epidermal Growth Factor (EGF) as bioactive protein were evaluated
in the in vivo model described in FIG. 14. The controlled released
of EGF from the scaffold generated a gradient of chemoattraction
for the pharmacological capture of tumor cells in the scaffold. One
of the scaffolds was fabricated by dissolving 25 mg of Xantana and
0.5 mL of EGF solution (40 mg/mL), before the addition of 750 mg
Pluronic F 127, as example of hydrogel technology. The second
scaffold was fabricated by dissolving 2.5 mg de poloxamin T1107+20
ug heparin+20 ug EGF in 300 .quadrature.l H.sub.2O, before
liophilization and resuspension in 400 ul de acetonitril+20 mg de
PLGA, further addition of 4 ml cottonseed oilde+0.5% de Lecitina
W/V, prior to 2 ml of petroleum eter for acetonitril removal,
filtration and lyophilization, as example of nanoparticle-based
technology. The efficacy of both technologies was evaluated after
surgical implantation in the peritoneal cavity, as described in
FIG. 14. As shown in FIG. 16, representative images and
quantification of captured tumor cells both by pluronic+EGF (FIG.
16A; n=2) and PLGA+EGF (FIG. 16B; n=4) devices resulted in a
partial capture of ovarian tumor cells metastasizing in the
peritoneal cavity. This demonstrated the competitive advantage of
M-Trap technology based on polyurethane scaffold+Type I collagen
coating for a complete capture of tumor cells in the peritoneal
cavity and the consequent focalization of the metastatic disease.
This study also demonstrated that the adhesive non-pharmacological
mode of action of M-Trap technology represents an improvement over
chemotactic pharmacological technologies for the capture of
metastatic tumor cells in the peritoneal cavity.
[0218] Sustainability of M-Trap Tumor Cell Capture Efficacy
[0219] Also related to the differential mode of action of M-Trap,
the duration of the effect and the capture ability of
pharmacological competitor devices are associated with the dynamics
of the release of chemoattractants. Theoretically, as the release
of these factors from the scaffold decreases, the gradient of
chemoattraction is reduced and the capture efficacy is gradually
lost. As M-Trap behaves differentially through a
non-pharmacological adhesive mode of action that is not altered,
its efficacy remains intact with time. This long-term durability
(sustainability) of the device to capture tumor cells has been
demonstrated by evaluating the efficacy of M-Trap to capture
ovarian cancer cells (SKOV3) disseminating in the peritoneal cavity
in a mouse model of ovarian cancer (SCID mouse) at one, three, and
six months post-implantation.
[0220] A description of the experimental groups is as follows:
[0221] M-Trap Group, One Month (n=4): M-Trap devices were
surgically implanted in the inner peritoneal wall of mice. One
month later, one million luciferase-expressing SKOV3 cells were
injected intraperitoneally. One week after tumor cell injection,
animals are sacrificed and the pattern of tumor cell dissemination
is evaluated by bioluminescence. [0222] M-Trap Group, Three Months
(n=4): M-Trap devices were surgically implanted in the inner
peritoneal wall of mice. Three months later, one million
luciferase-expressing SKOV3 cells were injected intraperitoneally.
One week after tumor cell injection, animals are sacrificed and the
pattern of tumor cell dissemination is evaluated by
bioluminescence. [0223] M-Trap Group, Six Months (n=4): M-Trap
devices were surgically implanted in the inner peritoneal wall of
mice. Six months later, one million luciferase-expressing SKOV3
cells are injected intraperitoneally. One week after tumor cell
injection, animals are sacrificed and the pattern of tumor cell
dissemination is evaluated by bioluminescence.
[0224] As shown in FIG. 17, the M-Trap device captured all
metastatic cells in all four (4) animals at each timepoint,
confirming the efficacy of the device at one, three and six months
post-implantation in an in-vivo model of metastatic ovarian
peritoneal dissemination.
[0225] The ability of M-Trap to focalize the peritoneal disease and
eradicate any new peritoneal metastasis linked to its particular
mode of action, was demonstrated in a model of sustained release
(M-Trap post-injection model in FIG. 18A-B). Moreover, the
focalization of the disease resulted in a benefit in survival as
demonstrated in the following preclinical study in the murine model
of ovarian cancer that simulates the intended clinical use of the
device. The endpoint of the study was defined as a decrease in the
Performance Status of the mice, according to the Directive
2010/63/EU guideline related to the appearance, body functions,
environment, behaviors, procedure-specific indicators, and free
observations. Once the study endpoint was reached, the specimen was
sacrificed and survival time recorded. Additionally, the pattern of
tumor cell dissemination was evaluated by bioluminescence and a
histological evaluation was performed.
[0226] A description of the experimental groups is schematically
represented in FIG. 18A, and as follows:
[0227] Control Group (n=5): 2.5 million luciferase-expressing SKOV3
cells were injected intraperitoneally to determine survival times
for the natural pattern of cancer cell dissemination and massive
peritoneal carcinomatosis, in the absence of M-Trap
intervention.
[0228] M-Trap Group (n=5): M-Trap devices were surgically implanted
in the inner peritoneal wall of mice. One week after surgical
implantation, 2.5 million luciferase-expressing SKOV3 cells were
injected intraperitoneally. This group represents survival benefits
attributable to M-Trap intervention and focalization of the
peritoneal disease.
[0229] Re-Operated Group (n=5): M-Trap devices were surgically
implanted in the inner peritoneal wall of mice. One week after
surgical implantation, 2.5 million luciferase-expressing SKOV3
cells were injected intraperitoneally. After one month following
tumoral cell injection, M-Trap devices were surgically removed.
This group represents survival benefits attributable to M-Trap
intervention and surgical removal, which is the intended clinical
use of the device.
[0230] M-Trap Post-Injection Group (n=5): 2.5 million
luciferase-expressing SKOV3 cells were injected intraperitoneally
and allowed to disseminate to their natural sites. One month later,
M-Trap devices were surgically implanted in the inner peritoneal
wall of mice. This group assesses the ability of the device to
capture tumor cells released from primary tumors, thereby
mitigating the normal pattern of cancer cell dissemination and
massive peritoneal carcinomatosis.
[0231] Representative in-vivo bioluminescence images in FIG. 18B
illustrate the different patterns of peritoneal dissemination at
three month follow-up in the four study groups. This interim view
provides evidence of the ability of M-Trap to effectively focalize
the disease (M-Trap group), and additionally illustrates that
eradication of peritoneal disease is achievable by surgical removal
of the device following metastatic cell capture (Re-operated
group). M-Trap is also able to capture cells disseminating from
primary lesions (M-Trap post-injection group), thereby mitigating
the massive peritoneal carcinomatosis seen in the Control group. As
shown in FIG. 18C, and Table 2, M-Trap has a significant impact on
survival outcomes; Kaplan-Meyer survival curves illustrate that
Control Group mice reproducibly reached the endpoint at 101 days
(.about.3.2 months). Animals in the M-TRAP Post-Injection Group
reached the study endpoint after 129 days on average (.about.4.3
months), demonstrating the ability of M-Trap to mitigate the
peritoneal carcinomatosis seen in the Control Group without any
additional intervention (i.e., reoperation). Animals in the M-Trap
Group reached the study endpoint after 161.5 days on average
(.about.5.4 months), further demonstrating the beneficial effect of
focalization of the disease. Finally, mice in the Re-Operated Group
had not reached the study endpoint at the five-month timepoint,
demonstrating the significant survival benefits associated with the
intended M-Trap clinical use. Histology in FIG. 18D confirmed the
eradication of peritoneal carcinomatosis associated with the
capture of metastatic tumor cells and the focalization of the
disease by M-Trap technology.
TABLE-US-00002 TABLE 2 Days 0 87 94 101 104 132 115 126 129 133 136
145 157 166 210 220 265 SUBJECT OF RISK Control 5 5 4 3 2 1
Post-Injection 5 5 4 3 2 1 M-Trap 4 4 3 2 1 Re-operated 2 2 1
SURVIVAL PROPORTIONS Control 100 80 60 40 20 0 Post-injection 100
80 60 40 20 0 M-Trap 100 75 50 25 0 Re-operated 100 50 50
[0232] M-Trap Tumor Cell Capture Efficacy in the Presence of
Chemotherapy
[0233] The efficacy of M-Trap to capture ovarian cancer cells
(SKOV3) disseminating in the peritoneal cavity in a mouse model of
ovarian cancer (SCID mouse), due to its differential mode of action
resulting in the focalization of the peritoneal disease, was also
demonstrated in the presence of IC50 dosage of standard
chemotherapy administered intraperitoneally (carbotaxol,
combination of paclitaxel+carboplatin). Because the device will be
implanted in patients while they are undergoing intraperitoneal
(IP) chemotherapy, this study was critical to verify device
efficacy in the presence of standard IP chemotherapy regimens.
[0234] A total of 16 mice were used for this study. A description
of the experimental groups is as follows:
[0235] Control Group (n=3): One million luciferase-expressing SKOV3
cells were injected intraperitoneally to evaluate the normal
pattern of cancer cell dissemination. One week after tumor cell
injection, the pattern of tumor cell dissemination was evaluated by
bioluminescence using an in-vivo imaging system.
[0236] Control IC50 Group (n=3): One million luciferase-expressing
SKOV3 cells were injected intraperitoneally. After 24 hours, IC50
dose of carbotaxol was administered. One week after tumor cell
injection and chemotherapy, the pattern of tumor cell dissemination
was evaluated.
[0237] M-Trap Group (n=5): M-Trap devices were surgically implanted
in the inner peritoneal wall of mice. One week after surgical
implantation, one million of luciferase-expressing SKOV3 cells were
injected intraperitoneally. One week after tumor cell injection,
the pattern of tumor cell dissemination was evaluated.
[0238] M-Trap IC50 Group (n=5): M-Trap devices were surgically
implanted in the inner peritoneal wall of mice. One week after
surgical implantation, one million luciferase-expressing SKOV3
cells were injected intraperitoneally. After 24 hours, IC50 dose of
carbotaxol was administered. One week after tumor cell injection
and chemotherapy, the pattern of tumor cell dissemination was
evaluated.
[0239] As shown, the study results demonstrated that neither the
pattern of metastasis (FIG. 19A) nor the percentage of survival
tumor cells (FIG. 19B) had been modified in the presence of
chemotherapy indicative of M-Trap efficacy in the presence of
standard intraperitoneal chemotherapy (paclitaxel+carboplatin) used
in the treatment of advanced ovarian cancer
[0240] In-Vivo Efficacy of M-Trap to Capture Different Ovarian
Cancer Cells
[0241] The efficacy of M-Trap to capture three additional ovarian
cancer cell types was evaluated in the murine model of ovarian
cancer peritoneal dissemination at one week post-implantation, in
addition to the SKOV3 adenocarcinoma cell line: TOV112 (serous
origin); OV90 (endometroid origin); and primary cancer cells
isolated from ascitic fluid of ovarian cancer patients.
[0242] A description of the experimental groups is as follows:
[0243] TOV112 Control Group (n=3): One million
luciferase-expressing TOV112 cells were injected intraperitoneally.
One week after tumor cell injection, the mice were sacrificed and
the normal pattern of TOV112 cell dissemination was evaluated by
bioluminescence.
[0244] TOV112 M-Trap Group (n=3): M-Trap devices were surgically
implanted in the inner peritoneal wall of mice. One week later, one
million luciferase-expressing TOV112 cells were injected
intraperitoneally. One week after tumor cell injection, the mice
were sacrificed and the pattern of TOV112 cell dissemination was
evaluated by bioluminescence.
[0245] OV90 Control Group (n=3): One million luciferase-expressing
OV90 cells were injected intraperitoneally. One week after tumor
cell injection, the mice were sacrificed and the normal pattern of
OV90 cell dissemination was evaluated by bioluminescence.
[0246] OV90 M-Trap Group (n=3): M-Trap devices were surgically
implanted in the inner peritoneal wall of mice. One week later, one
million luciferase-expressing OV90 cells were injected
intraperitoneally. One week after tumor cell injection, the mice
were sacrificed and the pattern of OV90 cell dissemination was
evaluated by bioluminescence.
[0247] Primary Cells Control Group (n=3): One million primary
culture cells isolated from an ascitic fluid of ovarian cancer
patients labeled with fluorescence marker Did were injected
intraperitoneally. One week after tumor cell injection, the mice
were sacrificed and the normal pattern of tumor cell dissemination
was evaluated by fluorescence.
[0248] Primary Cells M-Trap Group (n=3): M-Trap devices were
surgically implanted in the inner peritoneal wall of mice. One week
later, one million primary culture cells isolated from an ascitic
fluid of ovarian cancer patients labeled with fluorescence marker
Did are injected intraperitoneally. One week after tumor cell
injection, the mice are sacrificed and the pattern of tumor cell
dissemination is evaluated by fluorescence.
[0249] Representative images shown in FIG. 20 demonstrated the
universality of M-Trap technology to capture different clinically
relevant ovarian cancer cells. The M-Trap device (right panels)
completely remodeled the pattern of peritoneal dissemination shown
in the control groups for TOV112, OV90 and primary ovarian cancer
cells (left panels). Quantification of the
bioluminescence/fluorescence signal from each group confirms the
ability of M-Trap to capture all metastatic ovarian cells
disseminating in the peritoneal cavity.
[0250] M-Trap Tumor Proliferation Risk
[0251] The risk of tumor growth and proliferation due to use of the
M-Trap device was evaluated in a murine subcutaneous tumor model.
This study was a comparative in-vivo assay in which subcutaneous
SKOV3 cell tumors were generated in mice under three different
conditions, with quantification of the bioluminescence signal at 2
weeks and 4 weeks to assess tumor growth and proliferation. The
three different tumor conditions generated in each animal are
depicted in FIG. 21A and described as follows:
[0252] Negative Control Tumor (PBS): Injection of 2.5 million SKOV3
cells resuspended in 50 microliters of phosphate buffer saline
(PBS) into the right lower dorsal area of each specimen. The PBS
arm represents the natural basal environment and native tumorigenic
potential.
[0253] Positive Control Tumor (Matrigel): Injection of 2.5 million
SKOV3 cells resuspended in 50 microliters of Matrigel into the
upper dorsal area (neck) of each specimen. Matrigel is a standard
protein mixture resembling the complex extracellular environment
found in many tissues. The Matrigel arm represents the most
favorable condition for the promotion of tumor growth.
[0254] Test Device Tumor (M-Trap): Seeding of 2.5 million SKOV3
cells within a M-Trap device and subsequent implantation of the
seeded M-Trap device into the left lower dorsal area of each
specimen.
[0255] As shown in FIG. 21B, M-Trap does not contribute to tumor
growth upon cell capture in a murine subcutaneous tumor model.
After 2 and 4 weeks, quantification of tumor growth showed similar
proliferation to that of the negative control (PBS group), and
significantly lower than that of the positive control (Matrigel
group).
[0256] M-Trap Technology Efficiently Captures Metastatic Tumor
Cells in an In Vivo Model of Ovarian Cancer Dissemination
[0257] To translate these evidences into an in vivo mice model
mimicking ovarian cancer dissemination and peritoneal metastasis
implantation, 1.times.10.sup.6 SKOV3 cells stably expressing the
luciferase reporter gene (Steinkamp et al., 2013 Front Oncol 3, 97)
were intraperitoneally injected. One week later, the pattern of
major natural peritoneal dissemination evaluated by bioluminescence
showed the pancreas and gonadal fat pad as preferential sites of
SKOV3 cells implantation. To evaluate whether M-Trap might be
competing with the natural foci of peritoneal metastasis and
capturing cells disseminating within the peritoneal cavity, the
pattern of natural peritoneal implants was compared to that
generated upon implantation of the Biomerix 3D scaffold or the
Biomerix 3D scaffold coated with collagen as capture agent. For
this, the device (scaffold alone or M-trap) was inserted at the
inner wall of the peritoneum opposite to the pancreas and the
gonadal fat pad as natural sites of metastasis. One week later,
SKOV3 cells were intraperitoneally injected and the localization of
metastasis was assessed seven days after injection. Remarkably, the
pattern of dissemination of metastatic ovarian tumor cells in the
presence of M-Trap device composed by the Biomerix scaffold
decorated with 250 .mu.g collagen was completely remodeled, with
the eradication of the regular places of metastasis and the
focalization of metastasis in a unique focus within the scaffold
with collagen. The quantification of bioluminescence signal in a
series of three mice per group for natural pattern of SKOV3 cells
peritoneal implants (Control), and those generated by the Biomerix
scaffold without collagen (Scaffold), and M-Trap device with
collagen (M-Trap), confirmed the capacity of M-Trap to capture
tumor cells disseminating within the peritoneal cavity and to
completely remodel the pattern of metastasis in a mice model of
ovarian cancer dissemination (p<0.0001).
[0258] Similar results were obtained both in vitro and in vivo with
M-Trap device composed of the Biomerix scaffold coated with the
extracellular matrix protein involved in cell adhesion Fibronectin.
Increasing concentrations of fibronectin decorating the scaffold
were able to capture SKOV3 cells in the in vitro dynamic orbital
assay mimicking transcoelomic peritoneal flow, in a dose dependent
manner (FIG. 22, panel A). Similarly to the collagen adhesive
properties of the M-Trap device, fibronectin coating of a Biomerix
scaffold resulted in a complete remodeled pattern of peritoneal
implants in the in vivo model of ovarian dissemination, with almost
all metastatic tumor cells being captured within the M-Trap device
(bioluminescent image of SKOV3 cells implant at M-Trap device; FIG.
22 panel B), quantified in panel FIG. 22C.
* * * * *