U.S. patent application number 16/081654 was filed with the patent office on 2019-03-21 for methods of preventing or reducing a fibrotic response using csf1r inhibitors.
This patent application is currently assigned to Massachusetts Institute of Technology. The applicant listed for this patent is Massachusetts Institute of Technology. Invention is credited to Daniel G. Anderson, Joshua C. Doloff, Shady Farah, Robert S. Langer, Arturo J. Vegas, Omid Veiseh.
Application Number | 20190083495 16/081654 |
Document ID | / |
Family ID | 58549290 |
Filed Date | 2019-03-21 |
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United States Patent
Application |
20190083495 |
Kind Code |
A1 |
Doloff; Joshua C. ; et
al. |
March 21, 2019 |
Methods Of Preventing Or Reducing A Fibrotic Response Using CSF1R
Inhibitors
Abstract
Described herein are methods of preventing or reducing fibrosis
comprising administering CSF1R inhibitors, coating formulations
comprising CSF1R inhibitors, coatings comprising CSF1R inhibitors
for implantable medical devices, CSF1R inhibitor coated implantable
medical devices, as well as corresponding embodiments comprising
additional agents.
Inventors: |
Doloff; Joshua C.; (Quincy,
MA) ; Farah; Shady; (Boston, MA) ; Veiseh;
Omid; (Cambridge, MA) ; Vegas; Arturo J.;
(Cambridge, MA) ; Langer; Robert S.; (Newton,
MA) ; Anderson; Daniel G.; (Framingham, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Institute of Technology |
Cambridge |
MA |
US |
|
|
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
|
Family ID: |
58549290 |
Appl. No.: |
16/081654 |
Filed: |
April 4, 2017 |
PCT Filed: |
April 4, 2017 |
PCT NO: |
PCT/US17/25991 |
371 Date: |
August 31, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62317831 |
Apr 4, 2016 |
|
|
|
62318208 |
Apr 4, 2016 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0024 20130101;
A61K 31/416 20130101; C07B 2200/13 20130101; A61K 31/519 20130101;
C07D 239/49 20130101; A61K 31/5025 20130101; A61K 31/444 20130101;
A61K 31/5377 20130101; A61K 31/496 20130101; A61K 31/551 20130101;
A61K 31/553 20130101; A61K 31/4709 20130101; A61K 31/404 20130101;
A61K 31/4439 20130101; A61P 29/00 20180101 |
International
Class: |
A61K 31/519 20060101
A61K031/519; A61K 31/4709 20060101 A61K031/4709; A61K 31/4439
20060101 A61K031/4439; A61K 31/5377 20060101 A61K031/5377; A61K
31/416 20060101 A61K031/416; A61K 31/444 20060101 A61K031/444; A61K
31/496 20060101 A61K031/496; A61K 31/404 20060101 A61K031/404; A61K
31/553 20060101 A61K031/553; A61K 31/551 20060101 A61K031/551; A61K
31/5025 20060101 A61K031/5025 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under Grant
No. R01 DE016516 awarded by the National Institutes of Health and
under Contract No. W81XWH-13-1-0215 awarded by the U.S. Army
Medical Research and Material Command. The Government has certain
rights in the invention.
Claims
1. A method of preventing or reducing a fibrotic response to an
implanted synthetic material in a patient, the method comprising
administering to the patient an effective amount of a CSF1R
inhibitor selected from the group consisting of a compound of
structural formula ##STR00073## a compound represented by
structural formula ##STR00074## a compound represented by
structural formula ##STR00075## a compound represented by
structural formula ##STR00076## and AC708,
4-(3,4-Dimethylanilino)-7-(4-(methylsulfonyl)phenyl)quinoline-3-carboxami-
de,
(4-cyano-N-(2-cyclohexenyl-4-(1-(2-(dimethylamino)acetyl)piperidin-4-y-
l)phenyl)-1H-imidazole-2-carboxamide), a compound represented by
structural formula ##STR00077## ARRY-382, a compound represented by
structural formula ##STR00078## a 2'-aminoanilide, a
3-amido-4-anilinocinniline, an indoline-2-one, a
2-(alpha-methylbenzylamino)-pyrazine, an arylamide, a
3,4,6-substituted 2-quinolone, a pyrido[2,3-d]pyrimidin-5-one, a
3-amido-4-anilinoquinoline, a pyridyl bisamide, a thiazolyl
bisamide, a 1,4-disubstituted-pyrrolo-[3,2-c]-pyridine, a
substituted diphenylurea, a 5'-pyrimidine-2,4-diamine, a compound
represented by structural formula ##STR00079## a compound
represented by structural formula ##STR00080## anilinoquinazoline,
a compound represented by structural formula ##STR00081## a
compound represented by structural formula ##STR00082## DCC-3014, a
compound represented by structural formula ##STR00083## a compound
represented by structural formula ##STR00084## a compound
represented by structural formula ##STR00085## a compound
represented by structural formula ##STR00086## a compound
represented by structural formula ##STR00087## a compound
represented by structural formula ##STR00088## and a compound
represented by structural formula ##STR00089##
2. The method of claim 1, wherein the CSF1R inhibitor is
GW2580.
3. The method of claim 1, wherein the CSF1R inhibitor is
Ki20227.
4. The method of claim 1, wherein the CSF1R inhibitor is
4-(3,4-Dimethylanilino)-7-(4-(methylsulfonyl)phenyl)quinoline-3-carboxami-
de.
5. The method of claim 1, wherein the CSF1R inhibitor is
(4-cyano-N-(2-cyclohexenyl-4-(1-(2-(dimethylamino)acetyl)piperidin-4-yl)p-
henyl)-1H-imidazole-2-carboxamide).
6. The method of claim 1, wherein the foreign body is an ingested
foreign body or an inhaled foreign body.
7. The method of claim 1, wherein the patient has an implanted
medical device comprising the implanted material.
8. The method of claim 7, wherein the implanted medical device is
implanted intraperitoneally, subcutaneously, or intramuscularly in
the patient.
9. The method of claim 7, wherein the implanted medical device
comprises at least one of a polymer, a ceramic, a hydrogel, a
rubber, a metal, and glass.
10. The method of claim 9, wherein the polymer is a
polysaccharide.
11. The method of claim 10, wherein the polysaccharide is alginate
or chitosan.
12. The method of claim 9, wherein the polymer is
polytetrafluoroethylene, polystyrene, polycaprolactone (PCL), or
polydimethylsiloxane (PDMS).
13. The method of claim 9, wherein the metal is gold or stainless
steel.
Description
RELATED APPLICATION(S)
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/317,831, filed Apr. 4, 2016. This application
also claims the benefit of U.S. Provisional Application No.
62/318,208 filed Apr. 4, 2016. The entire teachings of the above
applications are incorporated herein by reference.
INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE
[0003] This application incorporates by reference the Sequence
Listing contained in the following ASCII text file being submitted
concurrently herewith:
[0004] a) File name: SEQLIST.txt; created Apr. 4, 2017, 7 KB in
size.
BACKGROUND
[0005] Implanted biomedical devices currently reside within tens of
millions of patients, both juvenile and adult, in the United States
alone, and are involved in millions of new as well as revisionary
surgeries every year (Kurtz S, Ong K, Lau E, Mowat F, Halpern M.
Projections of primary and revision hip and knee arthroplasty in
the United States from 2005 to 2030. The Journal of bone and joint
surgery American volume 2007, 89(4): 780-785.; Med I. Medical
Devices and the Public's Health: The FDA 510(k) Clearance Process
at 35 Years. Medical Devices and the Public's Health: The Fda
510(K) Clearance Process at 35 Years 2011: 1-298.). As such, they
comprise a major component of modern biomedicine, both in time and
cost, and are essential for many clinical applications ranging from
hip/knee replacement Cobelli N, Scharf B, Crisi G M, Hardin J,
Santambrogio L. Mediators of the inflammatory response to joint
replacement devices. Nature reviews Rheumatology 2011, 7(10):
600-608), tissue repair/reconstruction (Hubbell J A, Langer R.
Translating materials design to the clinic. Nature materials 2013,
12(11): 963-966), prosthesis and neural interfacing (Fattahi P,
Yang G, Kim G, Abidian M R. A review of organic and inorganic
biomaterials for neural interfaces. Advanced materials 2014,
26(12): 1846-1885), controlled drug release (Farra R, Sheppard N F,
Jr., McCabe L, Neer R M, Anderson J M, Santini J T, Jr., et al.
First-in-human testing of a wirelessly controlled drug delivery
microchip. Sci Transl Med 2012, 4(122): 122ra121), electronic
pacing (Rosen M R, Robinson R B, Brink P R, Cohen I S. The road to
biological pacing. Nat Rev Cardiol 2011, 8(11): 656-666), vital
sign monitoring (Nichols S P, Koh A, Storm W L, Shin J H,
Schoenfisch M H. Biocompatible materials for continuous glucose
monitoring devices. Chemical reviews 2013, 113(4): 2528-2549),
intraocular lens replacement (Perez-Cambrodi R J, Pinero D P,
Ferrer-Blasco T, Cervino A, Brautaset R. The posterior chamber
phakic refractive lens (PRL): a review. Eye 2013, 27(1): 14-21),
and cell encapsulation and transplantation (Kearney C J, Mooney D
J. Macroscale delivery systems for molecular and cellular payloads.
Nature materials 2013, 12(11): 1004-1017.). Unlike particulates,
which may be phagocytosed by the immune system and cleared via
circulation and excretion (Cobelli N, Scharf B, Crisi G M, Hardin
J, Santambrogio L. Mediators of the inflammatory response to joint
replacement devices. Nature reviews Rheumatology 2011, 7(10):
600-608), larger non-biodegradable macroscale devices cannot be
dislodged and extruded from the body. Instead, the host senses
these implants as foreign and mounts an immune-mediated rejection
response (Anderson J M, Rodriguez A, Chang D T. Foreign body
reaction to biomaterials. Semin Immunol 2008, 20(2): 86-100; Wynn T
A, Ramalingam T R. Mechanisms of fibrosis: therapeutic translation
for fibrotic disease. Nature medicine 2012, 18(7): 1028-1040.).
Immune cell adhesion leads to fibrosis, which encapsulates the
implants in layers of scar tissue and extracellular matrix (Wynn T
A, Ramalingam T R. Mechanisms of fibrosis: therapeutic translation
for fibrotic disease. Nature medicine 2012, 18(7): 1028-1040.).
Such sequestration can impair and eventually ruins device function
by enzyme, acid, or reactive oxygen species-based degradation. This
can also prevent necessary interaction with the surrounding
microenvironment, including sensing of biochemical stimuli such as
pH, oxygen, blood glucose levels, and/or obstructing nutrient flux
where internal device components are of biologic origin (Anderson J
M, Rodriguez A, Chang D T. Foreign body reaction to biomaterials.
Semin Immunol 2008, 20(2): 86-100; Kenneth Ward W. A Review of the
Foreign-body Response to Subcutaneously-implanted Devices: The Role
of Macrophages and Cytokines in Biofouling and Fibrosis. J Diabetes
Sci Technol Online 2008, 2: 768-777.). Furthermore, fibrotic scar
tissue can cause pain and discomfort by displacing or abrading
normal primary tissues (Bryers J D, Giachelli C M, Ratner B D.
Engineering biomaterials to integrate and heal: the
biocompatibility paradigm shifts. Biotechnol Bioeng 2012, 109(8):
1898-1911; Williams D F. On the mechanisms of biocompatibility.
Biomaterials 2008, 29(20): 2941-2953.). It would be useful to have
better methods to prevent or treat detrimental fibrosis.
SUMMARY OF THE INVENTION
[0006] The present invention relates to methods of treating or
preventing fibrosis using a CSF1R inhibitor.
[0007] In one example embodiment, the method is a method of
preventing or reducing a fibrotic response to an implanted
synthetic material in a patient. The method includes administering
to the patient an effective amount of a CSF1R inhibitor selected
from the group consisting of a compound of structural formula
##STR00001##
of a compound represented by structural formula
##STR00002##
a compound represented by structural formula
##STR00003##
a compound represented by structural formula
##STR00004##
AC708,
4-(3,4-Dimethylanilino)-7-(4-(methylsulfonyl)phenyl)quinoline-3-ca-
rboxamide,
(4-cyano-N-(2-cyclohexenyl-4-(1-(2-(dimethylamino)acetyl)piperi-
din-4-yl)phenyl)-1H-imidazole-2-carboxamide), a compound
represented by structural formula
##STR00005##
ARRY-382, a compound represented by structural formula
##STR00006##
a 2'-aminoanilide, a 3-amido-4-anilinocinniline, an indoline-2-one,
a 2-(alpha-methylbenzylamino)-pyrazine, an arylamide, a
3,4,6-substituted 2-quinolone, a pyrido[2,3-d]pyrimidin-5-one, a
3-amido-4-anilinoquinoline, a pyridyl bisamide, a thiazolyl
bisamide, a 1,4-disubstituted-pyrrolo-[3,2-c]-pyridine, a
substituted diphenylurea, a 5'-pyrimidine-2,4-diamine, a compound
represented by structural formula
##STR00007##
a compound represented by structural formula
##STR00008##
anilinoquinazoline, a compound represented by structural
formula
##STR00009##
a compound represented by structural formula
##STR00010##
DCC-3014, a compound represented by structural formula
##STR00011##
a compound represented by structural formula
##STR00012##
a compound represented by structural formula
##STR00013##
a compound represented by structural formula
##STR00014##
a compound represented by structural formula
##STR00015##
a compound represented by structural formula
##STR00016##
a compound represented by structural formula
##STR00017##
and combinations thereof.
[0008] In some embodiments, the CSF1R inhibitor is at least one
CSF1R inhibitor described herein. In some embodiments, for example,
the CSF1R inhibitor is one or more CSF1R inhibitors selected from
the group consisting of GW2580, Ki20227,
4-(3,4-Dimethylanilino)-7-(4-(methylsulfonyl)phenyl)quinoline-3-carboxami-
de,
(4-cyano-N-(2-cyclohexenyl-4-(1-(2-(dimethylamino)acetyl)piperidin-4-y-
l)phenyl)-1H-imidazole-2-carboxamide), BLZ945, Quizartinib, AC708,
Linifanib (a multitargeted receptor tyrosine kinase inhibitor),
ARRY-382, Pexidartinib, a 2'-aminoanilide, a
3-amido-4-anilinocinniline, an indoline-2-one, a
2-(alpha-methylbenzylamino)-pyrazine, an arylamide, a
3,4,6-substituted 2-quinolone, a pyrido[2,3-d]pyrimidin-5-one, a
3-amido-4-anilinoquinoline, a pyridyl bisamide, a thiazolyl
bisamide, a 1,4-disubstituted-pyrrolo-[3,2-c]-pyridine, a
substituted diphenylurea, a 5'-pyrimidine-2,4-diamine, CYC10268,
AZ683, anilinoquinazoline, OSI-930, DCC-2618, DCC-3014,
JNJ-40346527 (a macrophage colony stimulating factor receptor
agonist and CSF-1R inhibitor), Sunitinib, Lestaurtinib,
Midostaurin, Tandutinib, Sorafenib, Ponatinib and combinations
thereof. In one embodiment, the CSF1R inhibitor is GW2580. In some
embodiments, the CSF1R inhibitor is Ki20227. In some embodiments,
the CSF1R inhibitor is
4-(3,4-Dimethylanilino)-7-(4-(methylsulfonyl)phenyl)quinoline-3-carbox-
amide. In some embodiments, the CSF1R inhibitor is
(4-cyano-N-(2-cyclohexenyl-4-(1-(2-(dimethylamino)acetyl)piperidin-4-yl)p-
henyl)-1H-imidazole-2-carboxamide). In one embodiment, the CSF1R
inhibitor is at least one of GW2580, Ki20227,
4-(3,4-Dimethylanilino)-7-(4-(methylsulfonyl)phenyl)quinoline-3-carboxami-
de and
(4-cyano-N-(2-cyclohexenyl-4-(1-(2-(dimethylamino)acetyl)piperidin--
4-yl)phenyl)-1H-imidazole-2-carboxamide).
[0009] In some embodiments, the foreign body is an ingested foreign
body or an inhaled foreign body. In some embodiments, the foreign
body is a mineral or element.
[0010] In some embodiments, the patient has an implanted medical
device comprising the implanted material. In some embodiments, the
implanted medical device is implanted, for example,
intraperitoneally, subcutaneously, or intramuscularly in the
patient. In some embodiments, the implanted medical device
comprises, for example, at least one of a polymer, a ceramic, a
hydrogel, a rubber, a metal, and glass. In some embodiments, the
polymer is a polysaccharide. In some embodiments, the
polysaccharide is alginate or chitosan.
[0011] In some embodiments, the polymer is polytetrafluoroethylene,
polystyrene, polycaprolactone (PCL), or polydimethylsiloxane
(PDMS). In some embodiments, the metal is gold or stainless
steel.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] U.S. Provisional Application No. 62/317,831 ('831
application) contains color drawings which correspond to drawings
of the present invention. With regard to indications of color
within the instant description of the figures provided herein,
reference is made to those corresponding drawings and associated
descriptions of (1) the '831 application and (2) Doloff et al.,
"Colony stimulating factor-1 receptor is a central component of the
foreign body response to biomaterial implants in rodents and
non-human primates", Nature Materials, Advance Online Publication,
published online Mar. 20, 2017 (DOI:10.1038/NMAT4866), both
incorporated herein by reference in their entirety.
[0013] The foregoing will be apparent from the following more
particular description of example embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different views.
The drawings are not necessarily to scale, emphasis instead being
placed upon illustrating embodiments of the present invention.
[0014] FIGS. 1A-G depicts that numerous immune populations respond
and adhere to implanted biomaterial alginate spheres. SLG20
alginate 500 .mu.m diameter spheres (0.35 ml total implant volume)
were implanted into the intraperitoneal space of C57BL/6 mice,
where they were retained for 14 days and analyzed for degree of
fibrosis upon retrieval. FIG. 1A Dark field phase contrast image
and FIG. 1B DAPI immunofluorescence image obtained from retrieved
spheres reveal a significant level of cellular overgrowth; scale
bar=2000 .mu.m). FIG. 1C: qPCR analysis of innate and adaptive
immune population and fibrosis markers present on fibrosed alginate
spheres, A, or non-transplant, N, or mock transplant, M, omental
and epididymal fat pad tissue after 14 days post-implantation into
the IP space of C57BL/6 mice. M.PHI.=macrophages, DCs=Dendritic
cells, NKs=Natural Killer cells. Data: mean.+-.SEM, n=5. qPCR
statistical analysis: one-way ANOVA with Bonferroni multiple
comparison correction **: p<0.001, and ***: p<0.0001; ns=not
significantly different. FIG. 1D Confocal staining showing DAPI
(cellular nuclei), innate immune macrophage marker CD68 (green),
adaptive immune B cells (magenta), alpha smooth muscle actin
(.alpha.SMactin, myofibroblasts, red), and overlay making up the
fibrosis on 500 .mu.m alginate spheres. FIG. 1E: Confocal staining
showing DAPI (cellular nuclei), innate immune neutrophil marker
Ly6g/Gr1 (green), alpha smooth muscle actin (.alpha.SMactin,
myofibroblasts, red), fluorescent overlay, and brightfield image
for the fibrosis on 500 .mu.m alginate spheres. In vivo intravital
imaging of adaptive B cell behavior and accumulation at day 14
post-implant for mock transplant (FIG. 1F) or SLG20 sphere
implanted (FIG. 1G) C57BL/6-Ccr6 (EGFP) mice. For intravital
imaging: N=3 mice per treatment. For all others, N=5 mice per
group. Experiments repeated at least 3 times.
[0015] FIG. 2 depicts that the same immune responders adhere to
implanted biomaterial alginate spheres in both the intraperitoneal
(IP) and subcutaneous (SC) sites. SLG20 alginate 500 .mu.m diameter
spheres were implanted into the subcutaneous space of C57BL/6 mice,
where they were retained for 14 days and analyzed upon retrieval.
qPCR analysis of innate and adaptive immune population and fibrosis
markers present in mock transplanted, M, versus fibrosed implanted
alginate-embedded subcutaneous tissues, A. M.PHI.=macrophages,
DCs=Dendritic cells, NKs=Natural Killer cells. Data: mean.+-.SEM,
n=5. qPCR statistical analysis: one-way ANOVA with Bonferroni
multiple comparison correction *: p<0.05, **: p<0.001, and
***: p<0.0001; ns=not significantly different. Experiment run at
least twice.
[0016] FIGS. 3A-G depicts that the immune response to implanted
biomaterial alginate is long lived. Flow analysis, using specific
markers for responding host innate immune macrophage (red),
neutrophil (blue), and adaptive immune B cells (green) at 1, 4, 7,
14, and 28 days post-implantation among peritoneal exudate (lavage)
(FIG. 3A), peripheral fibrosed omental and epididymal fat pads
(through which host cells infiltrate and to which material spheres
become fibrosed, becoming part of the fibrosis themselves) (FIG.
3B), and directly on fibrosed alginate spheres (FIG. 3C). For
sphere-specific FACS, fixation and permeabilization was also
carried out to stain for fibrosis-depositing myofibroblasts
(.alpha.SMactin, white). NanoString-based analysis for expression
of macrophage (FIG. 3D) and neutrophil (FIG. 3E) associated markers
analyzed from deposited cell RNA extracts at 14 days post-implant,
presented on a base 2 logarithmic scale. (FIG. 3F) qPCR analysis on
either peripheral tissue (yellow) or spheres alone (blue), relative
to day 1 tissue for all, showing that there is a slightly delayed
mobilization of adaptive B cells out of peripheral tissue and onto
spheres after 1 week post-implantation. (FIG. 3G) qPCR analysis on
either peripheral tissue (yellow) or spheres alone (blue), relative
to day 1 for either tissue or spheres, respectively, showing that
there is no apparent response (due to high tissue background)
across time points for .alpha.SMactin, as opposed to a high dynamic
response if spheres alone are examined. Error bars, mean.+-.SEM.
For FACS and qPCR analysis N=5 mice per treatment; for NanoString
analysis N=4 per treatment. FACS and qPCR experiments were
performed twice and NanoString analysis was performed once. FACS
time point comparisons were performed by unpaired, two-tailed
t-test *: p<0.05, **: p<0.001, and ***: p<0.0001, vs mock
or non-implanted controls.
[0017] FIGS. 4A-E depict that kinetic profiling of 30 cytokines in
the blood shows no global response to alginate. Multiplexed Luminex
kinetic profiling of protein production of 30 inflammatory
cytokines in the serum of C57BL/6 mice at 1, 4, 7, 14, and 28 days
post-intraperitoneal implantation of 500 .mu.m biomaterial alginate
spheres. (FIG. 4A) Fold change numbers of all 30 cytokines at
individual points, relative to protein levels of the
mock/non-implanted (NT) control serum samples. (FIGS. 4B-E)
Individual kinetic plots of the only 4 cytokines (IL-5, IL-6,
G-CSF, and KC) that showed any significant responses (red squares
in (a)) in the blood of implanted C57BL/6 mice. Responses, however,
were transient and gone within 4-7 days post-implantation,
suggesting that these increases were instead surgery related. Error
bars, mean.+-.SEM. N=5 mice per treatment. Performed at least two
times.
[0018] FIG. 5 depicts a photo sequence for retrieval process. 500
.mu.m diameter alginate microspheres are retrieved from the
intraperitoneal (IP) space of wildtype C57BL/6 mice following a
2-week implantation. 1-2) Incisions were made first into the skin
and then the underlying peritoneum. 3-4) Once the skin and
peritoneal wall were successfully resected, the intestines were
moved to the side exposing both the IP omental and epididymal fat
pads. 5) It has been consistently and reliably observed that
alginate spheres are fibrosed to non-collagen-encapsulated adipose
tissues (omental, top blue inset square; and epididymal, bottom
left and right inset squares) within the IP space (and never to
tissues with collagen capsules, such as the liver, kidneys, etc.).
Implanted materials suffer immune attack from immune cell responses
that extravasate out of these microvessel rich adipose tissues (Med
I. Medical Devices and the Public's Health: The FDA 510(k)
Clearance Process at 35 Years. Medical Devices and the Public's
Health: The Fda 510(K) Clearance Process at 35 Years 2011: 1-298.).
5a & 5b) Large groups of fibrosed alginate microspheres are
found around and under both the left and right epididymal fat pads.
Black arrows, fibrosed alginate microsphere(s). 6) While numerous
microspheres are fibrosed directly to and embedded within adipose
tissues, with more adherence over extended implantation times, many
individual dirty (fibrosed) microspheres can be flushed out of the
IP space following a 2-week implantation (6b). Images,
representative across all mice; observed for countless (at least
100-200+) different implantations over the past 3-4 years (as of
filing date of the U.S. Provisional Application No.
62/317,831).
[0019] FIGS. 6A-C depict additional kinetic expression profiling
for immune-related factors associated with fibrotic cascade. qPCR
analysis (Macrophage marker CD68 (FIG. 6A) and Transforming growth
factor-beta (TGF.beta.) (FIG. 6B)) on either peripheral tissue
(yellow) or alginate spheres alone (blue), relative to day 1 for
either tissue or spheres, respectively, showing that there are
similar kinetic responses between surrounding fibrosed tissue and
embedded, fibrosed 500 .mu.m biomaterial alginate spheres,
implanted in the IP space of C57BL/6 mice. Error bars, mean.+-.SEM.
N=5 mice per treatment. Experiments were performed twice. qPCR
statistical analysis: one-way ANOVA with Bonferroni multiple
comparison correction *: p<0.5, **: p<0.001, and ***:
p<0.0001, vs non-implanted (NT) controls. MT=mock transplanted,
and N/A=not applicable, for spheres alone. (FIG. 6C) Western
blotting time course for chemokine CXCL13 expression in mock
implant tissue vs. day 1, 4, 7, 14, and 28 tissue and sphere
protein samples retrieved from the intraperitoneal space of C57BL/6
mice, showing similar kinetics to Cxcl13 gene expression as shown
by Nanostring analysis in FIG. 3D. Run at least twice.
[0020] FIG. 7 depicts knockouts and targeted depletion fibrosis
summary. Serial or combined immune perturbations were used across
various C57BL/6 strains to determine which cell populations are
necessary for immune-mediated fibrosis. After extensive
characterization, macrophages, and not neutrophils, are the only
cell population required for downstream fibrotic sequestration of
implanted alginate spheres. Right column: representative summary
responses based on phase contrast images showing fibrosis levels on
500 .mu.m alginate spheres retrieved from wild type C57BL/6 mice
(n=5/group), after 14-day intraperitoneal implantations. *, as
reported.
[0021] FIGS. 8A-G depict that innate immune macrophage function is
required for fibrosis of alginate. SLG20 alginate 500 .mu.m
diameter spheres (0.35 ml total implant volume) were implanted into
the intraperitoneal space of wild type and various knockout
(IghMnull, B cell deficiency; Rag2.sup.null, T and B cell
deficiency; and Rag2.sup.null/IL2r.gamma..sup.null, T, B, NK cell
deficiency, and M.PHI. and DC dysfunction) C57BL/6 mice, where they
were retained for 14 days and analyzed for degree of fibrosis upon
retrieval. (FIG. 8A) Dark field phase contrast images showing that
fibrosis, as compared to wild type (WT) control, was partially
decreased upon removal of adaptive B cells (B KO), increased when T
cells were also removed (TB KO), and completely removed with
additional M.PHI. dysfunction (Rag2/.gamma. KO). (FIG. 8B)
Semi-quantitative western blot analysis of .alpha.-SMact expression
in cell overgrowth on microspheres (bands correspond to 5
individual mice), confirming relative changes to the levels of
fibrosis observed due to the same immune perturbations in (a).
(FIG. 8C) Plot of analyzed band intensities from western blot
images shown in f (FIG. 8B). Error bars, mean.+-.SEM. N=5 mice per
treatment. All experiments were performed at least three times.
(FIG. 8D) q-PCR based expression analysis of fibrotic markers
.alpha.-SMactin, Collagen 1a1 (Col1a1), and Collagen 1a2 (Col1a2)
directly on retrieved spheres from WT, B KO, and Rag2/.gamma. KO
C57BL/6 strains, plotted normalized to relative expression levels
on spheres from WT mice. (FIG. 8E) Quantified IVIS live imaging
fluorescent ProSense 750 inflammation levels across Mock (saline),
WT, B KO, and Rag2/.gamma. KO mice 7 days post-subcutaneous
implant. (FIG. 8F) H&E and Masson's Trichrome stained
histological sections of excised subcutaneous tissue at 14 days
post-implant from various treatments, as noted (FIG. 8F, Scale
bar=500 .mu.m). (FIG. 8G) Flow analysis, using specific markers for
responding host innate immune macrophage, neutrophil, and adaptive
B cells dissociated directly from spheres (normalized to cell
counts from 100 .mu.l material) 14 days post-intraperitoneal (i.p.)
implantation (note: one WT subcutaneous sample is also included for
comparison). qPCR, western blot, IVIS, and FACS statistical
analysis: one-way ANOVA with Bonferroni multiple comparison
correction *: p<0.05, **: p<0.001, and ***: p<0.0001, vs
WT. N=5 mice/group. Experiments repeated at least 2-3 times.
[0022] FIGS. 9A-F depict a complete panel of phase contrast images
for all knockouts. Samples, retrieved Ba-crosslinked SLG20 spheres
of 500 .mu.m diameter spheres implanted into the intraperitoneal
space of wildtype (FIG. 9A) and various immune compromised knockout
(IghM.sup.null, B cell deficiency, (FIG. 9B); Rag2.sup.null, T and
B cell deficiency, (FIG. 9C); Rag2.sup.null/IL2r.gamma..sup.null,
T, B, NK cell deficiency, and M.PHI. and DC dysfunction, (FIG. 9D);
T cell knockout (T KO) nude (FIG. 9E) and complement (C3) knockout
(FIG. 9F)) C57BL/6 mice. Fibrosis, as compared to wildtype (WT)
control, was partially decreased upon losing adaptive B cells (B
KO), increased when T cells were also removed (TB KO), and gone
completely with additional M.PHI. dysfunction (Rag2/.gamma.).
Fibrosis was also not significantly affected by T cell loss alone,
nor dependent on C3 complement immune recognition. Images obtained
from all spheres retrieved from individual mice (n=5/group). The
same material volume of hydrogel spheres was implanted into each
mouse in all cases. Experiments were repeated twice.
[0023] FIGS. 10A-C depict additional B cell (IghM.sup.null)
knockout model characterization, for reduced B cell response and
corresponding reductions in fibrosis. (FIG. 10A) Additional in vivo
intravital imaging of adaptive B cell behavior and accumulation at
day 14 post-implant for SLG20 sphere implanted C57BL/6-Ccr6 (EGFP)
mice (extra images from those shown in FIG. 1G showing B cell
responses, extravasation from surrounding IP epididymal fat pads
and aggregation between quantum dot (pink) encapsulating SLG20
alginate microspheres. (FIG. 10B) IgM protein levels determined by
ELISA for both blood serum (left axis) and IP protein lysates
(right axis) taken from tissue and spheres 14 days post-implant
from mock (MT) and implanted wildtype (WT) and knockout strains
((IghM.sup.null, B cell knockout, B KO; and
Rag2.sup.null/IL2r.gamma..sup.null knockout (Rag2/.gamma. KO), with
T, B, NK cell deficiency, and M.PHI. and DC dysfunction). Loss of
IgM in both blood and IP lysates taken from B cell deficient
strains was confirmed. No IgM increases were detectable in wildtype
(WT) responses in the blood but were significantly increased
locally in the intraperitoneal space, suggesting that IgM is
present not as secreted antibody but as a B cell receptor (BCR) on
responding B cells. (FIG. 10C) Confocal staining showing DAPI
(cellular nuclei), innate immune macrophage marker CD68 (green),
alpha smooth muscle actin (.alpha.SMactin, myofibroblasts, red), B
cell marker CD19 (magenta), fluorescent overlay, and brightfield
image for the fibrosis on 500 .mu.m alginate spheres in wildtype
(WT) C57BL/6 mice. B cell CD19 staining was unsurprisingly lost in
B cell knockout (B KO) mice. More so, however, confocal imaging
confirmed decreased fibrotic overgrowth due to loss of B cells, as
seen by multiple imaging and staining methods (FIGS. 8A-G).
Experiments were repeated twice.
[0024] FIGS. 11A-D depict additional histology (H&E and
Masson's Trichrome staining) panels for subcutaneously implanted
wildtype and knockout mice. Mock (saline injected) (FIG. 11A) and
SLG20 500 .mu.m diameter alginate sphere implanted wildtype (WT)
(FIG. 11B), B cell knockout (B KO) (FIG. 11C), and M.PHI.
dysfunctional Rag2.sup.null/IL2r.gamma..sup.null (Rag2/.gamma.)
C57BL/6 mice (FIG. 11D). 2.times. and 20.times. magnifications are
shown in all cases to show both the scale as well as cellular
details of varying levels of fibrosis in each treatment group.
Arrows, showing implanted regions surrounding by an outer fibrosis
collagen capsule. *, denote individual fibrosed alginate spheres.
As shown in FIGS. 8A-G, B cell loss contributes to fibrotic
reduction, while M.PHI. dysfunction results in loss of fibrosis
(similar to mock), as compared to WT implanted controls.
[0025] FIGS. 12A-C depict additional FACS characterization of mock
(left), distant (middle), and implanted (right) subcutaneous sites.
Subcutaneous tissue groups, comparing immune population
compositions of mock implanted (saline injected) to that from
alginate-implanted C57BL/6 mice either at a site distant to (at
least 1 cm away) or directly at material delivery (Implanted). Flow
analysis, using specific markers for responding host innate immune
macrophage (CD68.sup.+CD11b.sup.+) (FIG. 12A), neutrophil
(Ly6g/Gr1.sup.+CD11b.sup.+) (FIG. 12B), and adaptive B cells
(CD19.sup.+IgM.sup.+) (FIG. 12C) from dissociated subcutaneous
tissue (as percent) 14 days post-subcutaneous (s.c.) alginate
sphere implantation. Interestingly, macrophage percentage is
slightly increased over that observed on intraperitoneally
implantated alginate spheres. Distant tissues, taken from the same
mice implanted s.c. with 500 .mu.m SLG20 alginate spheres, appear
no different than s.c. tissue taken from mock-implanted (saline
injected) mouse controls. Experiments were repeated twice.
[0026] FIGS. 13A-F depict that innate immune macrophages, and not
neutrophils, are necessary and sufficient for fibrosis of
biomaterial alginate spheres. SLG20 alginate 500 .mu.m diameter
spheres (0.35 ml total implant volume) were implanted into the
intraperitoneal space of different groups of wild type C57BL/6
mice, either treated with vehicle (WT) or targeted depletion agents
to eliminate macrophages and/or neutrophils over a 14 day
implantation and analyzed for degree of fibrosis upon retrieval.
(FIG. 13A) Dark field phase contrast images showing that fibrosis,
as compared to wild type (WT) control, was completely eliminated
following removal of innate immune macrophages (- M.PHI.) either
alone or in combination with neutrophil depletion (- Neutros &
M.PHI.). Neutrophil depletion alone (- Neutros) did not alleviate
fibrosis, and may have augmented sphere clumping. (FIG. 13B) Flow
analysis, using specific markers for responding host innate immune
macrophage, neutrophil, and adaptive B cells dissociated directly
from spheres (as percent composition) 14 days post-intraperitoneal
(i.p.) implantation, illustrating specificity of depletions. Shown
to be recruited downstream of macrophages, adaptive B cells also
decreased upon macrophage depletions. (FIG. 13C) Flow analysis for
host innate immune macrophage, neutrophil, and adaptive B cells
dissociated directly from spheres (normalized to cell counts from
100 .mu.l material) 14 days post-intraperitoneal (i.p.)
implantation, showing absolute cell presence, and the lack thereof
on spheres in mice which received macrophage depletion treatments.
NanoString analysis for expression of all known cytokine and
cytokine receptors (see FIG. 16A-C) for complete data set,
excerpted based on response here) for macrophage-specific factors
(by depletion and cell sorting) also inhibited by the CSF1R
inhibitor GW2580 (FIG. 13D), for macrophage-specific factors
(corroborated by cell sorting) removed by depletion but not
affected by CSF1R blockade, suggesting altered macrophage
polarization/phenotype and residual function (FIG. 13E), and for
macrophage-associated factors (by depletion, but NOT cell sorting)
also inhibited by the CSF1R inhibitor GW2580 (FIG. 13F), analyzed
from RNA extracts at 14 days post-implant, presented on a base 2
logarithmic scale. Since these factors were removed by macrophage
depletion, but not expressed by sorted macrophages
(decreased/diluted green expression profile as compared to mock
controls), they likely belong to cells that are recruited by
macrophages downstream in the fibrotic cascade (ie, CD19, B cell
marker and fibrosis protein collagen 1a1, Col1a1). FACS statistical
analysis: one-way ANOVA with Bonferroni multiple comparison
correction ***: p<0.0001, vs WT. ns=not significantly different.
N=5 mice/group. Experiments repeated at least 3 times.
[0027] FIGS. 14A-D depict a complete panel of phase contrast images
for targeted, serial innate immune depletions (as shown in FIG.
13). Images of retrieved 500 .mu.m diameter SLG20 spheres following
implantation into the intraperitoneal space of wildtype C57BL/6
mice treated with either saline vehicle (Veh) (FIG. 14A),
neutrophil depleting Gr1 antibody (- N) (FIG. 14B), macrophage
depleting clodrosome (- M.PHI.) (FIG. 14C), and both neutrophil and
macrophage depletion agents (- M.PHI. & N) (FIG. 14D).
Fibrosis, as compared to vehicle-treated wildtype (WT) controls,
was not decreased with neutrophil depletion (--N), increased when T
cells were also removed (TB KO), and gone completely with
additional M.PHI. dysfunction (Rag2/.gamma.). Images obtained from
all spheres retrieved from individual mice (n=5/group). The same
material volume of hydrogel spheres was implanted into each mouse
in all cases. Experiments were repeated twice.
[0028] FIG. 15A-C depict flow analysis plots, comparing wildtype
versus targeted innate immune depletion responses to implantation
of alginate. Flow analysis, using specific markers for responding
host innate immune macrophage (CD68.sup.+CD11b.sup.+) (FIG. 15A),
neutrophil (Ly6g/Gr1.sup.+CD11b.sup.+) (FIG. 15B), and adaptive B
cells (CD19.sup.+IgM.sup.+) (FIG. 15C) from cells dissociated from
fibrosed or clean tissue/spheres as well as spleens (as percent
composition) taken 14 days post-intraperitoneal (i.p.)
implantation, from wildtype C57BL/6 mice treated with either saline
vehicle or macrophage-depleting clodrosome, corresponding to the
fibrosis images shown in FIG. 12A-C. Interestingly, M.PHI.
depletion by clodrosome was specific, leaving neutrophil responses
intact. B cells were also decreased in M.PHI. depleted mice,
suggesting that macrophages are responsible for their recruitment.
These results combined with fibrosis images and data from FIG. 8
and FIG. 12A-C suggest that neutrophils alone are not capable of
fibrosing biomaterial alginate. Cell population percentages in the
spleen (global immune reservoir) were unaffected by i.p. injected
clodrosome treatment. Experiments were repeated twice.
[0029] FIG. 16A-C depict flow analysis plots, showing specificity
of serial innate immune depletions. Specific markers used are for
responding host innate immune macrophage (CD68.sup.+CD11b.sup.+)
(FIG. 16A), neutrophil (Ly6g/Gr1.sup.+CD11b.sup.+) (FIG. 16B), and
adaptive B cells (CD19.sup.+IgM.sup.+) (FIG. 16C) from cells
dissociated from fibrosed or clean tissue/spheres (as percent
composition) taken 14 days post-intraperitoneal (i.p.)
implantation, from wildtype C57BL/6 mice treated with either saline
(Vehicle, left column), neutrophil depleting Gr1 antibody (- N,
middle column), or both macrophage and neutrophil depletion agents
(- M.PHI. & N, right column), corresponding to the fibrosis
images shown in FIG. 12A-C. All depletion agents proved to be
population specific. Furthermore, neutrophil depletion neither
affected macrophage nor B cell presence on fibrosed spheres, again
suggesting their non-importance in a macrophage and B cell driven
fibrotic response. Related, B cells were once again decreased in
M.PHI. depleted mice. A CD11b.sup.loGr1.sup.lo/-CD68.sup.-
population, likely repopulating monocytes, was also apparent in
M.PHI. depleted mice. These results combined with fibrosis images
and data from FIG. 8 and FIG. 12A-C suggest that neutrophils alone
are not capable of fibrosing biomaterial alginate, nor are they
required. Experiments were repeated twice.
[0030] FIG. 17 depicts a complete NanoString analysis for
identification of inflammation and immune population-specific
factors. Expression of all known mouse (host) cytokine and cytokine
receptors, corresponding to sorted truncated heatmaps in FIGS.
13D-F for macrophage-specific or associated (downstream) factors,
based on removal by depletion and corroborated by cell sorting.
Subsets not affected by CSF1R blockade suggest altered macrophage
polarization/phenotype and residual function (corroborated by FIG.
17. All samples were analyzed from RNA extracts at 14 days
post-implant from each treatment group, presented on a base 2
logarithmic scale. White, within 2 standard deviations of the mean
background of the assay.
[0031] FIG. 18A-D depicts elucidation of upstream inflammation
induced by implanted biomaterial alginate, and peripheral
macrophage function spared by CSF1R inhibition. (FIG. 18A)
NanoString analysis heatmap was enriched from expression profiling
of all known cytokine and cytokine receptors for all C57BL/6
wildtype (unperturbed and perturbed) and knockout models for mock
and alginate material implanted treatment groups (see FIG. 18) for
factors not associated with any immune population (not affected
upon perturbations or CSF1R blockade), therefore likely associated
with an upstream inflammation response. (FIG. 18B) Interestingly,
some factors not associated with the removal of individual innate
or adaptive immune populations, were found to be decreased or
eliminated by CSF1R inhibition. These factors are likely induced
inflammation response genes that are being negated by peripheral
intact macrophage immune functions (ie., wound healing), implicated
by a subset of macrophage-specific factors, removed by macrophage
population depletion, but unaffected by CSF1R blockade (FIG. 13E),
suggesting altered macrophage polarization/phenotype and residual
preserved function. N=4 per treatment group. White boxes indicate
values within 2 standard deviations of the background noise of the
assay, indicating that they are statistically not detectable. (FIG.
18C) CXCL10 ELISA quantification for protein lysates derived from
various wildtype and knockout model fibrosed or clean alginate
spheres and tissue and retrieved at 14 days. CXCL10, an
immune-mobilizing chemokine presented on the surface during periods
of stress/damage, is increased upon B cell removal and even moreso
upon macrophage depletion during implantation of C57BL/6 mice with
500 .mu.m alginate spheres, suggesting that these cell populations
are not just involved in fibrosis initiation but also inflamed
tissue repair. Interestingly, the CSF1R inhibitor GW2580 (GW)
resulted in increased reduction of CXCL10 back to wildtype levels,
suggesting that residual would healing functions are intact in
CSF1R-inhibited monocyte/macrophages. CXCL10 protein levels also
matched RNA expression changes in panel (b). (FIG. 18D) VEGF
Luminex protein quantification for the same protein lysates used in
(c) wildtype (control and perturbed) and knockout models, as in
(c). VEGF, important for neovascularization and wound healing, is
significantly reduced in both macrophage depletion groups, but
returned to normal and not significantly different (ns) than levels
observed in fully functional wildtype (WT) immune competent and
implanted control mice. N=5/group. Luminex and ELISA run once
each.
[0032] FIGS. 19A-G depict that CSF1R-dependent macrophages recruit
fibrosis-potentiating adaptive B cells via chemokine CXCL13. CSF1R
inhibition prevents the entire immune response to implanted
biomaterials. Dark field phase contrast images showing that
fibrosis, as compared to wild type (WT) control, was partially
eliminated by CXCL13 neutralization, and completely eliminated with
continuous CSF1R inhibition (160 mg/kg BW GW2580 s.c.) over a 14
day implant period (FIG. 19A). Fibrosis, was reduced the same
extent as that of the B cell knockout (B KO) shown in FIG. 8a.
(FIG. 19B) Flow analysis for responding host innate immune
macrophage, neutrophil, and adaptive B cells dissociated directly
from spheres (normalized to cell counts from 100 .mu.l material) 14
days post-intraperitoneal (i.p.) implant, showing partial loss of
cell presence with CXCL13 neutralization, and complete loss in WT
mice treated with the CSF1R inhibitor GW2580. (FIG. 19C)
Brightfield images showing that fibrosis, as compared to vehicle
controls, was completely eliminated by both macrophage depletion (-
M.PHI.) and CSF1R inhibition (inh.). (FIG. 19D) Flow analysis for
responding host innate immune macrophages and neutrophils
dissociated directly from spheres (normalized to cell counts from
100 .mu.l of each material) 14 days post-intraperitoneal (i.p.)
implantation, showing the loss of immune adhesion with loss of
fibrosis due to either macrophage depletion (- M.PHI.) or CSF1R
inhibition (inh.). (FIG. 19E) NanoString analysis for expression of
all known cytokine and cytokine receptors (see FIG. 22A-C for
complete data set, excerpted here), showing similar unique factors
increased across all material (hydrogel alginate, ceramic glass,
and polymer polystyrene (PS)) groups. (FIG. 19F) Confocal staining
showing DAPI (cellular nuclei), innate immune macrophage marker
CD68 (green), adaptive immune B cell marker CD19 (magenta), alpha
smooth muscle actin (.alpha.SMactin, myofibroblasts, red), overlay,
and brightfield making up the fibrosis on 500 .mu.m alginate
spheres, showing that CXCL13 neutralization resulted in loss of B
cell recruitment. (FIG. 19G) qPCR based expression analysis of
fibrotic marker .alpha.-SMactin directly on retrieved spheres from
WT, B KO, and CXCL13 neutralized mice, plotted normalized to
relative expression levels on spheres from WT mice. qPCR and FACS
statistical analysis: one-way ANOVA with Bonferroni multiple
comparison correction ***: p<0.0001, vs Vehicle. N=5 mice/group.
Experiments repeated at least 2 times.
[0033] FIG. 20A-B depict CSF1R inhibition prevents fibrosis of IP
implanted alginate spheres (cont.). Complete phase contrast
fibrosis images for wildtype C57BL/6 mice treated with either
saline vehicle (Veh) (FIG. 20A) or CSF1R inhibitor GW2580 (FIG.
20B), corresponding to the images in FIG. 19D. Images obtained from
all spheres retrieved from individual mice (n=5/group). The same
material volume of hydrogel spheres was implanted into each mouse
in all cases. Experiments were repeated twice.
[0034] FIGS. 21A-C depict that macrophage elimination or,
minimally, CSF1R inhibition prevents fibrosis of IP implanted 500
.mu.m glass ceramic spheres. Complete brightfield fibrosis images
for wildtype C57BL/6 mice treated with either saline vehicle (Veh)
(FIG. 21A), macrophage depletion agent clodrosomes (FIG. 21B), or
CSF1R inhibitor GW2580 (FIG. 21C), corresponding to the images in
FIG. 19F. Images obtained from all spheres retrieved from
individual mice (n=4/group). The same material volume of hydrogel
spheres was implanted into each mouse in all cases. Experiments
were repeated twice.
[0035] FIG. 22A-C depict that macrophage elimination or, minimally,
CSF1R inhibition prevents fibrosis of IP implanted 500 .mu.m
polystyrene polymer spheres. Complete brightfield fibrosis images
for wildtype C57BL/6 mice treated with either saline vehicle (Veh)
(FIG. 22A), macrophage depletion agent clodrosomes (FIG. 22B), or
CSF1R inhibitor GW2580 (FIG. 22C), corresponding to the images in
FIG. 19F. Images obtained from all spheres retrieved from
individual mice (n=4/group). The same material volume of hydrogel
spheres was implanted into each mouse in all cases. Experiments
were repeated twice.
[0036] FIG. 23 depicts a NanoString analysis for complete cytokine
signaling common to host responses across multiple material
classes. Expression of all known mouse (host) cytokine and cytokine
receptors to identify common inflammation and immune signaling
across implanted hydrogel alginate, ceramic glass, and polymer
polystyrene spheres, all retrieved 14 days after IP implantation
into C57BL/6 mice. N=4/group. Presented on a base 2 logarithmic
scale. Corresponds to excerpted heat map in FIG. 19G. Green box in
Mock column, within 2 standard deviations of the mean background of
the assay.
[0037] FIGS. 24A-D depict FACS plots and phase contrast images
showing effects of CXCL13 antibody neutralization on B cell
recruitment and downstream fibrosis. Flow analysis, using specific
markers for responding host adaptive B cells (CD19.sup.+IgM.sup.+)
from cells dissociated from fibrosed tissue/spheres (as percent
composition) taken 14 days post-intraperitoneal (i.p.)
implantation, from wildtype C57BL/6 mice treated with either saline
(Vehicle) (FIG. 24A) or CXCL13-neutralizing antibody (- CXCL13)
(FIG. 24B). Complete phase contrast fibrosis images for wildtype
C57BL/6 mice treated with either saline vehicle (Veh) (FIG. 24C) or
CXCL13-neutralizing antibody (- CXCL13) (FIG. 24D), corresponding
to the images in FIG. 19. Images obtained from all spheres
retrieved from individual mice (n=5/group). The same material
volume of hydrogel spheres was implanted into each mouse in all
cases. Experiments were repeated twice.
[0038] FIGS. 25A-E. Essential fibrotic cascade players are also
increased in non-human primates. 0.5 mm-sized spheres of SLG20
hydrogels were implanted either intraperitoneally or subcutaneously
in the dorsal region of cynomolgus macaque monkeys and retrieved by
laparoscopy-guided tissue excision (control mock or implanted and
sphere-embedded omentum fat tissue, (FIG. 25A)) or biopsy punch
after 28 days (Veiseh O, Doloff J C, Ma M, Vegas A J, Tam H H,
Bader A R, et al. Size- and shape-dependent foreign body immune
response to materials implanted in rodents and non-human primates.
Nature materials 2015, 14(6): 643-651.). (FIG. 25B) H&E and
Masson's Trichrome stained histological sections of excised IP
omentum tissue at 28 days for mock and implanted groups, showing
clean non-fibrosed fat-laden (Mock) or heavily collagen-deposited
and sphere-embedded omental tissue (Implanted). (FIG. 25C) Despite
a limited amount of functional antibodies for the cynomolgus
species, we performed confocal staining showing DAPI (cellular
nuclei), innate immune macrophage marker CD68 (green), and
fibrosis-associated activated myofibroblast alpha smooth muscle
actin (.alpha.SMactin, myofibroblasts, red), overlaid together,
showing cellular infiltration around and fibrosis deposition on an
embedded 500 .mu.m alginate sphere (20.times. magnification). White
scale bars: both 200 um for each respective image. (FIG. 25D) Flow
analysis showing similar host innate immune macrophage
(CD68.sup.+CD11b.sup.+, top right quadrants) and neutrophil/myeloid
(CD68.sup.-CD11b.sup.+, bottom right quadrants) cells across
C57BL/6 mice and cynomolgus macaque monkeys, dissociated directly
from fibrosed spheres and adjacent fibrosed omentum tissue (as
percent composition) 28 days post-intraperitoneal (i.p.)
implantation. While the prominence of CD11b seems to be inverted
between macrophages and neutrophils in C57BL/6 mice vs cynomolgus
monkeys, population response percentages are similar 28 days
post-IP implantation. (FIG. 25E) NanoString analysis for immune
markers and cytokines, originally identified in C57BL/6 mice (Note:
CD66b is used here as a neutrophil marker, as Ly6g/Gr1 does not
exist in NHPs or humans). Significant increases are observed for
macrophage markers, as well as CSF1R and CXCL13 in both peritoneal
and subcutaneous implant sites, as compared to mock
(saline-injected) controls (there was no difference between SC and
IP mock controls). N=2 for IP implanted groups; N=4 for
subcutaneous (s.c.) treatment groups. These experiments were
performed once for s.c. and twice for IP delivery.
[0039] FIGS. 26A-F depict additional primate histology and
immunofluorescence panels showing similar foreign body responses.
(FIG. 26A) H&E (a) and Masson's Trichrome (FIG. 26B) (b)
stained histological sections of excised IP omentum tissue at 28
days for mock and implanted groups, showing clean non-fibrosed
fat-laden (Mock) or heavily collagen-deposited and sphere-embedded
omental tissue (Implanted). Corresponds to panels in FIG. 25.
Magnifications: 10 and 40.times.. Additional confocal images for
immunostained sections from implant alginate sphere embedded
omental tissue excised at 28 days from cynomolgus monkeys. Shown:
DAPI (cellular nuclei), innate immune macrophage marker CD68
(green), and alpha smooth muscle actin (.alpha.SMactin,
myofibroblasts, red; also refer to blue arrows)), overlayed
together, showing cellular infiltration around and fibrosis
deposition on embedded 500 .mu.m alginate spheres. 5.times. (c)
(FIG. 26C) and 20.times. (d) (FIG. 26D) magnifications. (FIG. 26E)
e) Single red channel images (corresponding to the same images in
(d)) showing material-bordering and more distant punctate
.alpha.SMactin-staining myofibroblasts (see blue arrows for
examples). It should also be noted, to not be confused, that
pericytes covering larger circular blood vessels are also positive
for .alpha.SMactin. (FIG. 26F) f) Single channels for DAPI
(cellular nuclei), colony stimulating factor-1 receptor (CSF1R)
(green), and brightfield views showing high CSF1R staining on both
fused foreign body giants cells (FBGCs) and individual macrophages
around embedded 500 .mu.m alginate spheres. 20.times.
magnification. n=2 NHPs/group. These experiments were performed
once for SC and twice for IP delivery.
[0040] FIGS. 27A-G depict CSF1R inhibition leaves many macrophage
functions intact. (FIG. 27A) a) Skin incisions (all 1.5 cm in
length, rulers are visible on left in all images) were made on day
0, and then wound clipped shut for both vehicle and daily
GW2580-treated C57BL/6 mice (top left). Wound clips were removed
and then replaced each imaging day up until day 7, after which
clips were left off completely. By day 7, upon stretching the skin
apart, incision sites on GW2580-treated mice were shut and not
pulling apart (top right, red inset). By day 14, there appeared to
be very little scarring in both vehicle and GW2580-treated groups
(bottom middle). (FIG. 27B) b) After both 4 and 14 days, IP immune
cells were taken by peritoneal lavage and analyzed by FACS for
innate immune macrophage phenotype (CD68 & CD11b staining). As
expected, at both time points, the mature tissue-resident
macrophage phenotype observed in vehicle-treated mice was shifted
(decreased CD68 & CD11b intensities) following daily GW2580
treatment. (FIG. 27C) c) Despite a phenotype shift, overall cell
numbers in the peritoneal exudate were unchanged across untreated,
vehicle-treated, or GW2580-treated groups. (FIG. 27 D) d)
Confirming visibly healing skin incisions, histological assessment
(H&E and Masson's Trichrome) show no significant (ns)
differences by width and depth measurements (FIG. 27E) (e) in wound
resolution and healing potential between vehicle or GW2580
treatment groups, by day 14 post-incision; scale bar: 400 .mu.m.
(FIG. 27F) f) Peritoneal exudate macrophages isolated by IP lavage
from (n=5) mice in each treatment group were immediately plated and
incubated with fluorospheres for 90 minutes to determine phagocytic
activity. Again, no significant differences were observed between
macrophages isolated from vehicle and GW2580-treated mice. (FIG.
27G) g) Protein lysates were prepared from alginate spheres
retrieved 14 days after IP implantation, and incubation with two
different reactive oxygen specie (ROS) substrate solutions. Once
again, no differences in ROS activity were observed between
untreated, vehicle-treated, and GW2580-treated mice. n=5 mice for
all assays. Error bars, mean+/-SE. Run 1-2 times, depending on the
assay.
DETAILED DESCRIPTION OF THE INVENTION
[0041] A description of example embodiments of the invention
follows below; additional description is found in International
application Ser. No. ______, entitled "Compositions Of Crystallized
Hydrophobic Compounds And Methods Of Making And Using Same" (HBSR
Attorney Docket No. 0050.2293-001), filed concurrently with the
instant application on Apr. 4, 2017, incorporated herein by
reference in its entirety.
[0042] Regardless of the specific stimulus that initiates device
rejection, aspects of the biology involved in the ensuing immune
response have been characterized. Macrophages have remarkable
plasticity, responding to numerous signals (Gordon S. Alternative
activation of macrophages. Nat Rev Immunol 2003, 3(1): 23-35), and
are a key component of material recognition, actively adhering to
the surface of foreign objects (Anderson J M, Rodriguez A, Chang D
T. Foreign body reaction to biomaterials. Semin Immunol 2008,
20(2): 86-100; Kenneth Ward W. A Review of the Foreign-body
Response to Subcutaneously-implanted Devices: The Role of
Macrophages and Cytokines in Biofouling and Fibrosis. . J Diabetes
Sci Technol Online 2008, 2: 768-777; Grainger D W. All charged up
about implanted biomaterials. Nat Biotechnol 2013, 31(6): 507-509;
Sussman E M, Halpin M C, Muster J, Moon R T, Ratner B D. Porous
implants modulate healing and induce shifts in local macrophage
polarization in the foreign body reaction. Annals of biomedical
engineering 2014, 42(7): 1508-1516.). They are increased locally
throughout the implant site within days and may persist at the
material surface even for the life of the implant (Anderson J M,
Rodriguez A, Chang D T. Foreign body reaction to biomaterials.
Semin Immunol 2008, 20(2): 86-100; Kenneth Ward W. A Review of the
Foreign-body Response to Subcutaneously-implanted Devices: The Role
of Macrophages and Cytokines in Biofouling and Fibrosis. J Diabetes
Sci Technol Online 2008, 2: 768-777; Sussman E M, Halpin M C,
Muster J, Moon R T, Ratner B D. Porous implants modulate healing
and induce shifts in local macrophage polarization in the foreign
body reaction. Annals of biomedical engineering 2014, 42(7):
1508-1516.). Devices too large to be cleared by phagocytosis
instead initiate macrophage fusion into foreign-body giant cells
(Anderson J M, Rodriguez A, Chang D T. Foreign body reaction to
biomaterials. Semin Immunol 2008, 20(2): 86-100; Kyriakides T R,
Foster M J, Keeney G E, Tsai A, Giachelli C M, Clark-Lewis I, et
al. The CC chemokine ligand, CCL2/MCP1, participates in macrophage
fusion and foreign body giant cell formation. Am J Pathol 2004,
165(6): 2157-2166), which recruit fibroblasts responsible for final
fibrous collagen and matrix protein deposition (Anderson J M,
Rodriguez A, Chang D T. Foreign body reaction to biomaterials.
Semin Immunol 2008, 20(2): 86-100; Kenneth Ward W. A Review of the
Foreign-body Response to Subcutaneously-implanted Devices: The Role
of Macrophages and Cytokines in Biofouling and Fibrosis. . J
Diabetes Sci Technol Online 2008, 2: 768-777; Rodriguez A, Meyerson
H, Anderson J M. Quantitative in vivo cytokine analysis at
synthetic biomaterial implant sites. Journal of biomedical
materials research Part A 2009, 89(1): 152-159.). Ultimately, this
fate is the same for many implanted materials of both natural and
synthetic origin, including polysaccharides, polymers, ceramics
such as silica and alumina, rubber, Teflon, and metals such as
gold, stainless steel and titanium (Kenneth Ward W. A Review of the
Foreign-body Response to Subcutaneously-implanted Devices: The Role
of Macrophages and Cytokines in Biofouling and Fibrosis. J Diabetes
Sci Technol Online 2008, 2: 768-777.).
[0043] One natural polysaccharide, alginate, is a multipurpose
biomaterial that has been evaluated for use in numerous biomedical
applications including biosensors, tissue regeneration, cell
encapsulation, and drug delivery (Kearney C J, Mooney D J.
Macroscale delivery systems for molecular and cellular payloads.
Nature materials 2013, 12(11): 1004-1017; Lee K Y, Mooney D J.
Alginate: properties and biomedical applications. Progress in
polymer science 2012, 37(1): 106-126.). While non-biodegradable
alginate capsules are used to immunoisolate transplanted islets for
type 1 diabetes therapy, host immune and fibrosis responses
directed to the encapsulating biomaterial results in device failure
(de Vos P, Faas M M, Strand B, Calafiore R. Alginate-based
microcapsules for immunoisolation of pancreatic islets.
Biomaterials 2006, 27(32): 5603-5617; Jacobs-Tulleneers-Thevissen
D, Chintinne M, Ling Z, Gillard P, Schoonjans L, Delvaux G, et al.
Sustained function of alginate-encapsulated human islet cell
implants in the peritoneal cavity of mice leading to a pilot study
in a type 1 diabetic patient. Diabetologia 2013, 56(7): 1605-1614;
Tuch B E, Keogh G W, Williams L J, Wu W, Foster J L, Vaithilingam
V, et al. Safety and viability of microencapsulated human islets
transplanted into diabetic humans. Diabetes care 2009, 32(10):
1887-1889; Weir G C. Islet encapsulation: advances and obstacles.
Diabetologia 2013, 56(7): 1458-1461.). The combination of biologic
and biomaterial complicates deconvoluting host immune responses,
but empty alginate microspheres alone elicit rejection (Dang T T,
Thai A V, Cohen J, Slosberg J E, Siniakowicz K, Doloff J C, et al.
Enhanced function of immuno-isolated islets in diabetes therapy by
co-encapsulation with an anti-inflammatory drug. Biomaterials 2013,
34(23): 5792-5801; Robitaille R, Dusseault J, Henley N, Desbiens K,
Labrecque N, Halle J P. Inflammatory response to peritoneal
implantation of alginate-poly-L-lysine microcapsules. Biomaterials
2005, 26(19): 4119-4127), thus immune attack of biomaterial
alginate occurs independently of an encapsulated tissue of foreign
origin. This response has been a fundamental barrier to translation
of encapsulated islets for several decades (Tuch B E, Keogh G W,
Williams L J, Wu W, Foster J L, Vaithilingam V, et al. Safety and
viability of microencapsulated human islets transplanted into
diabetic humans. Diabetes care 2009, 32(10): 1887-1889; Weir G C.
Islet encapsulation: advances and obstacles. Diabetologia 2013,
56(7): 1458-1461.).
[0044] Overcoming the rejection of implanted biomaterial devices
could allow for a range of medical advancements (Harding J L,
Reynolds M M. Combating medical device fouling. Trends in
biotechnology 2014, 32(3): 140-146; Langer R. Perspectives and
challenges in tissue engineering and regenerative medicine.
Advanced materials 2009, 21(32-33): 3235-3236.). Current approaches
for immune suppression or management of long-term biomedical device
implantation often involve broad-spectrum anti-inflammatories (Rhen
T, Cidlowski J A. Antiinflammatory action of glucocorticoids--new
mechanisms for old drugs. New England Journal of Medicine 2005,
353(16): 1711.). The major immunosuppressive agents used as
standard care for implantation procedures are rapamycin
(sirolimus), tacrolimus, everolimus, cyclosporine, and
corticosteroids, as well as azathioprine, mycophenolate mofetil
(MMF), mycophenolate sodium (Myfortic), and belatacept for
transplantation (Denton M D, Magee C C, Sayegh M H.
Immunosuppressive strategies in transplantation. Lancet 1999,
353(9158): 1083-1091; Halloran P F. Immunosuppressive drugs for
kidney transplantation. N Engl J Med 2004, 351(26): 2715-2729; Khan
W, Muntimadugu E, Jaffe M, Domb A J. Implantable Medical Devices.
Focal Controlled Drug Delivery. Springer US, 2014, pp 33-59; Wong
W, Venetz J P, Tolkoff-Rubin N, Pascual M. 2005 immunosuppressive
strategies in kidney transplantation: which role for the
calcineurin inhibitors? Transplantation 2005, 80(3): 289-296.).
However, many anti-inflammatory drugs, including rapamycin, FK-506
(tacrolimus), cyclosporine, and numerous glucocorticosteroids, are
not specific to individual immune populations, having multiple
targets and differential effects in vivo (Rhen T, Cidlowski J A.
Antiinflammatory action of glucocorticoids--new mechanisms for old
drugs. New England Journal of Medicine 2005, 353(16): 1711; Attur M
G, Patel R, Thakker G, Vyas P, Levartovsky D, Patel P, et al.
Differential anti-inflammatory effects of immunosuppressive drugs:
cyclosporin, rapamycin and FK-506 on inducible nitric oxide
synthase, nitric oxide, cyclooxygenase-2 and PGE 2 production.
Inflammation Research 2000, 49(1): 20-26.). Another anti-oxidant
and immunomodulatory agent, curcumin, has also been shown to
inhibit numerous immune cell populations (Dang T T, Thai A V, Cohen
J, Slosberg J E, Siniakowicz K, Doloff J C, et al. Enhanced
function of immuno-isolated islets in diabetes therapy by
co-encapsulation with an anti-inflammatory drug. Biomaterials 2013,
34(23): 5792-5801), decrease macrophage and natural killer cell
nitric oxide synthesis (Bhaumik S, Jyothi M D, Khar A. Differential
modulation of nitric oxide production by curcumin in host
macrophages and NK cells. FEBS Lett 2000, 483(1): 78-82), inhibit
dendritic cell stimulation and cytokine production (Kim G Y, Kim K
H, Lee S H, Yoon M S, Lee H J, Moon D O, et al. Curcumin inhibits
immunostimulatory function of dendritic cells: MAPKs and
translocation of NF-kappa B as potential targets. J Immunol 2005,
174(12): 8116-8124), and decrease T cell proliferation (Kim W, Fan
Y Y, Smith R, Patil B, Jayaprakasha G K, McMurray D N, et al.
Dietary curcumin and limonin suppress CD4+ T-cell proliferation and
interleukin-2 production in mice. J Nutr 2009, 139(5): 1042-1048),
thereby leading to functional decreases in multiple innate and
adaptive immune populations. In general, broad-spectrum immune
inhibition results in unwanted side effects. As such, improved drug
targets and corresponding inhibitory compounds, capable of specific
immune population inhibition or modulation, need to be identified.
However, to do so, deeper understanding of the host immune-mediated
foreign body rejection response must be achieved.
[0045] Foreign body responses are one of the largest impediments to
biomedical device success. Up until now, the reasons why they occur
are poorly understood. To identify key cell and cytokine targets,
we performed in-depth systems analysis of innate and adaptive
immune systems. While innate macrophages were indispensable to the
fibrotic cascade, contrary to established belief, neutrophils and
complement were not. Macrophages, via CXCL13, also led to
downstream recruitment of B cells, which further potentiated
fibrosis. Previously unimplicated, CSF1R was significantly
increased upon implantation of multiple biomaterial classes:
ceramic, polymer, and hydrogel. Its inhibition, like macrophage
depletion, led to complete loss of fibrosis. CSF1R blockade,
however, spared other macrophage function such as wound healing,
establishing a more selective method of fibrosis inhibition. Immune
cell and cytokine targets were additionally confirmed in non-human
primates.
[0046] Here, we sought to further examine the role of innate and
adaptive immunity on biomaterial biocompatibility in vivo. First,
we focused on interrogating immune and fibrosis responses to
implanted alginate hydrogels, then extended this characterization
to include other materials as well. It is important to note that
the fibrosis of alginate microspheres in rodents has been shown to
be strain dependent (King A, Sandler S, Andersson A. The effect of
host factors and capsule composition on the cellular overgrowth on
implanted alginate capsules. Journal of biomedical materials
research 2001, 57(3): 374-383; Manoury B, Caulet-Maugendre S,
Guenon I, Lagente V, Boichot E. TIMP-1 is a key factor of
fibrogenic response to bleomycin in mouse lung. International
journal of immunopathology and pharmacology 2006, 19(3): 471-487.).
Implantation of alginate into the intraperitoneal (IP) space of
immune compliant BALB/c mice yields little to no fibrosis (King A,
Sandler S, Andersson A. The effect of host factors and capsule
composition on the cellular overgrowth on implanted alginate
capsules. Journal of biomedical materials research 2001, 57(3):
374-383; Manoury B, Caulet-Maugendre S, Guenon I, Lagente V,
Boichot E. TIMP-1 is a key factor of fibrogenic response to
bleomycin in mouse lung. International journal of immunopathology
and pharmacology 2006, 19(3): 471-487), whereas alginate retrieved
from C57BL/6 mice, which have more aggressive innate immunity, is
densely covered with fibrous overgrowth (King A, Sandler S,
Andersson A. The effect of host factors and capsule composition on
the cellular overgrowth on implanted alginate capsules. Journal of
biomedical materials research 2001, 57(3): 374-383), mimicking the
foreign body response observed in humans and non-human primates
(NHPs). Therefore, we sought to further elucidate the immunity
involved in the foreign body response in various C57BL/6 strains,
with additional confirmation in cynomolgus monkeys, across which
our recent work has translated successfully (Veiseh O, Doloff J C,
Ma M, Vegas A J, Tam H H, Bader A R, et al. Size- and
shape-dependent foreign body immune response to materials implanted
in rodents and non-human primates. Nature materials 2015, 14(6):
643-651; Vegas A J, Veiseh O, Doloff J C, Ma M, Tam H H, Bratlie K,
et al. Combinatorial hydrogel library enables identification of
materials that mitigate the foreign body response in primates. Nat
Biotechnol 2016.).
[0047] In one example embodiment, the method is a method of
preventing or reducing a fibrotic response to a foreign body or to
an implanted material in a patient comprising administering to the
patient an effective amount of a CSF1R inhibitor. In some
embodiments, the foreign body and/or the implanted material is of
natural origin (e.g., natural). In some embodiments, it is of
synthetic origin (e.g., synthetic). In some embodiments, the
foreign body and/or the implanted material or device does not
comprise biologics, cells and/or tissues.
[0048] In one embodiment, the method is a method of preventing or
reducing a fibrotic response to a foreign body or an implanted
material in a patient, the method comprising administering to the
patient an effective amount of a CSF1R inhibitor selected from the
group consisting of GW2580, Ki20227, BLZ945, Quizartinib, AC708,
4-(3,4-Dimethylanilino)-7-(4-(methylsulfonyl)phenyl)quinoline-3-carboxami-
de,
(4-cyano-N-(2-cyclohexenyl-4-(1-(2-(dimethylamino)acetyl)piperidin-4-y-
l)phenyl)-1H-imidazole-2-carboxamide), Linifanib, ARRY-382,
Pexidartinib, a 2'-aminoanilide, a 3-amido-4-anilinocinniline, an
indoline-2-one, a 2-(alpha-methylbenzylamino)-pyrazine, an
arylamide, a 3,4,6-substituted 2-quinolone, a
pyrido[2,3-d]pyrimidin-5-one, a 3-amido-4-anilinoquinoline, a
pyridyl bisamide, and a thiazolyl bisamide, a
1,4-disubstituted-pyrrolo-[3,2-c]-pyridine, a substituted
diphenylurea, a 5'-pyrimidine-2,4-diamine, CYC10268, AZ683,
anilinoquinazoline, OSI-930, DCC-2618, DCC-3014, JNJ-40346527,
Sunitinib, Lestaurtinib, Midostaurin, Tandutinib, Sorafenib, and
Ponatinib.
[0049] The methods described herein pertain to preventing or
reducing a fibrotic response to a substance in a subject (e.g., a
human, e.g., a patient). In some embodiments, the substance is a
foreign body or an implanted material in a patient. Generally, a
foreign body refers to a substance that has entered the body, e.g.,
unintentionally, such as by ingestion (e.g., due to debris in
drinking water and/or food) or by inhalation (e.g., due to airborne
debris). In some instances, a foreign body cannot be extruded or
expelled by the body, either by responding immune cells or through
normal circulation and excretion. Such materials, including
nano-scale materials, can lead to downstream fibrosis and scar
tissue deposition in major organs. Implanted materials generally
refer to materials that have been introduced into the body, e.g.,
intentionally (e.g., by a surgeon or physician).
[0050] In some embodiments, the devices, materials and/or foreign
bodies can be organic or inorganic. In some embodiments, they are
synthetic.
[0051] The implanted medical devices can be implanted into a
patient in a variety of locations. For example, they can be
implanted intraperitoneally, subcutaneously, or intramuscularly.
The implanted medical device or material (e.g., the implanted
synthetic material) can be made from a variety of suitable
materials, such as polymers, ceramics, hydrogels, rubbers, metals,
and glasses. The implanted synthetic material can comprise a
plurality of materials or be made from a combination of materials,
such as such as polymers, ceramics, hydrogels, rubbers, metals, and
glasses. The polymer can be a natural polymer, which refers to
polymers that can be found in nature regardless of the method by
which the polymer was actually made. Examples of natural polymers
include polysaccharides, such as alginate or chitosan. For example,
alginate can be purified from biological sources (e.g., seaweed).
Alginate can also be synthetically derived. The polymer can also be
a synthetic polymer, which refers to polymer that are not typically
found in nature. Examples of synthetic polymers include
polytetrafluoroethylene and polystyrene. Other examples of polymers
include polycaprolactone (PCL) and polydimethylsiloxane (PDMS).
[0052] In some embodiments, the implanted material is not from a
biological source. For example, in some embodiments, the material
does not comprise a biologic (e.g., an antibody or fragment
thereof), cells, tissues, protein or a combination thereof.
[0053] As used herein, the term "preventing" or "reducing," such as
in the context of preventing or reducing a fibrotic response,
refers to obtaining desired pharmacological and/or physiological
effect. The effect can be prophylactic or therapeutic, which
includes achieving, partially or substantially, one or more of the
following results: partially or totally reducing the extent of the
disease, disorder or syndrome; ameliorating or improving a clinical
symptom or indicator associated with the disorder; delaying,
inhibiting or decreasing the likelihood of the progression of the
disease, disorder or syndrome; or partially or totally delaying,
inhibiting or reducing the likelihood of the onset or development
of disease, disorder or syndrome.
[0054] "Effective amount" means that amount of active compound
agent that elicits the desired biological response in a subject.
Such response includes alleviation of the symptoms of the disease
or disorder being treated. The effective amount of a compound of
the invention in such a therapeutic method is from about 0.01
mg/kg/day to about 1000 mg/kg/day, from about 0.1 mg/kg/day to
about 300 mg/kg/day, from about 50 mg/kg/day to about 250
mg/kg/day, from about 100 mg/kg/day to 200 mg/kg/day, or from about
125 mg/kg/day to about 175 mg/kg/day.
[0055] "Pharmaceutically acceptable carrier" means compounds and
compositions that are of sufficient purity and quality for use in
the formulation of a composition of the invention and that, when
appropriately administered to an animal or human, do not produce an
adverse reaction.
[0056] In certain embodiments, the compositions of the invention
described herein are formulated for therapeutic (e.g.,
pharmaceutical) use with one or more pharmaceutically-acceptable
carriers or excipients. The term "pharmaceutically acceptable
carrier" means a non-toxic solvent, dispersant, excipient, adjuvant
or other material which is mixed with the active ingredient in
order to permit the formation of a pharmaceutical composition,
i.e., a dosage form capable of administration to the patient.
Generally, pharmaceutically-acceptable carriers or excipients may
be present in amounts having no substantial effect on the stability
and release rate profiles of the hydrophobic compound(s) in the
composition. Suitable excipients/carriers are well known in the
art, including those described in Gennaro et al., Remington's
Pharmaceutical Sciences (18th ed., Mack Publishing Company, 1990,
see especially Part 8: Pharmaceutical Preparations and their
Manufacture), which is incorporated herein by reference in its
entirety. The compositions of the invention formulated for
therapeutic use may be used as is, or may be used as a
pharmaceutically acceptable salt thereof. The term
"pharmaceutically acceptable salt" means either an acid addition
salt or a basic addition salt which is compatible with the
treatment of patients/subjects.
[0057] In some embodiments, exemplary inorganic acids which form
suitable salts include, but are not limited thereto, hydrochloric,
hydrobromic, sulfuric and phosphoric acid and acid metal salts such
as sodium monohydrogen orthophosphate and potassium hydrogen
sulfate. Illustrative organic acids which form suitable salts
include the mono-, di- and tricarboxylic acids. Illustrative of
such acids are, for example, acetic, glycolic, lactic, pyruvic,
malonic, succinic, glutaric, fumaric, malic, tartaric, citric,
ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic,
phenylacetic, cinnamic, salicylic, 2-phenoxybenzoic,
p-toluenesulfonic acid and other sulfonic acids such as
methanesulfonic acid and 2-hydroxyethanesulfonic acid. Either the
mono- or di-acid salts can be formed, and such salts can exist in
either a hydrated, solvated or substantially anhydrous form. In
general, the acid addition salts of these compounds are more
soluble in water and various hydrophilic organic solvents, and
generally demonstrate higher melting points in comparison to their
free base forms. Other non-pharmaceutically acceptable salts e.g.
oxalates may be used.
[0058] The compositions of the invention can be in a solid form or
liquid form. Typically, they are in dosage unit form, such as
tablet, powder, sachet, bead, pellet, osmotic dosage form, etc.,
but they may as well be in a liquid, cream or aerosol form for use
in various applications, i.e., parenteral, oral, buccal,
ophthalmic, nasal, dermal, rectal, and pulmonary routes. In one
embodiment, the compositions provided in the present invention are
encapsulated. Non limiting examples of materials used for
encapsulation of the composition of the current invention include
materials composed of ceramic, glass, metal, poly
lactic-co-glycolic acid (PLGA) co-polymer, polymer (e.g.,
polystyrene beads) and alginate hydrogels. In a particular
embodiment, the compositions provided in the present invention are
encapsulated in a biocompatible polymer (e.g., alginate
hydrogel).
[0059] The compositions of the present invention can be formulated
for different modes of administration, including, but not limited
to, parenteral, oral, buccal, ophthalmic, nasal, dermal, rectal,
and pulmonary routes. In one embodiment, the compositions are in an
oral delivery form, such as a tablet, capsule or osmotic dosage
form. In another embodiment, the compositions are in a form
suitable for administration by injection. In another embodiment,
the compositions are in a form suitable for administration by
implantation.
[0060] Compositions and compounds of the invention suitable for
parenteral injection include sterile solutions.
[0061] The dosage form containing the composition of the invention
contains an effective amount of the active ingredient necessary to
provide a therapeutic effect. The composition may contain from
about 5,000 mg to about 0.5 mg (preferably, from about 1,000 mg to
about 0.5 mg) of a compound of the invention or salt form thereof
and may be constituted into any form suitable for the selected mode
of administration. The composition may be administered about 1 to
about 5 times per day. Daily administration or post-periodic dosing
may be employed.
[0062] In some embodiments, the compounds may be administered
parenterally via injection. A parenteral formulation may consist of
the active ingredient dissolved in or mixed with an appropriate
inert liquid carrier. Acceptable liquid carriers usually comprise
aqueous solvents and other optional ingredients for aiding
solubility or preservation. Such aqueous solvents include sterile
water, Ringer's solution, or an isotonic aqueous saline solution.
Other optional ingredients include vegetable oils (such as peanut
oil, cottonseed oil, and sesame oil), and organic solvents (such as
solketal, glycerol, and formyl). A sterile, non-volatile oil may be
employed as a solvent or suspending agent. The parenteral
formulation is prepared by dissolving or suspending the active
ingredient in the liquid carrier whereby the final dosage unit
contains from 0.005 to 10% by weight of the active ingredient.
Other additives include preservatives, isotonizers, solubilizers,
stabilizers, and pain-soothing agents. Injectable suspensions may
also be prepared, in which case appropriate liquid carriers,
suspending agents and the like may be employed.
[0063] A number of compounds can be administered to a patient in
order prevent or reduce a fibrotic response to a foreign body or to
an implanted material. In general, the compounds inhibit CSF1R.
Examples of compounds include GW2580; Ki20227;
4-(3,4-Dimethylanilino)-7-(4-(methylsulfonyl)phenyl)quinoline-3-carboxami-
de;
(4-cyano-N-(2-cyclohexenyl-4-(1-(2-(dimethylamino)acetyl)piperidin-4-y-
l)phenyl)-1H-imidazole-2-carboxamide).
[0064] The compound GW2580 has the following structure:
##STR00018##
[0065] The compound Ki20227 has the following structure:
##STR00019##
[0066] The compound
4-(3,4-Dimethylanilino)-7-(4-(methylsulfonyl)phenyl)quinoline-3-carboxami-
de, also known as cFMS Receptor Inhibitor III, has the following
structure:
##STR00020##
[0067] The compound
(4-cyano-N-(2-cyclohexenyl-4-(1-(2-(dimethylamino)acetyl)piperidin-4-yl)p-
henyl)-1H-imidazole-2-carboxamide), also known as JNJ-28312141, has
the following structure:
##STR00021##
[0068] A class of compounds that inhibit CSF1R include
2'-aminoanilides, of which the following compound is an
example:
##STR00022##
[0069] wherein NR.sup.1R.sup.2 is selected from the group
consisting of: piperidino; morpholino; piperazine;
N-ethyl-N-propylamino; N,N-dipropylamino; anilino; azetidino;
pyrrolidino; azepino; 2-methylpiperidino; 3-methylpiperidino;
3,5-dimethylpiperidino; 4-methylpiperidino; 4-methylpiperazino;
4-phenylpiperazino; 4-hydroxypiperidino; 4-hydroxymethylpiperidino;
4-(2-hydroxyethyl)piperidino; 3-hydroxypyrrolidino; and
3-hydroxymethylpyrrolidino. (See also Raymond J. Patch et al,
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6070-6074.)
For example, the compound of the following structural formula:
##STR00023##
[0070] A class of compounds that inhibit CSF1R include
3-amido-4-anilinocinnolines, of which the following compound is an
example:
##STR00024##
[0071] wherein R.sup.3 is selected from the group consisting of EtO
and MeO; and wherein each R.sup.4 is independently selected from
fluorine, chlorine, and methyl. (See also David A. Scott et al.
Bioorganic & Medicinal Chemistry Letters 21 (2011)
1382-1384.)
[0072] A class of compounds that inhibit CSF1R include
indoline-2-ones (e.g., of structural formula
##STR00025##
such as sunitinib, lestaurtinib, midostaurin, tandutinib,
sorafenib, ponatinib, and quizartinib. (See also Li Sun et al, J.
Med. Chem. 1998, 41, 2588-2603.) Other indoline-2-ones include
compounds having the following structure:
##STR00026##
[0073] wherein R.sup.5 is selected from the group consisting of
--H; 1'-CH.sub.3; 4'-CH.sub.3; 6'-F; 5'-Cl; 5'-Br; and wherein
R.sup.6 is selected from the group consisting of
4'-N(CH.sub.3).sub.2;
##STR00027##
4'-OH; 4'-OCH.sub.3; 4'-Br; 4'-COOH; 3'- and 5'-C(CH.sub.3).sub.3,
4-OH; 3'- and 5'-CH(CH.sub.3).sub.2, 4-OH; 3'-C(CH.sub.3).sub.3,
4-OCH.sub.3, and 5'-Br; 3'-C(CH.sub.3).sub.3 and 4'-OCH.sub.3;
4'-CH(CH.sub.3).sub.2; 3'- and 5'-CH(CH.sub.3).sub.2 and 4'-OH;
3'-C(CH.sub.3).sub.3 and 4'-OCH.sub.3; 4'-Br; and
3'-C(CH.sub.3).sub.3, 4'-OCH.sub.3, and 5'-Br; and wherein the
indicated stereochemistry can be either the E or Z
configuration.
[0074] Another indole-2-one compound is a compound having the
following structure:
##STR00028##
[0075] Other indoline-2-ones include compounds having the following
structure:
##STR00029##
[0076] wherein R.sup.7 is selected from the group consisting of
--H; 4'-CH.sub.3; 5'-CH.sub.3; 6'-F; 5'-Cl; and 5'-NO.sub.2; and
wherein R.sup.8 is selected from the group consisting of --H; 3'-
and 4'-CH.sub.3; 3'-CH.sub.2CH.sub.2COOH and 4'-CH.sub.3;
3'-CH.sub.3 and 4'-CH.sub.2CH.sub.2COOCH.sub.3; 3'-CH.sub.3 and
COOCH.sub.2CH.sub.3; 3'- and 5'-CH.sub.3; 3'- and 5' CH.sub.3,
4'-CH.sub.2CH.sub.3; 3'- and 5'-CH.sub.3, 4'-COOCH.sub.2CH.sub.3;
3'-CH.sub.2CH.sub.3, 4'- and 5'-CH.sub.3; 1'-CH.sub.3; and 3'- and
5'-CH.sub.3.
[0077] Other indoline-2-ones include compounds having the following
structure:
##STR00030##
[0078] wherein R.sup.9 is selected from the group consisting of --H
and 4'-CH.sub.3; and wherein R.sup.10 is selected from the group
consisting of 3'-Br; 4'-Br; 5'-SCH.sub.3; and
5'-CH.sub.2CH.sub.3.
[0079] Other indoline-2-ones include compounds having the following
structure:
##STR00031##
[0080] wherein R.sup.11 is --H; and wherein R.sup.12 is selected
from the group consisting of --H, -5'-CH.sub.3, and
5'-CH.sub.2CH.sub.3.
[0081] Other indoline-2-ones include compounds having the following
structure:
##STR00032##
[0082] wherein R.sup.13 is selected from the group consisting of
4'-Cl; and 1'-CH.sub.3 and 4'-Cl.
[0083] A class of compounds that inhibit CSF1R include
2-(.alpha.-methylbenzylamino) pyrazines, of which the following
compound is an example:
##STR00033##
(CYC10268). See also Christopher J. Burns et al, Bioorganic &
Medicinal Chemistry Letters 19 (2009) 1206-1209).
[0084] Other examples of 2-(.alpha.-methylbenzylamino) pyrazines
include compounds having the following structure:
##STR00034##
[0085] wherein R.sup.14 is selected from the group consisting
of
##STR00035##
[0086] Other examples of 2-(.alpha.-methylbenzylamino) pyrazines
include compounds having the following structure:
##STR00036##
[0087] wherein R.sup.15 is selected from the group consisting
of
##STR00037##
and wherein X is N or C.
[0088] A further class of compounds that inhibits CSF1R is
arylamides. For example, the compound of structural formula
##STR00038##
(See also Carl R. Illig et al, J. Med. Chem. 2011, 54, 7860-7883
and Hui Huang et al, J. Med. Chem. 2009, 52, 1081-1099)
[0089] A further class of compounds that inhibits CSF1R is
3,4,6-Substituted-2-quinolone, for example, the compound of
structural formula
##STR00039##
wherein R is a substituent (see Mark J. Wall et al. Bioorganic
& Medicinal Chemistry Letters 18 (2008) 2097-2102).
[0090] A further class of compounds that inhibits CSF1R is
3-amido-4-anilinoquinolines, for example, the compound of
structural formula
##STR00040##
wherein R.sup.1 is a substituent.
[0091] Further suitable compounds include cFMS Receptor Inhibitor
III--CAS 959861-21-3, represented by structural formula
##STR00041##
Ki20227, represented by structural formula
##STR00042##
AZ683, and OSI-930, represented by structural formula
##STR00043##
See also David A. Scott et al. Bioorganic & Medicinal Chemistry
Letters 19 (2009) 697-700.
[0092] A further class of compounds that inhibits CSF1R is the
class of pyridyl and thiazolyl bisamides, such as the compound
represented by structural formula
##STR00044##
wherein R.sup.1, R.sup.2 and R.sup.3 are independently selected
substituents; and the compound represented by structural formula
CSF-1R enzyme and cell activity--pyridal bisamides
##STR00045##
wherein R.sup.1 and R.sup.2 are independently selected
substituents, and the X stands for a ring atom, such as C or N; see
also David A. Scott et al. Bioorganic & Medicinal Chemistry
Letters 18 (2008) 4794-4797.)
[0093] Another suitable compound is Linifanib (ABT-869);
N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N1-(2-fluoro-5-methylphenyl)
urea, represented by structural formula
##STR00046##
See also Daniel H. Albert et al, Mol Cancer Ther 2006; 5(4)
995-1006).
[0094] Another suitable compound is the compound is ARRY-382 (Array
BioPharma); see also J. Bendell, et al., EORTC-NCI-AACR Symposium
on Molecular Targets and Cancer Therapeutics.
[0095] Another suitable compound is the compound is Pexidartinib,
PLX3397 (Plexxikon), represented by structural formula
##STR00047##
See also Nicholas Butowsk et al, Neuro-Oncology 18(4), 557-564,
2016.
[0096] A further class of compounds that inhibits CSF1R is
7-azaindoles, for example, the compound represented by structural
formula
##STR00048##
wherein R and R' stand are independently selected substituents (for
example, PLX3397; see also Chao Zhang et al, PNAS, 110, 14, 2013,
5689-5694.)
[0097] A further class of compounds that inhibits CSF1R is
1,4-disubstituted-pyrrolo-[3,2-c]-pyridines, for example, the
compound represented by structural formula
##STR00049##
wherein R and R' stand are independently selected substituents; see
also Chao Zhang et al, Bioorganic & Medicinal Chemistry Letters
22 (2012) 4362-4367.
[0098] A further class of compounds that inhibits CSF1R is
substituted diphenylurea compounds, for example, the compound
represented by structural formula
##STR00050##
wherein R and R' are independently selected substituents. For
example, Linifanib (ABT-869). See also Jun Guo et al, Mol Cancer
Ther 2006; 5(4) 1007-1013.
[0099] A further class of compounds that inhibits CSF1R is
5'-pyrimidine-2,4-diamines, for example, the compound represented
by structural formula
##STR00051##
wherein R is a substituent. For example GW2580. See also Vadim
Bernard-Gauthier et al, Bioorganic & Medicinal Chemistry
Letters 24 (2014) 4784-4790.
[0100] A further class of compounds that inhibits CSF1R is
pyrido[2,3-d]pyrimidin-5-ones, for example, the compound
represented by structural formula
##STR00052##
wherein R, R' and R'' are independently selected substituents. For
example, the compound represented by structural formula
##STR00053##
See also Hui Huang et al, J. Med. Chem. 2009, 52, 1081-1099.
[0101] Another suitable compound is AZ683 (a 3-amidoquinoline),
represented by structural formula
##STR00054##
[0102] Another suitable compound is anilinoquinazoline, represented
by structural formula
##STR00055##
[0103] In other embodiment of the present inventions a plurality of
CSF1R inhibitors are used in combination, for example, two, three,
or four of the CSF1R inhibitors described herein. For example, one
embodiment of the present invention is a method of preventing or
reducing a fibrotic response to an implanted synthetic material in
a patient, the method comprising administering to the patient an
effective amount of GW2580 and an effective amount of Ki20227.
[0104] In some embodiments (e.g., where fibrosis is a concern, the
CSF1R inhibitor compounds and compositions disclosed herein drugs
can be merged or integrated into already existing platforms (e.g.,
implants). Examples of implants and implanted medical devices
include, but are not limited to, the following: 1) Breast implants,
2) implanted sensors, for continuous sensing and monitoring of
physiological conditions (e.g., subcutaneously embedded continuous
glucose monitors, CGMs), 3) implants for nerve/muscle enervation
(STIMs, stimulation systems) for preventing muscle and/or nerve
atrophy following injury, 4) implants for any pacing/pacemaker, for
monitoring/regulating heart rhythm, 5) implants for hip/knee
replacement, 6) implants for tissue repair/reconstruction, 7)
implants for prosthesis and neural interfacing, 8) implants for
controlled drug release, 9) implants for vital sign monitoring, 10)
implants for intraocular lens replacement, 11) implants for cell
encapsulation and transplantation, and 12) implants for tissue
engineering/regeneration.
[0105] In another embodiment of the present invention, the CSF1R
inhibitor compounds disclosed herein (for example, GW2580) can be
incorporated into compositions for coating an implantable medical
device, such as prostheses, artificial valves, vascular grafts,
stents, or catheters. Suitable coatings and the general preparation
of coated implantable devices are known in the art. The coatings
are typically biocompatible polymeric materials such as a hydrogel
polymer, polymethyldisiloxane, polycaprolactone, polyethylene
glycol, polylactic acid, ethylene vinyl acetate, and mixtures of
any of the foregoing. The coatings can optionally be further
covered by a suitable topcoat of fluorosilicone, polysaccharides,
polyethylene glycol, phospholipids or combinations of any of the
foregoing to impart controlled release characteristics in the
composition.
[0106] Another embodiment of the present invention is an
implantable medical device having a coating comprising a CSF1R
inhibitor. In a specific aspect, the CSF1R inhibitor is GW2580. In
another specific aspect, the CSF1R inhibitor (for example, CSF1R)
and an effective amount of an additional agent (selected from the
ones set forth below) are comprised in one or more coatings of the
implantable medical device.
[0107] According to another embodiment, the invention provides a
method of impregnating an implantable medical device comprising the
step of contacting the device with a CSF1R inhibitor disclosed
herein or composition of this invention. Implantable drug release
devices include, but are not limited to, biodegradable polymer
capsules or bullets, non-degradable, diffusible polymer capsules
and biodegradable polymer wafers. In a specific aspect, the CSF1R
inhibitor is GW2580.
[0108] Another embodiment of the present invention, is a CSF1R
inhibitor, for use in preventing or reducing a fibrotic response to
an implanted synthetic material in a patient. In a specific aspect,
the CSF1R inhibitor is GW2580.
[0109] Yet another embodiment is the use of a CSF1R inhibitor for
the manufacture of a medicament for preventing or reducing a
fibrotic response to an implanted synthetic material in a patient.
In a specific aspect, the CSF1R inhibitor is GW2580.
[0110] In some embodiments, the CSF1R inhibitor can be administered
in combination with one or more additional agents, e.g., additional
therapeutic agents, either simultaneously or sequentially. In some
embodiments, the CSF1R inhibitor can be administered with one or
more additional agents in a composition, e.g., a pharmaceutical
composition. For example, the additional agent can be administered
in an effective amount, using the routes of administration
described herein.
[0111] In some embodiments, the additional agent is an
anti-inflammatory, e.g., a broad-spectrum anti-inflammatory (e.g.,
rapamycin/sirolimus, tacrolimus, everolimus, cyclosporine,
corticosteroids, other NSAIDS, etc.) for preventing (with one or
more CSF1R inhibitors) fibrosis either due to implant or damaged
tissues (i.e., due to cirrhosis of the liver, fatty liver disease,
torn muscle and wear and tear, following surgery procedures, etc.),
as well as, by the other anti-inflammatories, preventing rejection,
mediated by other immune cells, of tissue/organ or
implant-incorporated biologics (for repairing, supplementing,
replacing, or regenerating disease/condition-affected tissues).
[0112] In some embodiments, the additional agent is an
immunomodulatory agent (i.e., immune checkpoint inhibitors such as
PD 1, PDL1, CLTA4 antibodies or others) for preventing suppressive
immune cells (ie., T regulatory suppressor cells) from inhibiting
the anti-tumor actions of other immune populations (ie., cytotoxic
T, macrophage, or other cells) in addition to using CSF1R
inhibitors for reducing fibrosis/scarring following cytotoxic
cancer drug treatments, which can also damage normal tissues. CSF1R
inhibitors also have the added benefit of influencing macrophage
phenotype (moving them from pro-tumor to anti-tumor
function/behaviors) for cancer therapy. In some embodiments, the
CSF1R agents would also inhibit fibrosis from damage caused by
dysfunctional immune cells just prior to or during treatment. In
some embodiments, coadministration with other immunodulatory agents
can change osteoblast/osteoclast function to prevent and treat
osteoporosis (bone loss).
[0113] In some embodiments, the additional agent is an
anti-angiogenic agent (ie., anti-VEGFR1, 2, or 3 monoclonal
antibodies or small molecular tyrosine kinase inhibitors),
cytotoxic (chemotherapeutics), or anti-proliferative (ie.
Paclitaxel) agents to simultaneously treat cancer while also
preventing tissue damage-induced fibrosis (by the CSF1R
agents).
[0114] In some embodiments, the additional agent is an antibiotic,
e.g., to prevent infection following implant/transplant surgeries,
while also blocking fibrosis.
[0115] A further embodiment is a method of preventing or reducing a
fibrotic response to an implanted synthetic material in a patient,
the method comprising administering to the patient an effective
amount of a CSF1R inhibitor and an effective amount of one or more
additional agents set forth above. In a specific aspect, the CSF1R
inhibitor is GW2580 and the additional agent is an
anti-inflammatory agent, an immunomodulatory agent, an
anti-angiogenic agent, or an antibiotic. In a specific aspect, the
CSF1R inhibitor (e.g., GW2580) and an additional agent are
comprised in a coating of an implanted medical device.
[0116] A further embodiment of the present invention is a coating
formulation, comprising a CSF1R inhibitor (e.g., GW2580) and
optionally one or more additional agents selected from an
anti-inflammatory agent, an immunomodulatory agent, an
anti-angiogenic agent, and an antibiotic.
[0117] A further embodiment of the present invention is a coating
of an implantable medical device, comprising a CSF1R inhibitor
(e.g., GW2580) and optionally one or more additional agents
selected from an anti-inflammatory agent, an immunomodulatory
agent, an anti-angiogenic agent, and an antibiotic.
[0118] In some embodiments of the present invention, a CSF1R
inhibitor is a compound of structural formula
##STR00056##
a compound represented by structural formula
##STR00057##
4-(3,4-Dimethylanilino)-7-(4-(methylsulfonyl)phenyl)quinoline-3-carboxami-
de,
(4-cyano-N-(2-cyclohexenyl-4-(1-(2-(dimethylamino)acetyl)piperidin-4-y-
l)phenyl)-1H-imidazole-2-carboxamide), a compound represented by
structural formula
##STR00058##
ARRY-382, a compound represented by structural formula
##STR00059##
a 2'-aminoanilide, a 3-amido-4-anilinocinniline, an indoline-2-one,
a 2-(alpha-methylbenzylamino)-pyrazine, an arylamide, a
3,4,6-substituted 2-quinolone, a pyrido[2,3-d]pyrimidin-5-one, a
3-amido-4-anilinoquinoline, a pyridyl bisamide, a thiazolyl
bisamide, a 1,4-disubstituted-pyrrolo-[3,2-c]-pyridine, a
substituted diphenylurea, a 5'-pyrimidine-2,4-diamine, a compound
represented by structural formula
##STR00060##
a compound represented by structural formula
##STR00061##
anilinoquinazoline, a compound represented by structural
formula
##STR00062##
a compound represented by structural formula
##STR00063##
a compound represented by structural formula
##STR00064##
DCC-3014, a compound represented by structural formula
##STR00065##
a compound represented by structural formula
##STR00066##
a compound represented by structural formula
##STR00067##
a compound represented by structural formula
##STR00068##
a compound represented by structural formula
##STR00069##
a compound represented by structural formula
##STR00070##
a compound represented by structural formula
##STR00071##
a compound represented by structural formula
##STR00072##
or AC708.
EXEMPLIFICATION
Methods and Materials
[0119] In brief, all materials were implanted intraperitoneally or
subcutaneously into and retrieved at specified times from C57BL/6
(wild type, knockout, or serially immune depleted/perturbed) mice,
or non-human primate cynomolgus macaques in accordance with
approved protocols and federal guidelines. Sample processing,
staining, FACS, NanoString expression analysis, and imaging were
performed as detailed below. Shown are representative images in all
cases from n=5 mice per treatment group. Quantified data shown are
group mean values.+-.SEM.
[0120] a. Materials/Reagents
[0121] All in vitro reagents were obtained from Life Technologies
(Carlsbad, Calif.), unless otherwise noted. Antibodies: Alexa
Fluor-conjugated anti-mouse CD68, Ly-6G/Ly-6C (Gr-1), CD11b, CD19,
and IgM (described below) were purchased from BioLegend Inc. (San
Diego, Calif.). For primate immunostaining, anti-human CD68 Alexa
Fluor-conjugated antibody was purchased from Santa Cruz (Dallas,
Tex.). The same CD11b (anti-mouse/human) antibody (BioLegend) was
used for both primate and mouse staining. Cy3-conjugated anti-mouse
alpha smooth muscle actin antibody and glass spheres (acid washed)
of medium (500 .mu.m) size were purchased from Sigma Aldrich (St.
Louis, Mo.). Polystyrene spheres of medium (400-500 .mu.m) size
were purchased from Phosphorex (Hopkinton, Mass.). A sampling of
materials used in this study were submitted for endotoxin testing
by a commercial vendor (Charles River, Wilmington, Mass.) and the
results showed that spheres contained <0.05 EU/ml of endotoxin
levels (below detectable limits) (Table 1). Table 1. Negative
endotoxin and glucan results for all materials used in this study.
As determined by Charles River Labs sample submission, as well as
in-house testing, for bacterial pyrogen and endotoxin.
Specifically, E. coli and Limulus Amebocyte Lysates were used as
positive controls to test for the presence of general endotoxin.
BDL=below detectable limits. These negative results have also been
corroborated by others in our group, having now been published in
multiple studies (Veiseh O, Doloff J C, Ma M, Vegas A J, Tam H H,
Bader A R, et al. Size- and shape-dependent foreign body immune
response to materials implanted in rodents and non-human primates.
Nature materials 2015, 14(6): 643-651; Jhunjhunwala S,
Aresta-DaSilva S, Tang K, Alvarez D, Webber M J, Tang B C, et al.
Neutrophil Responses to Sterile Implant Materials. PloS one 2015,
10(9): e0137550; Vegas A J, Veiseh O, Doloff J C, Ma M, Tam H H,
Bratlie K, et al. Combinatorial hydrogel library enables
identification of materials that mitigate the foreign body response
in primates. Nat Biotechnol 2016).
TABLE-US-00001 Sample Endotoxin Test Glucan Test Saline control
<0.05 EU/mL (BDL) <10 .rho.g/ml (BDL) SLG20 alginate <0.05
EU/mL (BDL) <10 .rho.g/ml (BDL) 500 .mu.m spheres SLG20 alginate
<0.05 EU/mL (BDL) <10 .rho.g/ml (BDL) solution Glass spheres
<0.05 EU/mL (BDL) <10 .rho.g/ml (BDL) Polystyrene <0.05
EU/mL (BDL) <10 .rho.g/ml (BDL) spheres
[0122] b. Fabrication of Alginate Hydrogel Spheres
[0123] Alginate hydrogel spheres were made with an in-house
customized electro-jetting system: voltage generator, vertical
syringe pump (Harvard Apparatus), and a gelation bath basin.
Voltage was coupled to the syringe needle dispensing the alginate
and grounded to the gelling bath vessel. Spheres were made with a
1.4% solution of commercially available sterile alginate (PRONOVA
SLG20, NovaMatrix, Sandvika, Norway) dissolved in 0.9% saline
(pH.apprxeq.7.4, Osmotic pressure.apprxeq.290 mOsm), and
crosslinked with 250 mL of sterile BaCl.sub.2 gelling solution (20
mM BaCl.sub.2, 250 mM D-Mannitol, 25 mM HEPES, pH.apprxeq.7.4,
Osmotic pressure.apprxeq.290 mOsm) (see Morch Y A, Donati I, Strand
B L, Skjak-Braek G. Effect of Ca.sup.2+, Ba.sup.2+, and Sr.sup.2+
on alginate microbeads. Biomacromolecules 2006, 7(5):
1471-1480.)
[0124] Alginate hydrogel 500 .mu.m diameter microspheres were
generated with a 25G blunt needle, a voltage of 5 kV and a 200
.mu.l/min flow rate. Immediately after gelation, alginate spheres
were washed with HEPES buffer (25 mM HEPES, 1.2 mM
MgCl.sub.2.times.6H2O, 4.7 mM KCl, 132 mM NaCl.sub.2,
pH.apprxeq.7.4, .apprxeq.290 mOsm) 4 times and stored overnight at
4.degree. C. Immediately prior to implantation, spheres were washed
an additional 2 times with 0.9% saline. A sampling of the
fabricated hydrogels was submitted for endotoxin testing by a
commercial vendor (Charles River, Wilmington, Mass.) and the
results showed that SLG20 hydrogels contained <0.05 EU/ml of
endotoxin levels (below detectable limits).
[0125] c. Implantation Surgeries
[0126] All protocols were approved by the MIT Committee on Animal
Care, and all surgical procedures and post-operative care were
supervised by MIT Division of Comparative Medicine veterinary
staff. All mice, wild type male immune-competent (non-diabetic),
STZ-induced diabetic, as well as knockout C57BL/6 mice, were
ordered pathogen-free from Jackson Laboratory (Bar Harbor, Me.) or
Taconic (Hudson, N.Y.). The status of all mice was subsequently
verified by testing sentinel animals, shown to be negative for at
least 12 known mouse pathogens. Implanted mice were anesthetized
with 3% isoflurane in oxygen and had their abdomens shaved and
sterilized using betadine and isopropanol. C57BL/6-Nude (T KO,
B6NU), C57BL/6-Rag2.sup.null (T & B KO, RAGN12),
C57BL/6-Rag2.sup.null/Il2r.gamma..sup.null (Rag2/.gamma., 4111)
were ordered from Taconic (Hudson, N.Y.), and C57BL/6-IghM.sup.null
(BKO, 002288), (C57BL/6) B6.12956-Ccr6tm1(EGFP)Irw/J. (Ccr6,
013061), and C57BL/6-C3 KO (C3 KO, 003641) mice were ordered from
Jackson Laboratory, Bar Harbor, Me.). Preoperatively, all mice
received 0.05 mg/kg buprenorphine and 0.2 mL of 0.9% saline
subcutaneously for pre-surgery analgesia and dehydration
prevention. A midline (abdomen) incision (0.5 mm) was made and the
peritoneal lining was exposed using blunt dissection. The
peritoneal wall was then grasped with forceps and a 0.5-1 mm
incision was made along the linea alba. A desired volume of spheres
(all materials) were then loaded into a sterile pipette and
implanted into the peritoneal cavity. The incision was then closed
using 5-0 taper-tipped polydioxanone (PDS II) absorbable sutures,
and the skin was closed using a wound clip and VetBond tissue glue.
For subcutaneous implantation, .about.200-300 .mu.L of 500 .mu.m
SLG20 spheres were injected s.c. following anesthesia with
isofluorane. All primate implant samples were derived by excising
fibrosed, material-containing omentum and subcutaneous tissues 28
days following implantation of SLG20 500 .mu.m diameter spheres in
cynomolgus macaques, as described (see Veiseh O, Doloff J C, Ma M,
Vegas A J, Tam H H, Bader A R, et al. Size- and shape-dependent
foreign body immune response to materials implanted in rodents and
non-human primates. Nature materials 2015, 14(6): 643-651).
[0127] For targeted macrophage depletions, clodrosome (200
.mu.L/mouse) (Encapsula Nano Sciences, Nashville, Tenn.) were
injected intraperitoneally starting at -3 days (prior to
implantation, day 0), and for every 7 days thereafter (so days -3,
4, and 11). To achieve neutrophil depletion, affinity purified
anti-Ly6g (Clone 1A8) antibody (250 ug/mouse) (BioLegend, San
Diego, Calif.) was administered also starting at day -3, and every
3 days thereafter (except the day of implantation). For the
combination therapy, in order to avoid drug-antibody interactions,
each agent was administered 4 hours apart from one another. To
neutralize secreted CXCL13, anti-CXCL13 antibody (mAB470, R&D
Systems) was injected i.p. in sterile 1.times.PBS at a dose of 100
.mu.g/mouse, once every 3 days, starting 3 days prior to
implantation as well. For selective macrophage inhibition and
polarization, the CSF1R-targeted inhibitor GW2580 (LC Labs, Woburn,
Mass.), was dissolved in a 1:1 DMSO/PEG400 solution, and injected
daily (starting 1 day prior to implantation) at a dose of 160 mg/kg
BW subcutaneously (s.c.), to eliminate concerns of vehicle directly
perturbing responding immune cells as well as a recovering implant
site.
[0128] d. IVIS Imaging
[0129] C57BL/6 mice (6-8 weeks old) were utilized for this assay.
200-300 .mu.l of alginate spheres were resuspended in saline, and
injected subcutaneously into the mouse on both left and right sides
of upper back. The mice were shaved to get rid of the hair on the
entire back before injection, and fed on sterilized AIN-93G
purified rodent diet (TD 94045, Harlan) to minimize the fluorescent
background after injection. Six days later, 100 .mu.l (4 nmol) of
ProSence 750 FAST (NEV11171, PerkinElmer Inc.) per mouse was
injected intravenously via tail vein. At day 7 (24 hours post the
ProSense 750 FAST intravenous administration), the mice were
scanned by IVIS Spectrum system (Xenogen, Caliper LifeScience). The
mice were anesthetized using 3% isofluorane in oxygen and
maintained at the same rate throughout the procedure, the new grown
hair were removed by Nair hair removal lotion, and then the mice
were scanned by the IVIS Spectrum system at the settings of
Exposure=7.50, Binning=Medium, FStop=2, Excitation=605-640 nm and
Emission=660-760 nm. The images were analyzed with LivingImage
Software, and the duplicated ROI of lower back on the same mouse
was used as control during the signal quantification.
[0130] e. Retrieval of Cells, Tissues, and Materials
[0131] At desired time points post-implantation or transplantation
(with encapsulated islets), as specified in figures, mice were
euthanized by CO.sub.2 administration, followed by cervical
dislocation. In certain instances, 5 ml of ice cold PBS was first
injected in order perform an intraperitoneal lavage to rinse out
and collect free-floating intraperitoneal immune cells. An incision
was then made using forceps and scissors along the abdomen, and
intraperitoneal lavage volumes were pipetted out into fresh 15 ml
falcon tubes (each prepared with 5 ml of RPMI cell culture media).
Next, a wash bottle tip was inserted into the abdominal cavity.
KREBS buffer was then used to wash out all material spheres into
petri dishes for collection. After ensuring all the spheres were
washed out or manually retrieved (if fibrosed directly to
intraperitoneal tissues, in particular epididymal and omental fat
pads), they were transferred into 50 mL conical tubes for
downstream processing and imaging. In certain instances, after
intraperitoneal lavage, portions of fibrosed intraperitoneal
tissues and material spheres were also excised for downstream FACS
and expression analyses.
[0132] f. Imaging of the Retrieved Material Spheres
[0133] For phase contrast imaging retrieved materials were gently
washed using Krebs buffer and transferred into 35 mm petri dishes
for phase contrast microscopy using an Evos X1 microscope (Advanced
Microscopy Group). For bright-field imaging of retrieved materials,
samples were gently washed using Krebs buffer and transferred into
35 mm petri dishes for bright-field imaging using a Leica
Stereoscopic microscope.
[0134] g. Confocal Immunofluorescence
[0135] Immunofluorescence imaging was used to determine immune
populations attached to spheres. Materials were retrieved from mice
and fixed overnight using 4% paraformaldehyde at 4.degree. C.
Samples were then washed twice with KREBS buffer, permeabilized for
30 min using a 0.1% Triton X100 solution, and subsequently blocked
for 1 hour using a 1% bovine serum albumin (BSA) solution. Next,
the spheres were incubated for 1 hour in an immunostaining cocktail
solution consisting of DAPI (500 nM), specific marker probes (1:200
dilution) in BSA. After staining, spheres were washed three times
with a 0.1% Tween 20 solution and maintained in a 50% glycerol
solution. Spheres were then transferred to glass bottom dishes and
imaged using an LSM 700 point scanning confocal microscope (Carl
Zeiss Microscopy, Jena Germany) equipped with 5 and 10.times.
objectives. Obtained images were adjusted linearly for presentation
using Photoshop (Adobe Inc. Seattle, Wash.). For antigen-specific
immunostaining, non-human primate (cynomolgus macaque)
intraperitoneal sphere-embedded omenta were sectioned and stained
according to traditional antigen retrieval and immunofluorescent
methods, specifically looking at cellular nuclei (DAPI), macrophage
marker CD68-AF488 (Santa Cruz, Dallas, Tex.) and Cy3-conjugated
anti-mouse alpha smooth muscle actin (fibrosis) (Sigma Aldrich, St.
Louis, Mo.).
[0136] h. Histological Processing for H&E and Masson's
Trichrome Staining
[0137] Retrieved material-containing tissue (omentum and/or
subcutaneous) was fixed overnight using 4% paraformaldehyde at
4.degree. C. After fixation, alginate sphere or retrieved tissue
samples were washed using 70% alcohol. The materials were then
paraffin embedded, sectioned and stained according to either
standard histological (H&E or Masson's Trichrome) or
antigen-specific methods (as described above).
[0138] i. Western Blotting
[0139] Protein was extracted directly from materials for western
blot analysis. Retrieved materials were prepared by lysis of
covering cellular overgrowth in Pierce RIPA buffer (Cat. #89901,
Thermo Scientific) with protease inhibitors (Halt Protease
inhibitor single-use cocktail, Cat. #78430, Thermo Scientific) on
ice, and then lysed by sonication (for 30 seconds on, 30 seconds
off, twice at 70% amplitude). Samples were then agitated constantly
for 2 hours at 4.degree. C. Lysates were centrifuged for 20 min at
12,000 rpm at 4.degree. C., and protein-containing supernatants
were collected in fresh tubes, on ice. In samples from fatty
tissue, an excess of fat (a top layer on the supernatant) was
removed before supernatant transfer. 20 .mu.g protein (quantified
by BCA assay, Pierce BCA protein assay kit, Cat. #23225, Thermo
Scientific) for each lane was boiled at 95.degree. C. for 5 min and
electrophoresed on SDS-polyacrylamide gels (Any kD 15-well comb
mini-gel, BioRad, Cat. #456-9036) and then blotted onto
nitrocellulose membranes (BioRad, Cat. #162-0213). Blots were
probed with anti-alpha Smooth Muscle actin antibody (1:400
dilution, Rabbit polyclonal to alpha smooth muscle actin; Cat.
#ab5694, AbCam) and anti-.beta.-actin antibody (1:4000 dilution,
monoclonal anti-.beta.-actin antibody produced in mouse; Cat.
#A1978, Sigma Aldrich) as a loading control followed by donkey
anti-rabbit (1:15,000 dilution, Cat. #926-32213, Li-Cor) and goat
anti-mouse (1:15,000 dilution, Cat. #926-68070, Li-Cor)
fluor-conjugated secondary antibodies. Bands were visualized using
an Odyssey detector (Li-Cor, Serial No. ODY-2329) at 700 and 800 nm
wavelengths. For CXCL13 detection, a rabbit anti-CXCL13/BCA1
polyclonal antibody (Bioss, Woburn, Mass.) was used.
[0140] j. qPCR Analysis
[0141] Total RNA was isolated from tissue (peripheral epididymal
and omental adipose tissue alone for mock controls, or fibrosed
spheres with adhered adipose tissue and immune overgrowth, if
present), liquid nitrogen snap-frozen immediately following
excision, using TRIzol (Invitrogen, Carlsbad, Calif.) according to
the manufacturer's instructions. In addition, to help ensure
complete tissue disruption, we also employed strong mechanical
disruption with a Polytron homogenizer. Thus, gene expression
signatures shown throughout are proportional and representative of
the entire cell population present on and/or around retrieved
materials. Before reverse transcription using the High Capacity
cDNA Reverse Transcription kit (Cat. #4368814; Applied Biosystems,
Foster City, Calif.), all samples were first normalized for
comparison by loading the same input 1 .mu.g total RNA in a volume
of 20 .mu.l for each sample. cDNA (4.8 .mu.l; 1:20 dilution) in a
total volume of 16 .mu.l (including SYBR Green and PCR primers) was
amplified by qPCR with the following primers. Primers (see Table 2
below) were designed using Primer Express software (Applied
Biosystems, Carlsbad, Calif., USA) and evaluated using LaserGene
software (DNAStar, Madison, Wis., USA) to ensure either mouse
(host)-specificity. Samples were incubated at 95.degree. C. for 10
min followed by 40 cycles of 95.degree. C. for 15 sec and
60.degree. C. for 1 min in an ABI PRISM 7900HT Sequence Detection
System (Applied Biosystems). Results were analyzed using the
comparative C.sub.T (DDC.sub.T) method as described by the
manufacturer. Results were analyzed using the comparative C.sub.T
(.DELTA..DELTA.C.sub.T) method and are presented as relative RNA
levels, as compared to control cell samples as specified in figure
legends after normalization to the .beta.-actin RNA content of each
sample. To further ensure proper normalization and sample handling
across multiple retrieval time points, RNA for all samples for each
harvest condition (ie., ip lavage, spheres with or without adhered
cells and fibrosis, and peripheral tissues with infiltration, as
described), were quantified, reverse transcribed, and analyzed by
qPCR in parallel.
TABLE-US-00002 TABLE 2 Mouse (m)-specific (host) forward and
reverse oligonucleotide primer sets used for qPCR analysis of RNA
levels. Gene names are also shown in parentheses. Primers (5' to
3'): Gene Sequence Sense & Antisense Mouse Collagen 1 Forward:
1a1 (mCol1a1) 5'-CATGTTCAGCTTTGTGG ACCT-3' 2 Reverse:
5'-GCAGCTGACTTCAGGGA TGT-3' Mouse Collagen 3 Forward: 1a2 (mCol1a2)
5'-GCAGGTTCACCTACTCT GTCCT-3' 4 Reverse: 5'-CTTGCCCCATTCATTTG
TCT-3' Mouse Alpha 5 Forward: Smooth Muscle 5'-CGCTTCCGCTGCCCAGA
actin (mActa2) GACT-3' 6 Reverse: 5'-TATAGGTGGTTTCGTGG ATGCCCGCT-3'
Mouse 7 Forward: Inflammation 5'-CCTGAGTGGCTGTCTTT marker TGAC-3'
Transforming 8 Reverse: Growth Factor 5'-ACAAGAGCAGTGAGCGC beta1
(mTGFb1) TGAAT-3' Mouse 9 Forward: Macrophage 5'-GATACAGCAATGCCAAG
marker F4/80 CAGT-3' (mEmr1) 10 Reverse: 5'-TTGTGAAGGTAGCATTC
ACAAGTGTA-3' Mouse 11 Forward: Macrophage 5'-GCCCGAGTACAGTCTAC
marker CD68 CTGG-3' (mCd68) 12 Reverse: 5'-AGAGATGAATTCTGCGC CAT-3'
Mouse Myeloid 13 Forward: cell marker 5'-CCAAGAGAATGCAAAAG CD11b
GCTTT-3' (mItgam) 14 Reverse: 5'-GGGGGGCTGCAACAACC ACA-3' Mouse 15
Forward: neutrophil 5'-TGCCCCTTCTCTGATGG marker Gr1 ATT-3' (mLy6g)
16 Reverse: 5'-TGCTCTTGACTTTGCTT CTGTGA-3' Mouse B cell 17 Forward:
marker CD19 5'-GGAAACCTGACCATCGA (mCd19) GAG-3' 18 Reverse:
5'-TGGGACTATCCATCCAC CAGTT-3' Mouse 19 Forward: Dendritic cell
5'-CCCAGGACCATGTGATG (DC) marker CAT-3' CD74 (mCd74) 20 Reverse:
5'-CTTAAGATGCTTCAGAT TCTCT-3' Mouse 21 Forward: Langerhans
5'-GGACTACAGAACAGCTT Dendritic cell GGAGAATG-3' (DC) marker 22
Reverse: CD207 (Langerin) 5'-TACTTCCAGCCTCGAGC (mCd207) CAC-3'
Mouse Natural 23 Forward: killer (NK) cell 5'-GCAACCCCCTGAAACTG
marker NKp46 GTA-3' (mNcr1) 24 Reverse: 5'-AAGGTTACCTCAGGCTG
TGGATA-3' Mouse 25 Forward: Adaptive helper 5'-GAAGATTCTGGGGCAGC T
cell marker ATGGCAAAG-3' CD4 (mCd4) 26 Reverse:
5'-TTTGGAATCAAAACGAT CAA-3' Mouse 27 Forward: Cytotoxic T cell
5'-CTGCGTGGCCCTTCTGC marker CD8a TGTCCT-3' (mCd8a) 28 Reverse:
5'-GGGACATTTGCAAACAC GCT-3' Mouse 29 Forward: regulatory T
5'-GCCTTCAGACGAGACTT suppressor cell GGAA-3' marker FoxP3 30
Reverse: (mFoxP3) 5'-CTGGCCTAGGGTTGGGC ATT-3' Mouse .beta.-actin 31
Forward: (mActB) 5'-GCTTCTTTGCAGCTCCT TCGTT-3' 32 Reverse:
5'-CGGAGCCGTTGTCGACG ACC-3'
[0142] k. Luminex Multiplexed Cytokine and ELISA Analyses
[0143] Cytokine array analysis was performed using the Luminex
Bio-Rad Bio-Plex Pro mouse cytokine panel based on Luminex beads
(multiplexing both the 23 and 8-cytokine arrays) (Bio-Rad,
Hercules, Calif.), according to manufacturer's instructions. 1:4
diluted mouse sera samples were analyzed for the time course, and
protein lysates taken from fibrosed tissue and alginate
microspheres, loaded at a concentration of 500 .mu.g/mL (200 uL per
sample), were run to compare cytokine responses across mock
(saline) treated versus wildtype, knockout or serially
perturbed/depleted C57BL/6 mice implanted with 500 .mu.m SLG20
alginate spheres. Samples were added to the panel of beads bearing
capture antibodies for the analytes of interest, and agitated for
30 minutes. Sample plates were subsequently washed using an
automated magnetic plate washer (Bio-Rad), and a biotinylated
detection antibody for each of the 32 cytokines was added followed
by agitation for 30 minutes. After another wash, streptavidin-PE
was added, the plate was agitated for 10 minutes, washed, and
resuspended in assay buffer. The plate was then read on a
BioPlex-200 plate reader, with absolute concentrations obtained
through fitting a standard curve. For Luminex runs, protein lysates
were prepared using an NP-40-based lysis buffer, as opposed to the
SDS-based RIPA buffer used for western blotting (described above)
to maintain native state protein folding. For mouse CXCL10 (IP-10)
and IgM analyses, an ELISA from eBioscience (San Diego, Calif.) was
used, according to manufacturer's specifications, with protein
lysates for CXCL10 and both sera and protein lysates for IgM.
[0144] l. FACS Analysis
[0145] Single-cell suspensions of freshly excised tissues were
prepared using a gentleMACS Dissociator (Miltenyi Biotec, Auburn,
Calif.) according to the manufacturer's protocol. Single-cell
suspensions were prepared in a passive PEB dissociation buffer
(1.times.PBS, pH 7.2, 0.5% BSA, and 2 mM EDTA) and suspensions were
passed through 70 .mu.m filters (Cat. #22363548, Fisher Scientific,
Pittsburgh, Pa.). This process removed the majority of cells
adhered to the surface (>90%) (See Supplemental FIG. 17 from
Veiseh O, Doloff J C, Ma M, Vegas A J, Tam H H, Bader A R, et al.
Size- and shape-dependent foreign body immune response to materials
implanted in rodents and non-human primates. Nature materials 2015,
14(6): 643-651). All tissue and material sample-derived,
single-cell populations were then subjected to red blood cell lysis
with 5 ml of 1.times.RBC lysis buffer (Cat. #00-4333, eBioscience,
San Diego, Calif., USA) for 5 min at 4.degree. C. The reaction was
terminated by the addition of 20 ml of sterile 1.times.PBS. The
cells remaining were centrifuged at 300-400g at 4.degree. C. and
resuspended in a minimal volume (.about.50 .mu.l) of eBioscience
Staining Buffer (cat. #00-4222) for antibody incubation. All
samples were then co-stained in the dark for 25 min at 4.degree. C.
with two of the fluorescently tagged monoclonal antibodies specific
for the cell markers CD68 (1 .mu.l (0.5 .mu.g) per sample;
CD68-Alexa647, Clone FA-11, Cat. #11-5931, BioLegend), Ly-6G (Gr-1)
(1 .mu.l (0.5 .mu.g) per sample; Ly-6G-Alexa-647, Clone RB6-8C5,
Cat. #108418, BioLegend), CD11b (1 .mu.l (0.2 .mu.g) per sample; or
CD11b-Alexa-488, Clone M1/70, Cat. #101217, BioLegend). For alpha
smooth muscle actin (fibrosis) analysis, additional cell aliquots
were also fixed in 1% paraformaldehyde and permeabilized with 0.1%
triton X-100 before being stained with Cy3-conjugated anti-mouse
.alpha.SM actin antibody (1:100) (Sigma Aldrich, St. Louis, Mo.).
Two ml of eBioscience Flow Cytometry Staining Buffer (cat.
#00-4222, eBioscience) was then added, and the samples were
centrifuged at 400-500g for 5 min at 4.degree. C. Supernatants were
removed by aspiration, and this wash step was repeated two more
times with staining buffer. Following the third wash, each sample
was resuspended in 500 .mu.l of Flow Cytometry Staining Buffer and
run through a 40 .mu.m filter (Cat. #22363547, Fisher Scientific)
for eventual FACS analysis using a BD FACSCalibur (cat. #342975),
BD Biosciences, San Jose, Calif., USA). For proper background and
laser intensity settings, unstained, single antibody, and IgG
(labeled with either Alexa-488 or Alexa-647, BioLegend) controls
were also run.
[0146] m. Intravital Imaging--Ccr6-EGFP Mice
[0147] For intravital imaging, 500 .mu.m SLG20 hydrogel spheres
were loaded with Qdot 605 (Life technologies, Grand Island, N.Y.)
and surgically implanted into (C57BL/6) B6.129S6-Ccr6tm1(EGFP)Irw/J
(Ccr6, 013061, Jackson Laboratory, Bar Harbor, Me.) mice, as
described above. After 14 days post implantation, the mice were
placed under isoflurane anesthesia throughout and a small incision
was made at the site of the original surgery to expose beads. The
mice were placed on an inverted microscope and imaged using a
25.times., N.A. 1.05 objective on an Olympus FVB-1000 MP
multiphoton microscope at an excitation wavelength of 860 nm. While
Ccr6 can show EGFP in adaptive T and B cells as well as innate
immune dendritic cells (DCs), only B cells respond in our alginate
material model.
[0148] n. NanoString Analysis
[0149] RNAs for mock-implanted (saline) treated controls, or for
500 .mu.m alginate sphere-bearing C57BL/6 mouse strains
(n=4/group), including wild type, knockouts, and serially immune
depleted wild type mice, were isolated from tissue samples taken at
various time points after implantation, as described. For each of
the knockout strains, they were normalized to their own
(strain-specific) mock (saline) implanted controls, in order to
eliminate any artifacts due to shifts in immune homeostasis, since
gene knockouts were also present throughout development.
Furthermore, to corroborate macrophage-specific changes,
macrophages were also sorted from implanted wild type C57BL/6 mice,
and also plotted relative to expression levels from the entire mock
tissue cell population. Thus, macrophage-specific genes show
enriched (red, increased) expression fold changes, and
macrophage-independent gene show diluted (green, decreased)
expression fold changes. In general, respective RNAs were
quantified, normalized to the appropriate loading concentration
(100 ng/.mu.l), and then 500 ng of each sample was processed
according to NanoString manufacturer protocols for expression
analysis via our customized multiplexed total (known) mouse
cytokine and cytokine receptor expression panel, used for both
immune strain comparisons with alginate, and material class
comparisons just the wild type C57BL/6 strain. Another non-human
primate custom panel was also used to corroborate gene hits in our
cynomolgus macaque intraperitoneal and subcutaneous models. RNA
levels (absolute copy numbers) were obtained following nCounter
(NanoString Technologies Inc., Seattle, Wash.) quantification, and
group samples were analyzed using nSolver analysis software
(NanoString Technologies Inc., Seattle, Wash.).
[0150] o. Statistical Analysis
[0151] Data are expressed as mean.+-.SEM, and N=5 mice per time
point and per treatment group. These sample sizes were chosen based
on previous literature. All animals were included in analyses
except in instances of unforeseen sickness or morbidity. Animal
cohorts were randomly selected. Investigators were not blind to
performed experiments. For qPCR, western blot quantification, IVIS
imaging, or FACS, data were analyzed for statistical significance
either by unpaired, two-tailed t-test, or one-way ANOVA with
Bonferroni multiple comparison correction, unless indicated
otherwise, as implemented in GraphPad Prism 5; *: p<0.05, **:
p<0.001, and ***: p<0.0001. For Nanostring, data was
normalized using the geometric means of the NanoString positive
controls and background levels were established using the means of
the negative controls. Housekeeping genes Tubb5, Hprt1, Bact, and
Cltc were used to normalize between samples. Data was then
log-transformed
Example 1: Implanted Biomaterial Alginate Elicits a Multi-Immune
Population Host Rejection Response
[0152] Host immune-mediated foreign body rejection of commercially
purified biomaterial alginate, implanted as 500 .mu.m spheres, is
complex, engaging both innate and adaptive immune cell populations
(FIGS. 1A-G). Historically, responses were thought to be a function
of contaminating endotoxins (Paredes-Juarez G A, de Haan B J, Faas
M M, de Vos P. The role of pathogen-associated molecular patterns
in inflammatory responses against alginate based microcapsules. J
Control Release 2013, 172(3): 983-992.). However, the clinical
grade alginates used here do not contain detectable endotoxins
(Veiseh O, Doloff J C, Ma M, Vegas A J, Tam H H, Bader A R, et al.
Size- and shape-dependent foreign body immune response to materials
implanted in rodents and non-human primates. Nature materials 2015,
14(6): 643-651; Vegas A J, Veiseh O, Doloff J C, Ma M, Tam H H,
Bratlie K, et al. Combinatorial hydrogel library enables
identification of materials that mitigate the foreign body response
in primates. Nat Biotechnol 2016; Jhunjhunwala S, Aresta-DaSilva S,
Tang K, Alvarez D, Webber M J, Tang B C, et al. Neutrophil
Responses to Sterile Implant Materials. PloS one 2015, 10(9):
e0137550.). For thoroughness, both the alginate solution, prior to
use, as well as spheres post-gelation were again tested and
verified to be endotoxin-free (See Table 1 above).
[0153] Cellular adhesion and fibrotic overgrowth of alginate
microspheres implanted in the intraperitoneal space of C57/B6 mice
after 14 days is apparent as a white plaque by phase contrast
imaging (FIG. 1A), with individual cells resolvable by nuclear DAPI
staining (FIG. 1B). Specific markers for innate immune cell
macrophages (F4/80 and CD68) and neutrophils (Ly6g), adaptive
immune B cells (CD19), as well as fibrosis (alpha smooth muscle
actin and collagen 1a1) were identified by qPCR, and increased over
time on alginate spheres, as compared to non-implanted and mock
(saline injected) controls (FIG. 1C and FIG. 2). Immunofluorescent
staining and confocal imaging of spheres retrieved 14 days
post-intraperitoneal (IP) implantation reveals that all three of
these responding immune populations (macrophages: CD68;
neutrophils, Ly6g/Gr1; B cells, CD19) reside within the fibrotic
plaque directly enveloping implanted alginate spheres (FIGS. 1D-E).
In addition, intravital imaging at 14 days post-implant in
Ccr6-EGFP transgenic C57BL/6 mice, verified trafficking of adaptive
B cells onto implanted quantum-dot labeled (pink) alginate spheres
(FIGS. 1F-G).
Example 2: Local Immune Responses to Alginate are Complex and
Long-Lived
[0154] To determine whether the host immune response to implanted
biomaterial alginate was acute and short-lived or manifested over a
longer period of time, we performed FACS on cells obtained by both
peritoneal lavage and dissociation directly from the surface of 500
alginate spheres and associated peripheral fibrosed tissue
retrieved at a range of time points (1, 4, 7, 14, and 28) days
following IP implantation into C57BL/6 mice (FIGS. 3A-C). In
general, peritoneal exudate cell number reflected an increase in
neutrophils (as % total), B cells decreased and macrophages were
unchanged (FIG. 3A). However, these observable responses in the
peritoneal exudate do not depict earlier identified cell population
increases directly on the implanted material surface (FIGS. 1A-G
and FIG. 2).
[0155] Luminex analysis of serum levels of cytokines following
implantation showed an acute and transient increase of 4 out of 32
cytokines (FIGS. 4A-E), suggesting an early, perhaps
surgery-related, event that goes away within .about.1-3 days
post-implantation. This is in stark contrast to the previous
mention of longer-term evidence of immunogenicity or host
reactivity to implanted alginate spheres. Such long-term responses,
however, do not appear to be discernable by looking at global,
blood-circulating cytokines, which have likely become too dilute.
When FACS was performed on dissociated fibrosed IP tissue
(repeatedly observed to be only non-collagen-encapsulated
epididymal and omental fat pads) immediately adjacent to and often
stuck to the fibrosed capsules (FIG. 5), as well as specifically
cells taken directly from the surface of implanted alginate
spheres, immune cells increased over a 28-day period, with
macrophages the major responding cell population (FIGS. 3B-C). FACS
analysis of alginate-associated cells on day 1 show the presence of
immature CD68.sup.-Gr1.sup.lo/-CD11b.sup.+ monocytes, known for
being early responders patrolling out from the blood (Shi C, Pamer
E G. Monocyte recruitment during infection and inflammation. Nat
Rev Immunol 2011, 11(11): 762-774.). On days 4 and 7, this immature
population disappeared as mature CD68.sup.+CD11b.sup.+ macrophages
increased in number (FIG. 3C, purple vs red). Macrophage subtyping
carried out on surface-dissociated cells at 1, 4, and 7 days also
suggest this change is due to early-stage monocyte recruitment and
differentiation (Veiseh O, Doloff J C, Ma M, Vegas A J, Tam H H,
Bader A R, et al. Size- and shape-dependent foreign body immune
response to materials implanted in rodents and non-human primates.
Nature materials 2015, 14(6): 643-651.). Later stage (day 7 and
beyond) recruitment of both adaptive immune CD19.sup.+IgM.sup.+ B
cells and fibrosis-associated alpha smooth muscle actin
(.alpha.SMactin)-positive myofibroblasts was observed (FIG. 3C,
white). Multiplexed NanoString gene expression analysis showed
longer-term (up to 4 weeks) increases in macrophage and
neutrophil-associated markers (FIGS. 3D-E). Tissue immediately
adjacent to alginate capsules (FIG. 5, epididymal and omental fat
pads) as well as cellular material taken directly from the surface
of implanted spheres were both analyzed over a 28-day time course
to ascertain localization of various macrophage and inflammation
markers (FIGS. 6A-B). By contrast, qPCR-determined expression of B
cell marker CD19 showed a delayed (day 7 and later) mobilization
out of peripheral fibrosed epididymal and omental tissue and onto
alginate spheres (FIG. 3F), indicating later adaptive B cell
recruitment not just to the site but also directly onto implanted
alginate. Lastly, expression analysis of fibrosis marker
.alpha.SMactin was masked in peripheral tissue due to very high
background in IP fat pads (also the case in subcutaneous tissue).
However, we were able to ascertain an appropriate kinetic, with a
significant, delayed expansion at and beyond day 7, when fibrotic
material was instead taken directly from implanted spheres (FIG.
3G).
Example 3: Adaptive as Well as Innate Immune Cells are Involved in
the Downstream Fibrotic Cascade
[0156] To better understand the cellular requirements driving the
fibrotic response, we examined fibrosis to implanted 500 .mu.m
alginate spheres retrieved after 14 days, in a range of
C57BL/6-derived immune mutant rodents. A total of 7 strains were
studied, ranging from fully immune competent wild type versus
knockout (KO) strains to strains with varying levels of
immunodeficiency (FIG. 7). Phase contrast imaging was used to show
relative levels of fibrotic overgrowth across mice with varying
immune deficiency after 14 days (FIG. 8A and FIGS. 9A-F).
Implicating importance in the fibrotic cascade, loss of B cells
alone (IghM.sup.null, B KO) resulted in a partial loss of fibrosis
(FIG. 8A, and FIGS. 9A-F, and FIGS. 10A-C). Additional T cell loss
(Rag2.sup.null, T & B KO) made fibrosis worse and more
comparable to wild type (WT) levels (FIG. 8A and FIG. 9E), perhaps
due to the loss of the regulatory T cell subset important for
suppressing overreaching immune reactions (Wood K J, Bushell A,
Hester J. Regulatory immune cells in transplantation. Nat Rev
Immunol 2012, 12(6): 417-430.). Ultimately, only with innate immune
cell macrophage dysfunction in Rag2.sup.null/Il2r.gamma..sup.null
(Rag2/.gamma.KO) was a complete loss of fibrosis observed (FIG. 8A
and FIG. 9D). Individual T cell (T KO, nude) and complement C3
knockouts did not result in the loss of fibrosis (FIGS. 9E-F).
[0157] Western blot assays were performed on extracted proteins
from retrieved spheres in all groups exhibiting significant
decreases in fibrosis (B KO and Rag2/.gamma. KO), as compared to
wild type controls, to quantify changes in the fibrosis marker
.alpha.SMactin (FIG. 8B-C). Expression results were further
corroborated using qPCR analysis of RNA isolated from retrieved
spheres, where .alpha.SMactin and Collagens 1a1 and 1a2 showed
similar significant decreases across both knockout models, as
compared to WT (FIG. 8D).
[0158] In addition to study of spheres retrieved from the
peritoneal space, which is therapeutically relevant for cell
encapsulation and transplantation (Jacobs-Tulleneers-Thevissen D,
Chintinne M, Ling Z, Gillard P, Schoonjans L, Delvaux G, et al.
Sustained function of alginate-encapsulated human islet cell
implants in the peritoneal cavity of mice leading to a pilot study
in a type 1 diabetic patient. Diabetologia 2013, 56(7): 1605-1614;
Weir G C. Islet encapsulation: advances and obstacles. Diabetologia
2013, 56(7): 1458-1461; Veiseh O, Doloff J C, Ma M, Vegas A J, Tam
H H, Bader A R, et al. Size- and shape-dependent foreign body
immune response to materials implanted in rodents and non-human
primates. Nature materials 2015, 14(6): 643-651), alginate spheres
were also implanted into the subcutaneous compartment in the same
wild type and knockout strains (FIGS. 8E-F). Immune cell activity
at the site of implantation was analyzed using Prosense, a
fluorescent indicator of secreted immune inflammation cathepsins
(Bratlie K M, Dang T T, Lyle S, Nahrendorf M, Weissleder R, Langer
R, et al. Rapid biocompatibility analysis of materials via in vivo
fluorescence imaging of mouse models. PloS one 2010, 5(4):
e10032.). IVIS imaging at 7 days post-implant showed increasingly
significant decreases in alginate-induced inflammation across both
knock-out models (FIG. 8E). Following a 1-month implantation,
subcutaneous spheres were excised and processed for histology
(H&E and Masson's Trichrome). In general, spheres were
completely embedded and individually sequestered in WT mice, but
only had an outer, thin fibrotic capsule in B KO mice, while
showing no significant deposition in macrophage dysfunctional
(Rag2/.gamma. KO) mice (FIG. 8F and FIG. 11A-D). FACS analysis of
retrieved capsules and surrounding tissue verified that wildtype
subcutaneous immune responses are also localized to the implant
(FIG. 12A-C) and similar in composition to those in the IP space
(FIG. 8G). Lastly, FACS analysis corroborated earlier findings of
decreased immunity and fibrosis rejection across both knock out
strains, with an .about.60% decrease in macrophage and neutrophil
presence on spheres taken from B KO mice, and an essentially
complete loss of adherent cells in macrophage dysfunctional
(Rag2/.gamma. KO) mice (FIG. 8G). Loss of macrophage presence
resulted in complete B cell loss as well, suggesting that
macrophages may be required for B cell recruitment to the
capsules.
Example 4: Macrophages, not Neutrophils, are Necessary for Fibrosis
of Alginate Microcapsules
[0159] Both macrophage and neutrophil populations were present on
the surface of alginate spheres even 1 day following alginate
sphere implantation into wild type C57BL/6 mice (FIG. 3C). Our
observation that microcapsules implanted into the Rag2/.gamma. KO
model being fibrosis-free indicates that macrophages are necessary
for fibrosis (FIG. 8A-G). However, Rag2/.gamma. KO rodents still
have macrophages, although they are reported to be reduced in
number and dysfunctional (Ito M, Hiramatsu H, Kobayashi K, Suzue K,
Kawahata M, Hioki K, et al. NOD/SCID/gamma(c)(null) mouse: an
excellent recipient mouse model for engraftment of human cells.
Blood 2002, 100(9): 3175-3182) (FIG. 7). Since the Rag2/.gamma.
model also lacks adaptive T and B cells, innate immune natural
killer cells, as well as dendritic cell function, other unknown
consequences affecting immune signaling and homeostasis may be
possible. It has also recently been reported that neutrophils, and
then macrophages, are recruited as first and second line responders
in an initiated fibrotic cascade (Grainger D W. All charged up
about implanted biomaterials. Nat Biotechnol 2013, 31(6):
507-509.). To further clarify the role of macrophages and
neutrophils in fibrosis we induced depletion of macrophages,
neutrophils, or both together by clodronate liposome (clodrosome)
and/or targeted Ly6g (clone 1A8)-antibody depletion, in implanted
wild type C57BL/6 mice (FIG. 13A and FIG. 14A-D). Depletion was
started 3 days prior to implantation in all cases in order to
ensure immune population ablation prior to alginate microcapsule
exposure. The extent and specificity of all targeted depletions was
verified by FACS (FIGS. 13B-C, FIGS. 15A-C, and FIGS. 16A-C).
Interestingly, fibrosis was only eliminated with targeted
macrophage depletion, both alone and in combination with neutrophil
removal (FIG. 13A and FIGS. 14A-D). Neutrophil depletion caused
implanted alginate spheres to clump more aggressively, an effect
seen with exacerbated immunity in the case of smaller diameter
spheres (Veiseh O, Doloff J C, Ma M, Vegas A J, Tam H H, Bader A R,
et al. Size- and shape-dependent foreign body immune response to
materials implanted in rodents and non-human primates. Nature
materials 2015, 14(6): 643-651) (FIG. 13A and FIG. 14A-D). This
maybe a consequence of the loss of the Ly6g-granulocyte myeloid
derived suppressor cell (MDSC) subset, which like T.sub.regs,
prevent excessive immune reactions (Wood K J, Bushell A, Hester J.
Regulatory immune cells in transplantation. Nat Rev Immunol 2012,
12(6): 417-430.).
[0160] Lastly, in order to elucidate immune cell signaling, RNA
samples derived from all day 14 treatment groups (fibrosed
capsules+epididymal/omental fat): mock vs. alginate-implanted wild
type, and knockouts or depletions) were analyzed in parallel with a
multiplexed NanoString probe set for all known mouse cytokines and
cytokine receptors (FIGS. 13D-F and FIG. 17). Numerous genes,
induced in alginate-implanted over mock (saline injected), after a
14-day period in wild type C57BL/6 mice (2.sup.nd versus 1.sup.st
column), were eliminated upon macrophage depletion, both alone or
in combination with neutrophil depletion (3.sup.rd and 5.sup.th
columns). Many of these hits were then corroborated by expression
enrichment (increased in red) or dilution (either decreasing back
to mock levels, black, or below, green) upon sorting and analyzing
just macrophages from extracted samples, with comparison to levels
in the original mixed tissue population in mock implanted mice
(2.sup.nd to last vs. 1.sup.st column). Macrophage-specific gene
subsets were identified reflecting macrophage-specific factors,
concomitantly lost upon macrophage depletion and increased in
expression upon analysis of macrophages isolated and enriched by
cell sorting (FIGS. 13D-E, partial). In addition, genes lost upon
macrophage depletion, and not enriched, but instead reduced upon
macrophage sorting, were identified (FIG. 13E, partial, & FIG.
13F). This subset is associated with cells (ie., B cell marker
CD19) and recruitment events downstream of macrophages in the
fibrotic cascade. Furthermore, since expression of this second gene
category is lost upon macrophage depletion; they are therefore
dependent on and only occur downstream of successful macrophage
activation and recruitment. Additional genes, not affected by any
of the immune population depletions utilized in this study, were
also identified, and are likely associated with upstream
biomaterial-induced tissue damage and inflammation events prior to
immune cell recruitment (FIG. 18A-D).
Example 5: CSF1R Inhibition Prevents Immune and Fibrotic Responses
to Implanted Microcapsules
[0161] While elimination of macrophages by clodrosome treatment can
prevent fibrosis of hydrogel alginate (FIG. 13A), it is unlikely
this approach would be suitable for human use (Diel I J, Bergner R,
Grotz K A. Adverse effects of bisphosphonates: current issues. The
journal of supportive oncology 2007, 5(10): 475-482.). An ideal
anti-fibrotic drug therapy would only modulate and not eliminate
this immune population, thus avoiding unnecessary immune
suppression and resulting side effects. One macrophage-specific
factor identified by our NanoString analysis was the cytokine
receptor CSF1R (FIG. 13D). This receptor had been previously
reported to play a role in selectively polarizing and modulating
macrophage phenotypes in cancer (Pyonteck S M, Akkari L,
Schuhmacher A J, Bowman R L, Sevenich L, Quail D F, et al. CSF-1R
inhibition alters macrophage polarization and blocks glioma
progression. Nat Med 2013, 19(10): 1264-1272.). To test whether
inhibition of this receptor could prevent macrophage-dependent
biomaterial fibrosis, we examined the potential the inhibitory
small molecule GW2580 to inhibit CSF1R (Conway J G, McDonald B,
Parham J, Keith B, Rusnak D W, Shaw E, et al. Inhibition of
colony-stimulating-factor-1 signaling in vivo with the orally
bioavailable cFMS kinase inhibitor GW2580. Proceedings of the
National Academy of Sciences of the U.S. Pat. No. 2,005,102(44):
16078-16083) and the fibrotic response to implanted microspheres.
Treatment of C57BL/6 mice with GW2580 prevented the fibrotic
response to a range of implanted microcapsules, including 500 .mu.m
alginate spheres, and 500 .mu.m glass and polystyrene spheres, all
implanted for 14 days into the intraperitoneal space (FIGS. 19A-D,
FIGS. 20A-B, FIGS. 21A-C, and FIGS. 22A-C). CSF1R blockade by
GW2580 was as effective as clodrosome-based macrophage depletion at
inhibiting host immune attack to alginate, glass and polystyrene
spheres (FIG. 13C vs. FIG. 19B, and FIG. 19D). Uninhibited fibrotic
responses against all three materials importantly share the same
core immune marker, cytokine and cytokine receptor signaling,
including CSF1R (FIG. 19E and FIG. 23). This response was dependent
upon the implanted material, and not endotoxin, as analysis did not
detect the presence of general pyrogens, endotoxins or glucan (See
Table 1 above).
[0162] RNA samples derived from alginate-implanted C57BL/6 mice
also treated with the CSF1R small molecule inhibitor GW2580, were
also analyzed and their gene expression patterns were integrated
into the cytokine genetic profiling array. This was done to
determine which of the earlier identified macrophage-specific and
(non-associated) inflammation genes would or would not be affected
upon CSF1R blockade (FIG. 13D vs. FIG. 13E, and FIG. 18A vs. FIG.
18B). Specifically, we inspected all macrophage-associating genes
that were both significantly increased 2 weeks following
implantation into the intraperitoneal space (WT vs Mock controls),
as well as eliminated or significantly decreased following
macrophage depletion. Interestingly, the presence of many
macrophage-specific factors not affected by CSF1R blockade (FIG.
13E) suggests possible spared macrophage function, not observed
with more blunt macrophage depletion and removal. This theory is
supported by the presence of a large non-immune-associated (due to
the lack of removal following any of the earlier serial depletions)
and, as such, likely upstream inflammation-associated gene family
that was decreased back to mock implanted (saline) control levels
upon CSF1R inhibition by GW2580 treatment (FIG. 18B). Many of these
factors are known stress/inflammation response genes that are
decreased or turned off as tissue is repaired due to spared
residual macrophage repair function, as a consequence of their
polarization upon CSF1R inhibition (Pyonteck S M, Akkari L,
Schuhmacher A J, Bowman R L, Sevenich L, Quail D F, et al. CSF-1R
inhibition alters macrophage polarization and blocks glioma
progression. Nat Med 2013, 19(10): 1264-1272.). Further supporting
the hypothesis of preserved macrophage tissue repair function
(MacDonald K P, Palmer J S, Cronau S, Seppanen E, Olver S, Raffelt
N C, et al. An antibody against the colony-stimulating factor 1
receptor depletes the resident subset of monocytes and tissue- and
tumor-associated macrophages but does not inhibit inflammation.
Blood 2010, 116(19): 3955-3963), tissue/cell stress chemokine
CXCL10 levels were increased significantly in inflamed
intaperitoneal epididymal and omental fat pad tissue immediately
adjacent to implanted alginate spheres in both macrophage depletion
treatment groups, instead of being decreased back to low levels, as
normally seen in both non-depleted as well as CSF1R inhibited B6
mice (FIG. 18C). VEGF protein production, directly associated with
macrophage repair function (Laskin D L, Sunil V R, Gardner C R,
Laskin J D. Macrophages and tissue injury: agents of defense or
destruction? Annual review of pharmacology and toxicology 2011, 51:
267-288), was significantly diminished in both macrophage depletion
groups, but spared to normal levels with GW2580 treatment (FIG.
18D).
Example 6: Innate Immune Macrophages Recruit Fibrosis-Potentiating
Adaptive B Cells Via Chemokine CXCL13
[0163] The chemokine CXCL13 has been shown to be expressed by
monocytes/macrophages and responsible for B cell recruitment in a
model of lymphoid neogenesis (Carlsen H S, Baekkevold E S, Morton H
C, Haraldsen G, Brandtzaeg P. Monocyte-like and mature macrophages
produce CXCL13 (B cell-attracting chemokine 1) in inflammatory
lesions with lymphoid neogenesis. Blood 2004, 104(10): 3021-3027.).
Interestingly, CXCL13 was also identified by profiling the kinetics
of host immune adhesion and fibrotic sequestration (FIG. 3D and
FIG. 6C), as well as macrophage depletion, sorting and cytokine
NanoString characterization (FIG. 13D). CXCL13 was one of numerous
immune markers and cytokines that either disappeared or were
decreased in expression below background levels following
macrophage depletion. Conversely, CXCL13 was also enriched upon
sorting the macrophage population away from the bulk heterogeneous
host cell infiltrate. To determine whether it plays a role in B
cell recruitment in the observed biomaterial-induced fibrotic
cascade, neutralizing antibody was administered to
alginate-implanted wild type C57BL/6 mice. CXCL13 neutralization
resulted in loss of B cell recruitment and reduced fibrosis (FIG.
19A, FIG. 19F, and FIGS. 24A-D), similar to levels observed with B
cell loss (FIG. 19G and FIG. 8, B KO). These findings suggest a
role for B cells in potentiating fibrosis, perhaps due to their
ability to regulate macrophage phenotype and response (Affara N I,
Ruffell B, Medler T R, Gunderson A J, Johansson M, Bornstein S, et
al. B cells regulate macrophage phenotype and response to
chemotherapy in squamous carcinomas. Cancer cell 2014, 25(6):
809-821.). Interestingly, B cell loss was also observed with both
clodrosome macrophage depletion and CSF1R inhibition (FIG. 13B,
FIG. 13C, FIG. 13F, and FIG. 19B), once again suggesting that
macrophages are responsible for their downstream recruitment.
NanoString and western blot analysis for CXCL13 over 1, 4, 7, 14,
and 28-day alginate implantation showed initial expression at both
the RNA and protein levels by day 4 onward (FIG. 3D and FIG. 6C),
correlating with maturation of macrophages from immature monocytes
(FIG. 3C), and just preceding apparent day 7 B cell recruitment
(FIG. 3B and FIG. 3G). Taken together, these results demonstrate
that macrophages are required and responsible for downstream B cell
recruitment via the B cell chemoattractant CXCL13, and that, upon
arrival, these B cells enhance fibrotic deposition. Also
highlighting the translational significance of these findings,
CXCL13 was recently shown in human patients to also be a biomarker
for idiopathic pulmonary fibrosis (Vuga L J, Tedrow J R, Pandit K
V, Tan J, Kass D J, Xue J, et al. C-X-C motif chemokine 13 (CXCL13)
is a prognostic biomarker of idiopathic pulmonary fibrosis.
American journal of respiratory and critical care medicine 2014,
189(8): 966-974.).
Example 7: Immune Factors Identified in Response to Implanted
Alginate in C57BL/6 Mice are Also Preserved in Non-Human
Primates
[0164] Next, with potential implications for clinical translation,
we wanted to study if these findings were also relevant in higher
order non-human primates (NHPs). To evaluate this, we implanted 500
.mu.m diameter SLG20 alginate spheres, either intraperitoneally
(N=2) or subcutaneously into the dorsal regions (N=4) of cynomolgus
monkeys for 28 days. At 28 days post-implantation the 500 .mu.m
SLG20 alginate spheres were heavily embedded in host omental or
subcutaneous tissues upon minimally invasive laparoscopic surgery
or biopsy punch retrieval (FIG. 25A and Ref.: Veiseh O, Doloff J C,
Ma M, Vegas A J, Tam H H, Bader A R, et al. Size- and
shape-dependent foreign body immune response to materials implanted
in rodents and non-human primates. Nature materials 2015, 14(6):
643-651.). Excised tissue obtained from the IP implanted 500 .mu.m
SLG20 alginate spheres or mock control tissue were examined through
histological analysis with H&E and Masson's Trichrome staining
(FIG. 25B and FIGS. 26A-B). Extensive embedding and fibrosis
buildup (up to 100 .mu.m thick), similar to that seen in C57BL/6
mice, is visible enveloping the implanted spheres. Confocal imaging
of sections taken from retrieved 28-day IP omentum tissue confirmed
extensive macrophage cellular deposition and fibrosis-associated
activated myofibroblast coverage (FIG. 25C and FIGS. 26C-D). Immune
responses (macrophage and myeloid/neutrophil) were determined by
FACS to be similar between C57BL/6 mice and NHP cynomolgus monkeys
(FIG. 25D). Lastly, to confirm that the immune factors identified
in the C57BL/6 model are also relevant for both intraperitoneal as
well as subcutaneous alginate implants isolated previously (Veiseh
O, Doloff J C, Ma M, Vegas A J, Tam H H, Bader A R, et al. Size-
and shape-dependent foreign body immune response to materials
implanted in rodents and non-human primates. Nature materials 2015,
14(6): 643-651) from NHPs, NanoString analysis was performed on RNA
derived from excised fibrotic alginate implants. Results showed
increased expression of numerous factors common to the C57BL/6
model: cytokine receptors and cytokines, such as CSF1R and CXCL13,
and immune markers, such as CD68 and CD66b (in place of Ly6g/Gr1,
which does not exist in NHPs and humans) (FIG. 25E). Together,
these findings support the use of the C57BL/6 mouse model to
recapitulate the foreign body response seen in higher species such
as non-human primate cynomolgus monkeys and human patients. This is
relevant not only for choosing the right mouse strain for research
but also confirming findings in a higher model organism before
moving to human patients. Lastly, the fact that both models share
similar immunologic responses, not just in content but also
kinetics and magnitude, adds additional credence to their use for
research validation before translation into the clinic.
Example 8: Macrophage Functional Testing
[0165] For in vivo wound healing: C57BL/6 mice were anesthetized
(same as above) and shaved, and skin incisions 1.5 cm in length
were made along the midline. Each incision was then wound clipped
shut for both vehicle and daily GW2580-treated C57BL/6 mice. Wound
clips were removed and then replaced each imaging day up until day
7 (required to maintain them up until at least this time), after
which clips were left off completely. Photos were acquired on days
0, 1, 4, 7, 10, and 14 post-incision. After 14 days, mice were
sacrificed, and skin samples were prepared for histological
assessment (H&E and Masson's Trichrome) of both vehicle and
GW2580 treatment groups. Cross-sections through the incision line
were generated to assess the level of in vivo healing following a
14-day treatment regimen. For IP innate immune cell counts, FACS,
and phagocytosis assays: cells were isolated by peritoneal lavage
with 8 mL DPBS, diluted into RPMI, and then subsequently counted,
and aliquoted either for staining and FACS analysis (as described
above), or for phagocytosis assays. Peritoneal exudate macrophages
isolated by IP lavage from (n=5) mice in each treatment group were
immediately plated (400,000 cells/well in a 24-well plate) and
incubated with fluorospheres for 90 minutes to determine phagocytic
activity. 1 .mu.m orange fluorospheres (540/560) (Cat# F13082,
Invitrogen, Carlsbad, Calif.) were added to each well in a volume
of 500 .mu.L from an initial dilution of 20 .mu.L into 10 mL. After
addition, plates were spun down at 1,000 rpm for 1 min, and then
placed in an incubator for 90 minutes to allow IP macrophages time
to phagocytose the 1 .mu.m diameter fluorospheres. Following the
90-minute incubation, all wells were fixed with 4% paraformaldehyde
and stored at 4 C prior to imaging using a fluorescent and
brightfield-capable EVOS microscope (Advanced Microscopy Group).
For ROS activity assessment: protein lysates were prepared from
alginate spheres retrieved 14 days after IP implantation into
untreated, vehicle-treated, and daily GW2580-treated C57BL/6 mice.
50 .mu.g of each lysate were aliquoted twice and incubated for 30
min. at 37 C with two different reactive oxygen specie (ROS)
substrate solutions: 10 .mu.M APF (Cat# A36003, Invitrogen,
Carlsbad, Calif.) and 5 .mu.M CellROX (Cat# C10422, Invitrogen,
Carlsbad, Calif.) diluted in PBS. Following appropriate incubation
times, samples were read in a black-wall 96-well plate with a Tecan
fluorescent-capable Infinite M1000 plate reader. Results of the
macrophage functional testing are shown in FIGS. 27A-G.
SUMMARY
[0166] In summary, we have demonstrated that by inhibiting CSF1R,
as opposed to more blunt macrophage depletion, can eliminate host
immune-mediated recognition and propagation of foreign body
rejection responses for a broad spectrum of materials encompassing
hydrogels, ceramics, and plastics. We believe these findings have
important implications for the integration of a highly
macrophage-specific agent for localized delivery at the
host-material interface of implanted biomedical devices, to prevent
fibrosis and ensure long-term success for a vast range of
biomedical device applications. Lastly, similar host rejection
responses and immunobiology across C57BL/6 mice and cynomolgus
monkeys implicates the clinical importance of these findings.
[0167] The teachings of all patents, published applications and
references cited herein are incorporated by reference in their
entirety.
[0168] While this invention has been particularly shown and
described with references to example embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
32121DNAArtificial Sequenceoligonucleotide primer 1catgttcagc
tttgtggacc t 21220DNAArtificial Sequenceoligonucleotide primer
2gcagctgact tcagggatgt 20322DNAArtificial Sequenceoligonucleotide
primer 3gcaggttcac ctactctgtc ct 22420DNAArtificial
Sequenceoligonucleotide primer 4cttgccccat tcatttgtct
20521DNAArtificial Sequenceoligonucleotide primer 5cgcttccgct
gcccagagac t 21626DNAArtificial Sequenceoligonucleotide primer
6tataggtggt ttcgtggatg cccgct 26721DNAArtificial
Sequenceoligonucleotide primer 7cctgagtggc tgtcttttga c
21822DNAArtificial Sequenceoligonucleotide primer 8acaagagcag
tgagcgctga at 22921DNAArtificial Sequenceoligonucleotide primer
9gatacagcaa tgccaagcag t 211026DNAArtificial
Sequenceoligonucleotide primer 10ttgtgaaggt agcattcaca agtgta
261121DNAArtificial Sequenceoligonucleotide primer 11gcccgagtac
agtctacctg g 211220DNAArtificial Sequenceoligonucleotide primer
12agagatgaat tctgcgccat 201322DNAArtificial Sequenceoligonucleotide
primer 13ccaagagaat gcaaaaggct tt 221420DNAArtificial
Sequenceoligonucleotide primer 14ggggggctgc aacaaccaca
201520DNAArtificial Sequenceoligonucleotide primer 15tgccccttct
ctgatggatt 201623DNAArtificial Sequenceoligonucleotide primer
16tgctcttgac tttgcttctg tga 231720DNAArtificial
Sequenceoligonucleotide primer 17ggaaacctga ccatcgagag
201822DNAArtificial Sequenceoligonucleotide primer 18tgggactatc
catccaccag tt 221920DNAArtificial Sequenceoligonucleotide primer
19cccaggacca tgtgatgcat 202022DNAArtificial Sequenceoligonucleotide
primer 20cttaagatgc ttcagattct ct 222125DNAArtificial
Sequenceoligonucleotide primer 21ggactacaga acagcttgga gaatg
252220DNAArtificial Sequenceoligonucleotide primer 22tacttccagc
ctcgagccac 202320DNAArtificial Sequenceoligonucleotide primer
23gcaaccccct gaaactggta 202423DNAArtificial Sequenceoligonucleotide
primer 24aaggttacct caggctgtgg ata 232526DNAArtificial
Sequenceoligonucleotide primer 25gaagattctg gggcagcatg gcaaag
262620DNAArtificial Sequenceoligonucleotide primer 26tttggaatca
aaacgatcaa 202723DNAArtificial Sequenceoligonucleotide primer
27ctgcgtggcc cttctgctgt cct 232820DNAArtificial
Sequenceoligonucleotide primer 28gggacatttg caaacacgct
202921DNAArtificial Sequenceoligonucleotide primer 29gccttcagac
gagacttgga a 213020DNAArtificial Sequenceoligonucleotide primer
30ctggcctagg gttgggcatt 203122DNAArtificial Sequenceoligonucleotide
primer 31gcttctttgc agctccttcg tt 223220DNAArtificial
Sequenceoligonucleotide primer 32cggagccgtt gtcgacgacc 20
* * * * *