U.S. patent application number 16/097013 was filed with the patent office on 2019-09-26 for targeting the innate immune system to induce long-term tolerance and to resolve macrophage accumulation in atherosclerosis.
This patent application is currently assigned to Icahn School of Medicine at Mount Sinai. The applicant listed for this patent is ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI. Invention is credited to Mounia BRAZA, Raphael DUIVENVOORDEN, Francois FAY, Zahi FAYAD, Willem MULDER, Jordi OCHANDO.
Application Number | 20190290593 16/097013 |
Document ID | / |
Family ID | 60161161 |
Filed Date | 2019-09-26 |
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United States Patent
Application |
20190290593 |
Kind Code |
A1 |
MULDER; Willem ; et
al. |
September 26, 2019 |
TARGETING THE INNATE IMMUNE SYSTEM TO INDUCE LONG-TERM TOLERANCE
AND TO RESOLVE MACROPHAGE ACCUMULATION IN ATHEROSCLEROSIS
Abstract
Methods and compositions for inducing long-term tolerance by
hybrid nanoparticles are provided. Compositions and formulations
comprising hybrid nanoparticles with inherent affinity for innate
immune cells are provided.
Inventors: |
MULDER; Willem; (New York,
NY) ; OCHANDO; Jordi; (New York, NY) ; FAYAD;
Zahi; (Larchmont, NY) ; BRAZA; Mounia; (New
York, NY) ; DUIVENVOORDEN; Raphael; (New York,
NY) ; FAY; Francois; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI |
New York |
NY |
US |
|
|
Assignee: |
Icahn School of Medicine at Mount
Sinai
New York
NY
|
Family ID: |
60161161 |
Appl. No.: |
16/097013 |
Filed: |
May 1, 2017 |
PCT Filed: |
May 1, 2017 |
PCT NO: |
PCT/US2017/030444 |
371 Date: |
October 26, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62329676 |
Apr 29, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/1275 20130101;
A61K 9/5169 20130101; A61P 37/06 20180101; A61K 31/436 20130101;
A61K 45/06 20130101; A61K 9/5123 20130101; A61K 51/1224 20130101;
A61K 38/13 20130101; A61K 9/0019 20130101 |
International
Class: |
A61K 9/51 20060101
A61K009/51; A61K 38/13 20060101 A61K038/13; A61K 31/436 20060101
A61K031/436; A61K 9/00 20060101 A61K009/00; A61P 37/06 20060101
A61P037/06 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under grants
R01 HL118440, R01 HL125703, R01 CA155432, R01 EB009638, K25
EB016673, and P30 CA008748 awarded by the National Institutes of
Health. The government has certain rights in the invention.
Claims
1. A method of inducing immune tolerance comprising administering
to a patient an effective amount of (i) a composition comprising a
high-density lipoprotein-derived nanoparticle (HDL) which comprises
an mTOR inhibitor.
2. The method of claim 1, wherein the mTOR inhibitor is rapamycin
or a pharmaceutically acceptable salt, solvate, poly-morph,
tautomer or prodrug thereof, formulated as rapamycin nanoparticle
(mTOR-HDL).
3. The method of claim 1, wherein the administration promotes
Ly-6C.sup.lo Mo/M.PHI. development.
4. The method of claim 1, wherein the patient has an autoimmune
condition selected from the group consisting of coeliac disease,
type I diabetes, multiple sclerosis, thyroiditis, Grave's disease,
systemic lupus erythematosus, scleroderma, psoriasis, arthritis,
rheumatoid arthritis, alopecia greata, ankylosing spondylitis,
Churg-Strauss Syndrome, autoimmune hemolytic anemia, autoimmune
hepatitis, Behcet's disease, Crohn's disease, dermatomyositis,
glomerulonephritis, Guillain-Barre syndrome, irritable bowel
disease (IBD), lupus nephritis, myasthenia gravis, myocarditis,
pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa,
polymyositis, primary biliary cirrhosis, rheumatic fever,
sarcoidosis, Sjogren's syndrome, ulcerative colitis, uveitis,
vitiligo, and Wegener's granulomatosis.
5.-16. (canceled)
17. A method for prophylaxis of organ or tissue rejection, the
method comprising the step of administering to a patient in need
thereof an effective amount of a composition comprising a
high-density lipoprotein-derived nanoparticle (HDL) which comprises
an mTOR inhibitor.
18. The method of claim 17, wherein the mTOR inhibitor is rapamycin
or a pharmaceutically acceptable salt, solvate, poly-morph,
tautomer or prodrug thereof, formulated as rapamycin nanoparticle
(mTOR-HDL).
19. The method of claim 17, wherein the HDL comprises
1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and
1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC) and further
comprises ApoA-1.
20. The method of claim 17, wherein the patient has undergone an
organ or tissue transplant and the transplanted tissue is lung
tissue, heart tissue, kidney tissue, liver tissue, retinal tissue,
corneal tissue, skin tissue, pancreatic tissue, intestinal tissue,
genital tissue, ovary tissue, bone tissue, tendon tissue, bone
marrow, or vascular tissue.
21. The method of claim 17, wherein the composition is administered
intravenously or intra-arterially.
22. The method of claim 17, further comprising administering to the
patient one or more immunosuppressant agents.
23.-25. (canceled)
26. A composition comprising a high-density lipoprotein-derived
nanoparticle (HDL) which comprises an m-TOR inhibitor.
27. The composition of claim 26, wherein the HDL comprises
1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and
1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC) and further
comprises ApoA-1.
28. The composition of claim 27, wherein the weight ratio of DMPC
to MHPC is about 3:1.
29. The composition of claim 26, wherein the mTOR inhibitor is
rapamycin or a pharmaceutically acceptable salt, solvate,
poly-morph, tautomer or prodrug thereof, formulated as rapamycin
nanoparticle (mTOR-HDL or rapamycin-HDL).
30. A pharmaceutical composition comprising a) pharmaceutically
effective amount of the composition of claim 26 and b) a
pharmaceutically acceptable carrier, diluent, excipient and/or
adjuvant.
31. The pharmaceutical composition of claim 30, further comprising
one or more immunosuppressive agents or anti-inflammatory
agent.
32. The pharmaceutical composition of claim 31, wherein the
immunosuppressant agent is cyclosporine A or FK506.
33.-38. (canceled)
39. A kit comprising the composition of claim 26.
40. The kit of claim 39, wherein said m-TOR inhibitor is
rapamycin.
41. The kit of claim 39, further comprising one or more
immunosuppressive agents.
42. The kit of claim 41, wherein the immunosuppressant agent is
cyclosporine A, FK506 or rapamycin.
43.-59. (canceled)
60. A composition comprising a high-density lipoprotein-derived
nanoparticle (HDL) which comprises rapamycin or a pharmaceutically
acceptable salt, solvate, poly-morph, tautomer or prodrug thereof,
formulated as rapamycin nanoparticle (rapamycin-HDL) and wherein
the HDL comprises 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine
(DMPC) and 1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC),
and wherein the weight ratio of DMPC to MHPC is about 3:1.
61. The composition of claim 60, further comprising ApoA-1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This present application claims priority to U.S. Provisional
Patent Application Ser. No. 62/329,676 filed Apr. 29, 2016, which
is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] Methods and compositions for inducing long-term tolerance by
hybrid nanoparticles are provided. Compositions and formulations
comprising hybrid nanoparticles with inherent affinity for innate
immune cells are provided.
BACKGROUND
[0004] Indefinite allograft survival remains an elusive goal in
organ transplantation. Transplantation requires suppression of the
immune system to prevent organ rejection. Patients undergoing organ
transplantation usually receive an immunosuppressive drug mixture
that includes, but is not limited to, corticosteroids, tacrolimus,
cyclosporine and sirolimus (rapamycin).sup.1-3. Such
immunosuppressive therapy has dramatically improved the short-term
results of organ transplantation. However, all immunosuppressive
agents have serious adverse effects, such as infections, and
considerable metabolic toxicity.sup.4. There is, consequently, an
ongoing need to reduce toxicity derived from chronic
immunosuppressive treatment and, by extension, to improve long-term
survival. Despite efforts to use currently available
immunosuppressive agents in less toxic ways, no alternative regimen
has seriously challenged these drugs' almost universal use.
[0005] Historically, transplant immunologists have attempted to
develop novel tolerogenic protocols by targeting the adaptive
immune response mechanism. Such work has been based on the
observation that T cells are both necessary and sufficient to
induce allograft rejection. However, the induction of
transplantation tolerance achieved in murine models cannot be fully
explained by mechanisms that target only the adaptive immunity,
such as deletion of activated T cells.sup.5-7. Recent advances in
our understanding of how numerous non-specific responses influence
immune activity have revealed how the innate immune system (a)
reacts to organ transplantation and (b) critically influences the
adaptive immune response toward inducing allograft
tolerance.sup.8-14. However, the innate immune system is a
potential in vivo therapeutic target that has not been successfully
explored in organ transplantation.
[0006] Rapamycin is one of the most widely used immunosuppressive
drugs in transplantation. This drug blocks T and B lymphocyte
activation via mTOR inhibition and efficiently inhibits T cell
proliferation.sup.18. However, use of this drug is associated with
severe side effects 19,20, including increased infection
susceptibility.
[0007] In present treatments, allograft survival requires a
cocktail of immunosuppressive drugs. Experimental antibodies
targeting the innate immune system have been shown to induce
long-term tolerance, with severe side effects.
[0008] Thus, there is a need for therapeutics which can modulate
the innate immune system and induce long-term tolerance with few
side effects.
[0009] Atherosclerosis is one of the leading causes of death and
disability in the world. Atherosclerosis involves the deposition of
fatty plaques on the luminal surface of arteries, which in turn
causes stenosis, i.e., narrowing of the artery. Ultimately, this
deposition blocks blood flow distal to the lesion causing ischemic
damage.
[0010] There is still a need to develop more effective therapeutics
for atherosclerosis and novel ones which target plaque
inflammation.
DESCRIPTION OF THE DRAWINGS
[0011] FIGS. 1A-G are diagrams showing an overview of mTOR-HDL
nanoimmunotherapy, allograft model, biodistribution and immune cell
targeting. FIG. 1A is a diagram showing that mTOR-HDL
nanoparticles, synthesized from phospholipids, human APOA1 and
rapamycin, had a discoidal shape as evaluated by transmission
electron microscopy (TEM) and that they can be radiolabeled with
.sup.89Zr. FIG. 1B is a schematic showing BALB/c donor hearts (H2d)
transplanted into fully allogeneic C57BL/6 recipients (H2b)
receiving mTOR nanoimmunotherapy, which are either radiolabeled for
PET imaging and biodistribution, or fluorescently labeled for
distribution among cell subsets of the innate and adaptive immune
system. FIG. 1C are representative micro-PET/CT 3D fusion images of
mice 24 hours after intravenous administration of mTOR-HDL
radiolabeled with .sup.89Zr (.sup.89Zr-mTOR-HDL). The CT image was
used as anatomical reference to create regions of interest to
determine radioactivity concentration in the transplanted heart
(3D-movie is provided as S2 A). FIG. 1D is a graph of radioactivity
counting showing biodistribution of .sup.89Zr-mTOR-HDL in tissues
of interest (kidney, liver, spleen, blood, bone, skin, and muscle)
24 hours post injection. The radioactivity content was expressed as
percentage of injected dose per gram of tissue (% ID/g). Error bars
are standard error of the mean (SEM), n=3. FIG. 1E is
autoradiography determined radiotracer distribution in native (N)
vs. transplanted heart (Tx) at 24 h post-intravenous administration
of .sup.89Zr-mTOR-HDL in the same recipient. Quantification was
carried out using Image J software. Error bars are standard
deviations (SD), n=3. FIG. 1F are graphical representations of flow
cytometry gating strategy to distinguish myeloid cells in blood,
spleen and the transplanted heart. Grey histograms show immune cell
distribution in the mice injected with DiO-labeled mTOR-HDL
compared to control (black histogram). FIG. 1G are graphs showing
mean fluorescence intensity (MFI) of neutrophils,
monocytes/macrophages, Ly-6C.sup.lo and Ly-6C.sup.hi
monocytes/macrophages, dendritic cells and T cells in the blood,
spleen and the transplanted heart is shown. Error bars are standard
error of the mean (SEM), n=4; ANOVA *P.ltoreq.0.05;
**P.ltoreq.0.01.
[0012] FIG. 2A-C are images and graphs showing that mTOR-HDL
nanoimmunotherapy rebalances the innate immune system. FIG. 2A are
graphs showing total numbers of graft-infiltrating leukocytes,
neutrophils, macrophages and dendritic cells. Flow cytometric
analysis of different cell subsets in the transplanted heart of
placebo, Oral-Ra and mTOR-HDL-treated recipients at day 6
post-transplantation is shown (ANOVA *P.ltoreq.0.05;
**P.ltoreq.0.01). FIG. 2B are graphical representations showing
frequency of Ly-6C.sup.hi vs. Ly-6C.sup.lo macrophages in the
transplanted heart from placebo, Oral-Ra and mTOR-HDL-treated
recipients are shown. Data represents mean.+-.SEM; n=4 per group;
ANOVA *P.ltoreq.0.05; **P.ltoreq.0.01. FIG. 2C displays images of
GSEA gene array analysis. Results indicate that the mTOR pathway is
down-regulated in Ly-6C.sup.lo intra-graft macrophages from
mTOR-HDL treated recipients. Heatmaps derived from the GSEA data of
selected genes that achieve p<0.05 in Ly-6C.sup.lo macrophages
from the allografts of mTOR-HDL treated recipients at day 6
post-transplantation are shown (means of n=3 per group).
[0013] FIG. 3A-G are diagrams and graphs showing that HDL
nanoimmunotherapy induces accumulation of regulatory macrophages
and promotes graft acceptance. FIG. 3A are images showing
functional characterization of graft-infiltrating Ly-6C.sup.lo and
Ly-6C.sup.hi M.PHI. and Ly-6 G neutrophils from placebo and
mTOR-HDL treated mice 6 days post-transplantation. Representative
and quantitative flow cytometry results for Ly-6C and Ly-6 G
expression in CD45.sup.+CD11b.sup.+ allografts, myeloid cell
subsets from the placebo and mTOR-HDL-treated allograft recipients
(top). In vitro suppressive capacity of graft-infiltrating
Ly-6C.sup.lo M.PHI. from placebo and mTOR-HDL-treated mice was
measured. Quantitative flow cytometry results for CFSE CD8.sup.+ T
cells, with cell proliferation percentage measured by CSFE dilution
after 72 hours are shown (middle). In vitro T-reg expansion
capacity of graft-infiltrating Ly-6Clo M.PHI. from placebo and
mTOR-HDL-treated mice was evaluated. Flow cytometric analysis
indicates percentage of Foxp3 expression on CD4+ T cells after
co-culture for 72 hours (bottom). Data are shown as mean.+-.SEM;
n=4 per group; t-test **P.ltoreq.0.01. FIG. 3B are images showing
percentage of graft-infiltrating CD4.sup.+CD25.sup.+ vs.
CD4.sup.+CD25.sup.- T-cells from placebo and mTOR-HDL-treated
allograft recipients. Data are shown as mean.+-.SEM; n=4 per group;
t-test **P.ltoreq.0.01. FIG. 3C are scatter plots and graphs
showing phenotypic characterization of graft-infiltrating
Ly-6C.sup.lo and Ly-6C.sup.hi M.PHI. and Ly-6 G neutrophils, at day
6 post-transplantation, from mTOR-HDL-treated mice following
Ly-6Clo M.PHI. depletion. Representative and quantitative flow
cytometry results of graft-infiltrating CD45.sup.+CD11b.sup.+
myeloid cell subsets of mTOR-HDL-treated CD169-DTR recipients
receiving DT for Ly-6Clo M.PHI. depletion. Data are shown as
mean.+-.SEM; n=4 per group; t-test **P.ltoreq.0.01. FIG. 3D is a
Kaplan-Meier curve showing graft survival following Ly-6C.sup.lo
macrophage depletion in mTOR-HDL treated recipients. Results
indicate that adoptive transfer of wild type monocytes restore
tolerance in mTOR-HDL treated macrophage depleted recipients (n=4
mice in each group; Kaplan-Meier **P.ltoreq.0.01). FIG. 3E is a
box-plot of the gene array for the expression of CD40 in
Ly-6C.sub.lo macrophages obtained from the allografts of placebo
versus mTOR-HDL treated recipients (means of n=3 per group; t-test
**P.ltoreq.0.01). FIG. 3F is a Kaplan-Meier curve showing graft
survival of mTOR-HDL recipients receiving agonistic stimulatory
CD40 mAb in vivo with or without TRAF6i-HDL nanoimmunotherapy (n=5
mice in each group; Kaplan-Meier **P.ltoreq.0.01). FIG. 3G is a
Kaplan-Meier curve showing graft survival curves of placebo,
Oral-Ra, mTOR-HDL and mTOR-HDL/TRAF6i-HDL combination therapy (n=8
mice in each group, Kaplan-Meier survival analysis; P.ltoreq.0.001
placebo vs. mTOR-HDL, P.ltoreq.0.01 Oral-Ra vs. mTOR-HDL,
P.ltoreq.0.01 TRAF6i-HDL vs. mTOR-HDL/TRAF6i-HDL, P.ltoreq.0.01
mTOR-HDL vs. mTOR-HDL/TRAF6i-HDL).
[0014] FIG. 4 is a transmission electron micrograph showing the
discoidal morphology of mTOR-HDL.
[0015] FIGS. 5A-C are graphs and images showing physiological
biodistribution and mTOR-HDL targeting in C57/B16 wild type mice.
FIG. 5A shows representative near infrared fluorescence images
(NIRF) of organs injected with either PBS control (first row of
organs) or DiR-labeled mTOR-HDL 24 hours before transplantation
show accumulation in liver, spleen, lung, kidney, heart and muscle.
The right panel is a graph with bars representing the control to
mTOR-HDL-DiR accumulation ratio in each organ, calculated by
dividing the total signal of each organ in the control and
mTOR-HDL-DiR groups. Error bars are standard error of the means
(SEM.), n=4; *P.ltoreq.0.05; **P.ltoreq.0.01, ***P.ltoreq.0.001.
FIG. 5B is a graph showing myeloid cell distribution in blood and
spleen. Grey histograms (right) show distribution in mice injected
with DiO-labeled mTOR-HDL compared to distribution in control
animals (black histogram). FIG. 5C are graphs showing mean
fluorescence intensity (MFI) of neutrophils, monocyte/macrophage
pool, Ly-6Clo/Ly-6Chi monocytes and dendritic cells in blood and
spleen. Error bars are standard error of the means (SEM.), n=4;
*P.ltoreq.0.05; **P.ltoreq.0.01.
[0016] FIG. 6 is a graph showing PET-quantified uptake values
according to the mean % ID/g in transplanted heart, kidney, liver
and spleen, n=3.
[0017] FIGS. 7A-B are graphs and flow cytometry images showing
mTOR-HDL nanoimmunotherapy does not target T lymphocytes. FIG. 7A
is scatter plot showing flow cytometry gating strategy to
distinguish T cells in blood and the transplanted heart. Grey
histograms (right) show the T cell distribution in mice injected
with DiO-labeled mTOR-HDL compared to distribution in control
animals (black histogram). FIG. 7B are graphs showing mean
fluorescence intensity (MFI) of monocytes/macrophages, CD3.sup.+ T,
CD4.sup.+ T and CD8.sup.+ T-cells in blood and the transplanted
heart. Error bars are standard error of the mean (SEM.), n=4;
**P.ltoreq.0.01; ***P.ltoreq.0.001.
[0018] FIG. 8 is graphs showing flow cytometric analysis of cell
suspensions retrieved from blood and spleen of placebo, Oral-Ra and
mTOR-HDL-treated allograft recipients at day 6 post
transplantation. Data are shown as mean.+-.SEM; n=4 per group;
*P.ltoreq.0.05; **P.ltoreq.0.01.
[0019] FIGS. 9A-B are diagrams and graphs relating to the frequency
of Ly-6C.sup.hi vs. Ly-6C.sup.lo monocytes in the blood and spleen
from placebo, Oral-Ra and mTOR-HDL-treated allograft recipients.
FIG. 9B are graphs showing a ratio of Ly-6C.sup.hi to Ly-6C.sup.lo
monocytes in the blood, spleen and transplanted hearts of placebo,
Oral-Ra and mTOR-HDL-treated allograft recipients. Data are shown
as mean.+-.SEM; n=4 per group; *P.ltoreq.0.05; **P.ltoreq.0.01.
[0020] FIG. 10 is a graph showing TNF-.alpha. secretion 6 days
post-transplantation in sera from placebo, Oral-Ra and
mTOR-HDL-treated allograft recipients, as analyzed by ELISA.
[0021] FIGS. 11A-B are transmission electron micrographs showing
the discoidal morphology of TRAF6i-HDL. The nanoparticles had a
mean hydrodynamic radius of 19.2.+-.3.1 nm and a drug incorporation
efficiency of 84.6.+-.8.6%, as determined by DLS and HPLC
respectively. FIG. 11B shows that the disc shape of the TRAF6i-HDL
particles can be appreciated when particles are in stacked
formation, while the size of the nanoparticles can be evaluated
when observing particles from a top down perspective.
[0022] FIGS. 12A-B are images and a Kaplan-Meier curve showing that
mTOR-HDL nanoimmunotherapy dramatically prolongs skin allograft
survival. FIG. 12A are images showing skin allograft rejection in
control and mTOR-HDL-treated mice at different time points
post-transplantation, as documented by a microscope with a digital
camera. FIG. 12B is a Kaplan-Meier curve of skin allografts (n=4
mice in each group, P.ltoreq.0.01 between Placebo and
mTOR-HDL).
[0023] FIGS. 13A-B are graphs showing kidney and liver images (FIG.
13A) and heart immunohistochemistry (IHC) (FIG. 13B) for toxicity
evaluation. The kidney and liver representative images of IHC for
hematoxylin/eosin (H&E), Periodic acid-Schiff (PAS) and
Masson's Trichrome (Masson) show no signs of toxicity. Kidney and
liver from mTor/TRAF6i-HDL treated recipients were collected at day
100 after transplantation (n=4; magnification X200). In FIG. 13B
the representative images of IHC for H&E and Sirius Red show no
signs of chronic allograph vasculopathy (CAV). Heart allografts
from mTor-HDL/TRAFi-HDL treated recipients were collected at dat
100 after transplantation (n=4; magnification X200). For FIG. 13B,
the chronic allograft vasculopathy analysis, the sections show mild
cicumferential inflammation without arteritis and no signs of
intimal hyperplasia. Mouse aortic segments did not exhibit any
histological alteration with no intimal thickening, and no signs of
CAV.
[0024] FIGS. 14A-G are images, schematics and graphs showing
TRAF6i-HDL nanoparticle biodistribution and uptake. Eight week old
Apoe-/- mice were fed a high-cholesterol diet for 12 weeks and then
received an IV injection with either 89Zr-, DiR- or DiO-labeled
TRAF6i-HDL nanoparticles. Twenty-four hours later, mice were used
for PET/CT imaging or sacrificed for ex vivo NIRF imaging or flow
cytometry analysis. FIG. 14A is a schematic representation of
TRAF6i-HDL, which was created by combining human apoA-I, lipids
(DMPC and MHPC) and a small molecule inhibitor of the CD40-TRAF6
interaction. FIG. 14B is a study overview showing the subsequent
steps that were taken to investigate TRAF6i-HDL. FIG. 14C is a
graph showing pharmacokinetics of .sup.89Zr-labeled TRAF6i-HDL in
Apoe-\- mice, showing the blood decay curve (left panel) and whole
body 3D-rendered PET/CT fusion image at 24 hours post
administration (right panel) showing the highest uptake in the
liver, spleen and kidneys. FIG. 14D is a graph of gamma counting of
the distribution of .sup.89Zr-labeled TRAF6i-HDL at 24 hours post
administration. Autoradiography of the aorta shows visible
TRAF6i-HDL accumulation in the aortic root, which is the
preferential location of atherosclerosis development in the mouse
model. FIG. 14E shows NIRF imaging of DiR-labeled TRAF6i-HDL
distribution in mouse aorta (n=2), and corresponding graphs showing
accumulation of TRAF6i-HDL in the aortic root area. FIG. 14F are
flow cytometry data of whole mouse aortas (n=8) with DiO-labeled
TRAF6i-HDL, showing high targeting efficiency of macrophages and
Ly6C.sup.hi monocytes, while lineage positive CD11b negative cells
did not take up nanoparticles. *** p<0.001. FIG. 14G are images
of flow cytometry analysis of bone marrow, blood, spleen and aorta
cells, showing that Ly6C.sup.hi monocytes and macrophages took up
DiO labeled TRAF6i-HDL. Neutrophils, Ly6C.sup.lo monocytes and
dendritic cells also took up DiO-TRAF6i-HDL, while lineage positive
cells (all non-myeloid cells) did not. Bars represent the standard
error of the mean.
[0025] FIGS. 15A-B are images and graphs illustrating that
TRAF6i-HDL therapy decreased plaque macrophage content as assessed
by histology. Eight week old Apoe-/- mice were fed a
high-cholesterol diet for 12 weeks and subsequently received
treatment with four i.v. injections of either PBS (n=10), rHDL
(n=10) or TRAF6i-HDL (n=10), over the course of seven days.
Twenty-four hours after the last injection, aortic roots were
sectioned (4 .mu.M) and stained with immunohistochemistry methods.
FIG. 15A are images and graphs of aortic roots showing no
difference in plaque size (H&E), collagen content (Sirius Red),
or number of proliferating cells (Ki67 staining) between the
treatment groups. FIG. 15B are images and graphs showing Mac3
staining of aortic roots illustrating a marked decrease in
macrophage positive area and a lower macrophage to collagen ratio.
** p<0.01, and *** p<0.001.
[0026] FIGS. 16A-E are images and graphs showing that TRAF6i-HDL
decreases plaque inflammation due to impaired Ly6C.sup.hi monocyte
recruitment. Eight week old Apoe-/- mice on a high-cholesterol diet
for 12 weeks and were treated with four i.v. injections of either
placebo (PBS), rHDL or TRAF6i-HDL within a single week. FIG. 16A
are images and a graph of FMT/CT imaging showing markedly decreased
protease activity in the aortic root in the TRAF6i-HDL (n=7) as
compared to the placebo (n=8) treated group. FIG. 16B are images of
flow cytometry analysis of whole aortas shows a significant
reduction in the number of macrophages in the TRAF6i-HDL (n=27)
treated group, compared to placebo (n=27) and rHDL (n=26). The fact
that Ly6Chi monocytes are also markedly reduced in the TRAF6i-HDL
group indicates impairment of Ly6Chi monocyte recruitment. FIG. 16C
are images and graphs of flow cytometry analysis of bone marrow,
blood and spleen showed that the decrease in plaque Ly6C.sup.hi
monocyte content could not be attributed to systemic decreases in
Ly6C.sup.hi monocytes. (FIG. 16D are images of in vivo BrdU
incorporation experiments showing no effect of TRAF6i-HDL on plaque
macrophage proliferation. FIG. 16E are graphs from in vitro
experiments (n=3) of BrdU incorporation in RAW 264.7 macrophages
treated for 24 hours, with either placebo, rHDL, TRAF6i-HDL, bare
CD40-TRAF6 small molecule inhibitor or a combination of rHDL+bare
CD40-TRAF6 small molecule inhibitor, showed no effect on macrophage
proliferation. ** p<0.01, and ***p<0.001.
[0027] FIGS. 17A-D are graphs and diagrams reflecting data from
whole transcriptome analysis of plaque monocytes/macrophages
illustrating the effect of TRAF6i treatment on cell migration,
among other affected processes. Eight week old ApoE-/- mice were
fed a high-cholesterol diet for 12 weeks and were then treated with
four i.v. injections of either placebo (n=10) or TRAF6i-HDL (n=10)
over seven days. Twenty-four hours after the last injection, mice
were sacrificed and frozen sections of aortic roots were used for
the isolation of plaque macrophages by laser capture
microdissection, followed by RNA isolation and sequencing. FIG. 17A
is a Volcano plot, showing the distribution of differentially
expressed (DE) genes in plaque monocytes/macrophages. FIG. 17B is a
graph showing the total number of significantly up- and
down-regulated genes, according to cut-off values of an FDR
threshold of 0.2. The FDR<0.2 corresponds to a p-value
<0.009. (FIG. 17C shows the gene enrichment analysis of the DE
gene set within the gene ontology (GO) database, showing 15 GO
terms that are significantly enriched with DE genes. FIG. 17D is a
schematic representation of a macrophage showing two significantly
altered pathways (focal adhesion and endocytosis) identified by
mapping the 416 DE genes with the Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathway tool. Also depicted are the 8 most
significant DE genes with FDR<0.05 and their location inside the
cell (darker black genes are up-regulated, lighter gray genes are
down-regulated, the genes are listing in FIGS. 23-24).
[0028] FIGS. 18A-C are graphs and images illustrating that
TRAF6i-HDL therapy shows no toxic effects in non-human primates.
Six non-human primates were infused with either placebo (n=3) or
1.25 mg/kg TRAF6i-HDL (n=3). Blood was collected at multiple time
points and the animals were sacrificed 72 hours after infusion.
FIG. 18 A are graphs of complete blood counts showing no effects of
TRAF6i-HDL therapy on lymphocytes, erythrocytes and platelets. FIG.
18B are graphs of extensive blood chemistry analysis showing no
toxic effects of TRAF6i-HDL infusion on hepatic, renal, pancreatic
or muscle cell biomarkers. Lipids, glucose, protein (albumin and
globulin) and electrolytes were also unaffected. FIG. 18C are
images of specimens from liver, kidneys and spleen that were
sectioned and stained (H&E) for histological analysis and
evaluated by a pathologist. No signs of tissue damage or
disturbances in tissue architecture were found in any of the
tissues.
[0029] FIGS. 19A-D are images and graphs showing TRAF6i-HDL
biodistribution in non-human primates. Six non-human primates were
infused with either .sup.89Zr-labeled TRAF6i-HDL (1.25 mg/kg).
Dynamic PET images were acquired within 60 minutes after infusion.
Static PET/MRI scans were performed at 24, 48 and 72 hours. NHP
were sacrificed after 72 hours. Organs were collected for ex vivo
analysis. FIG. 19A are dynamic PET images at 1, 5, 15, 30 and 60
minutes. Images are split up to visualize liver and other organs
separately. The graph shows the quantified uptake in the
represented organs at the different time points. The rotating image
on the right shows a 3D representation of the distribution at 60
min. FIG. 19B are additional static PET/MR images at 24, 48 and 72
hours show the distribution and accumulation of TRAF6i-HDL. The
graph shows the quantified uptake in the represented organs at the
different time points. FIG. 19C includes graphs and images
reflecting gamma counting distribution in NHPs at 24 and 72 hours
post administration of .sup.89Zr-TRAF6i-HDL. FIG. 19D is a graph
showing blood time-activity curve for .sup.89Zr-TRAF6i-HDL in
NHPs.
[0030] FIG. 20 is a table showing complete blood count values of
placebo, HDL and TRAF6i-HDL treated Apoe-/- mice. P-values were
calculated with Kruskal Wallis tests.
[0031] FIG. 21 is a table showing blood chemistry values of placebo
and TRAF6i-HDL treated Apoe-/- mice. P-values were calculated by
Mann Whitney U tests. No significant differences between any of the
groups were observed, except for a minor increase in alkaline
phosphatase.
[0032] FIG. 22 is a table showing differential expression of genes
in Gene Ontology terms. CD68 positive cells from aortic sinus
plaques of Apoe-/- mice were isolated by laser capture
microdissection. 15 GO terms showed enrichment with differential
expressed genes. P-values are shown as adjusted p-values.
[0033] FIG. 23 is a table showing differential expression of genes
in two main identified KEGG pathways. CD68 positive cells from
aortic sinus plaques of Apoe-/- mice were isolated by laser capture
microdissection. Differential expression of genes in two
significant KEGG pathways, Focal adhesion and Endocytosis, between
placebo and TRAF6i-HDL treated Apoe-/- mice. P-values are shown as
unadjusted p-values.
[0034] FIG. 24 is a table showing differential expression of genes
with FDR<0.05. CD68 positive cells from aortic sinus plaques of
Apoe-/- mice were isolated by laser capture microdissection.
Differential expression of genes between placebo and TRAF6i-HDL
treated Apoe-/- mice are shown. P-values are shown as adjusted
p-values.
[0035] FIG. 25 is a table showing differential expression of genes
involved in proliferation, apoptosis and migratory egress. CD68
positive cells from aortic sinus plaques of Apoe-/- mice were
isolated by laser capture microdissection. Differential expression
of genes between placebo and TRAF6i-HDL treated Apoe-/- mice are
shown. Unadjusted p values are shown.
SUMMARY
[0036] Encompassed by the present disclosure is a method for
prolonging allograft survival in a patient, the method comprising
administering an effective amount of the present composition to a
patient in need thereof.
[0037] The present disclosure provides for a method for decreasing
dendritic cell stimulatory capacity in a patient, comprising
administering an effective amount of the present composition to a
patient in need thereof.
[0038] The present disclosure provides for a method for promoting
the development of regulatory macrophages in a patient, comprising
administering an effective amount of the present composition to a
patient in need thereof.
[0039] The present disclosure provides for a method of inducing
transplant tolerance in a patient comprising administering an
effective amount of the present composition to a patient in need
thereof.
[0040] The present disclosure provides for a method of targeting
myeloid cells in a patient comprising administering an effective
amount of the present composition to a patient in need thereof,
wherein the mTOR-HDL reduces Mo/M.PHI. numbers in the circulation
of the patient.
[0041] In certain embodiments, the present composition specifically
targets myeloid cells.
[0042] In certain embodiments, the patient has undergone a
transplant and the transplanted tissue is lung tissue, heart
tissue, kidney tissue, liver tissue, retinal tissue, corneal
tissue, skin tissue, pancreatic tissue, intestinal tissue, genital
tissue, ovary tissue, bone tissue, tendon tissue, bone marrow, or
vascular tissue. In certain embodiments, the transplanted tissue is
an intact organ.
[0043] In certain embodiments, the patient has received an
allogeneic tissue or organ transplant. In certain embodiments, the
present method is performed prior to performance of an allogeneic
tissue or organ transplant. In certain embodiments, the method is
performed in conjunction with an allogeneic tissue or organ
transplant. In certain embodiments, the method is performed within
at least two weeks after an allogeneic tissue or organ
transplant.
[0044] In certain embodiments, the subject or patient is human.
[0045] In certain embodiments, the composition is administered
intravenously or intra-arterially.
[0046] In certain embodiments, the present method further comprises
administering to the patient one or more immunosuppressant agents,
such as cyclosporine A or FK506.
[0047] The present disclosure provides for a method of inducing
immune tolerance comprising administering to a patient an effective
amount of (i) a composition comprising a high-density
lipoprotein-derived nanoparticle (HDL) which comprises an mTOR
inhibitor, and optionally (ii) a composition comprising a
high-density lipoprotein-derived nanoparticle (HDL) which comprises
a CD40-TRAF6 inhibitor. In certain embodiments, the mTOR inhibitor
is rapamycin or a pharmaceutically acceptable salt, solvate,
poly-morph, tautomer or prodrug thereof, formulated as rapamycin
nanoparticle (mTOR-HDL). In certain embodiments, the CD40-TRAF6
inhibitor is 6877002 or a pharmaceutically acceptable salt,
solvate, poly-morph, tautomer or prodrug thereof, formulated as
TRAF6i-HDL nanoparticle.
[0048] In certain embodiments, the administration promotes
Ly-6C.sup.lo Mo/M.PHI. development.
[0049] In certain embodiments, the patient has an autoimmune
condition selected from the group consisting of coeliac disease,
type I diabetes, multiple sclerosis, thyroiditis, Grave's disease,
systemic lupus erythematosus, scleroderma, psoriasis, arthritis,
rheumatoid arthritis, alopecia greata, ankylosing spondylitis,
Churg-Strauss Syndrome, autoimmune hemolytic anemia, autoimmune
hepatitis, Behcet's disease, Crohn's disease, dermatomyositis,
glomerulonephritis, Guillain-Barre syndrome, irritable bowel
disease (IBD), lupus nephritis, myasthenia gravis, myocarditis,
pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa,
polymyositis, primary biliary cirrhosis, rheumatic fever,
sarcoidosis, Sjogren's syndrome, ulcerative colitis, uveitis,
vitiligo, and Wegener's granulomatosis.
[0050] In certain embodiments, the patient is susceptible to or has
an atherosclerotic condition including: coronary atherosclerosis,
diabetic atherosclerosis, a sequela of atherosclerosis, such as
acute coronary syndrome, myocardial infarction, angina pectoris,
peripheral vascular disease, intermittent claudication, myocardial
ischemia, stroke, heart failure and combinations thereof.
[0051] The present disclosure provides for a method of treating
atherosclerosis, the method comprising administering to a patient
an effective amount of a composition comprising a high-density
lipoprotein-derived nanoparticle (HDL) which comprises a CD40-TRAF6
inhibitor. In certain embodiments, the CD40-TRAF6 inhibitor is
6877002 or a pharmaceutically acceptable salt, solvate, poly-morph,
tautomer or prodrug thereof, formulated as TRAF6i-HDL
nanoparticle.
[0052] In certain embodiments, the present method further comprises
administering to the patient an effective amount of a composition
comprising a high-density lipoprotein-derived nanoparticle (HDL)
which comprises an mTOR inhibitor. In certain embodiments, the mTOR
inhibitor is rapamycin or a pharmaceutically acceptable salt,
solvate, poly-morph, tautomer or prodrug thereof, formulated as
rapamycin nanoparticle (mTOR-HDL). In certain embodiments, the HDL
comprises 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC)
and 1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC) and
further comprises ApoA-1.
[0053] In certain embodiments, atherosclerosis includes: coronary
atherosclerosis, diabetic atherosclerosis, a sequela of
atherosclerosis, such as acute coronary syndrome, myocardial
infarction, angina pectoris, peripheral vascular disease,
intermittent claudication, myocardial ischemia, stroke, heart
failure and combinations thereof.
[0054] The present disclosure provides for a method of targeting
macrophages and/or monocytes in a plaque or a vascular inflammatory
site, the method comprising administering to a patient an effective
amount of a composition comprising a high-density
lipoprotein-derived nanoparticle (HDL) which comprises a CD40-TRAF6
inhibitor. In certain embodiments, the CD40-TRAF6 inhibitor is
6877002 or a pharmaceutically acceptable salt, solvate, poly-morph,
tautomer or prodrug thereof, formulated as TRAF6i-HDL
nanoparticle.
[0055] In certain embodiments, the present method further comprises
administering to the patient an effective amount of a composition
comprising a high-density lipoprotein-derived nanoparticle (HDL)
which comprises an mTOR inhibitor. In certain embodiments, the mTOR
inhibitor is rapamycin or a pharmaceutically acceptable salt,
solvate, poly-morph, tautomer or prodrug thereof, formulated as
rapamycin nanoparticle (mTOR-HDL). In certain embodiments, the HDL
comprises 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC)
and 1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC) and
further comprises ApoA-1.
[0056] The present disclosure provides for a method for prophylaxis
of organ or tissue rejection, the method comprising the step of
administering to a patient in need thereof an effective amount of a
composition comprising a high-density lipoprotein-derived
nanoparticle (HDL) which comprises an mTOR inhibitor. In certain
embodiments, the mTOR inhibitor is rapamycin or a pharmaceutically
acceptable salt, solvate, poly-morph, tautomer or prodrug thereof,
formulated as rapamycin nanoparticle (mTOR-HDL). In certain
embodiments, the HDL comprises
1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and
1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC) and further
comprises ApoA-1.
[0057] In certain embodiments, the patient has undergone an organ
or tissue transplant and the transplanted tissue is lung tissue,
heart tissue, kidney tissue, liver tissue, retinal tissue, corneal
tissue, skin tissue, pancreatic tissue, intestinal tissue, genital
tissue, ovary tissue, bone tissue, tendon tissue, bone marrow, or
vascular tissue.
[0058] In certain embodiments, the composition is administered
intravenously or intra-arterially.
[0059] In certain embodiments, the present method further comprises
administering to the patient one or more immunosuppressant
agents.
[0060] Also encompassed by the present disclosure is a method for
slowing the progression of atherosclerosis, the method comprising
the step of administering to a patient in need thereof an effective
amount of a composition comprising a high-density
lipoprotein-derived nanoparticle (HDL) which comprises a CD40-TRAF6
inhibitor. In certain embodiments, the CD40-TRAF6 inhibitor is
6877002 or a pharmaceutically acceptable salt, solvate, poly-morph,
tautomer or prodrug thereof, formulated as TRAF6i-HDL nanoparticle.
In certain embodiments, the HDL comprises
1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and
1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC) and further
comprises ApoA-1.
[0061] The present disclosure provides for a composition comprising
a high-density lipoprotein-derived nanoparticle (HDL) which
comprises an m-TOR inhibitor. In certain embodiments, the HDL
comprises 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC)
and 1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC) and
further comprises ApoA-1. In certain embodiments, the weight ratio
of DMPC to MHPC is about 3:1. In certain embodiments, the mTOR
inhibitor is rapamycin or a pharmaceutically acceptable salt,
solvate, poly-morph, tautomer or prodrug thereof, formulated as
rapamycin nanoparticle (mTOR-HDL or rapamycin-HDL).
[0062] In certain embodiments, the pharmaceutical composition
further comprises one or more immunosuppressive agents or
anti-inflammatory agent. In certain embodiments, the
immunosuppressant agent is cyclosporine A or FK506.
[0063] Also encompassed by the present disclosure is a composition
comprising a high-density lipoprotein-derived nanoparticle (HDL)
which comprises a CD40-TRAF6 inhibitor. In certain embodiments, the
HDL comprises 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine
(DMPC) and 1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC)
and further comprises ApoA-1. In certain embodiments, the weight
ratio of DMPC to MHPC ranges from about 8:1 to about 9:1. In
certain embodiments, the CD40-TRAF6 inhibitor is 6877002, or a
pharmaceutically acceptable salt, solvate, poly-morph, tautomer or
prodrug thereof, formulated as TRAF6i-HDL nanoparticle.
[0064] The present disclosure also provides for a pharmaceutical
composition comprising a) pharmaceutically effective amount of the
present composition, and b) a pharmaceutically acceptable carrier,
diluent, excipient and/or adjuvant.
[0065] The present disclosure provides for a pharmaceutical
composition comprising a) a composition comprising a high-density
lipoprotein-derived nanoparticle (HDL) which comprises an m-TOR
inhibitor, and b) a composition comprising a high-density
lipoprotein-derived nanoparticle (HDL) which comprises a CD40-TRAF6
inhibitor.
[0066] The present disclosure provides for a kit comprising the
present composition. In certain embodiments, the m-TOR inhibitor is
rapamycin. In certain embodiments, the kit further comprises one or
more immunosuppressive agents, such as cyclosporine A, FK506 or
rapamycin. In certain embodiments, the CD40-TRAF6 inhibitor is
6877002.
[0067] The present disclosure provides for use of a high-density
lipoprotein-derived nanoparticle (HDL) which comprises an mTOR
inhibitor, and optionally (ii) a high-density lipoprotein-derived
nanoparticle (HDL) which comprises a CD40-TRAF6 inhibitor, in the
preparation of a composition for inducing immune tolerance.
[0068] The present disclosure provides for use of a high-density
lipoprotein-derived nanoparticle (HDL) which comprises a CD40-TRAF6
inhibitor, in the preparation of a composition for treating
atherosclerosis.
[0069] The present disclosure provides for use of a high-density
lipoprotein-derived nanoparticle (HDL) which comprises a CD40-TRAF6
inhibitor, in the preparation of a composition for targeting
macrophages and/or monocytes in a plaque or a vascular inflammatory
site.
[0070] The present disclosure provides for use of a high-density
lipoprotein-derived nanoparticle (HDL) which comprises an mTOR
inhibitor, in the preparation of a composition for prophylaxis of
organ or tissue rejection.
[0071] The present disclosure provides for use of a high-density
lipoprotein-derived nanoparticle (HDL) which comprises a CD40-TRAF6
inhibitor, in the preparation of a composition for slowing the
progression of atherosclerosis.
[0072] The present disclosure provides for use of the present
nanoparticles in the preparation of a composition for prolonging
allograft survival in a patient in need thereof.
[0073] The present disclosure provides for use of the present
nanoparticles in the preparation of a composition for decreasing
dendritic cell stimulatory capacity in a patient in need
thereof.
[0074] The present disclosure provides for use of the present
nanoparticles in the preparation of a composition for promoting the
development of regulatory macrophages in a patient in need
thereof.
[0075] The present disclosure provides for use of the present
nanoparticles in the preparation of a composition for inducing
transplant tolerance in a patient in need thereof.
[0076] The present disclosure provides for use of the present
nanoparticles in the preparation of a composition for targeting
myeloid cells in a patient in need thereof. In certain embodiments,
the mTOR-HDL reduces Mo/M.PHI. numbers in the circulation of the
patient.
DETAILED DESCRIPTION
[0077] A high-density lipoprotein-derived nanoparticle (HDL) has
been developed to deliver rapamycin to innate immune cells. A
hybrid HDL nanoparticle, named Rapamycin-HDL (an exemplary
mTOR-HDL), which encapsulates rapamycin in a corona of natural
phospholipids and apolipoprotein A-I (APOA1) was developed to
prolong allograft survival. HDL-nanoparticles contain APOA1, which
efficiently bind to macrophages cells through the scavenger
receptor type B-1 (sr-b1) and adenosine triphosphate-binding
cassette transporter A1 (ABCA1).sup.21,22. As a result, mTOR-HDL
nanoparticles specifically deliver rapamycin to innate immune cells
in vivo. mTOR-HDL nanoparticles, --15 nm in diameter, had a high
rapamycin encapsulation efficiency of .about.65%. Radiolabeled
mTOR-HDL was observed to specifically accumulate in the
transplanted heart and to be mainly associated with myeloid cells.
The results demonstrate a significant reduction of
Ly-6C.sup.hi/Ly-6C.sup.low as well as CD25.sup.-/CD25.sup.+ cells
in the transplanted heart. This treatment also resulted in a
dramatic enhancement of allograft survival.
[0078] Additionally, the inventors developed an HDL nanobiologic
that incorporates a small molecule inhibitor (TRAF-STOP) directed
against the binding domain of CD40 on TRAF6 (referred to hereafter
as TRAF6i-HDL). The 6877002 inhibitor was used for the development
of this TRAF6i-HDL (the 6877002 inhibitor is described in
Chatzigeorgiou et al. 2014, and also U.S. Pat. No. 9,408,829, as
well as other inhibitors). The TRAF6i-HDL nanoparticles had a mean
hydrodynamic radius of 19.2.+-.3.1 nm and a drug incorporation
efficiency of 84.6.+-.8.6%. The TRAF6i-HDL nanoparticles can be
used alone or in combination with the mTOR-HDL nanoparticles
described herein.
[0079] In alternative embodiments, other CD40-TRAF6 inhibitors such
as SMI 6860766 (described in Van der Berg et al. 2015) can be used
to form alternative TRAF6i-HDLs. These inhibitors can be used alone
or in combination with any of the other nanobiologics as described
herein. Additional suitable compounds for blocking the CD40-TRAF6
interaction are described in U.S. Pat. No. 9,408,829.
[0080] Using an experimental heart transplantation model in
combination with molecular imaging and immunological techniques,
the present data demonstrate that mTOR-HDL restricts dendritic
cells' potent stimulatory capacity, promotes the development of
regulatory macrophages, and prolongs heart allograft survival
indefinitely. The regimen comprised only three intravenous tail
vein injections of 5 mg/kg equivalent rapamycin during the first
week after transplantation. Using a combination of in vivo positron
emission tomography with computed tomography (PET-CT) imaging and
an array of immunological assays, we evaluated heart allograft
targeting and cellular specificity. We subsequently and extensively
studied innate immune response, allograft survival and therapeutic
mechanisms. Our data demonstrate that mTOR-HDL nanoparticle
treatment promotes indefinite heart allograft survival.
Additionally, the inventors were able to extend these results in a
skin transplant model. These results provide critical information
about how to manipulate the immune response toward inducing
donor-specific non-response in the clinic and identify new
therapeutic targets that may prevent allograft rejection in
humans.
[0081] Furthermore, the present data demonstrate that a short-term
therapeutic treatment with mTOR-HDL in combination with an
inhibitory CD40-TRAF6 specific nanoimmunotherapy (TRAF6i-HDL)
synergistically promote organ transplant acceptance leading to
indefinite allograft survival.
[0082] Together, the results demonstrate that HDL-based nanotherapy
represents an effective treatment paradigm for the induction of
transplantation tolerance. This study provides the foundation for
developing novel therapeutic nanomedicinal compounds and treatments
that generate tolerance-inducing immune regulatory macrophages.
Additionally, the TRAF6i-HDL treatment has been shown to resolve
macrophage accumulation in atherosclerosis and to exhibit a
desirable safety and efficacy profile in non-human primates.
Definitions and Methods
[0083] In certain embodiments, compositions of the present
invention include a high-density lipoprotein-derived nanoparticle
(HDL) which comprises an m-TOR inhibitor (indicated as
mTOR-inhibitor-HDL), wherein an example of such as m-TOR inhibitor
is rapamycin or a pharmaceutically acceptable salt, solvate,
poly-morph, tautomer or prodrug thereof, formulated as rapamycin
nanoparticle (an exemplary mTOR-HDL). In alternative embodiments,
the composition may comprise one or more rapamycin derivatives and
potential targets of the rapamycin signaling cascade (S6K).
[0084] In certain embodiments, the composition may further comprise
a pharmaceutically acceptable carrier, diluent, excipient and/or
adjuvant.
[0085] In certain embodiments, the HDL composition can be
administered in combination with one or more additional
immunosuppressive agents such as cyclosporine A, FK506, or
azathioprine, mycophenolate mofetil, and any analogues thereof
(e.g., everolimus, ABT-578, CCI-779, and AP23573).
[0086] In an embodiment, "patient" or "subject" refers to mammals
and includes human and veterinary subjects. In an embodiment, the
subject is mammalian.
[0087] In an embodiment, the compound is administered in a
composition comprising a pharmaceutically acceptable carrier.
[0088] In certain embodiments, the invention relates to a method of
the treatment or prophylaxis of a disorder or disease mediated by
allograft rejection, comprising administering to a patient in need
thereof a therapeutically effective amount of a high-density
lipoprotein-derived nanoparticle (HDL) which comprises an m-TOR
inhibitor, wherein the m-TOR inhibitor is rapamycin or a
pharmaceutically acceptable salt, solvate, poly-morph, tautomer or
prodrug thereof, formulated as rapamycin nanoparticle (mTOR-HDL),
or the pharmaceutical composition thereof. In an embodiment, the
subject is at risk for allograft rejection and the method is for
preventing (i.e., prophylaxis) or inhibiting allograft
rejection.
[0089] Additionally, since any transplant is at risk of rejection,
embodiments include adjuvant therapy using any of the methods or
compositions described herein to prevent any transplant
rejection.
[0090] Diseases mediated by allograft rejection include, but are
not limited to heart transplant, skin transplant, liver transplant,
lung transplant, bronchiolitis-obliterans syndrome (BOS), kidney
transplant, pancreas transplant, pancreatic islets transplant,
intestinal transplant, bone transplant, retinal transplant, bone
marrow transplant, islet transplantation and corneal transplant. In
certain embodiments, treatments are facilitated by administering
mTOR-HDL. In other embodiments, treatments are facilitated by
administering a combination of mTOR-HDL and TRAF6i-HDL, either in a
single HDL or in two separate HDL compositions.
[0091] In certain embodiments, the invention relates to a method of
the treatment or prophylaxis of a disorder or disease mediated by
allograft rejection, comprising administering to a patient in need
thereof a therapeutically effective amount of a (i) high-density
lipoprotein-derived nanoparticle (HDL) which comprises an m-TOR
inhibitor, wherein the m-TOR inhibitor is rapamycin or a
pharmaceutically acceptable salt, solvate, poly-morph, tautomer or
prodrug thereof, formulated as rapamycin nanoparticle (mTOR-HDL),
or the pharmaceutical composition thereof and optionally (ii)
TRAFi-HDL nanoparticles which comprise a CD40-TRAF6 inhibitor,
wherein the CD40-TRAF6 inhibitor is 6877002, or a pharmaceutically
acceptable salt, solvate, poly-morph, tautomer or prodrug thereof,
formulated as an HDL nanoparticle (TRAFi-HDL), or the
pharmaceutical composition thereof. In certain embodiments, the
mTOR-HDL and TRAFi-HDL nanoparticles are administered in
combination, or in sequence to a patient in need thereof. In an
embodiment, the subject is at risk for allograft rejection and the
method is for preventing (i.e., prophylaxis) or inhibiting
allograft rejection. Diseases mediated by allograft rejection
include, but are not limited to heart transplant, skin transplant,
liver transplant, lung transplant, bronchiolitis-obliterans
syndrome (BOS), kidney transplant, pancreas transplant, pancreatic
islets transplant, intestinal transplant, bone transplant, retinal
transplant, and corneal transplant.
[0092] In additional embodiments, the invention relates to a method
of treatment or prophylaxis of an autoimmune disease. Examples of
autoimmune disease include coeliac disease, type I diabetes,
multiple sclerosis, thyroiditis, Grave's disease, systemic lupus
erythematosus, scleroderma, psoriasis, arthritis, rheumatoid
arthritis, alopecia greata, ankylosing spondylitis, Churg-Strauss
Syndrome, autoimmune hemolytic anemia, autoimmune hepatitis,
Behcet's disease, Crohn's disease, dermatomyositis,
glomerulonephritis, Guillain-Barre syndrome, IBD, lupus nephritis,
myasthenia gravis, myocarditis, pemphigus/pemphigoid, pernicious
anemia, polyarteritis nodosa, polymyositis, primary biliary
cirrhosis, rheumatic fever, sarcoidosis, Sjogren's syndrome,
ulcerative colitis, uveitis, vitiligo, and Wegener's
granulomatosis.
[0093] Conditions that may also be treated using the present
compositions and methods include diseases which are associated with
increased inflammation. Schwarz et al., Identification of
differentially expressed genes induced by transient ischemic
stroke, Brain Res Mol Brain Res. 2002; 101(1-2):12-22.
[0094] The present compositions and methods may be used to treat or
prevent a cardiovascular disease, such as atherosclerosis,
stenosis, restenosis, hypertension, heart failure, left ventricular
hypertrophy (LVH), myocardial infarction, acute coronary syndrome,
stroke, transient ischemic attack, impaired circulation, heart
disease, cholesterol and plaque formation, ischemia, ischemia
reperfusion injury, peripheral vascular disease, myocardial
infection, cardiac disease (e.g, risk stratification of chest pain
and interventional procedures), cardiopulmonary resuscitation,
kidney failure, thrombosis (e.g., venous thrombosis, deep vein
thrombosis, portal vein thrombosis, renal vein thrombosis, jugular
vein thrombosis, cerebral venous sinus thrombosis, arterial
thrombosis, etc.), thrombus formation, thrombotic event or
complication, Budd-Chiari syndrome, Paget-Schroetter disease,
coronary heart disease, coronary artery disease, need for coronary
revascularization, peripheral artery disease, a pulmonary
circulatory disease, pulmonary embolism, a cerebrovascular disease,
cellular proliferation and endothelial dysfunction, graft occlusion
or failure, need for or an adverse clinical outcome after
peripheral bypass graft surgery, need for or an adverse clinical
outcome after coronary artery bypass (CABG) surgery, failure or
adverse outcome after angioplasty, internal mammary artery graft
failure, vein graft failure, autologous vein grafts, vein graft
occlusion, ischemic diseases, intravascular coagulation,
cerebrovascular disease, or any other cardiovascular disease
related to obesity or an overweight condition.
[0095] Any type of atherosclerotic lesion may be treated, such as
coronary atherosclerosis, diabetic atherosclerosis, atherosclerosis
and its sequelae (e.g., acute coronary syndrome, myocardial
infarction, angina pectoris, peripheral vascular disease,
intermittent claudication, myocardial ischemia, stroke, heart
failure, etc.).
[0096] In certain embodiments, hydrophobicity of a compound (e.g.,
rapamycin, or any compound described herein) can be modified by
adding a long alkyl chain to the molecule.
[0097] The compounds used in the methods of the present invention
include all hydrates, solvates, and complexes of the compounds used
by this invention. If a chiral center or another form of an
isomeric center is present in a compound of the present invention,
all forms of such isomer or isomers, including enantiomers and
diastereomers, are intended to be covered herein. Compounds
containing a chiral center may be used as a racemic mixture, an
enantiomerically enriched mixture, or the racemic mixture may be
separated using well-known techniques and an individual enantiomer
may be used alone. The compounds described in the present invention
are in racemic form or as individual enantiomers. The enantiomers
can be separated using known techniques, such as those described in
Pure and Applied Chemistry 69, 1469-1474, (1997) IUPAC. In cases in
which compounds have unsaturated carbon-carbon double bonds, both
the cis (Z) and trans (E) isomers are within the scope of this
invention. In cases wherein compounds may exist in tautomeric
forms, such as keto-enol tautomers, each tautomeric form is
contemplated as being included within this invention whether
existing in equilibrium or predominantly in one form.
[0098] When the structure of the compounds used in this invention
includes an asymmetric carbon atom such compound can occur as
racemates, racemic mixtures, and isolated single enantiomers. All
such isomeric forms of these compounds are expressly included in
this invention. Each stereogenic carbon may be of the R or S
configuration. It is to be understood accordingly that the isomers
arising from such asymmetry (e.g., all enantiomers and
diastereomers) are included within the scope of this invention,
unless indicated otherwise. Such isomers can be obtained in
substantially pure form by classical separation techniques and by
stereochemically controlled synthesis, such as those described in
"Enantiomers, Racemates and Resolutions" by J. Jacques, A. Collet
and S. Wilen, Pub. John Wiley & Sons, N Y, 1981. For example,
the resolution may be carried out by preparative chromatography on
a chiral column.
[0099] The subject invention is also intended to include use of all
isotopes of atoms occurring on the compounds disclosed herein.
Isotopes include those atoms having the same atomic number but
different mass numbers. By way of general example and without
limitation, isotopes of hydrogen include tritium and deuterium.
Isotopes of carbon include carbon-13 and carbon-14.
[0100] It will be noted that any notation of a carbon in structures
throughout this application, when used without further notation,
are intended to represent all isotopes of carbon, such as .sup.12C,
.sup.13C, or .sup.14C. Furthermore, any compounds containing
.sup.13C or .sup.14C may specifically have the structure of any of
the compounds disclosed herein.
[0101] It will also be noted that any notation of a hydrogen in
structures throughout this application, when used without further
notation, are intended to represent all isotopes of hydrogen, such
as .sup.1H, .sup.2H, or .sup.3H. Furthermore, any compounds
containing .sup.2H or .sup.3H may specifically have the structure
of any of the compounds disclosed herein.
[0102] Isotopically-labeled compounds can generally be prepared by
conventional techniques known to those skilled in the art or by
processes analogous to those described in the Examples disclosed
herein using an appropriate isotopically-labeled reagents in place
of the non-labeled reagents employed.
[0103] The compounds of the instant invention may be in a salt
form. As used herein, a "salt" is salt of the instant compounds
which has been modified by making acid or base, salts of the
compounds. In the case of compounds used for treatment of cancer,
the salt is pharmaceutically acceptable. Examples of
pharmaceutically acceptable salts include, but are not limited to,
mineral or organic acid salts of basic residues such as amines;
alkali or organic salts of acidic residues such as phenols. The
salts can be made using an organic or inorganic acid. Such acid
salts are chlorides, bromides, sulfates, nitrates, phosphates,
sulfonates, formates, tartrates, maleates, malates, citrates,
benzoates, salicylates, ascorbates, and the like. Phenolate salts
are the alkaline earth metal salts, sodium, potassium or lithium.
The term "pharmaceutically acceptable salt" in this respect, refers
to the relatively non-toxic, inorganic and organic acid or base
addition salts of compounds of the present invention. These salts
can be prepared in situ during the final isolation and purification
of the compounds of the invention, or by separately treating a
purified compound of the invention in its free base or free acid
form with a suitable organic or inorganic acid or base, and
isolating the salt thus formed. Representative salts include the
hydrobromide, hydrochloride, sulfate, bisulfate, phosphate,
nitrate, acetate, valerate, oleate, palmitate, stearate, laurate,
benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,
succinate, tartrate, napthylate, mesylate, glucoheptonate,
lactobionate, and laurylsulphonate salts and the like. (See, e.g.,
Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci.
66:1-19).
[0104] As used herein, "alkyl" includes both branched and
straight-chain saturated aliphatic hydrocarbon groups having the
specified number of carbon atoms and may be unsubstituted or
substituted. The Alkyls are C1-C10 alkyls, or a subset or
individual thereof. In a non-limiting example, where the alkyl is
C1-05 as in "C1-C5 alkyl", it is defined to include groups having
1, 2, 3, 4 or 5 carbons in a linear or branched arrangement and
specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl,
t-butyl, and pentyl. Alkyl may optionally be substituted with
phenyl or substituted phenyl to provide substituted or
unsubstituted benzyl.
[0105] Heterocyclyl means a saturated or partially unsaturated
monocyclic radical containing 3 to 8 ring atoms and preferably 5 to
6 ring atoms selected from carbon or nitrogen but not limited to
pyrrolidine.
[0106] As used herein the term "aryl" refers to aromatic monocyclic
or multicyclic groups containing from 5 to 15 carbon atoms. Aryl
groups include, but are not limited to groups such as unsubstituted
or substituted phenyl. When referring to said aryl being
substituted, said substitution may be at any position on the ring,
other than the point of attachment to the other ring system of a
compound of the invention. Therefore, any hydrogen atom on the aryl
ring may be substituted with a substituent defined by the
invention. In embodiments where the aryl is a phenyl ring, said
substitution may be at the meta- and/or ortho- and/or para-position
relative to the point of attachment. Aryl may optionally be
substituted with a heterocyclyl-C(O)-- moiety which includes a
pyrrolidinyl-C(O)-- moiety.
[0107] The term "heteroaryl" as used herein, represents a stable
monocyclic, bicyclic or polycyclic ring of up to 10 atoms in each
ring, wherein at least one ring is aromatic and contains from 1 to
4 heteroatoms or particularly 1 to 2 heteroatoms selected from the
group consisting of O, N and S. Bicyclic aromatic heteroaryl groups
include phenyl, pyridine, pyrimidine or pyridazine rings that are
(a) fused to a 6-membered aromatic (unsaturated) heterocyclic ring
having one nitrogen atom; (b) fused to a 5- or 6-membered aromatic
(unsaturated) heterocyclic ring having two nitrogen atoms; (c)
fused to a 5-membered aromatic (unsaturated) heterocyclic ring
having one nitrogen atom together with either one oxygen or one
sulfur atom; or (d) fused to a 5-membered aromatic (unsaturated)
heterocyclic ring having one heteroatom. selected from 0, N or S.
Heteroaryl groups within the scope of this definition include but
are not limited to: benzoimidazolyl, benzofuranyl, benzofurazanyl,
benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,
carbazolyl, carbolinyl, cinnolinyl, furanyl, indolinyl, indolyl,
indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl,
isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl,
oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl,
pyridazinyl, pyridopyridinyl , pyridazinyl, pyridyl, pyrimidyl,
pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrazolyl,
tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl,
azetidinyl, aziridinyl, 1,4-dioxanyl, hexahydroazepinyl,
dihydrobenzoimidazolyl, dihydrobenzofuranyl,
dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl,
dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,
dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,
dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,
dihydropyriinidinyl, dihydropyrrolyl, dihydroquinolinyl,
dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,
dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,
methylenedioxybenzoyl , tetrahydrofuranyl, tetrahydrothienyl,
acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl,
indolyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl,
isothiazolyl, furanyl, thienyl, benzothienyl, benzofuranyl,
quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl,
pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl,
tetra-hydroquinoline. In cases where the heteroaryl substituent is
bicyclic and one ring is non-aromatic or contains no heteroatoms,
it is understood that attachment is via the aromatic ring or via
the heteroatom containing ring, respectively. If the heteroaryl
contains nitrogen atoms, it is understood that the corresponding
N-oxides thereof are also encompassed by this definition.
[0108] In the compounds of the present invention, the alkyl, aryl,
or heteroaryl groups can be further substituted by replacing one or
more hydrogen atoms be alternative non-hydrogen groups. These
include, but are not limited to, 1-4 groups selected from alkyl,
alkoxy, halo, hydroxy, mercapto, amino, carboxy, cyano and
carbamoyl.
[0109] The term "substituted" refers to a functional group as
described above in which one or more bonds to a hydrogen atom
contained therein are replaced by a bond to non-hydrogen or
non-carbon atoms, provided that normal valencies are maintained and
that the substitution results in a stable compound. Substituted
groups also include groups in which one or more bonds to a carbon
(s) or hydrogen (s) atom are replaced by one or more bonds,
including double or triple bonds, to a heteroatom. Examples of
substituent groups include the functional groups described above,
and, in particular, halogens (i.e., F, Cl, Br, and I); alkyl
groups, such as methyl, ethyl, n-propyl, isopropryl, n-butyl,
tert-butyl, and trifluoromethyl; hydroxyl; alkoxy groups, such as
methoxy, ethoxy, n-propoxy, and isopropoxy; aryloxy groups, such as
phenoxy; arylalkyloxy, such as benzyloxy (phenylmethoxy) and
p-trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy);
heteroaryloxy groups; sulfonyl groups, such as
trifluoromethanesulfonyl, methanesulfonyl, and p-toluenesulfonyl;
nitro, nitrosyl; mercapto; sulfanyl groups, such as methylsulfanyl,
ethylsulfanyl and propylsulfanyl; cyano; heterocyclyl-C(O)-moiety;
amino groups, such as amino, methylamino, dimethylamino,
ethylamino, and diethylamino; and carboxyl. Where multiple
substituent moieties are disclosed or claimed, the substituted
compound can be independently substituted by one or more of the
disclosed or claimed substituent moieties, singly or plurally. By
independently substituted, it is meant that the (two or more)
substituents can be the same or different.
[0110] It is understood that substituents and substitution patterns
on the compounds of the instant invention can be selected by one of
ordinary skill in the art to provide compounds that are chemically
stable and that can be readily synthesized by techniques known in
the art, as well as those methods set forth below, from readily
available starting materials. If a substituent is itself
substituted with more than one group, it is understood that these
multiple groups may be on the same carbon or on different carbons,
so long as a stable structure results.
[0111] In choosing the compounds of the present invention, one of
ordinary skill in the art will recognize that the various
substituents, i.e. R.sub.1, R.sub.2, etc. are to be chosen in
conformity with well-known principles of chemical structure
connectivity. Moreover, where hydrogens are not shown in the
carbon-based structures herein, implicit hydrogens are understood
to complete valences as required.
[0112] The compounds of the instant invention may be in a salt
form. As used herein, a "salt" is salt of the instant compounds
which has been modified by making acid or base, salts of the
compounds. In the case of compounds used for treatment of cancer,
the salt is pharmaceutically acceptable. Examples of
pharmaceutically acceptable salts include, but are not limited to,
mineral or organic acid salts of basic residues such as amines;
alkali or organic salts of acidic residues such as phenols. The
salts can be made using an organic or inorganic acid. Such acid
salts are chlorides, bromides, sulfates, nitrates, phosphates,
sulfonates, formates, tartrates, maleates, malates, citrates,
benzoates, salicylates, ascorbates, and the like. Phenolate salts
are the alkaline earth metal salts, sodium, potassium or lithium.
The term "pharmaceutically acceptable salt" in this respect, refers
to the relatively non-toxic, inorganic and organic acid or base
addition salts of compounds of the present invention. These salts
can be prepared in situ during the final isolation and purification
of the compounds of the invention, or by separately reacting a
purified compound of the invention in its free base or free acid
form with a suitable organic or inorganic acid or base, and
isolating the salt thus formed. Representative salts include the
hydrobromide, hydrochloride, sulfate, bisulfate, phosphate,
nitrate, acetate, valerate, oleate, palmitate, stearate, laurate,
benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,
succinate, tartrate, napthylate, mesylate, glucoheptonate,
lactobionate, and laurylsulphonate salts and the like. (See, e.g.,
Berge et al. (1977) "Pharmaceutical Salts", J''. Pharm. Sci.
66:1-19).
[0113] Where a numerical range is provided herein for any
parameter, it is understood that all numerical subsets of that
numerical range, and all the individual integer values contained
therein, are provided as part of the invention. Thus, C1-C10 alkyl
includes the subset of alkyls which are 1-3 carbon atoms, the
subset of alkyls which are 2-5 carbon atoms etc. as well as an
alkyl which has 1 carbon atom, an alkyl which has 3 carbon atoms,
an alkyl which has 10 carbon atom, etc.
[0114] In an embodiment, the purines discussed herein are one or
more of adenosine, inosine, hypoxanthine, or adenine. In an
embodiment, "determining" as used herein means experimentally
determining.
[0115] The term "composition", as in pharmaceutical composition, is
intended to encompass a product comprising the active
ingredient(s), and the inert ingredient(s) (pharmaceutically
acceptable excipients) that make up the carrier, as well as any
product which results, directly or indirectly, from combination,
complexation or aggregation of any two or more of the ingredients,
or from dissociation of one or more of the ingredients, or from
other types of reactions or interactions of one or more of the
ingredients. Accordingly, the pharmaceutical compositions of the
present invention encompass any composition made by admixing a
compound of a high-density lipoprotein-derived nanoparticle (HDL)
compound which comprises an m-TOR inhibitor, wherein the m-TOR
inhibitor is rapamycin or a pharmaceutically acceptable salt,
solvate, poly-morph, tautomer or prodrug thereof, formulated as
rapamycin nanoparticle (mTOR-HDL), and pharmaceutically acceptable
excipients.
[0116] As used herein, the term "optionally" means that the
subsequently described event(s) may or may not occur, and includes
both event(s), which occur, and events that do not occur.
[0117] As used herein, the term "substituted with one or more
groups" refers to substitution with the named substituent or
substituents, multiple degrees of substitution, up to replacing all
hydrogen atoms with the same or different substituents, being
allowed unless the number of substituents is explicitly stated.
Where the number of substituents is not explicitly stated, one or
more is intended.
[0118] As used herein, "a compound of the invention" means a
compound of formula or a salt, solvate or physiologically
functional derivative thereof.
[0119] As used herein, the term "solvate" refers to a complex of
variable stoichiometry formed by a solute (e.g., a compound of
formula I, or a salt thereof) and a solvent. Such solvents for the
purpose of the invention may not interfere with the biological
activity of the solute. Examples of suitable solvents include, but
are not limited to, water, acetone, methanol, ethanol and acetic
acid. Preferably the solvent used is a pharmaceutically acceptable
solvent. Examples of suitable pharmaceutically acceptable solvents
include water, ethanol and acetic acid. Most preferably the solvent
is water.
[0120] In certain embodiments, the term "physiologically functional
derivative" refers to a compound (e.g., a drug precursor) that is
transformed in vivo to yield a compound of a high-density
lipoprotein-derived nanoparticle (HDL) which comprises an m-TOR
inhibitor, wherein the m-TOR inhibitor is rapamycin or a
pharmaceutically acceptable salt, solvate, poly-morph, tautomer or
prodrug thereof, formulated as rapamycin nanoparticle (mTOR-HDL),
or a pharmaceutically acceptable salt, hydrate or solvate of the
compound. The transformation may occur by various mechanisms (e.g.,
by metabolic or chemical processes), such as, for example, through
hydrolysis in blood. Prodrugs are such derivatives, and a
discussion of the use of prodrugs is provided by T. Higuchi and W.
Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the
A.C.S. Symposium Series, and in Bioreversible Carriers in Drug
Design, ed. Edward B. Roche, American Pharmaceutical Association
and Pergamon Press, 1987. Additionally, the term may encompass a
compound (e.g., a drug precursor) that is transformed in vivo to
yield a compound of HDL which encompasses a CD40-TRAF6 inhibitor,
e.g. TRAF6i-HDL.
[0121] All combinations of the various elements described herein
are within the scope of the invention unless otherwise indicated
herein or otherwise clearly contradicted by context. Whilst the
embodiments for each variable have generally been listed above
separately for each variable, this invention also includes those
compounds in which several or each embodiment for compounds of a
high-density lipoprotein-derived nanoparticle (HDL) which comprises
an m-TOR inhibitor, wherein the m-TOR inhibitor is rapamycin or a
pharmaceutically acceptable salt, solvate, poly-morph, tautomer or
prodrug thereof, formulated as rapamycin nanoparticle (mTOR-HDL),
selected from each of the embodiments listed above. Therefore, this
invention is intended to include all combinations of embodiments
for each variable.
[0122] In certain embodiments, the present invention also includes
compounds which further comprise a TRAF6i-HDL (also called
CD40-TRAF6 inhibitor), wherein the inhibitor is 6877002 (described
in Zarzycka, T. et al, J. Chem. Inf. Model. 55:294-307 (2015) or a
pharmaceutically acceptable salt, solvate, poly-morph, tautomer or
prodrug thereof, formulated as TRAF6i-HDL nanoparticle
(TRAF6i-HDL), selected from any of the embodiments listed above.
Therefore, all combinations of embodiments for each variable are
contemplated herein.
[0123] The high-density lipoprotein-derived nanoparticle (HDL)
compound which comprises an m-TOR inhibitor, wherein the m-TOR
inhibitor is rapamycin or a pharmaceutically acceptable salt,
solvate, poly-morph, tautomer or prodrug thereof, formulated as
rapamycin nanoparticle (mTOR-HDL), and salts, solvates and
physiologically functional derivatives thereof are believed to be
useful for treating a subject at risk for allograft rejection and
the method is for preventing (i.e., prophylaxis) or inhibiting
allograft rejection. It is noted that any transplant is at risk for
allograft rejection, and thus the compositions and methods
described herein are contemplated for therapeutic use for any
transplant condition. Furthermore, combining TRAF6i-HDL composition
with the mTOR-HDL treatment regimen provides synergistic effects in
preventing (i.e., prophylaxis) or inhibiting allograft
rejection.
[0124] In a further embodiment, the present invention provides for
the use of a compound of a high-density lipoprotein-derived
nanoparticle (HDL) which comprises an m-TOR inhibitor, wherein the
m-TOR inhibitor is rapamycin or a pharmaceutically acceptable salt,
solvate, poly-morph, tautomer or prodrug thereof, formulated as
rapamycin nanoparticle (mTOR-HDL), or a pharmaceutically acceptable
salt or solvate thereof, or a physiologically functional derivative
thereof, in the preparation of a medicament for the treatment of a
disorder mediated by certain levels of immune reactants that
indicate a likelihood of immune intolerance.
[0125] Accordingly, the invention further provides a pharmaceutical
composition, which comprises a compound of a high-density
lipoprotein-derived nanoparticle (HDL) which comprises an m-TOR
inhibitor, wherein the m-TOR inhibitor is rapamycin or a
pharmaceutically acceptable salt, solvate, poly-morph, tautomer or
prodrug thereof, formulated as rapamycin nanoparticle (mTOR-HDL),
and salts, solvates and physiological functional derivatives
thereof, and one or more pharmaceutically acceptable carriers,
diluents, or excipients. The compounds of a high-density
lipoprotein-derived nanoparticle (HDL) which comprises an m-TOR
inhibitor, wherein the m-TOR inhibitor is rapamycin or a
pharmaceutically acceptable salt, solvate, poly-morph, tautomer or
prodrug thereof, formulated as rapamycin nanoparticle (mTOR-HDL),
and salts, solvates and physiological functional derivatives
thereof, are as described above. The carrier(s), diluent(s) or
excipient(s) must be acceptable in the sense of being compatible
with the other ingredients of the formulation and not deleterious
to the recipient thereof. In accordance with another aspect of the
invention there is also provided a process for the preparation of a
pharmaceutical composition including admixing a compound of a
high-density lipoprotein-derived nanoparticle (HDL) which comprises
an m-TOR inhibitor, wherein the m-TOR inhibitor is rapamycin or a
pharmaceutically acceptable salt, solvate, poly-morph, tautomer or
prodrug thereof, formulated as rapamycin nanoparticle (mTOR-HDL),
or salts, solvates and physiological functional derivatives
thereof, with one or more pharmaceutically acceptable carriers,
diluents or excipients.
[0126] The invention further provides a pharmaceutical composition,
which comprises a compound of a high-density lipoprotein-derived
nanoparticle (HDL) which comprises an CD40-TRAF6 inhibitor, wherein
the CD40-TRAF6 inhibitor is 6877002, or a pharmaceutically
acceptable salt, solvate, poly-morph, tautomer or prodrug thereof,
formulated as CD40-TRAF6 nanoparticle (TRAF6i-HDL), and salts,
solvates and physiological functional derivatives thereof, and one
or more pharmaceutically acceptable carriers, diluents, or
excipients. The compounds of a high-density lipoprotein-derived
nanoparticle (HDL) which comprises a CD40-TRAF6 inhibitor, wherein
the CD40-TRAF6 inhibitor is 6877002 or a pharmaceutically
acceptable salt, solvate, poly-morph, tautomer or prodrug thereof,
formulated as CD40-TRAF6 nanoparticle (TRAF6i-HDL), and salts,
solvates and physiological functional derivatives thereof, are as
described above. The carrier(s), diluent(s) or excipient(s) must be
acceptable in the sense of being compatible with the other
ingredients of the formulation and not deleterious to the recipient
thereof. In accordance with another aspect of the invention there
is also provided a process for the preparation of a pharmaceutical
composition including admixing a compound of a high-density
lipoprotein-derived nanoparticle (HDL) which comprises a CD40-TRAF
inhibitor, wherein the CD40-TRAF6 inhibitor is 6877002 or a
pharmaceutically acceptable salt, solvate, poly-morph, tautomer or
prodrug thereof, formulated as CD40-TRAF6 nanoparticle
(TRAF6i-HDL), or salts, solvates and physiological functional
derivatives thereof, with one or more pharmaceutically acceptable
carriers, diluents or excipients.
[0127] Additionally, in certain embodiments, a combination
composition comprising both the CD40-TRAF6 inhibitor and m-TOR
inhibitor formulated as a combined HDL nanoparticle formulation is
contemplated. In such a combination composition, the active
agent/compound can be as described above, but any suitably charged
CD40-TRAF6 inhibitor or m-TOR inhibitor can be formulated as a
combined HDL nanoparticle formulation.
[0128] Pharmaceutical compositions of the present invention may be
presented in unit dose forms containing a predetermined amount of
active ingredient per unit dose. Such a unit may contain, for
example, 5 .mu.g to 1 g, preferably 1 mg to 700 mg, more preferably
5 mg to 100 mg of a compound of a high-density lipoprotein-derived
nanoparticle (HDL) which comprises an m-TOR inhibitor, wherein the
m-TOR inhibitor is rapamycin or a pharmaceutically acceptable salt,
solvate, poly-morph, tautomer or prodrug thereof, formulated as
rapamycin nanoparticle (mTOR-HDL), depending on the condition being
treated, the route of administration and the age, weight and
condition of the patient. Such unit doses may therefore be
administered more than once a day. Preferred unit dosage
compositions are those containing a daily dose or sub-dose (for
administration more than once a day), as herein above recited, or
an appropriate fraction thereof, of an active ingredient.
Furthermore, such pharmaceutical compositions may be prepared by
any of the methods well known in the pharmacy art. Exemplary dosage
includes 5 mg/kg in mice.
[0129] Pharmaceutical compositions of the present invention may be
adapted for administration by any appropriate route, for example by
the oral (including buccal or sublingual), inhaled, nasal, ocular,
or parenteral (including intravenous and intramuscular) route. Such
compositions may be prepared by any method known in the art of
pharmacy, for example by bringing into association the active
ingredient with the carrier(s) or excipient(s).
[0130] A therapeutically effective amount of a compound of the
present invention will depend upon a number of factors including,
for example, the age and weight of the animal, the precise
condition requiring treatment and its severity, the nature of the
formulation, and the route of administration, and will ultimately
be at the discretion of the attendant physician or veterinarian
However, an effective amount of a compound of a high-density
lipoprotein-derived nanoparticle (HDL) which comprises an m-TOR
inhibitor, wherein the m-TOR inhibitor is rapamycin or a
pharmaceutically acceptable salt, solvate, poly-morph, tautomer or
prodrug thereof, formulated as rapamycin nanoparticle (mTOR-HDL)
for the treatment of diseases or conditions associated with
allograft rejection--including heart transplant, skin transplant,
liver transplant, lung transplant, bronchiolitis-obliterans
syndrome (BOS), kidney transplant, pancreas transplant, pancreatic
islets transplant, intestinal transplant, bone transplant, retinal
transplant, and corneal transplant will generally be in the range
of 5 .mu.g to 100 mg/kg body weight of recipient (mammal) per day
and more usually in the range of 5 .mu.g to 10 mg/kg body weight
per day. This amount may be given in a single dose per day or more
usually in a number (such as two, three, four, five or six) of
sub-doses per day such that the total daily dose is the same. An
effective amount of a salt or solvate, thereof, may be determined
as a proportion of the effective amount of the compound of a
high-density lipoprotein-derived nanoparticle (HDL) which comprises
an m-TOR inhibitor, wherein the m-TOR inhibitor is rapamycin or a
pharmaceutically acceptable salt, solvate, poly-morph, tautomer or
prodrug thereof, formulated as rapamycin nanoparticle (mTOR-HDL),
per se.
[0131] Compounds of the present invention, and their salts and
solvates, and physiologically functional derivatives thereof, may
be employed alone or in combination with other therapeutic agents
for the treatment of diseases and conditions related to allograft
rejection--including heart transplant, skin transplant, liver
transplant, lung transplant, bronchiolitis-obliterans syndrome
(BOS), kidney transplant, pancreas transplant, pancreatic islets
transplant, intestinal transplant, bone transplant, retinal
transplant, and corneal transplant.
[0132] Combination therapies according to the present invention
thus comprise the administration of at least one compound of a
high-density lipoprotein-derived nanoparticle (HDL) which comprises
an m-TOR inhibitor, wherein the m-TOR inhibitor is rapamycin or a
pharmaceutically acceptable salt, solvate, poly-morph, tautomer or
prodrug thereof, formulated as rapamycin nanoparticle (mTOR-HDL),
or a pharmaceutically acceptable salt or solvate thereof, or a
physiologically functional derivative thereof, and the use of at
least one other pharmaceutically active agent. The compound(s) of
compound of a high-density lipoprotein-derived nanoparticle (HDL)
which comprises an m-TOR inhibitor, wherein the m-TOR inhibitor is
rapamycin or a pharmaceutically acceptable salt, solvate,
poly-morph, tautomer or prodrug thereof, formulated as rapamycin
nanoparticle (mTOR-HDL) and the other pharmaceutically active
agent(s) may be administered together or separately and, when
administered separately this may occur simultaneously or
sequentially in any order. The amounts of the compound(s) of a
high-density lipoprotein-derived nanoparticle (HDL) which comprises
an m-TOR inhibitor, wherein the m-TOR inhibitor is rapamycin or a
pharmaceutically acceptable salt, solvate, poly-morph, tautomer or
prodrug thereof, formulated as rapamycin nanoparticle (mTOR-HDL)
and the other pharmaceutically active agent(s) and the relative
timings of administration will be selected in order to achieve the
desired combined therapeutic effect.
[0133] In certain embodiments, combination therapies according to
the present invention thus comprise the administration of (i)
high-density lipoprotein-derived nanoparticle (HDL) which comprises
an m-TOR inhibitor, wherein the m-TOR inhibitor is rapamycin or a
pharmaceutically acceptable salt, solvate, poly-morph, tautomer or
prodrug thereof, formulated as rapamycin nanoparticle (mTOR-HDL),
or the pharmaceutical composition thereof and (ii) TRAF6i-HDL
nanoparticles which comprises CD40-TRAF6 inhibitor, wherein the
CD40-TRAF6 inhibitor is 6877002, or a pharmaceutically acceptable
salt, solvate, poly-morph, tautomer or prodrug thereof, formulated
as TRAF6i-HDL nanoparticle (also referred to generally as
CD40-HDL), or the pharmaceutical composition thereof.
[0134] It will be clear to a person skilled in the art that, where
appropriate, the other therapeutic ingredient(s) may be used in the
form of salts, for example as alkali metal or amine salts or as
acid addition salts, or prodrugs, or as esters, for example lower
alkyl esters, or as solvates, for example hydrates, to optimize the
activity and/or stability and/or physical characteristics, such as
solubility, of the therapeutic ingredient. It will be clear also
that, where appropriate, the therapeutic ingredients may be used in
optically pure form.
[0135] The combinations referred to above may conveniently be
presented for use in the form of a pharmaceutical composition and
thus pharmaceutical compositions comprising a combination as
defined above together with a pharmaceutically acceptable diluent
or carrier represent a further aspect of the invention.
[0136] The individual compounds of such combinations may be
administered either sequentially or simultaneously in separate or
combined pharmaceutical compositions. Preferably, the individual
compounds will be administered simultaneously in a combined
pharmaceutical composition. Appropriate doses of known therapeutic
agents will be readily appreciated by those skilled in the art.
[0137] The compounds of this invention may be made by a variety of
methods, including standard chemistry. Any previously defined
variable will continue to have the previously defined meaning
unless otherwise indicated. Illustrative general synthetic methods
are set out below and then specific compounds of the invention are
prepared in the Working Examples.
[0138] Compounds of the present invention may be prepared by
methods known in the art of organic synthesis as set forth in part
by the following synthesis schemes. In all of the schemes described
below, it is well understood that protecting groups for sensitive
or reactive groups are employed where necessary in accordance with
general principles of chemistry. Protecting groups are manipulated
according to standard methods of organic synthesis (T. W. Green and
P. G. M. Wuts (1991) Protecting Groups in Organic Synthesis, John
Wiley & Sons). These groups are removed at a convenient stage
of the compound synthesis using methods that are readily apparent
to those skilled in the art. The selection of protecting groups as
well as the reaction conditions and order of reaction steps shall
be consistent with the preparation of compounds of the present
invention. Those skilled in the art will recognize if a
stereocenter exists in compounds of the present invention.
Accordingly, the present invention includes all possible
stereoisomers and includes not only mixtures of stereoisomers (such
as racemic compounds) but the individual stereoisomers as well.
When a compound is desired as a single enantiomer, it may be
obtained by stereospecific synthesis or by resolution of the final
product or any convenient intermediate. Resolution of the final
product, an intermediate, or a starting material may be effected by
any suitable method known in the art. See, for example,
Stereochemistry of Organic Compounds by E. L. Eliel, S. H. Wilen,
and L. N. Mander (Wiley-Interscience, 1994).
[0139] A "transplantable graft" refers to a biological material,
such as cells, tissues and organs (in whole or in part) that can be
administered to a subject. Transplantable grafts may be autografts,
allografts, or xenografts of, for example, a biological material
such as an organ, tissue, skin, bone, nerves, tendon, neurons,
blood vessels, fat, cornea, pluripotent cells, differentiated cells
(obtained or derived in vivo or in vitro), etc. In some
embodiments, a transplantable graft is formed, for example, from
cartilage, bone, extracellular matrix, or collagen matrices.
Transplantable grafts may also be single cells, suspensions of
cells and cells in tissues and organs that can be transplanted.
Transplantable cells typically have a therapeutic function, for
example, a function that is lacking or diminished in a recipient
subject. Some non-limiting examples of transplantable cells are
islet cells, beta.-cells, hepatocytes, hematopoietic stem cells,
neuronal stem cells, neurons, glial cells, or myelinating cells.
Transplantable cells can be cells that are unmodified, for example,
cells obtained from a donor subject and usable in transplantation
without any genetic or epigenetic modifications. In other
embodiments, transplantable cells can be modified cells, for
example, cells obtained from a subject having a genetic defect, in
which the genetic defect has been corrected, or cells that are
derived from reprogrammed cells, for example, differentiated cells
derived from cells obtained from a subject.
[0140] "Transplantation" refers to the process of transferring
(moving) a transplantable graft into a recipient subject (e.g.,
from a donor subject, from an in vitro source (e.g., differentiated
autologous or heterologous native or induced pluripotent cells))
and/or from one bodily location to another bodily location in the
same subject.
[0141] "Undesired immune response" refers to any undesired immune
response that results from exposure to an antigen, promotes or
exacerbates a disease, disorder or condition provided herein (or a
symptom thereof), or is symptomatic of a disease, disorder or
condition provided herein. Such immune responses generally have a
negative impact on a subject's health or is symptomatic of a
negative impact on a subject's health.
[0142] In an embodiment, the transplanted tissue is lung tissue,
heart tissue, kidney tissue, liver tissue, retinal tissue, corneal
tissue, skin tissue, pancreatic tissue, intestinal tissue, genital
tissue, ovary tissue, bone tissue, tendon tissue, or vascular
tissue.
[0143] In an embodiment, the transplanted tissue is transplanted as
an intact organ.
[0144] As used herein a "recipient subject" is a subject who is to
receive, or who has received, a transplanted cell, tissue or organ
from another subject.
[0145] As used herein a "donor subject" is a subject from whom a
cell, tissue or organ to be transplanted is removed before
transplantation of that cell, tissue or organ to a recipient
subject.
[0146] In an embodiment the donor subject is a primate. In a
further embodiment the donor subject is a human. In an embodiment
the recipient subject is a primate. In an embodiment the recipient
subject is a human. In an embodiment both the donor and recipient
subjects are human. Accordingly, the subject invention includes the
embodiment of xenotransplantation.
[0147] As used herein "rejection by an immune system" describes the
event of hyperacute, acute and/or chronic response of a recipient
subject's immune system recognizing a transplanted cell, tissue or
organ from a donor as non-self and the consequent immune
response.
[0148] The term "allogeneic" refers to any material derived from a
different animal of the same species as the individual to whom the
material is introduced. Two or more individuals are said to be
allogeneic to one another when the genes at one or more loci are
not identical.
[0149] The term "autologous" refers to any material derived from
the same individual to whom it is later to be re-introduced into
the same individual.
[0150] As used herein an "immunosuppressant pharmaceutical" is a
pharmaceutically-acceptable drug used to suppress a recipient
subject's immune response. Non-limiting examples include
cyclosporine A, FK506 and rapamycin.
[0151] As used herein, a "prophylactically effective" amount is an
amount of a substance effective to prevent or to delay the onset of
a given pathological condition in a subject to which the substance
is to be administered. A prophylactically effective amount refers
to an amount effective, at dosages and for periods of time
necessary, to achieve the desired prophylactic result. Typically,
since a prophylactic dose is used in subjects prior to or at an
earlier stage of disease, the prophylactically effective amount
will be less than the therapeutically effective amount.
[0152] As used herein, a "therapeutically effective" amount is an
amount of a substance effective to treat, ameliorate or lessen a
symptom or cause of a given pathological condition in a subject
suffering therefrom to which the substance is to be
administered.
[0153] In one embodiment, the therapeutically or prophylactically
effective amount is from about 1 mg of agent/kg subject to about 1
g of agent/kg subject per dosing. In another embodiment, the
therapeutically or prophylactically effective amount is from about
10 mg of agent/kg subject to 500 mg of agent/subject. In a further
embodiment, the therapeutically or prophylactically effective
amount is from about 50 mg of agent/kg subject to 200 mg of
agent/kg subject. In a further embodiment, the therapeutically or
prophylactically effective amount is about 100 mg of agent/kg
subject. In still a further embodiment, the therapeutically or
prophylactically effective amount is selected from 50 mg of
agent/kg subject, 100 mg of agent/kg subject, 150 mg of agent/kg
subject, 200 mg of agent/kg subject, 250 mg of agent/kg subject,
300 mg of agent/kg subject, 400 mg of agent/kg subject and 500 mg
of agent/kg subject.
[0154] "Treating" or "treatment" of a state, disorder or condition
includes: [0155] (1) preventing or delaying the appearance of
clinical symptoms of the state, disorder, or condition developing
in a person who may be afflicted with or predisposed to the state,
disorder or condition but does not yet experience or display
clinical symptoms of the state, disorder or condition; or [0156]
(2) inhibiting the state, disorder or condition, i.e., arresting,
reducing or delaying the development of the disease or a relapse
thereof (in case of maintenance treatment) or at least one clinical
symptom, sign, or test, thereof; or [0157] (3) relieving the
disease, i.e., causing regression of the state, disorder or
condition or at least one of its clinical or sub-clinical symptoms
or signs.
[0158] The benefit to a subject to be treated is either
statistically significant or at least perceptible to the patient or
to the physician.
[0159] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically, since a prophylactic
dose is used in subjects prior to or at an earlier stage of
disease, the prophylactically effective amount will be less than
the therapeutically effective amount.
[0160] Acceptable excipients, diluents, and carriers for
therapeutic use are well known in the pharmaceutical art, and are
described, for example, in Remington: The Science and Practice of
Pharmacy. Lippincott Williams & Wilkins (A. R. Gennaro edit.
2005). The choice of pharmaceutical excipient, diluent, and carrier
can be selected with regard to the intended route of administration
and standard pharmaceutical practice.
[0161] As used herein, the phrase "pharmaceutically acceptable"
refers to molecular entities and compositions that are "generally
regarded as safe", e.g., that are physiologically tolerable and do
not typically produce an allergic or similar untoward reaction,
such as gastric upset, dizziness and the like, when administered to
a human. Preferably, as used herein, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopoeia or other
generally recognized pharmacopeias for use in animals, and more
particularly in humans.
[0162] "Patient" or "subject" refers to mammals and includes human
and veterinary subjects. Certain veterinary subjects may include
avian species.
[0163] This invention will be better understood from the
Experimental Details, which follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described
more fully in the claims that follow thereafter.
General Methods
[0164] Standard methods in molecular biology are described
Sambrook, Fritsch and Maniatis (1982 & 1989 2.sup.nd Edition,
2001 3.sup.rd Edition) Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Sambrook
and Russell (2001) Molecular Cloning, 3.sup.rd ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Wu (1993)
Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif.).
Standard methods also appear in Ausbel, et al. (2001) Current
Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons,
Inc. New York, N.Y., which describes cloning in bacterial cells and
DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast
(Vol. 2), glycoconjugates and protein expression (Vol. 3), and
bioinformatics (Vol. 4).
[0165] Methods for protein purification including
immunoprecipitation, chromatography, electrophoresis,
centrifugation, and crystallization are described (Coligan, et al.
(2000) Current Protocols in Protein Science, Vol. 1, John Wiley and
Sons, Inc., New York). Chemical analysis, chemical modification,
post-translational modification, production of fusion proteins,
glycosylation of proteins are described (see, e.g., Coligan, et al.
(2000) Current Protocols in Protein Science, Vol. 2, John Wiley and
Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in
Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp.
16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life
Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia
Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391).
Production, purification, and fragmentation of polyclonal and
monoclonal antibodies are described (Coligan, et al. (2001) Current
Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New
York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane,
supra). Standard techniques for characterizing ligand/receptor
interactions are available (see, e.g., Coligan, et al. (2001)
Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New
York).
[0166] Rapamycin is a known macrolide antibiotic produced by
Streptomyces hygroscopicus. Suitable derivatives of rapamycin
include e.g. compounds of formula I wherein R.sub.1 is CH.sub.3 or
C.sub.3-6alkynyl, R.sub.2 is H or --CH.sub.2-CH.sub.2-OH, and X is
.dbd.O, (H,H) or (H.sub.2OH) provided that R.sub.2 is other than H
when X is .dbd.O and R.sub.1 is CH.sub.3. The structure of
rapamycin is shown below:
##STR00001##
[0167] Compounds of formula I are disclosed e.g. in U.S. Pat. Nos.
5,665,772; 6,440,990; 5,985,890; and 6,200,985, which are
incorporated herein by reference. They may be prepared as disclosed
or by analogy to the procedures described in these references.
[0168] Preferred compounds are 32-deoxorapamycin,
16-pent-2-ynyloxy-32-deoxorapambycin,
16-pent-2-ynyloxy-32(S)-dihydro-rapamycin,
16-pent-2-ynyloxy-32(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin
and, more preferably, 40-O-(2-hydroxyethyl)-rapamycin (referred
thereafter as Compound A), disclosed as Example 8 in U.S. Pat. Nos.
5,665,772 and 6,440,990.
[0169] Compounds of formula I have, on the basis of observed
activity, e.g. binding to macrophilin-12 (also known as FK-506
binding protein or FKBP-12), e.g. as described in WO 94/09010, WO
95/16691 or WO 96/41807, been found to be useful e.g. as
immunosuppressant, e.g. in the treatment of acute allograft
rejection.
[0170] Embodiments also include nanoparticles comprising rapamycin
derivatives with improved hydrophobicity and/or miscibility. For
example, rapamycin may be conjugated with an alkyl chain as
described in Zhao et al., Augmenting drug-carrier compatibility
improves tumour nanotherapy efficacy, Nature Communications 7,
Article number: 11221 (2016) doi:10.1038/ncomms11221.
[0171] In certain embodiments, the addition of cholesterol has
stabilized the formulation as well as improved entrapment
efficiency. In certain embodiments, the weight percentage of
cholesterol ranges from about 0% to about 10% (w/w), from about 1%
(w/w) to about 10% (w/w), from about 2% (w/w) to about 10% (w/w),
from about 3% (w/w) to about 10% (w/w), from about 4% (w/w) to
about 10% (w/w), from about 5% (w/w) to about 10% (w/w), from about
6% (w/w) to about 10% (w/w), from about 7% (w/w) to about 10%
(w/w), from about 8% (w/w) to about 10% (w/w), from about 1% (w/w)
to about 9% (w/w), from about 1% (w/w) to about 8% (w/w), from
about 1% (w/w) to about 7% (w/w), or from about 1% (w/w) to about
6% (w/w), of the nanoparticle, of the lipids, or of the
composition.
[0172] Delivery vehicles such as liposomes, nanocapsules,
microparticles, microspheres, lipid particles, vesicles, and the
like, may be used for the introduction of the compositions of the
present invention targeting the innate immune system, e.g.
targeting macrophages to induce transplantation tolerance. In
addition to being formulated as a nanotherapy as mTOR-HDL, the
compounds targeting macrophages may be formulated for delivery in a
number of different forms and methods including either encapsulated
in a lipid particle, a liposome, a vesicle, a nanosphere, or a
nanoparticle or the like.
[0173] The formation and use of liposomes is generally known to
those of skill in the art. Recently, liposomes were developed with
improved serum stability and circulation half-times (U.S. Pat. No.
5,741,516). Further, various methods of liposome and liposome like
preparations as potential drug carriers have been described (U.S.
Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and
5,795,587).
[0174] Liposomes have been used successfully with a number of cell
types that are normally resistant to transfection by other
procedures. In addition, liposomes are free of the DNA length
constraints that are typical of viral-based delivery systems.
Liposomes have been used effectively to introduce genes, drugs,
radiotherapeutic agents, viruses, transcription factors and
allosteric effectors into a variety of cultured cell lines and
animals. In addition, several successful clinical trials examining
the effectiveness of liposome-mediated drug delivery have been
completed.
[0175] Liposomes are formed from phospholipids that are dispersed
in an aqueous medium and spontaneously form multilamellar
concentric bilayer vesicles (also termed multilamellar vesicles
(MLVs). MLVs generally have diameters of from 25 nm to 4 .mu.m.
Sonication of MLVs results in the formation of small unilamellar
vesicles (SUVs) with diameters in the range of 200 to 500 .ANG.,
containing an aqueous solution in the core.
[0176] Alternatively, nanocapsule formulations of the compositions
of the present invention targeting the innate immune system may be
used. Nanocapsules can generally entrap substances in a stable and
reproducible way. To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
.mu.m) should be designed using polymers able to be degraded in
vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet
these requirements are contemplated for use.
[0177] "Synthetic nanocarrier(s)" means a discrete object that is
not found in nature, and that possesses at least one dimension that
is less than or equal to 5 microns in size. Albumin nanoparticles
are generally included as synthetic nanocarriers, however in
certain embodiments the synthetic nanocarriers do not comprise
albumin nanoparticles. In certain embodiments, synthetic
nanocarriers do not comprise chitosan. In other embodiments,
inventive synthetic nanocarriers are not lipid-based nanoparticles.
In further embodiments, synthetic nanocarriers do not comprise a
phospholipid.
[0178] A synthetic nanocarrier can be, but is not limited to, one
or a plurality of lipid-based nanoparticles (also referred to
herein as lipid nanoparticles, i.e., nanoparticles where the
majority of the material that makes up their structure are lipids),
polymeric nanoparticles, metallic nanoparticles, surfactant-based
emulsions, dendrimers, buckyballs, nanowires, virus-like particles
(i.e., particles that are primarily made up of viral structural
proteins but that are not infectious or have low infectivity),
peptide or protein-based particles (also referred to herein as
protein particles, i.e., particles where the majority of the
material that makes up their structure are peptides or proteins)
(such as albumin nanoparticles) and/or nanoparticles that are
developed using a combination of nanomaterials such as
lipid-polymer nanoparticles. Synthetic nanocarriers may be a
variety of different shapes, including but not limited to
spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and
the like. Synthetic nanocarriers according to the invention
comprise one or more surfaces. Exemplary synthetic nanocarriers
that can be adapted for use in the practice of the present
invention comprise: (1) the biodegradable nanoparticles disclosed
in U.S. Pat. No. 5,543,158 to Gref et al., (2) the polymeric
nanoparticles of Published US Patent Application 20060002852 to
Saltzman et al., (3) the lithographically constructed nanoparticles
of Published US Patent Application 20090028910 to DeSimone et al.,
(4) the disclosure of WO 2009/051837 to von Andrian et al., (5) the
nanoparticles disclosed in Published US Patent Application
2008/0145441 to Penades et al., (6) the protein nanoparticles
disclosed in Published US Patent Application 20090226525 to de los
Rios et al., (7) the virus-like particles disclosed in published US
Patent Application 20060222652 to Sebbel et al., (8) the nucleic
acid coupled virus-like particles disclosed in published US Patent
Application 20060251677 to Bachmann et al., (9) the virus-like
particles disclosed in WO2010047839A1 or WO2009106999A2, (10) the
nanoprecipitated nanoparticles disclosed in P. Paolicelli et al.,
"Surface-modified PLGA-based Nanoparticles that can Efficiently
Associate and Deliver Virus-like Particles" Nanomedicine.
5(6):843-853 (2010), or (11) apoptotic cells, apoptotic bodies or
the synthetic or semisynthetic mimics disclosed in U.S. Publication
2002/0086049. In embodiments, synthetic nanocarriers may possess an
aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or
greater than 1:10.
[0179] Synthetic nanocarriers may have a minimum dimension of equal
to or less than about 100 nm, preferably equal to or less than 100
nm, do not comprise a surface with hydroxyl groups that activate
complement or alternatively comprise a surface that consists
essentially of moieties that are not hydroxyl groups that activate
complement. In certain embodiments, synthetic nanocarriers may have
a minimum dimension of equal to or less than about 100 nm,
preferably equal to or less than 100 nm, do not comprise a surface
that substantially activates complement or alternatively comprise a
surface that consists essentially of moieties that do not
substantially activate complement. Synthetic nanocarriers in some
embodiments have a minimum dimension of equal to or less than about
100 nm, preferably equal to or less than 100 nm, do not comprise a
surface that activates complement or alternatively comprise a
surface that consists essentially of moieties that do not activate
complement. In embodiments, synthetic nanocarriers exclude
virus-like particles. In embodiments, synthetic nanocarriers may
possess an aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3,
1:5, 1:7, or greater than 1:10.
[0180] In certain embodiments, the present composition comprises
(consists essentially of, or consists of) one or more types of
phospholipids.
[0181] Examples of suitable phospholipids include, without
limitation, phosphatidylcholines, phosphatidylethanolamines,
phosphatidylinositol, phosphatidylserines, sphingomyelin or other
ceramides, as well as phospholipid-containing oils such as lecithin
oils. Combinations of phospholipids, or mixtures of a
phospholipid(s) and other substance(s), may be used.
[0182] Non-limiting examples of the phospholipids that may be used
in the present composition include, dimyristoylphosphatidylcholine
(DMPC), soy lecithin, dipalmitoylphosphatidylcholine (DPPC),
distearoylphosphatidylcholine (DSPC),
dilaurylolyphosphatidylcholine (DLPC), dioleoylphosphatidylcholine
(DOPC), dilaurylolylphosphatidylglycerol (DLPG),
dimyristoylphosphatidylglycerol (DMPG),
dipalmitoylphosphatidylglycerol (DPPG),
distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol
(DOPG), dimyristoyl phosphatidic acid (DMPA), dimyristoyl
phosphatidic acid (DMPA), dipalmitoyl phosphatidic acid (DPPA),
dipalmitoyl phosphatidic acid (DPPA), dimyristoyl
phosphatidylethanolamine (DMPE), dipalmitoyl
phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylserine
(DMPS), dipalmitoyl phosphatidylserine (DPPS), dipalmitoyl
sphingomyelin (DPSP), distearoyl sphingomyelin (DSSP), and mixtures
thereof.
[0183] In certain embodiments, when the present composition
comprises (consists essentially of, or consists of) two or more
types of phospholipids, the weight ratio of two types of
phospholipids may range from about 1:10 to about 10:1, from about
2:1 to about 4:1, from about 1:1 to about 5:1, from about 2:1 to
about 5:1, from about 6:1 to about 10:1, from about 7:1 to about
10:1, from about 8:1 to about 10:1, from about 7:1 to about 9:1, or
from about 8:1 to about 9:1. For example, the weight ratio of two
types of phospholipids may be about 1:10, about 1:9, about 1:8,
about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2,
about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1,
about 7:1, about 8:1, about 9:1, or about 10:1.
[0184] In one embodiment, the present high-density lipoprotein
(HDL)-derived nanoparticle comprises (consists essentially of, or
consists of) 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine
(DMPC), and 1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC).
The weight ratio of DMPC to MHPC may range from about 1:10 to about
10:1, from about 2:1 to about 4:1, from about 1:1 to about 5:1,
from about 2:1 to about 5:1, from about 6:1 to about 10:1, from
about 7:1 to about 10:1, from about 8:1 to about 10:1, from about
7:1 to about 9:1, or from about 8:1 to about 9:1. The weight ratio
of DMPC to MHPC may be about 1:10, about 1:9, about 1:8, about 1:7,
about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1,
about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1,
about 8:1, about 9:1, or about 10:1.
mTor Inhibitors and Combinations with Other Pharmaceutically Active
Components
[0185] Examples of mTOR inhibitors include rapamycin and analogs
thereof (e.g., CCL-779, RAD001, AP23573, C20-methallylrapamycin
(C20-Marap), C16-(S)-butylsulfonamidorapamycin (C16-BSrap),
C16-(S)-3-methylindolerapamycin (C16-iRap) (Bayle et al. Chemistry
& Biology 2006, 13:99-107)), AZD8055, BEZ235 (NVP-BEZ235),
chrysophanic acid (chrysophanol), deforolimus (MK-8669), everolimus
(RAD0001), KU-0063794, PI-103, PP242, temsirolimus, and WYE-354
(available from Selleck, Houston, Tex., USA).
[0186] In certain embodiments, one or more additional or
alternative active ingredients targeting PIRb+ macrophages and
promoting allograft survival may be utilized in combination. Any
one or more of these active ingredients may be formulated in one
dosage unit, or in a combination of forms such as an mTOR-HDL
nanoparticle could be administered in combination with a lipid
particle, a liposome, a vesicle, a nanosphere comprising a second
or third active ingredient. Other suitable active agents include
one or more immunosuppressive agents.
Treatment Regimens/Options
[0187] The mTOR-HDL can be used in combination with other induction
therapies that target the adaptive immune response such as T and B
cell depletion. For example, for kidney living donors, transplant
recipients can be treated before and shortly after transplantation.
Patients under current immunosuppressive therapy can be switched to
the mTOR-HDL therapy, or combination mTOR-HDL/TRAF6i-HDL therapy.
In additional scenarios, mTOR-HDL treatment is administered to the
patient prior to and shortly after transplantation, which can be
repeated every 6 or 12 months, with the goal to eliminate or
strongly reduce immunosuppressive therapy. In additional scenarios,
patients are administered the mTOR-HDL therapy, or combination
mTOR-HDL/TRAF6i-HDL therapy without any additional
immunosuppressive therapy.
[0188] Exemplary immunosuppressants include, but are not limited
to, statins; mTOR inhibitors, such as rapamycin or a rapamycin
analog; TGF-.beta. signaling agents; TGF-.beta. receptor agonists;
histone deacetylase (HDAC) inhibitors; corticosteroids; inhibitors
of mitochondrial function, such as rotenone; P38 inhibitors;
NF-.kappa.beta. inhibitors; adenosine receptor agonists;
prostaglandin E2 agonists; phosphodiesterase inhibitors, such as
phosphodiesterase 4 inhibitor; proteasome inhibitors; kinase
inhibitors; G-protein coupled receptor agonists; G-protein coupled
receptor antagonists; glucocorticoids; retinoids; cytokine
inhibitors; cytokine receptor inhibitors; cytokine receptor
activators; peroxisome proliferator-activated receptor antagonists;
peroxisome proliferator-activated receptor agonists; histone
deacetylase inhibitors; calcineurin inhibitors; phosphatase
inhibitors and oxidized ATPs. Immunosuppressants also include IDO,
vitamin D3, cyclosporine A, aryl hydrocarbon receptor inhibitors,
resveratrol, azathiopurine, 6-mercaptopurine, aspirin, niflumic
acid, estriol, tripolide, interleukins (e.g., IL-1, IL-10),
cyclosporine A, siRNAs targeting cytokines or cytokine receptors
and the like. Examples of statins include atorvastatin
(LIPITOR.RTM., TORVAST.RTM.), cerivastatin, fluvastatin
(LESCOL.RTM., LESCOL.RTM. XL), lovastatin (MEVACOR.RTM.,
ALTOCOR.RTM., ALTOPREV.RTM.), mevastatin (COMPACTIN.RTM.),
pitavastatin (LIVALO.RTM., PIAVA.RTM.), rosuvastatin
(PRAVACHOL.RTM., SELEKTINE.RTM., LIPOSTAT.RTM.), rosuvastatin
(CRESTOR.RTM.), and simvastatin (ZOCOR.RTM., LIPEX.RTM.).
[0189] The mTOR-HDL nanotherapy was tested in an allogeneic heart
transplantation mouse model. The regimen comprised only three
intravenous tail vein injections of 5 mg/kg equivalent rapamycin
during the first week after transplantation. Using a combination of
in vivo positron emission tomography with computed tomography
(PET-CT) imaging and an array of immunological assays, the heart
allograft targeting and cellular specificity were evaluated.
Subsequently, the innate immune response was analyzed along with
allograft survival and therapeutic mechanisms. These results
demonstrate that mTOR-HDL nanoparticle treatment promotes
indefinite heart allograft survival. Additionally, similar results
were also observed for a skin transplant model. These results
demonstrate how to manipulate the immune response toward inducing
donor-specific non-response in the clinic and identify new
therapeutic targets that may prevent allograft rejection in
humans.
EXAMPLES
Example 1
[0190] Development of mTOR-HDL Nanoparticles
[0191] mTOR-HDL nanoparticles (see FIG. 1A) were synthesized by
hydrating a lipid film, containing rapamycin and phospholipids,
with APOA1 in PBS. Subsequently, and after vigorous homogenization,
the sample was sonicated to generate mTOR-HDL nanoparticles with
62.+-.11% rapamycin encapsulation efficiency and a mean
hydrodynamic diameter of 12.7.+-.4.4 nm, as determined by high
performance liquid chromatography and dynamic light scattering,
respectively. The size of the nanoparticles can vary, but will
typically be from about 10 nm to about 250 nm.
[0192] As revealed by transmission electron microscopy (FIG. 4),
the mTOR-HDL had the discoidal structure that is typical of
HDL-based nanoparticles.sup.16. The biodistribution and cellular
specificity of
1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide
(DiR)-labeled mTOR-HDLs were evaluated in C57Bl/6 wild type mice
using ex vivo near infrared fluorescence (NIRF) imaging and flow
cytometry. mTOR-HDL was shown to accumulate primarily in the liver,
spleen and kidneys (FIG. 5A) while displaying a higher affinity for
monocytes than either dendritic cells (DC) or neutrophils in the
blood and spleen (FIG. 5 B, C) (respectively, P.ltoreq.0.001,
P.ltoreq.0.01 and P.ltoreq.0.01, P.ltoreq.0.01).
[0193] mTOR-HDL treatment was utilized in a heart transplant mouse
model (FIG. 1B). mTOR-HDL's biodistribution, allograft targeting,
and cellular specificity were determined using in vivo PET-CT
imaging (FIG. 1B) and ex vivo techniques. Subsequently, an array of
immunological readouts, including flow cytometry, enzyme-linked
immunosorbent assay and mixed lymphocyte reaction were utilized, to
evaluate the effects of a short-term mTOR-HDL nanotherapy regimen
(FIG. 1B).
mTOR-HDL Nanotherapy Targets the Innate Immune System
[0194] To quantitatively study tissue targeting and specificity,
mTOR-HDL nanoparticles were radiolabeled with
.sup.89Zr(.sup.89Zr-mTOR-HDL). Six days after having hearts
transplanted into their abdomens, mice received .sup.89Zr-mTOR-HDL
intravenously. The nanoparticles were allowed to circulate and
distribute for 24 hours before mice underwent in vivo PET-CT
imaging. Marked .sup.89Zr-mTOR-HDL presence was observed in the
heart allografts (FIG. 1C). After imaging, mice were sacrificed,
and the organs were collected for .sup.89Zr-mTOR-HDL quantification
by ex vivo autoradiography. Allograft heart (Tx) activity
(25.2.+-.2.4.times.10.sup.3 counts/unit area) was determined to be
2.3 fold higher than in native hearts (N)
(11.1.+-.1.9.times.10.sup.3 count/unit area) (FIG. 1F). Gamma
counting assessed .sup.89Zr-mTOR-HDL's full biodistribution. The ex
vivo autoradiography indicates that .sup.89Zr-mTOR-HDL target many
tissues (FIG. 1D), suggesting a systemic biodistribution of the
drug, consistent with the typical pattern of distribution for
drug-loaded HDL nanoparticles.sup.17.
[0195] The favorable organ distribution pattern and heart allograft
uptake, lead the inventors to evaluate mTOR-HDL targeting and
specificity at the cellular level in the heart allograft, blood,
spleen and bone marrow. mTOR-HDL nanoparticles were labeled with
the fluorescent dye 3,3'-Dioctadecyloxacarbocyanine Perchlorate
(DiO), intravenously administered and allowed to circulate for 24
hours. Drawing on several tissue types, myeloid cells were
extracted, including neutrophils; the monocyte/macrophage
(Mo/M.PHI.) pool, including Ly-6C.sup.lo and Ly-6C.sup.hi
monocytes, DCs, and T cells for analysis by flow cytometry.
[0196] Myeloid cell targeting was observed in the heart allograft,
blood and spleen (FIGS. 1F and 1G). Importantly, the inventors
observed cellular specificity towards the Mo/M.PHI. pool and
neutrophils, with significantly higher mTOR-HDL uptake by the
Mo/M.PHI. pool than either DC or neutrophils in the heart, blood
and spleen (respectively: P.ltoreq.0.01, P.ltoreq.0.01,
P.ltoreq.0.05 and P.ltoreq.0.01, P.ltoreq.0.01, P.ltoreq.0.05). In
contrast, the DiO-labeled mTOR-HDL uptake by T cells was virtually
absent (FIG. 1F, 1G), indicative for the nanotherapy's innate
immune cell specificity. Overall, the data demonstrate that
mTOR-HDL exhibits high specificity for inflamed sites, such as the
heart allograft, and is avidly taken up by myeloid cells including
monocytes, DC and neutrophils.
mTOR-HDL Significantly Decreases the Myeloid Cell Compartment
[0197] The leukocyte population was profiled and, more extensively,
the myeloid cell compartment, including neutrophils, Mo/M.PHI. and
DC, in the blood, spleens and allografts of mice receiving placebo,
oral rapamycin (Oral-Ra) and mTOR-HDL treatments, where the
treatment regimen involved three intravenous mTOR-HDL injections,
on the day of, as well as at two and five days post
transplantation. In line with the targeting data, significantly
decreased total leukocytes were observed in the blood, spleens and
allografts (FIG. 2A and FIG. 8) of mTOR-HDL-treated recipients as
compared to either placebo (P.ltoreq.0.05 and P.ltoreq.0.01) or
Oral-Ra-treated recipients. Additionally, these data show that
mTOR-HDL treatment lowered neutrophil levels in the blood, spleen
and allograft, as compared to both placebo (P.ltoreq.0.05,
P.ltoreq.0.05 and P.ltoreq.0.05) and Oral-Ra-treated recipients
(P.ltoreq.0.05). In addition, mTOR-HDL treatment dramatically
reduced Mo/M.PHI. numbers in the circulation, spleen and heart
allografts, as compared to placebo (P.ltoreq.0.05, P.ltoreq.0.01
and P.ltoreq.0.05) or Oral-Ra-treated recipients (P.ltoreq.0.05).
Finally, mTOR-HDL treatment dramatically decreased DC in the
circulation, spleen and allograft, as compared to placebo
(P.ltoreq.0.05, P.ltoreq.0.01 and P.ltoreq.0.05) or Oral-Ra-treated
recipients (P.ltoreq.0.05). All together, these results demonstrate
that mTOR-HDL treatment limits the alloreactive immune response by
interfering with myeloid cell accumulation in the transplanted
allograft.
[0198] Following these myeloid cell investigations, the effects of
mTOR-HDL nanotherapy on Mo/M.PHI. tissue distribution were
evaluated. Mo/M.PHI. comprise two different subsets (Ly-6C.sup.hi
and) Ly-6C.sup.lo with district migratory properties.sup.23. Six
days after transplantation, untreated and Oral-Ra-treated mice had
increased numbers of accumulated myeloid cells in their blood,
spleen and heart allografts (FIG. 2B and FIG. 9A). Further, the
elevated Mo/M.PHI. populations contained high percentages of
inflammatory Ly-6C.sup.hi monocytes (FIGS. 9A and 2B). By contrast,
mTOR-HDL recipients accumulated significantly more Ly-6C.sup.lo
monocytes than placebo and Oral-Ra-treated animals in blood (60%
vs. 12% and 13%), spleen (55% vs. 29% and 44%) and heart allografts
(56% vs. 20% and 18%) (FIG. 2B, FIG. 9A). Accordingly, notably
fewer circulating Ly-6C.sup.hi monocytes were identified in the
mTOR-HDL-treated group than in either the placebo or the
Oral-Ra-treated recipients (P.ltoreq.0.05 and P.ltoreq.0.05,
respectively). The Mo/M.PHI. subset proportions in the spleen and
transplanted organs reflected the levels in peripheral blood (FIG.
1E). The data indicate that while Ly-6C.sup.hi monocytes dominate
the myeloid response in transplant rejection, Ly-6C.sup.lo
monocytes dominate the myeloid response during tolerance. This
suggests mTOR-HDL treatment promotes the accumulation of regulatory
Ly-6C.sup.lo M.PHI., and can rebalance the myeloid compartment in
favor of homeostasis.
mTOR Pathway is Negatively Regulated by mTOR-HDL
[0199] Molecular pathways targeted by mTOR-HDL nanoimmmunotherapy
were studied using Gene Set Enrichment Analysis (GSEA) of mRNA
isolated from flow sorted M.PHI. from the allografts of either
placebo or mTOR-HDL treated recipients. Gene array results
indicated that the mTOR (FIG. 2C) pathway is negatively regulated
by mTOR-HDL.
mTOR-HDL Treatment Favor the Induction of Transplantation Tolerance
by Promoting the Development of Regulatory Ly-6C.sup.lo M.PHI.
[0200] Next, the suppressive function of graft-infiltrating
Ly-6C.sup.lo Mo/M.PHI. allografts were evaluated in vitro.
Ly-6C.sup.lo M.PHI.'s regulatory suppressive function was assessed
by the capacity to inhibit in vitro proliferation of
carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled
CD8.sup.+ T cells. The results of the present invention indicate
that regulatory Ly-6C.sup.lo M.PHI. obtained from the allografts of
mTOR-HDL treated recipient mice prevent T cell proliferation in
vitro (FIG. 3A). The inventors also observed that, unlike
Ly-6C.sup.lo M.PHI. obtained from the allografts of placebo
recipient mice, Ly-6C.sup.lo M.PHI. obtained from the allografts of
mTOR-HDL treated recipients expand immunosuppressive
Foxp3-expressing T-regs (FIG. 3A). In agreement with these data,
the inventors observed a significant increase in the number of
allograft CD4.sup.+CD25.sup.+ T-cells (FIG. 3B; FIG. 10). This
suggests that mTOR-HDL treatment may favor the induction of
transplantation tolerance by promoting the development of
regulatory Ly-6C.sup.lo M.PHI..
mTOR-HDL Nanotherapy Prevents Potent T Cell Stimulation by
Dendritic Cells
[0201] Since dendritic cells (DC) take up mTOR-HDL nanoparticles
(FIGS. 1F-G), the effects of mTOR-HDL on immune cell activation,
antigen presentation and DC-mediated T cell stimulation were
investigated. First, enzyme-linked immunosorbent assay (ELISA) were
utilized to assess the expression of tumor necrosis factor alpha
(TNF-.alpha.). These data indicate that mTOR-HDL treatment
significantly reduces the serum TNF-.alpha. levels, as compared to
placebo and Oral-Ra (FIG. 10; P.ltoreq.0.05 and P.ltoreq.0.01).
Next, the expression of co-stimulatory and adhesion molecules that
are upregulated during acute rejection.sup.25,26 were examined.
Flow cytometry indicates that both CD40 and CD54 molecules are
significantly reduced in leukocytes from mTOR-HDL treated
recipients compared to the placebo and Oral-Ra-treated recipients
(FIG. 10). Using the Y-Ae monoclonal antibody (mAb), which
recognizes a donor-derived I-E.sup.d peptide presented by recipient
MHC class II I-A.sup.b molecules, mTOR-HDL's effects on antigen
presentation were evaluated. Significantly fewer antigen-presenting
Y-Ae.sup.+ cells were observed in the para-aortic lymph nodes and
spleens of mTOR-HDL-treated recipients than those from either
placebo or Oral-Ra. Next, the capacity of DC obtained from mTOR-HDL
recipients was evaluated to stimulate antigen-specific T cells in
vitro. CD11c.sup.+MHC-II.sup.+ DC extracted from the spleens of
placebo and mTOR-HDL-treated mice were used as initiators to
stimulate a mixed lymphocyte reaction (MLR) in vitro.
Antigen-specific TEa CD4.sup.+ T cells were isolated as responders,
as these T cells recognize the same I-E.sup.d-I-A.sup.b complex of
peptide and MHC as do Y-Ae mAb, labeled the cells with
carboxyfluoroscein succinimidyl ester (CFSE) and cultured with
CD11c.sup.+MHC-II.sup.+ splenic DC as previously described.sup.27.
The stimulatory properties of CD11c.sup.+MHC-II.sup.+ splenic DC
were tested by measuring CFSE dilution in T cells by flow
cytometry. These data indicate that DC from mTOR-HDL recipients are
significantly less capable of stimulating naive T cell
proliferation in vitro than DC obtained from control mice. Next,
the proliferative capabilities of T cells obtained from
transplanted mice were tested. These data indicate that T cells
from mTOR-HDL recipients are able to mount in vitro immune
responses similar to T cells obtained from placebo rejecting mice.
Overall, these results illustrate that mTOR-HDL nanoparticle
treatment prevents DC-mediated graft-reactive T cell immune
responses.
mTOR-HDL Nanotherapy Promotes the Development of Suppressive
Macrophages
[0202] Having determined that mTOR-HDL nanoparticles target
Mo/M.PHI. (FIGS. 1F and 1G) and affect their tissue distribution),
the functional properties of Ly-6C.sup.lo Mo/M.PHI. that accumulate
in the allograft during tolerance induction, were tested. Donor
heart allografts were harvested six days after transplantation and
the myeloid compartment was analyzed by flow cytometry. By focusing
on live CD45.sup.+CD11b.sup.+ recipient graft-infiltrating myeloid
cells, we discerned three major populations based on differential
expression patterns of Ly-6C.sup.hi Mo/M.PHI., Ly-6C.sup.lo
Mo/M.PHI. and Ly-6 G neutrophils (FIG. 1D). Flow cytometric
analysis confirmed the presence of more Ly-6C.sup.lo than
Ly-6C.sup.hi Mo/M.PHI. in the allografts of mTOR-HDL-treated mice
as compared to placebo recipients (FIG. 1D). There were no
differences in Ly-6 G neutrophil frequency between the groups.
[0203] Gene array characterization of Ly-6C.sup.lo macrophages that
accumulate in the allografts of mTOR-HDL treated recipients
revealed that the mTORC1 pathway is negatively regulated in these
mice. This confirms that mTOR-HDL treatment targets
graft-infiltrating macrophages.
[0204] A comprehensive analysis of the costimulatory molecules that
prevent successful organ transplantation revealed that mTOR-HDL
treatment increased CD40 expression. In line with this observation,
the inventors found agonistic CD40 mAb treatment to abrogate the
prolonged allograft survival in mTOR-HDL treated recipients FIG.
3F). This suggests that CD40L expressing T cells may stimulate CD40
signaling in recipient M.PHI., resulting in eventual graft loss. To
suppress detrimental CD40 signaling, the inventors developed a
second nanoimmunotherapy treatment consisting of a CD40-TRAF6
inhibitory HDL (referred to as CD40-HDL or TRAF6i-HDL; FIG. 11A-B).
The small molecule inhibitor CD40-TRAF6 is directed against the
binding domain of CD40 on TRAF6 and blocks CD40 signaling,
resulting in Ly6C.sup.hi inflammatory macrophage polarization
towards an anti-inflammatory phenotype.
mTOR-HDL Prolongs Allograft Survival Indefinitely
[0205] Lastly, nanoimmunotherapy treatment's capacity to prevent
organ rejection and prolong allograft survival was evaluated.
Balb/c (H2.sup.d) donor cardiac allografts were transplanted into
fully allogeneic C57Bl/6 (H2.sup.b) recipients treated with: (1)
placebo, (2) Oral-Ra, (3) mTOR-HDL, (4) TRAF6i-HDL, or (4)
mTOR-HDL+TRAF6i-HDL. To assess graft survival, recipients underwent
abdominal palpation until cardiac contractions completely ceased.
The present data indicate that mTOR-HDL nanotherapy dramatically
prolongs graft survival with more than 85% allograft survival over
a 50-day period (FIG. 3G) By contrast, the oral rapamycin treatment
only prolonged allograft survival by 35% during the same period
(P.ltoreq.0.01, P.ltoreq.0.01). This is a remarkable result,
especially considering the regimen involved only three doses during
the first week post-transplantation.
[0206] As a secondary endpoint, we evaluated the histology of the
allografts 100 days after combined treatment (FIGS. 13A-B). FIG.
13B shows mild circumferential inflammation without arteritis and
no signs of intimal hyperplasia. Mouse aortic segments did not
exhibit any histological alteration with no intimal thickening and
no signs of chronic allograft vasculopathy (CAV). Furthermore, the
inventors evaluated a combined treatment regimen involving three
injections of both mTOR-HDL and TRAF6i-HDL within the first five
days post transplantation using the heart allograft model. As shown
in FIG. 3G, combined mTOR-HDL/TRAF6i-HDL treatment promotes organ
transplant synergistically, resulting in a more than 70% survival
over 100 days post-transplantation, significantly outperforming
mTOR-HDL and TRAF6i-HDL monotherapies.
[0207] The timing of treatment can vary and can commence either
before the transplantation, concomitant with the transplantation,
or following transplantation. In one embodiment, the mTOR-HDL or
combined mTOR-HDL/TRAF6i-HDL treatment is initiated 1-2 days before
organ transplantation.
[0208] To test whether in vitro suppressive Ly-6C.sup.lo Mo/M.PHI.
mediate prolonged graft survival in mTOR-HDL-treated recipients,
the inventors depleted Ly-6C.sup.lo Mo/M.PHI. in vivo, as recently
described.sup.9. Briefly, Balb/c (H2.sup.d) donor cardiac
allografts were transplanted into fully allogeneic CD169 diphtheria
toxin (DT) receptor (DTR) (H2.sup.b) recipient mice on the day of
transplantation to deplete recipient Ly-6C.sup.lo macrophages.
Graft-infiltrating leukocytes were examined by flow cytometry six
days after transplantation to confirm the specific depletion of
Ly-6C.sup.lo in vitro suppressive macrophages (FIG. 3B). Subsequent
graft survival experiments showed that Ly-6C.sup.lo Mo/M.PHI.
depletion resulted in graft rejection by day 15 (12.3.+-.1.8)
despite mTOR-HDL treatment (FIG. 3D). Adoptive transfer of
wild-type monocytes restored allograft survival, demonstrating that
the nanoimmunotherapy exerts its effects through regulatory M.PHI..
These experiments suggest that mTOR-HDL treatment stimulates in
vivo development of regulatory Ly-6C.sup.lo macrophages that
prevent T cell-mediated immune responses and thereby promotes
prolonged allograft survival.
[0209] To further investigate the general therapeutic applicability
of mTOR-HDL, mTOR-HDL nanotherapy described here was applied to a
fully allogeneic skin transplant model in which rejection was
macroscopically monitored (FIGS. 12A AND 12B). Using the same
three-dose regimen, the mTOR-HDL nanomedicine treatment
dramatically enhanced graft survival. The mean survival time
significantly increased in mTOR-HDL-treated recipients, with more
than 75% survival at day 50; the placebo group, on the other hand,
had a 100% rejection rate (P.ltoreq.0.01) (10.5.+-.2.9 days).
Overall, these experiments and results show that mTOR-HDL
nanotherapy prevents DC-mediated T cell stimulation, promotes
Ly-6C.sup.lo Mo/M.PHI. development and dramatically prolongs
allograft survival.
[0210] FIGS. 13A-B are graphs showing toxicity associated with
Oral-Ra compared with mTOR-HD treatment. Recipient mice either
received the mTOR-HDL treatment regimen or were given an Oral-Ra
treatment for which the dose was increased to achieve the same
therapeutic outcome (n=4, grey) or (n=4, black). mTOR-HDL has no
significant effects on blood urea nitrogen (BUN, shown in FIG. 13A)
or serum creatinine (shown in FIG. 13B), but kidney toxicity
parameters show statistical differences between Oral-Ra and
mTOR-HDL, while no differences between syngeneic and mTOR-HDL
recipients 30 days after infusion were observed (ANOVA
*P.ltoreq.0.05,**P.ltoreq.0.01).
[0211] Histology sections from kidneys, stained by H&E, PAS and
Masson Trichrome and examined by a renal pathologist show no
significant changes in the three compartments of kidney parenchyma
(FIG. 13A). There is normal appearing glomeruli, with no evidence
of glomerulosclerosis. The tubules show no significant atrophy or
any evidence of epithelial cell injury including vacuolization,
loss of brush border or mitosis. Arteries and arterioles show no
evidence of intimal fibrosis or arteriolar hyalinosis,
respectively. Liver sections stained by H&E, PAS and Masson
Trichrome and examined by a liver pathologist demonstrate normal
acinar and lobular architecture. There is no evidence of
inflammation or fibrosis in the portal tract and hepatic
parenchyma. Hepatocytes are normal with no evidence of cholestasis,
inclusions or apoptosis (FIG. 13A). In FIG. 13B the section shows
mild circumferential inflammation without arteritis and no signs of
intimal hyperplasia. Mouse aortic segments did not exhibit any
histological alteration with no intimal thickening, and no signs of
chronic allograft vasculopathy (CAV).
Discussion
[0212] Transplant patients are treated with immunosuppressive drugs
to avoid organ rejection.sup.30. Immunosuppressants target the
adaptive immune system and have serious side effects.sup.31,32.
Current transplant immunology research seeks to develop novel
tolerogenic protocols using different experimental transplantation
models. Combining basic immunology with innovative nanomedicine is
a promising new approach to encourage immune tolerance. The use of
animal models plays an essential role in this research.
Unfortunately, while some experimental tolerogenic protocols can
induce indefinite allograft survival in mice and
primates.sup.33,34, thromboembolic complications have prevented
these methods from being translated into clinical
treatments.sup.35. Consequently, there is an ongoing need for
alternative approaches to immune regulation, such as targeting the
innate immune system, to prevent transplant
rejection.sup.11,12,36.
[0213] In the current study, the data demonstrate that
conservatively-dosed HDL-encapsulated rapamycin prolongs graft
survival. This indicates that only encapsulated rapamycin--i.e. not
the free form--may be used to induce immunological tolerance, as
recently described.sup.37. The data also mechanistically show that
mTOR-HDL decreases leukocytes in the blood, spleen and allograft.
Reduced leukocyte adhesion and migration is associated with better
graft survival, in agreement with previous studies.sup.38-41. More
specifically, significantly lower Mo/M.PHI. and neutrophil counts
accompanied by less myeloid cell infiltration in allografts were
observed. In contrast to the present mTOR-HDL nanotherapy approach
which targets the myeloid compartment, 95% of absorbed oral
rapamycin binds to erythrocytes.sup.42. Therefore, the present
nanotherapy delivery strategy presents an innovative way to
dramatically increase the drug's bioavailability.
[0214] In association with its capacity to decrease cellular
infiltration in the transplanted organ, in vivo mTOR-HDL
administration markedly reduces production of pro-inflammatory
molecules and diminishes the ability of DC to induce T cell
proliferation. These results accord with a previous report showing
that DC conditioned in vitro with rapamycin reduce pro-inflammatory
mediators and prolong allograft survival.sup.43. Additionally,
these data indicate that mTOR-HDL nanotherapy further affects DC by
inhibiting their stimulatory function, thereby suggesting
alloantigen-specific T cell activation can be therapeutically
modulated. The present data also demonstrate that mTOR-HDL
treatment reduces alloantigen presentation to CD4.sup.+ T cells.
These immune regulatory effects are of pivotal importance during
transplantation, in which antigen-presenting cells mediate the
specific alloreactivity against the transplanted organ.sup.44.
[0215] The present data illustrate that mTOR-HDL treatment mediates
the accumulation of suppressive macrophages that inhibit cytotoxic
T cell responses. In addition, Ly-6C.sup.lo macrophages from
HDL-treated recipients expand Foxp3.sup.+Treg in vitro and
correlate with intra-graft Foxp3.sup.+Treg accumulation in vivo.
Regulatory Ly-6C.sup.lo macrophage accumulation in the transplanted
organ appears to be critical to prolonged allograft survival as
mediated by TOR-HDL, since depleting Ly-6C.sup.lo macrophages
prevents tolerance induction despite mTOR-HDL treatment. These
results are consistent with studies showing that Ly-6C.sup.lo
macrophages inhibit cytotoxic T cell proliferation, mediate Treg
expansion and promote transplantation tolerance.sup.9. The results
demonstrate that HDL-based nanoparticles represent novel a
therapeutic approach to develop drug delivery systems that target
macrophages in vivo.
[0216] Collectively, these data show that HDL nanoparticle
technology effectively delivers immunosuppressive drugs to the
innate immune system. mTOR-HDL prevents DC activation, promotes the
regulatory macrophage development and induces indefinite allograft
survival. The mTOR-HDL technology is an innovative, effective, and
a potentially translational therapeutic approach that targets
innate immune cells to induce long-term allograft survival.
Clinical testing and implementation of an optimized GMP protocol
will confirm long-term safety and efficacy. As mTOR-HDL combines
existing FDA approved agents, its development--or the development
of HDL nanoparticles systems that release other FDA-approved
immunosuppressive agents--may have an immediate path to
translation.
Materials and Methods
Nanoparticle Synthesis
[0217] The present targeted approach delivers the drug rapamycin
using a novel synthetic high-density lipoprotein nanoparticle
platform. mTOR-HDL nanoparticles were synthesized using a modified
lipid film hydration method. Briefly,
1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC),
1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC) (both
purchased from Avanti Polar Lipids) and rapamycin (Selleckchem)
were dissolved in a chloroform/methanol (10:1 v/v) mixture at a
3:1:0.5 weight ratio. After evaporating the solvents, human APOA1
in PBS was added to hydrate the lipid film, in a phospholipid to
APOA1 5:1 weight ratio and left to incubate for 20 minutes in an
ice-bath. The resulting mixture was homogenized using a probe
sonicator in an ice-bath for 15 minutes to yield mTOR-HDL
nanoparticles. mTOR-HDL was washed and concentrated by centrifugal
filtration using 10 kDa molecular weight cut-off (MWCO) filter
tubes. Aggregates were removed using centrifugation and filtration
(0.22 .mu.m). Oral rapamycin solution (Oral-Ra) consisted of 4%
ethanol, 5% PEG300 and 5% TWEEN80 in PBS, while intravenous
rapamycin solution (i.v.-Ra) included 4% ethanol and 5% TWEEN80 in
PBS. The animals received oral doses or intravenous tail injections
(for mTOR-HDL or i.v.-Ra) at a rapamycin dose of 5 mg/kg on the day
of transplantation as well as days two and five
posttransplantation.
[0218] CD40-HDL nanoparticles were synthesized according to a
similar procedure as described above. DMPC, MHPC and the
TRAF6-inhibitor
(2E)-1-phenyl-3-(2,5-dimethylanilino)-2-propen-lone1 were dissolved
in a chloroform/methanol mixture (10:1 v/v) at a 8.7:1:0.6 weight
ratio, and then dried under vacuum to create a thin lipid film. PBS
containing APOA1 was added to the lipid film, in a phospholipid to
APOA1 9.5:1 weight ratio, and left to incubate on ice for 3 hours
until the film was hydrated and a homogenous solution was formed.
The solution was then sonicated for 1 hour to form CD40-HDL
nanoparticles. Subsequently, the solution was purified by multiple
centrifugation and filtration steps.
Mice
[0219] Female C57BL/6J (B6 WT, H-2b), BALB/c (H-2d) mice were
purchased from the Taconic Laboratory. 8 week old C57BL/6J
(Foxp3tm1Flv/J) mice were purchased from The Jackson Laboratory.
The C57BL/6J CD169.sup.DTR mice were from Masato Tanaka (Kawaguchi,
Japan). C57BL/6J CD4.sup.+ transgenic TEa mice that recognize a
peptide representing residues 52 to 68 of the I-E.alpha. chain
(E.alpha. peptide) bound to class II I-A.sup.b molecules were from
Alexander Rudensky (New York, USA). Animals were enrolled at 8 to
10 weeks of age (body weight, 20-25 g). All experiments were
performed with 8 to 12 week old female matched mice in accordance
with protocols approved by the Institutional Animal Care and
Utilization Committee.
Vascularized Heart Transplantation
[0220] BALB/c hearts were transplanted as fully vascularized
heterotopic grafts into C57BL/6 mice as previously
described.sup.45. Hearts were transplanted into recipients'
peritoneal cavities by establishing end-to-side anastomosis between
the donor and recipient aortae and end-to-side anastomosis between
the donor pulmonary trunk and the recipient inferior vena cava.
Cardiac allograft survival was subsequently assessed through daily
palpation. Rejection was defined as the complete cessation of
cardiac contraction and was confirmed by direct visualization at
laparotomy. Graft survival was compared among groups using
Kaplan-Meier survival analysis.
Micro-PET/CT Imaging and Biodistribution Studies
[0221] Mice (n=6; 3 with heart transplants and 3 with skin grafts)
[weight: 18.8.+-.1.0 g]) were injected with .sup.89Zr-mTOR-HDL
(0.17.+-.0.01 mCi, .about.0.25 mg APOA1) in 0.2 mL PBS solution via
their lateral tail vein. At 24 h, animals were anesthetized with
isoflurane (Baxter Healthcare, Deerfield, Ill., USA)/oxygen gas
mixture (2% for induction, 1% for maintenance), and a scan was then
performed using an Inveon PET/CT scanner (Siemens Healthcare
Global, Erlangen, Germany). Whole body PET static scans, recording
a minimum of 30 million coincident events, were performed for 15
min. The energy and coincidence timing windows were 350-700 keV and
6 ns, respectively. The image data were normalized to correct for
PET response non-uniformity, dead-time count losses, positron
branching ratio and physical decay to the time of injection, but no
attenuation, scatter or partial-volume averaging correction was
applied. The counting rates in the reconstructed images were
converted to activity concentrations (percentage injected dose [%
ID] per gram of tissue) using a system calibration factor derived
from imaging a mouse-sized water-equivalent phantom containing
.sup.89Zr. Images were analyzed using ASIPro VM.TM. software
(Concorde Microsystems, Knoxville, Tenn., USA). Whole body standard
low magnification CT scans were performed with the X-ray tube setup
at a voltage of 80 kV and current of 500 .mu.A. The CT scan was
acquired using 120 rotational steps for a total of 220 degrees
yielding an estimated scan time of 120 s with an exposure of 145 ms
per frame. Immediately after the PET/CT scan, animals were
sacrificed and tissues of interest--kidney, heart, liver, spleen,
blood, bone, skin, and muscle--were collected, weighed and counted
on a Wizard2 2480 automatic gamma counter (Perkin Elmer, Waltham,
Mass.) to determine radioactivity content. The values were
decay-corrected and converted to percentage of injected dose per
gram (% ID/g). To determine radioactivity distribution within the
transplanted hearts, the native and grafted specimens were placed
in a film cassette against a phosphorimaging plate (BASMS-2325,
Fujifilm, Valhalla, N.Y.) for 4 hours at -20.degree. C. The plate
was read at a pixel resolution of 25 .mu.m with a Typhoon 7000IP
plate reader (GE Healthcare, Pittsburgh, Pa.). The images were
analyzed using ImageJ software.
Isolating Graft-Infiltrating Leukocytes
[0222] Mouse hearts were rinsed in situ with HBSS with 1% heparin.
Explanted hearts were cut into small pieces and digested for 40
minutes at 37.degree. C. with 400 U/ml collagenase IV
(Sigma-Aldrich), 10 mM HEPES (Cellgro) and 0.01% DNase I (MP
Biomedicals) in HBSS (Cellgro). Digested suspensions were passed
through a nylon mesh and centrifuged, and the cell pellet was
re-suspended in complete HBSS, stained and analyzed by flow
cytometry (BD LSR-II; BD Biosciences).
Flow Cytometry and Cell Sorting
[0223] For myeloid cell staining, fluorochrome-conjugated mAbs
specific to mouse CD45 (clone 30-F11), CD11b (clone M1/70), CD11c
(clone N418), F4/80 (clone CI:A3.1), Ly-6C (clone HK1.4) and
corresponding isotype controls were purchased from eBioscience.
Ly-6 G (clone 1A8) mAb was purchased from Biolegend. F4/80 (clone
CI:A3.1) was purchased from AbD Serotec. For T cell staining,
antibodies against CD45 (clone 30-F11), CD3 (clone 2C11), CD4
(clone GK1.5), CD8 (clone 53-6.7), CD25 (clone PC61.5), CD40 (clone
1C10) and CD54 (clone YN1/1.7.4) were purchased from eBioscience.
The absolute cell counting was performed using countbright beads
(Invitrogen). To detect antigen presentation, the Y-Ae mAb was
purchased from eBioscience. Flow cytometric analysis was performed
on LSR II (BD Biosciences) and analyzed with FlowJo software (Tree
Star, Inc.). Results are expressed as percentage of cells staining
or cells counting (cells per milliliter) above background. mAbs
were titered at regular intervals during the course of these
studies to ensure the use of saturating concentrations. To purify
graft-infiltrating myeloid cells, donor heart single cell
suspensions were sorted with an InFlux cell sorter (BD) to achieve
>96% purity at the Flow Cytometry Shared Resource Facility at
Icahn School of Medicine at Mount Sinai.
Mixed Lymphocyte Reaction
[0224] Spleens of antigen-specific TE.alpha. (H-2.sup.b) mice were
gently dissociated into single-cell suspensions, and red blood
cells were removed using hypotonic ACK lysis buffer. Splenocytes
were labeled with CFSE cell proliferation marker at 5 .mu.M
concentration (molecular probes from Invitrogen) followed by
staining with anti-CD4 mAb for 30 minutes on ice. Responder
CFSE.sup.+CD4.sup.+ T cells were sorted using FACS Aria II sorter
(BD Biosciences) with a purity of >98%. Splenocytes from
mTOR-HDL- and placebo-treated recipients were enriched for
CD11c.sup.+ cells using the EasySep Mouse CD11c positive selection
Kit (StemCell). Enriched CD11c.sup.+ splenocytes were stained with
anti-mouse CD11c mAb for 30 minutes on ice. CD11c.sup.+ cells were
sorted using FACS Aria II sorter (BD Biosciences) and then used to
stimulate responder CFSE.sup.+CD4.sup.+ T cells. Cells were
cultured for 4 days at 37.degree. C. in a 5% CO.sub.2 incubator,
and CFSE.sup.+CD4.sup.+ T cells proliferation was measured by flow
cytometric analysis of CFSE dilution on CD4.sup.+ T cells.
In Vitro Suppression Assay
[0225] Spleens of C57BL/6 (H-2.sup.b) mice were gently dissociated
into single-cell suspensions, and red blood cells were removed
using hypotonic ACK lysis buffer. Splenocytes were labeled with
CFSE at 5 .mu.M concentration (molecular probes from Invitrogen)
followed by staining with anti-CD8 mAb for 30 minutes on ice.
Responder CFSE.sup.+CD8.sup.+ T cells were sorted using FACS Aria
II (BD Biosciences) with >98% purity. CFSE.sup.+CD8.sup.+ T
cells were used together with anti-CD3/CD28 microbeads as
stimulators. Stimulated CFSE.sup.+CD8.sup.+ T cells were cultured
with graft-infiltrating Ly-6C.sup.lo macrophages, mTOR-HDL or
placebo for 72 hours at 37.degree. C. in a 5% CO.sub.2 incubator. T
cell proliferation was measured by flow cytometric analysis of CFSE
dilution on CD8.sup.+ T cells.
Treg Expansion Assay
[0226] Spleens of C57BL/6-Foxp3tm1F1v/J (H-2.sup.b) mice were
gently dissociated into single-cell suspensions, and red blood
cells were removed using hypotonic ACK lysis buffer. Splenocytes
were stained with anti-CD4 mAb for 30 minutes on ice. Responder
CD4.sup.+ were sorted using FACS Aria II (BD Biosciences) with a
purity of >98%. CD4.sup.+ T cells were used together with
anti-CD3/CD28 microbeads as stimulators. Stimulated CD4.sup.+ T
cells were cultured with graft-infiltrating Ly-6C.sup.lo
macrophages, mTOR-HDL or placebo for 72 hours at 37.degree. C. in a
5% CO.sub.2 incubator. Treg expansion was measured by flow
cytometric analysis of Foxp3-RFP on CD4.sup.+ T cells.
Microarray.
[0227] Graft infiltrating recipient Ly-6C.sup.lo macrophages were
sorted from mTOR-HDL treated and placebo rejecting recipients at
day 6 after transplantation. Cells were sorted twice with a FACS
Aria II sorter (BD Biosciences) to achieve >98% purity.
Microarray analysis of sorted cells was performed with a total of 6
Affymetrix Mouse Exon GeneChip 2.0 arrays were run in triplicate
with the samples of interest. Raw CEL file data from Affymetrix
Expression Console were background corrected, normalized, and
summarized using RMA. The summary expression scores were computed
at the transcript meta-probeset level using annotation files
supplied by the manufacturer. Gene expression was filtered based on
IQR (0.25) filter using gene filter package. The log 2 normalized
and filtered data (adjusted P.ltoreq.0.05) was used for further
analysis. Gene signature comparisons were performed between
intra-graft Ly6C.sup.lo macrophages from mTOR-HDL and placebo
treated recipients. GSEA was performed using GSEA version 17 from
Gene pattern version 3.9.6. Parameters used for the analysis were
as follows. Gene sets c2.cp.biocarta.v5.1.symbols.gmt;
c2.cp.kegg.v5.1.symbols.gmt; c2.cp.reactome.v5.1.symbols.gmt;
c6.all.v5.1.symbols.gmt (Oncogenic Signatures),
c7.all.v5.1.symbols.gmt (Immunologic signatures); and
h.all.v5.1.symbols.gmt (Hallmarks) were used for running GSEA and
1000 permutations were used to calculate p value and permutation
type was set to gene set. Each gene set was run separately. All
basic and advanced fields were set to default. To select the
significant pathways from each gene set result, fdr q-value of 0.25
was set as cutoff. Only genes that contributed to core enrichment
were considered.
In Vivo Macrophage Depletion
[0228] To deplete CD169-expressing Ly-6C.sup.lo macrophages,
heterozygous CD169-DTR recipients were injected intraperitoneally
with 10 ng/g body weight of DT (Sigma-Aldrich) 24, 48 and 72 hours
after transplantation.sup.46.
Statistical Analyses
[0229] Results are expressed as mean.+-.SEM. Statistical
comparisons between 2 groups were evaluated using the Mann Whitney
tests. Kaplan-Meier survival graphs were performed, and a log-rank
comparison of the groups calculated P values. A value of
P.ltoreq.0.05 was considered statistically significant. IBM SPSS
statistics 22 were utilized for statistical analysis.
Near Infrared Fluorescence Imaging
[0230] C57/B6 wild type mice received a single intravenous
injection of 5 mg/kg mTOR-HDL labeled with either DiR dye or
phosphate-buffered saline (PBS). After 24 hours, the mice were
sacrificed and perfused with PBS. Liver, spleen, lung, kidney,
heart and muscle tissues were collected for NIRF imaging.
Fluorescent images were acquired with the IVIS 200 system (Xenogen)
with a 2 second exposure time using a 745 nm excitation filter and
a 820 nm emission filter. Both the average radiant efficiency
within each tissue and the ratio to control have been
quantified.
Radiolabeling mTOR-HDL Nanoparticles
[0231] .sup.89Zr-mTOR-HDL was prepared according to previously
described procedures [15]. Briefly, ready-to-label mTOR-HDL was
obtained by adding 1 mol % of the phospholipid chelator
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-1,8-Diazafluoren-9-one
(DSPE-DFO) [44] at the expense of
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in the initial
formulation. Radiolabeling with .sup.89Zr was achieved by reacting
the DFO-bearing nanoparticles with .sup.89Zr-oxalate in PBS
(pH=7.1) at 37.degree. C. for 1 h. .sup.89Zr-mTOR-HDL was isolated
by centrifugal filtration using 10 kDa molecular weight cut-off
tubes. The radiochemical yield was 75.+-.2% (n=2).
Biodistribution of .sup.89Zr-mTOR-HDL
[0232] Immediately after the PET/CT scan, mice were sacrificed and
tissues of interest (blood, heart, kidneys, lungs, liver, spleen,
bone, skin, muscle and graft) harvested, blotted and weighed before
radioactivity counting on a Wizard2 2480 automatic gamma counter
(Perkin Elmer, Waltham, Mass., USA). The radioactivity content was
then converted to radioactivity concentration and expressed as
percentage of injected dose per gram of tissue (% ID/g).
Enzyme-Linked Immunosorbent Assay (ELISA)
[0233] Blood was harvested at day 6 post-transplantation, and sera
were purified using 1.1 ml Z-Gel microtubes (Sarstedt) after
incubation at room temperature and a brief centrifugation.
TNF-.alpha. secretion in sera was assessed by ELISA (eBiosciences)
according to the manufacturer protocol (n=4 in each group).
Allograft cytokine production was determined in supernatants using
commercial ELISA kits for IL-6 and TNF.alpha. according to the
manufacturer guidelines (R&D systems).
Ultrasound Imaging
[0234] Cardiac allograft transplant rate (beats per minute, BPM)
was monitored using a short axis cross sectional B-Mode image of
the transplanted heart, with M-mode cursor line through its largest
dimension and tracing of the left ventricular wall.
Skin Transplantation
[0235] Full-thickness trunk skin allografts were placed as
previously described [42]. Skin was harvested from BALB/C, cut into
0.5-cm pieces and placed in C57BL/6 recipients. The skin allograft
was placed in a slightly larger graft bed prepared over the chest
of the recipient and secured using Vaseline, gauze and a bandage.
The grafts were visually scored daily for evidence of rejection.
Skin allograft rejection was monitored by digital microscope
photography and was considered fully rejected when it was >90%
necrotic. Graft survival was compared among groups using
Kaplan-Meier survival analysis.
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Example 2
Targeted CD40-TRAF6 Inhibition Resolves Macrophage Accumulation in
Atherosclerosis
[0285] In atherosclerosis, macrophage accumulation is directly
linked to destabilization and rupture of plaque, causing acute
atherothrombotic events. Circulating monocytes enter the plaque and
differentiate into macrophages, where they are activated by CD4+ T
lymphocytes through CD40-CD40 ligand signaling. Here we show that
interruption of this signaling pathway in monocytes/macrophages
exerts rapid anti-inflammatory effects in an ApoE.sup.-/- mouse
model of atherosclerosis. For this purpose we developed an
infusible reconstituted high-density lipoprotein nanoparticle
carrying a small molecule inhibitor of the interaction of CD40 and
tumor necrosis factor receptor-associated factor 6. We show
monocyte/macrophage specific targeting of our nanoimmunotherapy,
which impairs their migratory capacity. Rapid reduction of plaque
inflammation by this therapy represents a novel strategy in the
treatment of atherosclerosis, with high potential for clinical
translation, as illustrated by the favorable toxicity profile in
non-human primates.
[0286] The recruitment of circulating monocytes that differentiate
into macrophages is a key contributing process in aggravating
atherosclerotic plaque inflammation [1]. This dynamic macrophage
accumulation in plaque is directly linked to the development of
atherothrombotic events [1].
[0287] As early as in the 1990s it was recognized that the
activation of plaque macrophages by CD4+ T-lymphocytes via
CD40-CD40 ligand (CD40-CD40L) signaling plays a central role in
abetting plaque inflammation [2]. Genetic disruption of CD40L in
apolipoprotein e knockout (Apoe.sup.-/-) mice drastically decreases
atherosclerotic lesion development and diminishes plaque
T-lymphocyte and macrophage content [3]. Treatment of low density
lipoprotein receptor knockout (LDLr.sup.-/-) mice and Apoe.sup.-/-
with an anti-mouse CD40L antibody had similar atheroprotective
effects [4-6]. Further studies revealed that tumor necrosis factor
receptor-associated factor 6 (TRAF6) is of specific importance in
propelling CD40's signaling cascade inside macrophages [7]. TRAFs
are adaptor proteins that can bind the cytoplasmic domain of CD40
and couple the receptor complex to several different signal
transduction pathways [8]. In fact, deficiency of CD40-TRAF6
interactions in myeloid cells has been shown to decrease monocyte
recruitment to plaques and abolish atherosclerotic plaque formation
in Apoe.sup.-/- mice [7].
[0288] Although the CD40-TRAF6 interaction provides a promising
therapeutic target, major limitations are associated with its
inhibition. In addition to CD40-TRAF6 interaction's role in myeloid
cells, it partly controls the maturation of B-lymphocytes and
generation of long-lived plasma cells [9]. Therefore, long-term
inhibition of the CD40-TRAF6 interaction will likely cause immune
deficiencies, rendering it an unfeasible therapeutic approach for
atherosclerosis.
[0289] To address this issue we developed a targeted immunotherapy
with the ability to block the CD40-TRAF6 interaction specifically
in monocytes/macrophages. For this purpose, we incorporated a
recently developed small molecule inhibitor of the CD40-TRAF6
interaction in reconstituted high density lipoprotein (TRAF6i-HDL)
[10, 11]. We show in an Apoe.sup.-/- mouse model of atherosclerosis
that TRAF6i-HDL targets monocytes/macrophages, while lymphocytes do
not take up nanoparticles. After a single week of TRAF6i-HDL
immunotherapy a rapid decrease in plaque inflammation and decreased
monocyte recruitment was observed. In line with these findings,
whole transcriptome analysis indicated that cell migration was
among the affected cellular processes. Finally, to assess its
translational potential, we evaluated TRAF6i-HDL's
pharmacokinetics, biodistribution and safety in non-human primates
(NHPs).
Results
TRAF6i-HDL Characteristics.
[0290] The aim of the study was to decrease plaque inflammation by
specifically inhibiting the CD40-TRAF6 interaction in
monocytes/macrophages via targeted nanoimmunotherapy (TRAF6i-HDL).
The TRAF6i-HDL nanoparticle was constructed from human
apolipoprotein A-I (apoA-I), and the phospholipids
1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC) and
1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), in which a
lipophilic small molecule inhibitor of CD40-TRAF6 interaction (SMI
6877002) was encapsulated [8, 11]. Because apoA-I can have
modulatory effects by itself, the nanoimmunotherapy was designed
with a low apoA-I to drug ratio. The resulting TRAF6i-HDL
nanoparticle, schematically shown in FIG. 14A, measured 22.6+/-12
nm in diameter (PDI=0.3), as determined by dynamic light-scattering
and transmission electron microscopy (TEM). TRAF6i-HDL variants,
incorporating fluorescent dyes (DiO or DiR) or Zirconium-89
(.sup.89Zr) radiolabeled phospholipids, were synthesized to allow
detection by fluorescence techniques, positron emission tomography
(PET), gamma counting and autoradiography.
Schematic Overview
[0291] A schematic overview of the study design is shown in FIG.
14B. The first part of the study was performed in mice with
atherosclerosis (Apoe.sup.-/- mice on a high cholesterol diet). In
these mice, we first studied TRAF6i-HDL's toxicity,
pharmacokinetics, biodistribution, and atherosclerotic plaque
monocyte/macrophage targeting efficiency. Subsequently, we assessed
plaque regression efficacy of a one-week TRAF6i-HDL regimen
involving four intravenous infusions. Next, we investigated the
mechanism by which TRAF6i-HDL affects plaque monocytes/macrophages
using whole transcriptome analysis. The second part of the study
focused on the translatability of TRAF6i-HDL nanoimmunotherapy. For
this purpose we investigated TRAF6i-HDL's toxicity and
pharmacokinetics, while in vivo positron emission tomography with
magnetic resonance (PET/MRI) was performed to longitudinally study
biodistribution and vessel wall targeting in non-human
primates.
Toxicity, Pharmacokinetics, and Biodistribution Studies in Apoe-/-
Mice.
[0292] One week TRAF6i-HDL treatment had no effect on erythrocytes,
platelets or leucocyte levels (FIG. 20). The number of
reticulocytes and lymphocytes was somewhat increased when compared
to placebo. The numbers of T cells and B cells in bone marrow blood
and spleen were not affected by TRAF6i-HDL therapy. No toxic
effects were observed on kidney and hepatic function, although
alkaline phosphatase was somewhat increased (FIG. 21). Lipids,
glucose, protein and electrolytes were unaffected.
[0293] To investigate its pharmacokinetics and biodistribution,
Apoe.sup.-/- mice received a single infusion of
.sup.89Zr-radiolabeled TRAF6i-HDL. Blood radioactivity clearance of
.sup.89Zr-TRAF6i-HDL was measured over 24 hours and data were
fitted using a two-phase decay non-linear regression. The weighted
blood half-life (t.sup.1/2) was finally calculated to be 124.4 min
based on a Ph-fast of 13.7 min and a Ph-slow of 195 min (FIG. 14C).
Biodistribution was evaluated by in vivo PET/CT imaging (FIG. 14C)
and validated by ex vivo gamma counting, the latter expressed as a
percentage of injected dose per gram tissue (% ID/g; FIG. 14D). As
expected, PET/CT imaging showed that TRAF6i-HDL primarily
accumulated in the liver, spleen and kidney, organs known to take
up and metabolize HDL. Gamma counting data confirmed these results,
showing nanoparticle uptake of 12.8% ID/g in the liver, 8.9% ID/g
in the spleen, and 7.9% ID/g in the kidneys. In comparison, the
heart, a similar sized organ, only contained 1.1% ID/g (FIG. 14D).
Ex vivo near infrared fluorescence (NIRF) imaging, 24 hours after
infusion, corroborated the PET/CT and gamma counting observations,
showing that TRAF6i-HDL accumulates mostly in the liver, spleen and
kidneys.
[0294] Flow cytometry analysis revealed that Ly6Chi monocytes and
macrophages in blood, bone marrow, and spleen took up DiO labeled
TRAF6i-HDL. Neutrophils, Ly6Clo monocytes and dendritic cells also
took up DiO-TRAF6i-HDL, while lineage positive CD11b negative cells
(all non-myeloid cells) did not (FIG. 14G), indicative of myeloid
cell specificity.
TRAF6i-HDL Accumulation in Atherosclerotic Lesions.
[0295] Ex vivo gamma counting of whole the aortas showed that 1.3%
ID/g of 89Zr-TRAF6i-HDL had accumulated 24 hours after infusion
(FIG. 14D). Looking specifically at TRAF6i-HDL nanoparticle
distribution throughout the aorta, the uptake was highest in the
aortic sinus area, which is the preferential site of plaque
development in this mouse model. While only accounting or 6.4% of
the total area, the aortic sinus area generated approximately 29%
of the signal, corresponding to 5.9% ID/g (FIG. 1d). NIRF imaging
showed similar preferential accumulation of DiR-labeled TRAF6i-HDL
in the aortic sinus area (FIG. 14E). Cell specificity of DiO
labeled TRAF6i-HDL uptake in aortic plaque was assessed by flow
cytometry. We found that 86% of macrophages and 81% of Ly6Chi
monocytes had taken up DiO-TRAF6i-HDL, while lineage positive cells
(all non-myeloid cells) had taken up virtually none (FIG. 14F).
Furthermore, the majority of neutrophils (64%) and dendritic cells
(61%) in the aortic plaque were found to contain labeled
nanoparticles (FIG. 14G). These results mirror our findings in
blood, bone marrow and spleen, showing that cells of the myeloid
lineage, and in particular the Ly6Chi monocyte subset and
macrophages, are preferentially targeted by TRAF6i-HDL
nanoparticles.
In Vivo Effects of TRAF6i-HDL on Plaque Inflammation.
[0296] To assess the therapeutic efficacy of TRAF6i-HDL, we used 20
week old Apoe.sup.-/- mice that had been on a high cholesterol diet
for 12 weeks in order to develop atherosclerotic lesions. While all
mice remained on a high-cholesterol diet, they received four
intravenous infusions of placebo, control HDL nanoparticles without
payload, or TRAF6i-HDL over a period of 7 days. The CD40-TRAF6
inhibitor dose administered per infusion was 5 mg/kg. To limit a
dominant therapeutic effect of apoA-I itself, we used a low apoA-I
dose of 9 mg/kg. All mice were sacrificed 24 hours after the final
infusion.
[0297] For the first experiment we performed quantitative
histologic analysis of plaques in the aortic sinus area in mice
treated with placebo, HDL or TRAF6i-HDL (n=10 per group).
Cross-sections were stained with Hematoxylin and Eosin (H&E)
and Sirius Red (collagen), and immunostained for Mac3 (macrophages)
and Ki67 (proliferating cells). No significant difference in plaque
size or collagen content was observed across the groups (FIG. 15A).
The percentage of Mac3 positive area was however markedly decreased
by 36% (p=0.001) and 37% (p<0.001) as compared to the placebo
and HDL groups, respectively (FIG. 15B). As a result, also the Mac3
to collagen ratio in the plaque was favorably affected towards a
more stable plaque phenotype in the TRAF6i-HDL group, as the ratio
was decreased by 31% (p<0.001) and 36% (p=0.004) compared to the
placebo and HDL groups (FIG. 15B). The number of proliferating
macrophages was similar in all groups (FIG. 15A), indicating that
the observed decrease in plaque macrophages was not caused by a
decrease in local proliferation of macrophages. Previous studies
showed that in addition to monocyte recruitment, local macrophage
proliferation plays a pivotal role in fueling plaque inflammation
[12].
[0298] Subsequently, we performed fluorescence molecular tomography
fused with computed tomography (FMT/CT) imaging to visualize
protease activity in the aortic sinus area. Placebo (n=8) and
TRAF6i-HDL (n=7) treated Apoe.sup.-/- mice all received one
injection of an activatable pan-cathepsin protease sensor 24 hours
before imaging. The protease sensor is taken up by activated
macrophages, followed by cleavage of the protease sensor within the
endolysosome, yielding fluorescence as a function of enzyme
activity. TRAF6i-HDL therapy decreased protease activity by 60%
(p=0.002, FIG. 16A). Next, we focused on quantification of aorta
macrophage content by flow cytometry of whole aortas. Again, 20
week old Apoe.sup.-/- on a high cholesterol diet were treated with
either placebo (n=27), HDL (n=27) or TRAF6i-HDL (n=27). Aorta
macrophage content decreased markedly in the TRAF6i-HDL treated
group, by 66% and 67% (p<0.001 for both comparisons), as
compared to the placebo and HDL groups (FIG. 16B). These results
corroborate the observations made by histologic analysis and
FMT-CT. Furthermore, in the TRAF6i-HDL treated group aorta T
lymphocyte content was decreased by 65% and 49% when compared to
placebo and HDL respectively. Altogether these data indicate a
potent anti-inflammatory effect of TRAF6i-HDL in atherosclerotic
plaques after only a single week of therapy.
[0299] Since we had already observed that the number of
proliferating Ki67+ macrophages was not affected by therapy, we
hypothesized that the decrease in plaque macrophages content and
inflammation might be caused by decreased monocyte recruitment
instead [13,14]. To further investigate this, we first quantified
aortic Ly6C.sup.hi monocytes in the same flow-cytometry experiment
as the one in which we measured macrophage content. We observed
that the decrease in macrophages was paralleled by a 49% and 52%
(p<0.001 for both comparisons) decrease in Ly6C.sup.hi monocytes
in the aorta, as compared to the placebo and HDL groups
respectively (FIG. 16B). Interestingly, the reduction in aortic
Ly6C.sup.hi monocyte content could not be explained by a systemic
decrease in Ly6C.sup.hi monocytes (FIG. 16C).
[0300] Secondly, we performed an experiment in which the thymidine
analogue 5-bromo-2'-deoxyuridine (BrdU) was injected
intraperitoneally 2 hours prior to sacrificing the mice. BrdU
incorporates into newly synthesized DNA, and therefore can be used
as a marker for proliferation. FIG. 16D shows that the percentage
of plaque macrophages that had incorporated BrdU was not decreased
by TRAF6i-HDL therapy. This result is in line with the histology
observation on Ki67 expression. In an in vitro experiment with RAW
264.7 cell line of murine macrophages, characterized by a high
proliferation rate [15], incubation with the CD40-TRAF6 inhibiting
compound or TRAF6i-HDL did not affect the proliferation rate (FIG.
16E).
[0301] Taken together, these data indicate that plaque macrophage
content as well as protease activity was decreased by TRAF6i-HDL
therapy. The mechanism of action by which TRAF6i-HDL decreases
plaque inflammation is likely mediated through the abatement of
monocyte recruitment, while local macrophage proliferation is not
affected.
Comparative Whole Transcriptome Analysis of Plaque
Monocytes/Macrophages.
[0302] In order to gain insight into the effects of TRAF6i-HDL on
gene expression of plaque monocytes/macrophages, we isolated CD68
positive cells from aortic sinus plaques by laser capture
microdissection of mice either treated with placebo or TRAF6i-HDL.
Whole RNA of these cells was isolated for sequencing.
[0303] We identified genes that were differentially expressed (DE)
between placebo and TRAF6i-HDL treated mice. Correction for
multiple testing was performed with a false discovery rate
(FDR)<0.2 (FIG. 17A). A total of 416 DE genes were identified,
of which 209 genes were down-regulated and 207 up-regulated (FIG.
17B). Gene ontology (GO)-function was used to annotate the DE
genes, and to find cellular components that significantly enriched
with DE genes (FIG. 17C). In the 15 enriched GO terms that
significantly enriched with DE genes, "focal adhesion" is of most
interest. Other enriched GO terms, such as "cell-substrate adherent
junction", "cell-substrate junction", "adherence junction", and
"anchoring junction" are closely related to "focal adhesion" and
the genes in these GO terms overlapped to a high degree (FIG. 22).
Focal adhesion is a dynamic process in which protein complexes
connect to the extracellular matrix, and plays a central role in
monocyte/macrophage migration [16]. In a subsequent analysis, the
same 416 DE genes were mapped with the Kyoto Encyclopedia of Genes
and Genomes (KEGG) pathway tool, by which we identified two
significantly altered pathways, namely "focal adhesion" and
"endocytosis" (FIG. 17D, FIG. 23).
[0304] The most significant DE genes, all with FDR<0.05 (FIG.
17D, FIG. 24) were Adcy3, Lgals3bp, Pltp and Stab1 (up-regulated)
and Impad1, Sept2, Slc4a7 and Spcs2 (down-regulated). Among these
genes, macrophage derived PLTP is known to exert
antiatherosclerotic effects [17], and Stab1 (encodes for
Stabilin-1) has functions in lymphocyte homing and cell adhesion
and is associated with an atheroprotective macrophage phenotype
[18, 19]. Sept2 (encoding for Septin2) is known to be abundantly
expressed in macrophages and is required for phagosome formation
[20]. Together, the transcriptome data analyses indicate that among
various affected processes, focal adhesion is significantly
affected by TRAF6i-HDL therapy. The fact that focal adhesion, a
process involved in cell migration, is importantly affected is
consistent with our aforementioned observation of decreased
Ly6C.sup.hi monocyte recruitment in TRAF6i-HDL treated mice. We did
not observe an effect on gene expression related to macrophage
proliferation, apoptosis or migratory egress (FIG. 25).
TRAF6i-HDL Toxicity, Pharmacokinetics, and Biodistribution Studies
in Non-Human Primates.
[0305] In order to assess the translatability of TRAF6i-HDL
therapy, we performed comprehensive blood testing, histological
analysis, and advanced pharmacokinetics and biodistribution studies
in TRAF6i-HDL treated non-human primates (NHP). Six NHPs were used
for complete haematological analyses and post mortem histological
analysis and another six for biodistribution imaging (PET/MRI) and
blood chemistry analysis. The NHPs were injected with either
placebo or a single dose of TRAF6i-HDL (1.25 mg/kg) and either
sacrificed after 72 hours or imaged at multiple time points and
then sacrificed.
[0306] Complete blood count data from 7 time points within 72 hours
after injection showed no differences between placebo and
TRAF6i-HDL treated animals in white blood cells, monocytes,
neutrophils, lymphocytes, red blood cells, platelets or any of the
other indices. (FIG. 18A). Additionally, blood chemistry analysis
showed no signs of hepatic, renal, pancreatic or muscle cell
toxicity in the TRAF6i-HDL treated group as compared to the placebo
group (FIG. 18B). Furthermore, lipid, glucose, and protein (albumin
and globulin) levels were equal in both groups (FIG. 18B).
Electrolytes were also unaffected. Specimens from liver, kidneys
and spleen were sectioned and stained (H&E) for histology and
evaluated by a pathologist. No signs of tissue damage or
disturbances in tissue architecture were found (FIG. 18C).
[0307] To assess biodistribution, six NHPs were subjected to full
body PET/MR imaging after intravenous administration of
89Zr-labeled TRAF6i-HDL. The animals were dynamically imaged over
the course of the first hour post administration, while subsequent
static scans were performed at 1, 24, 48 and 72 hours. Dynamic PET
imaging showed rapid radioactivity accumulation in the liver,
spleen and kidneys, followed by a significant uptake in the bone
marrow (FIG. 19A). One hour post injection, PET images were
dominated by the strong signal from the kidneys, followed by the
liver and spleen at the 1 hour time point (FIG. 19A). At 24, 48 and
72 hours, radioactivity accumulated mostly in the liver and spleen
(FIG. 19B). After sacrificing the animals at the 72 hour time
point, tissue gamma counting showed that the largest amount of the
injected dose (% ID/g) could be traced back to the liver and
spleen, followed by the kidneys, which corroborates the findings of
the PET/MRI imaging (FIG. 19C). Blood was collected at different
time points and the data were fitted using a two-phase decay
non-linear regression. The t1/2-fast was 14.2 min and the Ph-slow
was 513 min, resulting in a weighted blood half-life (t1/2) of 272
min (FIG. 19D).
Discussion
[0308] In the current study we describe the development of an HDL
based nanoimmunotherapy targeted against the CD40-TRAF6 interaction
in monocytes/macrophages. Our data show that TRAF6i-HDL accumulates
in atherosclerotic lesions, and has a strong affinity for
monocytes/macrophages. A single week of therapy rapidly reduces
plaque macrophage content, which can in part be attributed to the
inhibition of monocyte recruitment. The fact that TRAF6i-HDL proved
to be safe in non-human primates illustrates the translational
potential of this therapy.
[0309] The CD40-CD40L signaling axis has long been recognized to
play an imperative role in eliciting immune responses in
atherosclerosis [2-5]. While its identification gave rise to high
anticipation, therapeutic targeting of this costimulatory
receptor-ligand pair proved cumbersome. An anti-CD40L antibody was
effective in diminishing atherosclerosis development in mice [3-5],
but thromboembolic complications due to CD40 expressed on platelets
prohibited its application in humans [21, 22]. Furthermore, CD40 is
expressed on B lymphocytes, and prolonged blocking would impair
their maturation causing immunodeficiency [9]. In the current
study, we addressed these issues by targeting TRAF6's interaction
with the cytoplasmic domain of CD40 specifically in
monocytes/macrophages. This was accomplished by using HDL as a
nanoparticle carrier loaded with a small molecule inhibitor of
CD40-TRAF6 interaction. These data show that our HDL based
nanoparticles exposed over 80% of monocytes and macrophages to its
cargo, while lymphocytes did not take up any nanoparticles.
[0310] In addition to restricting the delivery of CD40-TRAF6
inhibitor to the monocyte/macrophage population, we also aimed to
minimize systemic immunosuppressive effects by using short duration
of therapy of only a single week. Previous therapeutic studies
targeting the CD40-CD40L signaling axis used prolonged treatment
times [3-5]. The fact that we found a 49% and 66% decrease in
plaque Ly6Chi monocyte and macrophage content within one week
indicates the high potency of TRAF6i-HDL therapy. Of note, we
proved the contribution of apoA-I to the therapeutic effect of
TRAF6i-HDL to be minor. We used 4 infusions of 9 mg/kg apoA-I,
which is relatively low compared to previously published studies
[24], and we found no effects of empty HDL on plaque
monocyte/macrophage content compared to placebo.
[0311] The mechanism by which TRAF6i-HDL decreased plaque
inflammation on such a short timescale can in part be explained by
decreased monocyte recruitment. In general, plaque macrophage
content is determined by a balance of monocyte recruitment as well
as macrophage proliferation, apoptosis and migratory egress. The
first two processes are considered the most important determinants
[25-28]. Our data did not reveal an effect on macrophage
proliferation, apoptosis or migratory egress, while we did observe
a decrease in plaque Ly6Chi monocyte content, suggestive of
decreased monocyte recruitment. Moreover, we did not find a
decrease in blood monocytes that could account for the decreased
number of monocytes in the plaque. Previous studies showed high
kinetics of monocytes [13, 14, 26-28], and decreased recruitment
was shown to cause over 70% reduction in plaque macrophage content
within 4 weeks [26]. Vice versa, a sudden increase in monocyte
recruitment, induced by myocardial infarction, caused a marked
increase in plaque macrophage content within 1-3 weeks [27]. These
observations are in line with our findings of decreased monocyte
recruitment causing a 66% decrease of plaque macrophage content
within one week.
[0312] Our transcriptome analysis data support that monocyte
recruitment is affected. The analyses did not show a clear role for
chemokine receptors or ligands. However, the GO function analysis
showed that "focal adhesion", a pivotal process in cell migration,
was significantly enriched with DE genes. The KEGG pathway analysis
also showed enrichment for "focal adhesion". Genes in the "focal
adhesion" pathway of specific interest are Rhoa, Rap1b and Rap1b,
which play a central role in the regulation of monocyte migration
by activating integrins [16]. They were all significantly down
regulated. This is in line with previous observations in a knockout
mouse model with defective CD40-TRAF6 signaling, in which luminal
adhesion of circulating monocytes to carotid arteries was impaired
in vivo as assessed by intravital microscopy [7]. Also, the
migratory capacity of macrophages was markedly affected [7].
[0313] The effects of TRAF6i-HDL are not limited to "focal
adhesion", as attested by various other gene expressions that were
shown to be affected. Together, the present data indicate that
TRAF6i-HDL affects various biological processes in plaque
monocytes/macrophages, including impairment of monocyte/macrophage
migration. The extensive experiments on pharmacokinetics,
biodistribution and safety in non-human primates (NHPs) illustrate
the translatability of this treatment. The use of reconstituted HDL
has previously proved to be safe in humans with apoA-I doses of 40
mg/kg [24]. Since we used 9 mg/kg apoA-I, this poses no safety
issues. The small molecule inhibitor of CD40-TRAF6 interaction that
was recently developed, has not been evaluated in humans to date.
Biodistribution of .sup.89Zr labeled TRAF6i-HDL was similar to
previous observations with .sup.89Zr labeled HDL in murine, rabbit,
and porcine atherosclerosis models [29]. We observed the highest
accumulation in the liver, spleen and kidneys. The liver and
kidneys are the main sites of apoA-I and HDL catabolism, and the
spleen is the major secondary lymphoid organ containing many
myeloid cells that clear the nanoparticles from the circulation.
There were no signs of toxic effects in the liver, kidney or spleen
and all tissues showed normal tissue architecture on histological
analysis. Furthermore, complete blood count did not show any
effects on the numbers of platelets, lymphocytes, monocytes,
neutrophils or red blood cells. Safety data was assessed up until
72 post administration. Long term safety was not assessed in the
current study.
[0314] Currently there are no specific therapies available that
address plaque inflammation, although chronic therapy with an
anti-interleukin-1.beta. antibody and low dose methotrexate is
currently being investigated in large Phase III clinical trials
[30-32]. The challenge with immunosuppression in a chronic disease
such as atherosclerosis is balancing the risk against the benefit.
In contrast to the aforementioned strategy of chronic
immunosuppression, we conceive that a short term induction
nanotherapy with immune modulating properties can be used to
rapidly suppress plaque inflammation in patients at high risk of
cardiovascular events. While targeted delivery enhances the local
efficacy of the drug, its short term application minimizes the
risks associated with prolonged immunosuppression. Patients
admitted for an acute coronary syndrome may be an appropriate
population for such induction therapy of inflammation since they
have a markedly increased risk of recurrent myocardial infarction
of up to 17.4% within the first year [33]. Recent studies have
proposed that it is the initial myocardial infarction itself that
evokes monocyte recruitment to atherosclerotic plaques causing them
to become inflamed and vulnerable for plaque rupture [27]. In this
pathophysiological context, our concept of rapid suppression of
monocyte recruitment in the vulnerable phase is expected to be
relevant. This study provides an innovative therapeutic approach of
a rapid induction therapy to treat inflammation in atherosclerosis,
by targeting CD40-TRAF6 signaling in monocytes/macrophages. The
infusible TRAF6i-HDL nanoimmunotherapy has promising potential for
translation as attested by the favorable safety data in non-human
primates.
[0315] In view of these results, it is expected that the TRAF6i-HDL
nanoparticles will also be useful in conditions associated with or
related to obesity and insulin resistance. Such conditions and
complications include: insulin resistance, type 2 diabetes mellitus
and cardiovascular disease. It is expected that blocking the
CD40-TRAF pathway will lead to a lack of insulin resistance and a
reduction in both adipose tissue (AT) inflammation and
hepatosteatosis in diet-induced obesity, and similar conditions. It
will further be expected that the TRAF6i-HDL nanoparticles of the
present invention will be able to protect against AT inflammation
and metabolic complications associated with obesity. Thus,
administering the TRAF6i-HDL nanoparticles, alone or in combination
with other standard of care treatments, may improve patient
outcomes and prevent or reverse damage associated with these
conditions.
Methods
[0316] Synthesis of rHDL Based Nanoparticles.
[0317] The synthesis of TRAF6i-HDL was based on a previously
published method [34, 23]. In short, the CD40-TRAF6 inhibitor
6877002 [10] was combined with
1-myristoyl-2-hydroxysn-glycero-phosphocholine (MHPC) and
1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) (Avanti
Polar Lipids) in a chloroform/methanol mixture (9:1 by volume) and
then dried in a vacuum, yielding a thin lipid film. A PBS solution
of human apolipoprotein A1 (apoA-I) was added to the lipid film.
The mixture was incubated on ice for 1 hour or until the film was
hydrated and a homogenous solution was formed. The solution was
then sonicated for 20 minutes to form TRAF6i-HDL nanoparticles.
Subsequently, the solution was purified by multiple centrifugal
filtration steps. For targeting, imaging and biodistribution
experiments, analogs of TRAF6i-HDL were prepared through
incorporation of the fluorescent dyes DiR or DiO (Invitrogen), or
the phospholipid chelator DSPE-DFO (1 mol % at the expense of
DMPC), which allows radiolabeling with .sup.89Zr [35].
Animals and Diet for the Mouse Studies.
[0318] Female Apoe.sup.-/- mice (B6.129P2-Apoe.sup.tmlUnc, n=103)
were used for this study. All animal care and procedures were based
on an approved institutional protocol from Icahn School of Medicine
at Mount Sinai. Eight-week-old Apoe.sup.-/- mice were purchased
from The Jackson Laboratory. All mice were fed a high-cholesterol
diet (HCD) (0.2% weight cholesterol; 15.2% kcal protein, 42.7% kcal
carbohydrate, 42.0% kcal fat; Harlan TD. 88137) for 12 weeks.
[0319] The treatment protocol in each experiment was identical:
twenty-week-old Apoe.sup.-/- mice were randomly assigned to either
placebo (saline), empty rHDL or TRAF6i-HDL (5 mg/kg) groups. Mice
were treated with 4 intravenous injections over 7 days, while kept
on a HCD during treatment. Animals were sacrificed 24 hours after
the last injection.
Flow Cytometry.
[0320] Apoe.sup.-/- mice were euthanized and perfused with PBS,
after which the aorta from the aortic root to the iliac bifurcation
was gently cleaned from fat and collected. Whole aortas were put in
an enzymatic digestion solution containing liberase TH (4 U/mL)
(Roche), deoxyribonuclease (DNase) I (40 U/ml) (Sigma-Aldrich) and
hyaluronidase (60 U/mL) (Sigma-Aldrich), minced and placed in a
37.degree. C. incubator for 60 min. Cells were run through a 70
.mu.m strainer, and twice spun down and resuspended in serum
containing media. Spleens were weighed and pushed through a 70
.mu.m cell-strainer, spun down, resuspended in red cell lysis
buffer for 4 minutes, and then inactivated using serum containing
media, spun down and resuspended in 1000 .mu.L serum containing
media per 100 mg of spleen tissue. EDTA treated blood was spun
down, resuspended in red cell lysis buffer for 4 minutes, and then
inactivated using serum containing media, spun down and resuspended
in 100 .mu.l of serum containing media. Bone marrow was obtained
from a single femur. The intact femurs were rinsed with 70% ethanol
followed by three subsequent washes in ice-cold sterile PBS. The
epiphyses were cut off and the bone marrow was flushed out with
PBS. Cells were run through a 70 .mu.m strainer, spun down and
resuspended in red cell lysis buffer for 30 seconds, and then
inactivated using serum containing media, spun down and resuspended
in 1000 .mu.L of serum containing media. The following antibodies
were used: F4/80-PE-Cy7 (clone BM8, BioLegend); CD11b-PerCP/Cy5.5
(clone M1/70, BioLegend); CD11c-APC (clone N418, BioLegend);
CD45-brilliant violet 510 (clone 30-F11, BioLegend); Ly-6C-PE
(clone AL-21, BD Biosciences); Ly6CFITC (clone AL-21), BD
Biosciences); CD90.2-eFluor 450 (clone 53-2.1, eBioscience);
CD90.2-PE (clone 53-2.1, BD Biosciences); Ter119-eFluor 450 (clone
TER-119, eBioscience); NK1.1-eFluor 450 (clone PK136, eBioscience);
NK1.1-PE (clone PK136, BD Biosciences); CD49b-eFluor 450 (clone
DX5, eBioscience); CD45R-eFluor450 (clone RA3-6B2, eBioscience);
Ly-6 G-Pacific Blue (clone 1A8, BioLegend); Ly-6 G-PE (clone 1A8,
BD Biosciences); CD3-PE (clone 17A2; Biolegend); CD19-PE (clone
1D3, BD Bioscience).
[0321] The antibody dilutions ranged from 1:200 to 1:100.
Contribution of newly made cells to different populations was
determined by in vivo labeling with bromodeoxyuridine (BrdU).
Incorporation was measured using APC-conjugated anti-BrdU
antibodies according to the manufacturer's protocol (BD APC-BrdU
Kit, 552598). Monocytes and macrophages were identified using a
method similar to one described previously [28]. Specifically,
Ly6C.sup.hi monocytes were identified as CD11b.sup.hi,
CD11c.sup.low, Lin.sup.-/low (with Lin defined as CD90.2+, CD45R+,
CD49b+, NK1.1+, Ly-6 G+, Ter119+ or CD90.2+, NK1.1+, Ly-6 G+,
CD19+, CD3+) F4/80.sup.low that were also Ly-6C.sup.hi. Macrophages
were identified as CD11b.sup.hi, CD11c.sup.low, Lin.sup.-/low,
F4/80.sup.hi, CD11.sup.-/low. Data were acquired on an LSRII flow
cytometer (BD Biosciences) and analyzed with FlowJo v10.0.7 (Tree
Star, Inc.).
Histology and Immunohistochemistry.
[0322] Tissues for histological analysis were collected and fixed
overnight in formalin and embedded in paraffin. Aortic roots were
sectioned into 4 .mu.m slices, generating a total of 90-100
cross-sections per root. Eight cross-sections were stained with
hematoxylin and eosin (HE) and used for atherosclerotic plaque size
measurement. Other sections were deparaffinized, blocked, incubated
in 95.degree. C. antigen-retrieval solution (DAKO), and
immunolabeled with either MAC-3 rat monoclonal antibody (1:30; BD
Biosciences) or anti-Ki67 rabbit polyclonal antibody (1:200,
Abcam). Sirius red staining was used for analysis of collagen
content. Antibody staining was visualized by either Immpact AMEC
red (Vectorlabs) or diaminobenzidine (DAB). Sections were analyzed
using a Leica DM6000 microscope (Leica Microsystems) or the VENTANA
iScan HT slide scanner (Ventana).
Laser Capture Microdissection and RNA Sequencing.
[0323] LCM was performed on 24 aortic root sections (6 .mu.m) as
previously described (20). In short, frozen sections were
dehydrated in graded ethanol solutions (70% twice, 95% twice, 100%
once), washed with DEPC treated water, stained with Mayer's
hematoxylin, eosin and cleared in xylene. For every 8 sections, 1
section was used for CD68 staining (Abdserotec, 1:250 dilution)
which was used to guide the LCM. CD68 rich areas within the plaques
were identified and cut out using the ArcturusXT LCM System. The
collected CD68 positive cells were used for RNA isolation (PicoPure
RNA Isolation Kit, Arcturus) and subsequent RNA amplification and
cDNA preparation according to the manufacturers protocols (Ovation
Pico WTA System, NuGEN). Quality and concentration of the collected
samples were measured with the Agilent 2100 Bioanalyzer.
[0324] RNA Sequencing.
[0325] Pair-end libraries were prepared and validated. The purity,
fragment size, yield and concentration were determined. During
cluster generation, the library molecules were hybridized onto an
Illumina flow cell. Subsequently, the hybridized molecules were
amplified using bridge amplification, resulting in a heterogeneous
population of clusters. The data set was obtained using an Illumina
HiSeq 2500 sequencer.
Differential Expression and Function Annotation Analysis.
[0326] The pair-ended sequencing reads were aligned to human genome
hg19 using tophat aligner (bowtie2) [36]. Following read alignment,
HTSeq [37] was used to quantify gene expression at the gene level
based on GENCODE gene model release 22 [38]. Gene expression raw
read counts were normalized as counts per million using trimmed
mean of M-values normalization method to adjust for sequencing
library size difference among samples [39]. Differential expressed
genes between drug treatments and placebo were identified using the
Bioconductor package limma [40]. In order to correct the multiple
testing problem, limma was used to calculate statistics and
p-values in random samples after a permutation of labels. This
procedure was repeated 1,000 times to obtain null t-statistic and
p-value distribution for estimating the false discovery rate (FDR)
of all genes. The differentially expressed (DE) genes were
identified by a cutoff of corrected p-value less than 0.2.
GO-function [41] was used to annotate the DE genes, and to find
cellular components that significantly enriched with the DE genes.
DE genes were also mapped to the Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathway with KEGG Mapper[42].
Fluorescence Molecular Tomography with CT.
[0327] Female Apoe.sup.-/- mice fed a high-fat diet for 12 weeks,
were treated with either four TRAF6i-HDL infusions (5 mg/kg, n=7)
or saline (n=8) over 7 days. Five nanomoles of pancathepsin
protease sensor (ProSense 680, PerkinElmer, Cat no. NEV10003) was
intravenously administered 24 hours prior to imaging. For the
FMT/CT imaging, animals were placed in a custom-built imaging
cartridge, which was equipped for isoflurane administration during
imaging. Animals were first scanned with high-resolution computed
tomography (CT; Inveon PET-CT, Siemens), with a continuous infusion
of CT-contrast agent (isovue-370, Bracco Diagnostics) at a rate of
55 .mu.L/min through a tail vein catheter. Animals were
subsequently scanned with an FMT scanner (PerkinElmer) in the same
cartridge. The CT X-ray source with an exposure time of 370-400 ms
was operated at 80kVp and 500 mA. Contrast-enhanced high-resolution
CT images were used to localize the aortic root, which was used to
guide the placement of the volume of interest for the quantitative
FMT protease activity map. Image fusion relied on fiducial markers.
Image fusion and analysis was performed with OsiriX v.6.5.2 (The
Osirix Foundation, Geneva).
Radiolabeling of HDL Nanoparticles.
[0328] Ready-to-label HDL nanoparticles were prepared by including
1 mol % the phospholipidchelat or DSPE-DFO (35) in the formulation
mix at the expense of DMPC. The DFO-containing nanoparticles were
then labeled with Zirconium-89 (.sup.89Zr) as previously described
(35). Briefly, the nanoparticles were reacted with
.sup.89Zr-oxalate in phosphate buffered saline (PBS, pH 7.1) at
37.degree. C. for 1 hour. Purification was carried out by
centrifugal filtration using 10 kDa molecular weight cut-off filter
tubes, and washing twice with fresh sterile PBS. The radiochemical
yield was 90.+-.4% (n=3) and radiochemical purity >97%, as
determined by size exclusion chromatography.
Pharmacokinetics, Biodistribution and PET/CT Imaging Studies in
Mice.
[0329] Female Apoe.sup.-/- mice fed a high-fat diet for 12 weeks
(n=4, 25.5.+-.2.6 g body weight) were injected with
.sup.89Zr-TRAF6i-HDL nanoparticles (183.+-.16 .mu.Ci, 5 mg
TRAF6i-HDL/kg). At predetermined time points (2, 15 and 30 min, and
1, 4, 8 and 24 hours) blood samples were taken, weighed and
measured for radioactivity content using a 2470 Wizard automatic
gamma counter (Perkin Elmer). Data were converted to percentage of
injected dose per gram tissue [% ID/g], plotted in a time-activity
curve and fitted using a non-linear two phase decay regression in
Prism GraphPad (GraphPad Software inc, USA). A weighted blood
radioactivity half-life (t1/2) was finally calculated.
[0330] Twenty-four hours after injection, the animals were scanned
on an Inveon PET/CT scanner (Siemens Healthcare Global) under
isoflurane/oxygen gas mixture anesthesia (2% for induction, 1% for
maintenance). The PET static scan recorded a minimum of 25 million
coincident events and lasted 10 min. The energy and coincidence
timing windows were 350-700 keV and 6 ns, respectively. Image data
were normalized to correct for nonuniformity of response of the
PET, dead-time count losses, positron branching ratio, and physical
decay to the time of injection, but no attenuation, scatter, or
partial-volume averaging correction was applied. The counting rates
in the reconstructed images were converted to activity
concentrations (% ID/g) by use of a system calibration factor
derived from the imaging of a mouse-sized water-equivalent phantom
containing .sup.89Zr. Images were analyzed using ASIPro VM.TM.
(Concorde Microsystems) and Inveon Research software (Siemens
Healthcare Global). Quantification of activity concentration was
done by averaging the maximum values in at least 5 ROIs drawn on
adjacent slices of the tissue of interest. Whole body standard low
magnification CT scans were performed with the X-ray tube setup at
a voltage of 80 kV and current of 500 .mu.A. The CT scan was
acquired using 120 rotational steps for a total of 220 degrees
yielding and estimated scan time of 120 s with an exposure of 145
ms per frame. Immediately after the PET/CT scan, animals were
sacrificed and perfused with PBS. Tissues of interest (liver,
kidneys, spleen, lungs, muscle, heart, aorta, bone and brain) were
collected, blotted and weighed. Radioactivity was measured by gamma
counting and radioactivity concentration expressed as percentage of
injected dose per gram [% ID/g].
Autoradiography.
[0331] Following radioactivity counting, aortas were placed in a
film cassette against a phosphorimaging plate (BASMS-2325,
Fujifilm, Valhalla, N.Y.) for 24 hours at -20.degree. C. in order
to determine radioactivity distribution. The plates were read at a
pixel resolution of 25 .mu.m with a Typhoon 70001P plate reader (GE
Healthcare, Pittsburgh, Pa.).
Ex Vivo Near Infrared Fluorescence Imaging (NIRF).
[0332] Female Apoe.sup.-/- mice fed a high-fat diet for 12 weeks,
received a single IV injection with DiR (0.5 mg/kg) labeled
TRAF6i-HDL (5 mg/kg, n=2) or saline (n=1). Mice were sacrificed 24
hours after the injection and perfused with 60 mL PBS. Liver,
spleen, lung, kidneys, heart and muscle tissue were collected for
NIRF imaging. Fluorescent images were acquired with the IVIS 200
system (Xenogen), with a 2 second exposure time, using a 745 nm
excitation filter and a 820 nm emission filter. ROIs were drawn on
each tissue with software provided by the vendor, after which a
quantitative analysis was done with the average radiant efficiency
within these ROIs.
Blood Tests.
[0333] In mice blood was collected by heart puncture at the time of
sacrifice. Serum was sent to IDEXX laboratories (Totowa, N.J., USA)
and analyzed with an Olympus AU400 chemistry analyzer. Whole blood
was collected in EDTA containing tubes and analyzed with an IDEXX
procyte DX hematology analyzer for complete blood count analysis.
In non-human primates blood was collected at 0 and 15 minutes and
6, 12, 24, 28, 48 and 72 hours after infusion. Serum was analyzed
with an Olymus AU400 chemistry analyzer. Whole blood samples were
also analyzed with an IDEXX procyte DX hematology analyzer.
Non-Human Primate Studies
[0334] Adult male cynomolgus monkeys (Macaca fascicularis) were
used for the non-human primate studies conducted at the University
of Kentucky and Icahn School of Medicine at Mount Sinai. Animals
were on average 7.3 years of age, and their weight was 7.3.+-.1.98
kg (mean.+-.SD). All animal care, procedures and experiments were
based on approved institutional protocols from Icahn School of
Medicine at Mount Sinai and the University of Kentucky
Institutional Animal Care and Use Committee. Monkeys were
pair-housed when possible in climate-controlled conditions with
12-hour light/dark cycles. Monkeys were provided water ad libitum
and fed Teklad Global 20% Protein Primate Diet. For the experiment
at the University of Kentucky, the six male monkeys were used.
After an overnight fast, monkeys were anesthetized with ketamine (5
mg/kg) and dexmedetomidine (0.0075-0.015 mg/kg), and blood was
collected from the femoral vein. The monkeys were then injected IV
via the saphaneous vein with either vehicle (PBS, USP grade) or
TRAF6i-HDL such that the dose of CD40-TRAF6 inhibitor 6877002 was
1.25 mg/kg. Blood was collected 15 minutes, 6, 12, 24, and 48 hours
postinjection. Following the blood draw anesthesia was reversed
with atipamezole (0.075-0.15 mg/kg). 72 hours postinjection, fasted
monkeys were anesthetized with ketamine (25 mg/kg), bled a final
time, and euthanized by exsanguination with whole-body saline
perfusion while anesthetized with isoflurane (3-5% induction, 1-2%
maintenance). Tissues were promptly removed and fixed in 10%
neutral-buffered formalin. Blood was subjected to complete blood
count (CBC) test (ANTECH Diagnostics).
[0335] For the experiment at Icahn School of Medicine at Mount
Sinai six female monkeys were used. For the .sup.89Zr-PET/MRI
imaging, animals were infused with 58.9.+-.17.9 MBq of
.sup.89Zr-labeled TRAF6i-HDL (1.25 mg/kg) and imaged by PET/MRI at
different time points. Dynamic PET imaging was performed during the
first 60 minutes after infusion. Additional PET/MRI scans were
performed at 24, 48 and 72 hours. PET and MR images were acquired
on a combined 3T PET/MRI system (Biograph mMR, Siemens
Healthineers, Erlangen, Germany). On day 1, dynamic PET imaging was
performed for 60 minutes using one bed position covering the chest
and abdomen, directly after injection with .sup.89Zr-labeled
TRAF6i-HDL. Simultaneously, anatomical vessel wall MR images were
acquired using a proton density (PD) weighted Sampling Perfection
with Application optimized Contrasts using different flip angle
Evolution (SPACE) sequence. MR imaging parameters were: acquisition
plane, coronal; repetition time (TR), 1000 ms; echo time (TE), 79
ms; field of view (FOV), 300.times.187 mm2; number of slices, 144;
number of averages, 4; bandwidth, 601 Hz/pixel; turbo factor (TF),
51; echo trains per slice, 4; echo train length, 192 ms; echo
spacing, 3.7 ms; acquisition duration, 33 minutes and 36 seconds.
After dynamic PET acquisition, static whole-body PET imaging was
acquired from the cranium to the pelvis, using 3 consecutive bed
positions, of 10 minutes each. Simultaneously with each bed, MR
images were acquired as described above, except using only 1.4
signal average (acquisition duration, 11 min 44 seconds per bed).
Whole-body PET and MR imaging was also performed at 24, 48 and 72
hours after injection, using 3 bed positions (PET duration per bed,
30 min; MR duration per bed, 33 min and 36 s). Whole-body MR images
from each bed were automatically collated together by the scanner.
After acquisition, PET raw data from each bed were reconstructed
and collated together offline using the Siemens proprietary e7tools
with an Ordered Subset Expectation Maximization (OSEM) algorithm
with Point Spread Function (PSF) correction. A dual-compartment
(soft tissue and air) attenuation map was used for attenuation
correction.
Statistical Analysis.
[0336] Continuous variables are expressed as means.+-.standard
deviation, unless otherwise stated. Significance of differences was
calculated by use of the nonparametric Mann-Whitney U test and
Kruskal-Wallis test. Probability values of P.ltoreq.0.05 were
considered significant. Statistical analyses were done using
Statistical Package for the Social Sciences (SPSS) version
22.0.0.0.
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[0379] All references cited herein are incorporated by reference to
the same extent as if each individual publication, database entry
(e.g. Genbank sequences or GenelD entries), patent application, or
patent, was specifically and individually indicated to be
incorporated by reference. This statement of incorporation by
reference is intended by Applicants, pursuant to 37 C.F.R. .sctn.
1.57(b)(1), to relate to each and every individual publication,
database entry (e.g. Genbank sequences or GenelD entries), patent
application, or patent, each of which is clearly identified in
compliance with 37 C.F.R. .sctn. 1.57(b)(2), even if such citation
is not immediately adjacent to a dedicated statement of
incorporation by reference. The inclusion of dedicated statements
of incorporation by reference, if any, within the specification
does not in any way weaken this general statement of incorporation
by reference. Citation of the references herein is not intended as
an admission that the reference is pertinent prior art, nor does it
constitute any admission as to the contents or date of these
publications or documents.
[0380] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0381] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. Various modifications of the invention in addition to
those shown and described herein will become apparent to those
skilled in the art from the foregoing description and fall within
the scope of the appended claims.
* * * * *