U.S. patent application number 10/159280 was filed with the patent office on 2003-02-13 for oxidant scavengers for treatment of diabetes or use in transplantation or induction of immune tolerance.
Invention is credited to Crapo, James D., Day, Brian J., Flores, Sonia C., Gammans, Richard, Gill, Ronald G., Haskins, Kathryn, Patel, Manisha, Piganelli, Jon D..
Application Number | 20030032634 10/159280 |
Document ID | / |
Family ID | 26968620 |
Filed Date | 2003-02-13 |
United States Patent
Application |
20030032634 |
Kind Code |
A1 |
Piganelli, Jon D. ; et
al. |
February 13, 2003 |
Oxidant scavengers for treatment of diabetes or use in
transplantation or induction of immune tolerance
Abstract
The present invention relates, in one embodiment, to a method of
preventing or treating diabetes using low molecular weight
antioxidants. In a further embodiment, the invention relates to a
method of protecting and/or enhancing viability of
cells/tissues/organs during isolation (harvesting), preservation,
expansion and/or transplantation. In yet another embodiment, the
present invention relates to a method of inducing immune tolerance.
The invention also relates to compounds and compositions suitable
for use in such methods.
Inventors: |
Piganelli, Jon D.;
(Pittsburgh, PA) ; Haskins, Kathryn; (Denver,
CO) ; Flores, Sonia C.; (Denver, CO) ; Crapo,
James D.; (Denver, CO) ; Day, Brian J.;
(Denver, CO) ; Gill, Ronald G.; (Denver, CO)
; Gammans, Richard; (Research Triangle Park, NC) ;
Patel, Manisha; (Denver, CO) |
Correspondence
Address: |
NIXON & VANDERHYE P.C.
8th Floor
1100 North Glebe Road
Arlington
VA
22201
US
|
Family ID: |
26968620 |
Appl. No.: |
10/159280 |
Filed: |
June 3, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60294604 |
Jun 1, 2001 |
|
|
|
60328398 |
Oct 12, 2001 |
|
|
|
Current U.S.
Class: |
514/185 ;
514/410 |
Current CPC
Class: |
A61P 3/10 20180101; A61P
9/14 20180101; A61P 37/06 20180101; A61P 39/06 20180101; A61K
31/409 20130101; A61K 31/555 20130101; A61P 9/10 20180101; A61P
37/00 20180101; A61P 37/02 20180101 |
Class at
Publication: |
514/185 ;
514/410 |
International
Class: |
A61K 031/555; A61K
031/409 |
Claims
What is claimed is:
1. A method of preventing or treating diabetes comprising
administering to a patient in need of such prevention or treatment
an amount of a low molecular weight antioxidant sufficient to
effect said prevention or treatment.
2. The method according to claim 1 wherein said antioxidant is a
porphine or a tetrapyrrole, or pharmaceutically acceptable salt
thereof.
3. The method according to claim 2 wherein said porphine or
tetrapyrrole is bound to a metal selected from the group consisting
of manganese, iron, copper, cobalt or nickel.
4. The method according to claim 3 wherein said metal is
manganese.
5. The method according to claim 4 wherein said compound is
manganese substituted AEOL 10113 or 10150.
6. The method according to claim 1 wherein said diabetes results
from death of pancreatic islet cells due to autoimmune disease.
7. The method according to claim 1 wherein said diabetes results
from death of pancreatic islet cells due to free radical induced
toxicity.
8. A method of preventing or treating diabetes-specific
microvascular disease comprising administering to a patient in need
of such prevention or treatment an amount of an antioxidant
sufficient to effect said prevention or treatment, wherein said
antioxidant is a porphine or a tetrapyrrole, or pharmaceutically
acceptable salt thereof
9. The method according to claim 8 wherein said porphine or
tetrapyrrole is bound to a metal selected from the group consisting
of manganese, iron, copper, cobalt or nickel.
10. The method according to claim 9 wherein said metal is
manganese.
11. A method of preventing or treating diabetes-accelerated
macrovascular atherosclerosis comprising administering to a patient
in need of such prevention or treatment an amount of an antioxidant
sufficient to effect said prevention or treatment, wherein said
antioxidant is a porphine or a tetrapyrrole, or pharmaceutically
acceptable salt thereof
12. The method according to claim 11 wherein said porphine or
tetrapyrrole is bound to a metal selected from the group consisting
of manganese, iron, copper, cobalt or nickel.
13. The method according to claim 12 wherein said metal is
manganese.
14. A method of inducing immune tolerance comprising comprising
administering to a patient in need of such induction an amount of a
low molecular induction.
15. The method according to claim 14 wherein said antioxidant is a
porphine or a tetrapyrrole, or pharmaceutically acceptable salt
thereof.
16. The method according to claim 15 wherein said porphine or
tetrapyrrole is bound to a metal selected from the group consisting
of manganese, iron, copper, cobalt or nickel.
17. The method according to claim 16 wherein said metal is
manganese.
18. The method according to claim 14 wherein said patient is a
transplant recipient and said antioxidant renders said patient
tolerant to said transplant.
19. A method of enhancing viability of a cell, tissue or organ
comprising contacting said cell, tissue or organ with a low
molecular weight antioxidant under conditions such that said
viability is enhanced.
20. The method according to claim 19 wherein said cell, tissue or
organ is transplanted into a host and viability of said
transplanted cell, tissue or organ is enhanced.
21. The method according to claim 19 wherein said antioxidant is a
porphine or tetrapyrrole.
22. The method according to claim 21 wherein said porphine or
tetrapyrrole is bound to a metal selected from the group consisting
of manganese, iron, copper, cobalt or nickel.
23. The method according to claim 22 wherein said metal is
manganese.
24. The method according to claim 19 wherein said cell is a
neuronal cell.
Description
[0001] This application claims priority from Provisional
Application No. 60/294,604, filed Jun. 1, 2001, and Provisional
Application No. 60/328,398, filed Oct. 12, 2001, the contents of
both applications being incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates, in one embodiment, to a
method of preventing or treating diabetes using low molecular
weight antioxidants. In a further embodiment, the invention relates
to a method of protecting and/or enhancing viability of
cells/tissues/organs during isolation (harvesting), preservation,
expansion and/or transplantation. In yet another embodiment, the
present invention relates to a method of inducing immune tolerance.
The invention also relates to compounds and compositions suitable
for use in such methods.
BACKGROUND
[0003] Diabetes is characterized by chronic hyperglycemia. There
are 2 forms of the disease, insulin dependent (Type I) and
non-insulin dependent (Type II). The disease process associated
with both Type I and Type II includes a microvascular pathology
that can result, for example, in blindness, renal failure and nerve
damage. In addition, an accelerated atherosclerotic macrovascular
pathology can affect arteries supplying the heart, brain and lower
extremities. (See, for example, Brownlee, Nature 414:813
(2001).)
[0004] Type I diabetes is caused by the autoimmune destruction of
insulin-producing pancreatic .beta. cells. A large body of evidence
supports the concept that the antigen-specific, T cell-mediated
infiltration of inflammatory cells to the pancreas leads to the
generation of reactive oxygen species (ROS) [superoxide,
(O.sub.2.sup..cndot.-), hydroxyl radical (.sup..cndot.OH), nitric
oxide (NO.sup..cndot.),peroxynitrite (ONOO.sup.-)], and
pro-inflammatory cytokines (TNF-.alpha., IL-1.beta. (interleukin
1.beta.) and IFN-.gamma. (interferon .gamma.) (Rabinovitch et al,
Endocrinology 137:2093-2099 (1996), Mandrup-Poulsen, Diabetologia
39:1005-1029 (1996), Eizirik et al, Diabetologia 39:875-890 (1996),
Mandrup-Poulsen et al, Eur. J. Endocrinol. 134:21-30 (1996)).
Synergistic interaction between ROS (reactive oxygen species) and
these cytokines results in the ultimate destruction of the
pancreatic .beta. cells.
[0005] Locally produced ROS are involved in the effector mechanisms
of .beta. cell destruction (Rabinovitch et al, Endocrinology
137:2093-2099 (1996), Mandrup-Poulsen, Diabetologia 39:1005-1029
(1996), Eizirik et al, Diabetologia 39:875-890 (1996), Grankvist et
al, Biochem. J. 182:17-25 (1979), Kroncke et al, Biochem. Biophys.
Res. Commun. 175:752-758 (1991), Corbet et al, J. Clin. Invest.
90:2384-2391 (1992)). In vitro, T cell and macrophage cytokines
such as IFN-.gamma., IL-1.beta. and TNF-.alpha. (tumor necrosis
factor-.alpha.) induce the production of ROS by .beta. cells. In
addition, ROS either given exogenously or elicited in .beta. cells
by cytokines lead to .beta. cell destruction (Lortz et al, Diabetes
49:1123-1130 (2000)). This destruction appears to ultimately be
caused by an apoptotic mechanism (Kurrer et al, Proc. Natl. Acad.
Sci. USA 94:213-218 (1993), O'Brien et al, Diabetes 46:750-757
(1997), Chervonski et al, Cell 89:17-24 (1997), Itoh et al, J. Exp.
Med. 186:613-618 (1997)). .beta. cells engineered to over-express
antioxidant proteins have been shown to be resistant to ROS and
NO.sup..cndot. (Grankvist et al, Biochem. J. 199:393-398 (1981),
Malaisse et al, Proc. Natl. Acad. Sci. USA 79:927-930 (1982),
Lenzen et al, Free Radic. Biol. Med. 20:463-466 (1996), Tiedge et
al, Diabetes 46:1733-1742 (1997), Benhamou et al, Diabetologia
41:1093-1100 (1998), Tiedge et al, Diabetes 47:1578-1585 (1998),
Tiedge et al, Diabetologia 42:849-855 (1999)). Furthermore, stable
expression of manganese superoxide dismutase (Mn-SOD) in insulinoma
cells prevented IL-1.beta.-induced cytotoxicity and reduced nitric
oxide production (Hohmeier et al, J. Clin. Invest. 101:1811-1820
(1998)). Finally, others have shown that transgenic mice with
.beta. cell-targeted over-expression of copper, zinc SOD or
thioredoxin are resistant to autoimmune and streptozotocin-induced
diabetes (Kubisch et al, Proc. Natl. Acad. Sci. USA 91:9956-9959
(1994), Kubisch et al, Diabetes 46:1563-1566 (1997), Hotta et al,
J. Exp. Med. 188:1445-1451 (1998)).
[0006] SOD mimics have been designed with a redox-active metal
center that catalyzes the dismutation of O.sub.2.sup.- in a manner
similar to the active metal sites of the mammalian Cu, Zn- or Mn-
containing SODs (Fridovich, J. Biol. Chem. 264:7761-7764 (1989),
Pasternack et al, J. Inorg. Biochem. 15:261 (1981), Faulkner et al,
J. Biol. Chem. 269:23471-2347(1994), Batinic-Haberle et al, J.
Biol. Chem. 273:24521-24528 (1998), Patel et al, Trends Pharmacol.
Sci. 20:359-364 (1999), Spasojevic et al, Inorg. Chem. 40:726
(2001)). The manganese porphyrins have a broad antioxidant
specificity, which includes scavenging O.sub.2.sup.-
(Batinic-Haberle et al, Inorg. Chem. 38:4011 (1999)),
H.sub.2O.sub.2 (Spasojevic et al, Inorg. Chem. 40:726 (2001), Day
et al, Arch. Biochem. Biophys 347:256-262 (1997)), ONOO.sup.-,
(Ferrer-Sueta et al, Chem. Res. Toxicol. 12:442-449 (1999)),
NO.sup..cndot. (Spasojevic et al, Nitric Oxide: Biology and
Chemistry 4:526 (2000)) and lipid peroxyl radicals (Day et al, Free
Radic. Biol. Med. 26:730-736 (1999)). SOD mimics have recently been
found to rescue vascular contractility in endotoxic shock
(Zingarelli et al, Br. J. Pharmacol. 120:259-267 (1997)), protect
neuronal cells from excitotoxic cell death (Patel et al, Neuron
16:345-355 (1996)) and apoptosis (Patel, J. Neurochem. 71:1068-1074
(1998)), inhibit lipid-peroxidation (Day et al, Free Radic. Biol.
Med. 26:730-736 (1999), Bloodsworth et al, Free Radic. Biol. Med.
28:1017-1029 (2000)), block hydrogen peroxide-induced mitochondrial
DNA damage (Milano et al, Nucleic Acids Res. 28:968-973 (2000)),
and partially rescue a lethal phenotype in a manganese superoxide
dismutase knockout mouse (Melov et al, Nat. Genet. 18:159-163
(1998)). The ability of the SOD mimics to scavenge a broad range of
ROS allows for there utilization in inflammatory diseases. The
present invention provides a pharmacological approach to protect
.beta. cells from the T cell mediated ROS and cytokine destruction
associated with autoimmune diabetes by employing a synthetic
metalloporphyrin-based superoxide dismutase mimic. The invention
also provides a method of improving survival of pancreatic .beta.
islet cells following transplantation.
SUMMARY OF THE INVENTION
[0007] The present invention relates, in one embodiment, to a
method of preventing or treating diabetes using low molecular
weight antioxidants. In accordance with this embodiment, low
molecular weight antioxidants can be used to treat or prevent
diabetes-specific microvascular disease of, for example, the
retina, renal glomerulus and peripheral nerve (e.g., resulting in
oedema, ischaemia and hypoxia-induced neovascularization in the
retina, proteinuria, mesangial matrix expansion and
glomerulosclerosis in the kidney, and multifocal axonal
degeneration in peripheral nerves). In addition, low molecular
weight antioxidants can be used to treat or prevent accelerated
atherosclerotic macrovascular disease affecting arteries supplying
the heart, brain and lower extremities. In a further embodiment,
the invention relates to a method of protecting and/or enhancing
viabiltity of cel s/tissues,/organs during isolation (harvesting),
preservation, expansion and/or transplantation. In yet another
embodiment, the present invention relates to a method of inducing
immune tolerance. The invention also relates to compounds and
compositions suitable for use in such methods.
[0008] Objects and advantages of the present invention will be
clear from the description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIGS. 1A and 1B. SOD mimic administration delays or prevents
T cell-mediated diabetes in young NOD (nonobese diabetic). scid
(severe combined immuno-deficient) recipients after diabetogenic T
cell clone BDC-2.5 transfer. FIG. 1A. NOD.scid mice 9-14 days of
age were injected i.p., one day prior to adoptive transfer of
1.times.10.sup.7 BDC-2.5 T cell clones, with 10 mg/kg of the SOD
mimic .circle-solid. or HBSS control .box-solid.. The SOD mimic was
then given every other day for a total of 5 days. The data
represented in FIG. 1A is the combination of 3 separate
experiments. FIG. 1B. Representative pancreatic histology from
young NOD.scid mice treated with SOD mimic or control after
adoptive transfer of the T cell clone BDC-2.5. FIG. 1Ba.
Hematoxylin and Eosin (H&E) staining of a heavily infiltrated
pancreas from the positive control, a young NOD.scid mouse after
adoptive transfer of BDC-2.5 FIG. 1Bb. c. H & E staining of
pancreas from young NOD.scid treated with SOD mimic (10 mg/kg)
after adoptive transfer of BDC-2.5. FIG. 1Bd. Aldehyde-fuchsin
(A/F) staining of pancreas from SOD mimic-treated NOD.scid
mouse.
[0010] FIGS. 2A-2C. Production of IFN-.gamma. by BDC-2.5 treated
cells with SOD mimic in vitro using three types of T cell
stimulation. FIG. 2A. 96-well round bottom plates were pre-coated
with 0.125 .mu.g/ml .alpha.-CD3 and 1 .mu.g/ml .alpha.-CD28 for 1
hr at 37.degree. C. The plates were washed twice with sterile HBSS
and then blocked with complete medium (CM) at 37.degree. C. for 1
hour. Blocking solution was removed and 2.times.10.sup.4 BDC-2.5 T
cell clones were added to the wells in the presence or absence of
the SOD mimic at concentrations of 34 and 17 .mu.M; the negative
control was BDC-2.5 without .alpha.-CD3 and .alpha.-CD28. Cultures
were incubated at 37.degree. C. for 48 hr before the supernatants
were harvested and assayed by sandwich ELISA for IFN-.gamma.
production. Data are the mean and SEM of 3 separate experiments; p
values are shown for conditions where statistical significance was
noted. FIG. 2B. BDC-2.5 T cells were plated at 2.times.10.sup.4
cells/well in 96-well flat-bottom plates with or without
5.times.10.sup.5 irradiated syngeneic spleen cells as APC (antigen
presenting cell/s) and Con A (2.5 .mu.g/ml final concentration), in
the presence or absence of the SOD mimic at concentrations of 34
.mu.M and 17 .mu.M. Cultures were incubated at 37.degree. C. or 24
hr before the supernatants were harvested and assayed by sandwich
ELISA for IFN-.gamma. production. Data are the mean and SEM of 3
separate experiments. FIG. 2C. BDC-2.5 T cell clones were cultured
in 96-well flat-bottom plates at a density of 2.times.10.sup.4
cells/well, with 5000 islet-cells as antigen and 2.5.times.10.sup.4
APC, in the presence or absence of SOD mimic at 34 and 17 .mu.M.
Cultures were incubated at 37.degree. C for 48 hr before the
supernatants were harvested and assayed by sandwich ELISA for
IFN-.gamma. production. Data are the mean and SEM of 3 separate
experiments.
[0011] FIG. 3. In vivo treatment of 2.5 TCR Tg (transgenic)/NOD
mice with the SOD mimic. 2.5 TCR-Tg/NOD mice were treated for 7
days with 10 mg/ml SOD mimic or HBSS. Spleen cells were harvested
from the animals on day 8 and the T cells were purified from SOD
mimic or control mice and plated (6.times.10.sup.4 cells/well) with
APC (3.times.10.sup.5 cell/well) from either SOD mimic or control
mice in a criss-cross fashion. The cultures were pulsed with 1
.mu.M of HRPI-RM peptide and on day 4 of culture, the plate was
pulsed with (1 .mu.Ci .sup.3H-TdR) for 6 hr before harvest. Values
are the mean and SEM of triplicate wells. Data are representative
of duplicate experiments.
[0012] FIGS. 4A and 4B. LPS (lipopolysaccharide)-induced
respiratory burst and cytokine production by peritoneal
macrophages. FIG. 4A. Peritoneal macrophages (PC) were harvested
from unprimed NOD mice and plated (5.times.10.sup.5 cells/well) in
24-well plates in CM with E. coli LPS (055:B5) at 200 ng/ml in the
presence or absence of the SOD mimic at 34 .mu.M or 3.4 .mu.M final
concentration. Cultures were incubated at 37.degree. C. for 48 hr;
the cells were trypsinized, and washed to remove the trypsin, and
subsequently transferred to microfuge tubes. PMA was added to a
final concentration of 50 ng/ml. After incubation at 37.degree. C.
for 20 min, superoxide production was assessed
spectrophotometrically by ferricytochrome c reduction using an
.epsilon.=20,000 M.sup.1 cm.sup.1. The reduction was monitored over
a period of 10 min. Data are mean and SEM or triplicate wells and
representative of duplicate experiments. FIG. 4B. Peritoneal
macrophages were harvested from unprimed NOD mice by washing the
cavity of each animal with 7 ml of HBSS. The cells were then washed
2.times. in sterile HBSS and adjusted to 5.times.10.sup.5
cells/well in a 24 well plate in CM with E. coli LPS (05:B5) at 200
ng/ml in the presence or absence of 34 .mu.M or 17 .mu.M SOD mimic.
Cultures were incubated at 37.degree. C. for 48 hr before the
supernatants were harvested and assayed by specific sandwich ELISA
for TNF-.alpha.. The data are the mean and SEM of 3 separate
experiments.
[0013] FIGS. 5A and 5B. Alloxan and cytokine cytotoxicity of SOD
mimic treated NIT-1 cells. FIG. 5A. NIT-1 cells were grown to
confluence in 12-well tissue culture dishes. Media was removed and
replaced with PBS alone or PBS containing 34 .mu.M SOD mimic. All
solutions were supplemented with 4% FCS. After 1 hour incubation,
10 mM alloxan was added to the appropriate wells, and cells were
incubated for an additional 2 hours. Cells were washed, collected
by trypsinization and processed for viability via ethidium
bromide/acridine orange fluorescence .quadrature. live, .box-solid.
live apoptotic. Data are representative of duplicate experiments.
FIG. 5B. NIT-1 cells were grown to 80% confluence in 12-well
plastic tissue culture dishes. Growth media was removed and
replaced with 500 .mu.l/well of either media alone or media+34
.mu.M SOD mimic. After 1 hour incubation, 500 .mu.l/well of media
alone, media or 20 ng/ml IL-1 (10 ng/ml final concentration) +/-34
uM SOD mimic were added. Cells were incubated an additional 48 hr,
and assessed for viability via ethidium bromide/acridine orange
fluorescence .quadrature. live, .box-solid. live apoptotic. Values
are the mean and SEM of triplicate wells per treatment.
[0014] FIG. 6. Protection from streptozotocin-induced diabetes by
in vivo treatment with SOD mimetic (AEOL 10113).
[0015] FIG. 7. Protection of islet transplants from
streptozotocin-induced diabetes by in vitro culture with SOD
mimetic.
[0016] FIG. 8. Facilitation of islet engraftment in spontaneously
diabetic NOD mice by in vitro pre-treatment with SOD mimetic.
[0017] FIGS. 9A-F. Structures of specific prophyrins mimetics.
[0018] FIG. 10. Percent of islet cell mass preserved measured by
DNA content from day 2 to day 7 in the presence or absence of AEOL
10113.
[0019] FIG. 11. Addition of the SOD mimic AEOL 10150 to liberase
during digestion procedure increases human islet cell mass as
compared to control.
[0020] FIGS. 12A-12C. Accelerated neuronal death in cerebrocortical
cultures from SOD2 knockout mice. FIG. 12A. Time-course of cell
death in cortical cultures from SOD2 knockout (+/+, +/- or -/-)
mice after serum withdrawal in ambient oxygen levels. n=16-20,
*p<0.01. FIGS. 12B and 12C. Effect of AEOL compounds to inhibit
cell death 2 and 3 days after serum withdrawal in SOD2 -/-
cultures. n=10-16 cultures.
[0021] FIGS. 13A-13C. 5-Day rescue of SOD2-deficient and normal
neurons. AEOL compounds increase neuronal survival of +/- and
+/+(normal) neurons 5 days after media change to serum-free
conditions.
[0022] FIG. 14. Percentage of preserved islet cell mass between day
2-7 during culture, in control (closed bars) and SOD-mimic (open
bars) treated groups (n=9). DNA content measured at day 2 (24 hours
after isolation) has been arbitrarily considered as the starting
reference value. Differences between groups were statistical
significant (p=0.02; by Mann-Whitney test).
[0023] FIGS. 15A and B. A) Immunocytochemical characterization of
islet preparations from five human pancreata before and after
culture. The number of positive cells was counted and expressed as
percentage of the total. The percentage of pro-insulin positive
cells was maintained over the culture period in all preparations
irrespective of the treatment. After culture, a statistically
significant (*p<0.01; by t test) decrease in amylase positive
cells was observed in both control and SOD-mimic treated
preparations. B) Bar graphs show the fold-increase in preserved
beta cell mass in SOD-mimic-treated preparations compared to
control over the culture time. Beta cell mass was calculated by
combining DNA content as indicator of cell number with
immunocytochemical analysis (Keymeulen et al, Diabetologia
41:452-459 (1998)).
[0024] FIG. 16. Survival rate for islet preparations of the
SOD-mimic group: ----- and Control group: -The donor pancreata
(n=3) were divided in two fractions and treated with or without
SOD-mimic during isolation. From the Kaplan and Meier analysis, it
can be inferred that islet cell loss reduced in SOD-treated
pancreatic tissue as compared to control tissue (log rank,
p=0.0001). Remarkably this difference was seen after 24 hours.
[0025] FIGS. 17A-C. Metabolic capacity of islet grafts in vivo.
Glycemia normalization time following islet transplantation of
variable islet mass: A) Islet grafts: 200-220 IEQ. Control n=4,
SOD-mimic n=5. B) Islet grafts: .about.400 IEQ Control n=4,
SOD-mimic n=4. C) Islet grafts: 700-1000 IEQ Control n=3, SOD-mimic
n=3. -black triangles- Control; -white circles- SOD-mimic.
[0026] FIGS. 18A-K. Structures of certain generic and specific
definitions of compounds suitable for use in the invention (in free
or metal-bound forms). With reference to FIG. 18C, mimetics of the
invention can be of Formula I or II, or dimeric forms thereof, an
example being shown in FIG. 18D.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention relates to methods of preventing or
treating diabetes using low molecular weight antioxidants (eg
mimetics of scavengers of reactive oxygen species, including
mimetics of SODs, catalases and peroxidases). In accordance with
the invention, the present mimetics can be used to prevent, delay
the onset of and/or limit the severity of diabetes resulting, for
example, from the death of pancreatic islet cells due to autoimmune
diseases or free radical induced toxicity, or toxins or drugs the
effects of which are mediated by free radical damage. Low molecular
weight antioxidants can be used to treat or prevent
diabetes-specific microvascular disease of, for example, the
retina, renal glomerulus and peripheral nerve (e.g., resulting in
oedema, ischaemia and hypoxia-induced neovascularization in the
retina, proteinuria, mesangial matrix expansion and
glomerulosclerosis in the kidney, and multifocal axonal
degeneration in peripheral nerves). In addition, low molecular
weight antioxidants can be used to treat or prevent accelerated
atherosclerotic macrovascular disease affecting arteries supplying
the heart, brain and lower extremities.
[0028] The invention further relates to a method of enhancing cell
survival (for example, .beta. islet cell survival) following
transplantaion. The invention further relates to formulations
suitable for use in such methods.
[0029] Mimetics of scavengers of reactive oxygen species
appropriate for use in the present methods include methine (ie
meso) substituted porphines and substituted tetrapyrroles, or
pharmaceutically acceptable salts thereof (eg chloride or bromide
salts). The invention includes both metal-free and metal-bound
porphines and tetrapyrroles. In the case of metal-bound porphines
and tetrapyrroles, manganic derivatives are preferred, however,
metals other than manganese such as iron (II or III), copper (I or
II), cobalt (II or III), or nickel (I or II), can also be used. It
will be appreciated that the metal selected can have various
valence states, for example, manganese II, III, IV or V can be
used. Zinc (II) can also be used even though it does not undergo a
valence change and therefore will not directly scavenge superoxide.
The choice of the metal can affect selectivity of the oxygen
species that is scavenged. Examples of such mimetics are shown in
FIG. 9, FIG. 18 and/or are described in U.S. Pat. Nos. 5,994,339,
6,103,714 and U.S. Pat. No. 6,127,356 and in U.S. application Ser.
Nos. 09/184,982, 09/296,615, 09/490,537, 09/880,075 and 60/211,857
(these applications are incorporated in their entirety by
reference). Appropriate methods of synthesis are described in these
patents and applications.
[0030] In addition to the mimetics described in the above
identified patents and applications, other nonproteinaceous
catalytic antioxidants can also be used, including manganese salen
compounds (Baudry et al, Biochem. Biophys. Res. Commun. 192:964
(1993)), manganese macrocyclic complexes, such as those described
by Riley et al (Inorg. Chem. 35:5213 (1996)), Deune et al (Plastic
Reconstr. Surg. 98:712 (1996)), Lowe et al (Eur. J. Pharmacol.
304:81 (1996)) and Weiss et al (J. Biol. Chem. 271:26149 (1996)),
nitroxides (Zamir et al, Free Radic. Biol. Med. 27:7-15 (1999)),
fullerenes (Lai et al, J. Autonomic Pharmacol. 17:229-235 (1997);
Huang et al, Free Radic. Biol. Med. 30:643-649 (2001), Bensasson et
al, Free Radic. Biol. Med. 29:26-33 (2000)), CuPUPY (Steinkuhler et
al, Biochem. Pharmacol. 39:1473-1479 (1990)) and CuDIPS
(Steinkuhler et al, Biochem. Pharmacol. 39:1473-1479 (1990)). (See
also U.S. Pat. Nos. 6,084,093, 5,874,421, 5,637,578, 5,610,293,
6,177,419, 6,046,188, 5,834,509, 5,827,880, 5,696,109, and
5,403,834).
[0031] The compounds of the invention can be used alone or in
combination with other agents to induce immune tolerance. As shown
in the Examples that follow, the present mimetics can be used to
alter events that occur during antigen presentation to lymphocytes
to create a condition of immune tolerance. This process can be used
in the treatment of diseases involving reaction to foreign antigens
e.g., transplantation) or self antigens (e.g., autoimmune diseases
such as diabetes, multiple sclerosis, glomerulonephritis,
rheumatoid arthritis and collagen vascular diseases).
[0032] The mimetics of the invention (including those in FIGS. 9
and 18, as well as those disclosed in the publications cited
herein) can also be used to protect or enhance viability of
cells/tissues/organs, e.g., mammalian cells/tissues/organs,
including stem cells, pancreatic .beta. cells, liver progenitor
cells, and progenitor cells isolated from adult tissue harvested
from cadavers. The mimetics can be used during the processes of
isolation (harvesting), preservation (e.g., freezing and thawing
(or "cryopreservation" which encompasses both freezing and
thawing)), expansion and/or transplantation. Cells/tissues/organs
treated with the present mimetics show enhanced potential in
transplantation therapy. Specifically, cells/tissues/organs treated
with the mimetics can be used in transplant therapy to treat, for
example, diabetes, liver failure, and inherited metabolic
conditions. Cells/tissues/organs used in such therapies
(particularly treatment of metabolic disorders) can be genetically
engineered. (By way of example, it is noted that AEOL 10112 (see
FIG. 9) has been used in connection with hepatic progenitors. In
this regard, the crryopreservation buffer can be supplemented with
trace elements (selenium (10.sup.-9M), copper (10.sup.-7M), zinc
(5.times.10.sup.-11M)) and an antioxidant (e.g., a porphyrin SOD
mimetic at 10 mcg/ml; ascorbate acid, used at about 0.1
mg/ml)).
[0033] Further, the mimetics of the invention (including those in
FIGS. 9 and 18, as well as those disclosed in the publications
cited herein) can be used to protect cells/tissue/organs from
toxicity, including free radical induced toxicity, during
harvesting, preservation and transport. For example, livers, hearts
and kidneys for transplant can be treated with the present
mimetics.
[0034] The compounds described above, metal bound and metal free
forms, can be formulated into pharmaceutical compositions suitable
for use in the present methods. Such compositions include the
active agent (mimetic) together with a pharmaceutically acceptable
carrier, excipient or diluent. The composition can be present in
dosage unit form for example, tablets, capsules or suppositories.
The composition can also be in the form of a sterile solution,
e.g., a solution suitable for injection (e.g., subcutaneous, i.p.
or i.v.) or nebulization. Compositions can also be in a form
suitable for opthalmic use. The invention also includes
compositions formulated for topical administration, such
compositions taking the form, for example, of a lation, cream, gel
or ointment. The concentration of active agent to be included in
the composition can be selected based on the nature of the regimen
and the result sought. The compounds can also be encapsulated in
lysosomes and thereby targeted to enhance delivery.
[0035] The dosage of the composition of the invention to be
administered can be determined without undue experimentation and
will be dependent upon various factors including the nature of the
active agent (including whether metal bound or metal free), the
route of administration, the patient, and the result sought to be
achieved. A suitable dosage of mimetic to be administered IV or
topically can be expected to be in the range of about 0.01 to 50
mg/kg/day, preferably, 0.1 to 10 mg/kg/day, more preferably 0.1 to
6 mg/kg/day. For aerosol administration, it is expected that doses
will be in the range of 0.001 to 5.0 mg/kg/day, preferably, 0.01 to
1 mg/kg/day. Suitable doses will vary, for example, with the
compound and with the result sought. The concentration of mimetic
presentation in a solution used to treat cells/tissues/organs in
accordance with the methods of the invention can also be readily
determined and will vary with the mimetic, the cell/tissue/organ
and the effect sought.
[0036] Certain aspects of the invention can be described in greater
detail in the non-limiting Example that follows
EXAMPLE 1
Inhibition of Autoimmune Diabetes by Metalloporphyrin-Based
Superoxide Dismutase
Experimental Details
Mice
[0037] NOD.scid breeding pairs were obtained either from The
Jackson Laboratory (Bar Harbor, Me.) or the breeding colony at the
Barbara Davis Center. NOD, NOD.scid, and BDC-2.5-TCR-Tg/NOD (2.5
TCR Tg/NOD) mice were bred and housed under specific pathogen-free
conditions in the Center for Laboratory Animal Care (CLAC) at the
University of Colorado Health Sciences Center.
Expansion Cultures of BDC-2.5
[0038] Expansion cultures for in vivo transfers were produced by
culturing 3-6.times.10.sup.6 T cells from 4-day restimulation
cultures (Haskins et al, Diabetes 37:1444-1448 (1988), Haskins et
al, Proc. Natl. Acad. Sci. USA 86:8000-8004 (1989)) in 60 ml
complete medium (CM) and 14 U/ml IL-2. CM is DMEM supplemented with
44 mM sodium bicarbonate, 0.55 mM L-arginine, 0.27 mM L-asparagine,
1.5 mM L-glutamine, 1 mM sodium pyruvate, 50 mg/L gentamicin
sulfate, 50 .mu.M 2-ME, 10 mM HEPES, and 10% FCS. Cells were
cultured in 75-cm.sup.2 flasks for 4 days at 37.degree. C. and 10%
CO.sub.2. T cells were harvested, washed three times, resuspended
in HBSS, and injected into young (<15 days of age) NOD.scid
recpients.
Metalloporphyrin Superoxide Dismutase Mimic (MnTE2PyP5.sup.+)
[0039] The SOD mimic Mn(III)
tetrakis(M-ethylpyridinium-2-y1)porphyrin (MnTE2PyP5.sup.+) (AEOL
10113) (SOD mimic) was obtained from Incara Pharmaceuticals. Stock
solutions of 600 .mu.g/ml in sterile HBSS for in vivo use, or 680
.mu.M in sterile CM for in vitro experiments were prepared.
Adoptive Transfer of BDC-2.5 T Cell Clones
[0040] Experimental mice were young NOD.scid mice 3-14 days of age.
The recipient mice were given one i.p. injection with BDC-2.5
(1.times.10.sup.7 cells) 1 day after the administration of either
the SOD mimic or HBSS as a control. The mimic or HBSS was
administered every other day for a total of five treatments. Urine
glucose was monitored daily and when animals became diabetic, blood
glucose measurements were taken. Overt diabetes was defined as a
positive urine glucose (>1%), followed by a positive blood
glucose test of >250 mg/dl (14 mM). Recipients were sacrificed
when blood glucose readings were 320 mg/dl (18 mM) or higher. At
sacrifice, the pancreata were removed for histological
analysis.
Histology
[0041] At sacrifice, pancreata were removed and placed in formalin
for at least 24 hr. Pancreata were subsequently embedded in
paraffin, sectioned, and stained with hematoxylin-eosin (H&E)
to detect mononuclear cell infiltration or aldehyde fuchsin (A/F)
to detect insulin.
Preparation of Purified CD4+T Cells from 2.5 TCR-Tg/NOD Mice
[0042] 2.5 TCR-Tg/NOD mice were injected i. p. with either 10 mg/kg
SOD mimic or HBSS every day for 7 days. At day 8, animals were
sacrificed, and the spleens were removed for isolation of CD4.sup.+
T cells by immunomagnetic positive selection using the MACs
magnetic cell separation kit (Miltenyi Biotec, Auburn Calif.)
according to the manufacturer's protocol. The purified T cells were
then plated in 96-well round-bottom plates, pre-coated with 50.mu.
of a 1 .mu.M solution of a BDC-2.5 peptide mimotope, HRPI-RM, as
antigen. Antigen-presenting (APC), treated with either the SOD
mimic or HBSS, were added to the T cells in a crisscross fashion.
The assay plates were incubated for 4 days, and then pulsed with 1
.mu.Ci of .sup.3H-TdR for 6 hrs before harvesting.
T cell and Macrophage Functional Assays
[0043] IFN-.gamma. production by BDC 2.5 was assessed by sandwich
ELISA analysis of responder T cells stimulated with .alpha.-CD3 and
.alpha.-CD26, Con-A or islet cell antigen. For
.alpha.-CD3/.alpha.-CD28 stimulation, 96-well round bottom plates
were pre-coated with 0.125 .mu.g/ml .alpha.-CD3 and 1 .mu.g/ml
.alpha.-CD28 for 1 hr at 37.degree. C. After washing the plates
with sterile HBSS and blocking with CM at 37.degree. C. for 1 hr,
the blocking solution was removed and the BDC-2.5 T cell clone
(2.times.10.sup.4 cells ) was added to the wells in the presence or
absence of the SOD mimic at concentrations of 17 and 34 .mu.M. The
negative control was BDC-2.5 alone without .alpha.-CD3 and
.alpha.-CD28. For Con-A stimulation, BDC-2.5 T cells were plated at
2.times.10.sup.4 cells/well in 96-well flat-bottom plates with or
without 5.times.10.sup.5 irradiated syngeneic spleen cells as APC
and Con A (2.5 .mu.g/ml final concentration), in the presence or
absence of the SOD mimic at concentrations of 17 and 34 .mu.M.
Cultures were incubated at 37.degree. C. for 24 hr before the
supernatants were harvested and assayed by sandwich ELISA for
IFN-.gamma. production. For antigen-specific recall assays, BDC-2.5
T cells were cultured in 96-well flat-bottom plates at a density of
2.times.10.sup.4 cells/well, with 5000 islet-cells as antigen and
2.5.times.10.sup.4 APC, in the presence or absence of 17 and 34
.mu.M SOD mimic. Cultures were incubated at 37.degree. C. for 48 hr
before the supernatants were harvested and assayed for IFN-.gamma..
For macrophage assays, peritoneal macrophages (PC) were harvested
from unprimed NOD mice by lavage, washed 2.times. in sterile HBSS,
and then adjusted to 5.times.10.sup.5 cells/well in a 24-well plate
in CM with E. coli LPS (055:B5) at 200 ng/ml in the presence or
absence of 17 or 34 .mu.M SOD mimic. Cultures were incubated at
37.degree. C. for 48 hr before the supernatants were harvested and
assayed by specific sandwich ELISA for TNF-.alpha. production,
following the manufacturer's protocol (R&D Systems). The
remaining cells were collected by trypsinization, and washed
3.times. in sterile PBS and 4% FCS.
Respiratory Burst of Peritoneal Macrophages
[0044] Peritoneal macrophages (PC), harvested as described above,
were washed 2.times. in sterile HBSS and then plated
(5.times.10.sup.5 cells/well) in 24-well plates in CM medium with
E. coli LPS (055:B5) at 200 ng/ml in the presence or absence of the
SOD mimic at 34 or 3.4 .mu.M. Cultures were incubated at 37.degree.
C. for 48 hr. Cells were trypsinized and then washed to remove the
trypsin and subsequently transferred to microfuge tubes. PMA was
added to a final concentration of 50 ng/ml. After incubation at
37.degree. C. for 20 min, superoxide production was assessed
spectrophotometrically by ferricytochrome c reduction using an
.epsilon.=20,000 M.sup.-1cm.sup.-1, monitoring the reduction over a
period of 10 min.
Determination of Beta Cell Apoptosis
[0045] In vitro apoptosis studies were conducted using the
.beta.-cell adenoma line NIT-1 (Hamaguchi et al, Diabetes
40:842-849 (1991)). Tumor cells were propagated in 75 cm.sup.2
flasks at 37.degree. C. in CM. Cell lines were re-fed with new
medium every other day and were grown to confluence in the 75
cm.sup.2 tissue culture flasks, at which time they were harvested
using non-enzymatic Cell Dissociation Buffer (Gibco, BRL; Grand
Island, N.Y.) and transferred to the appropriate culture dishes for
either expansion or for the experiments described. Alloxan
monohydrate (Sigma) was prepared fresh as a 0.5 M stock solution in
PBS adjusted to pH 2 with hydrochloric acid. IL-1.beta. was
purchased from R & D Systems (Minneapolis Minn.). NIT-1 cells
were grown to confluence in 12-well tissue culture dishes. Media
was removed and replaced with PBS alone or PBS containing 34 .mu.M
mimic. All solutions were supplemented with 4% FCS. After 1 hr
incubation, 10 mM alloxan was added to the appropriate wells, and
cells were incubated for an additional 2 hr. For cytokine
cytotoxicity assays NIT-1 cells were grown to 80% confluence in
12-well plastic tissue culture dishes. Growth media was removed and
replaced with 500 .mu.l/well of either media alone or media+34
.mu.M mimic. After 1 hr incubation, 500 .mu.l/well of media alone,
or 20 ng/ml IL-1 (10 ng/ml final concentration)+/-34 uM SOD mimic
were added. Cells were incubated an additional 48 hr, and
processed. Alloxan or cytokine-treated NIT-1 cells were harvested
by brief trypsinization (200 .mu.l/well of a 12-well dish) followed
by addition of 50 .mu.l FCS to inhibit trypsin. Cells were
transferred to a microcentrifuge tube and centrifuged for 5 min at
200.times.g. Supernatants were aspirated very carefully, leaving
approximately 25 .mu.l to allow resuspension of the cell pellets by
gentle shaking of the tube. After addition of 1.3 .mu.l of dye mix
(100 .mu.g/ml Acridine Orange+100 .mu.g/ml of EtBr in PBS), 10
.mu.l of cell suspension was transferred to a clean microscope
slide and a coverslip placed on the suspension. Cells were scored
for morphological evidence of apoptosis as described (Squier et al,
Assays of Apoptosis. Humana Press, Totowa) using a fluorescence
microscope with an excitation of 450-490 nm.
Statistical Analysis
[0046] Statistical significance within experiments was determined
using JMP analysis software (SAS Institute, Cary, N.C.). Survival
analysis was done using the product-limit (Kaplan-Meier) method.
The endpoint of the experiment was defined as diabetes. Data on
animals that did not become diabetic by the end of the experiment
were censored. The p values shown were determined by Log-Rank test.
All other statistical analysis was done by Oneway analysis of
variance Anova (Wilcoxon/Kruskal-Wallis Rank Sums). If p values
were .ltoreq. to 0.05, they were considered significant.
Results
[0047] In vivo treatment of young NOD.scid mice with the SOD mimic
prevents adoptive transfer of T cell mediated diabetes.
[0048] SOD mimic was delivered paraenterally to NOD.scid recipients
and 24 hr later, mice were adoptively transferred with the
diabetogenic T cell clone BDC-2.5. The SOD mimic or HBSS was then
given every other day for a total of 5 treatments. Treatment with
the SOD mimic significantly delayed (p<0.0002) onset of diabetes
(FIG. 1A), with 50% of the treated mice still normoglycemic after
28 days at which time all animals were sacrificed for histological
examination. Pancreatic tissue from positive control animals
(BDC-2.5, no SOD mimic) showed a disseminated infiltrate resembling
pancreatitis, and the pancreatic architecture was almost absent
(FIG. 1B-a). In contrast, the SOD mimic-treated animals showed an
intact pancreatic architecture with few or no infiltrating
mononuclear cells (FIG. 1B-b,c), as well as healthy and
well-granulated islets (FIG. 1B-d). These data clearly demonstrate
that the SOD mimic is inhibiting the infiltration by BDC-2.5 T
cells and mononuclear cells to the pancreas. Remarkably, in these
experiments, the animals were still protected on day 21, even
though the SOD mimic was stopped on day 9, sugesting that this
compound prevents priming and subsequent activation of the APC, the
T-cell, or both. Longer administration of the SOD mimic may prove
to be even more protective.
[0049] Interferon-gamma production by BDC-2.5 is inhibited by the
SOD mimic in vitro: indirect effect on the APC leading to
inhibition of T cell priming.
[0050] In vivo, BDC-2.5 must be primed by its antigen via
presentation by APC in order to become activated and produce
IFN-.gamma.. Therefore, the SOD mimic could be directly inhibiting
T cell activation or the interaction between the APC and the T cell
or both. In order to elucidate the mechanism of inhibition of
disease transfer, priming of BDC-2.5 was studied in vitro, in the
presence or absence of APC. To determine if the SOD mimic has a
direct effect on IFN-.gamma. production by the T cell, BDC-2.5 was
cultured with plate bound .alpha.-CD3 and .alpha.-CD28. This type
of activation substitutes for both signals 1 and 2 of T cell
activation (Mueller et al, J. Immunol. 142:2617-2628 (1989),
Mueller et al, Annu. Rev. Immunol. 7:445-480 (1989), Schwartz et
al, Cold Spring Harb. Symp. Quant. Biol. 54:605-610 (1989), June et
al, Immunology Today 15 (1994)), thus removing the contribution of
the APC. FIG. 2A shows that .alpha.-CD3 and .alpha.-CD28
stimulation resulted in no significant difference in IFN-.gamma.
production by the BDC-2.5 clone, whether or not the SOD mimic was
present. These results demonstrate that when plate-bound antibodies
substitute for signals 1 and 2, the SOD mimic has no direct effect
on the ability of BDC-2.5 to be stimulated to effector function and
produce IFN-.gamma.. Although primed T cells can directly respond
to Con A, optimal Con A-induced T-cell cytokine production requires
the participation of accessory cells (e.g., macrophages) (Ahmann et
al, J. Immunol. 121:1981-1989 (1978), Hunig et al, Eur. J. Immunol.
13:1-6 (1983), Hunig, Eur. J. Immunol. 13:596-601 (1983), Hunig,
Eur. J. Immunol. 14:483-489 (1984), Bekoff et al, J. Immunol.
134:1337-1342 (1985), Roosnek et al, Eur. J. Immunol. 15:652-656
(1985), Hoffmann et al, Lymphokine Res. 5:1-9 (1986)). In order to
determine if the SOD mimic could inhibit APC-mediated Con A
stimulation of T cells, BDC-2.5 cells were incubated with Con A and
APC in the presence or absence of SOD mimic. FIG. 2B shows that 34
or 17 .mu.M SOD mimic inhibited IFN-.gamma. production by 47 or
30%, respectively. The levels of IFN-.gamma. produced in the
presence of the SOD mimic were similar to levels seen when BDC-2.5
was incubated with Con A alone. These results indicate that the SOD
mimic inhibits the ability of the APC to optimally stimulate Con
A-dependent T cell activation and IFN-.gamma. production. To
further study the SOD mimic's effect on APC-T cell interactions,
IFN-.gamma. production was measured in the presence of macrophages
as APC and islet cells as a source of antigen. FIG. 2C shows that
when this more physiological in vitro assay was done, the ability
of BDC-2.5 to make IFN-.gamma. was reduced: the 17 .mu.M
concentration of SOD mimic inhibited by 46% (p<0.05), while the
34 .mu.M concentration inhibited by 66% (p<0.05).
[0051] In vivo treatment of 2.5 TCR Tg/NOD mice with the SOD mimic
affects T cell proliferation by inhibiting APC function.
[0052] In order to determine if the SOD mimic can influence T cell
priming in vivo, 2.5 TCR-Tg/NOD mice, which carry the rearranged
TCR genes of the BDC-2.5 T cell clone (Katz et al, Cell
74:1089-1100 (1993)), were treated with either the SOD mimic (10
mg/kg) or HBSS each day for 7 days. The T cells and APC were
purified from SOD mimic-treated and control mice and cultured in a
crisscross proliferation assay using a peptide mimotope HRPI-RM
that acts as a stimulating antigen for the 2.5 TCR-Tg cells. FIG. 3
demonstrates that APC from SOD mimic-treated mice showed a reduced
ability to support T cell proliferation whether they are presenting
the peptide to SOD mimic-treated or untreated T cells. Notably,
when control APC were used as presenters, the proliferative
response in SOD mimic-treated T cells approached the level achieved
with control APC and T cells. These data demonstrate that in vivo
SOD mimic treatment inhibits the response in TCR-Tg mice primed to
a specific self-peptide and indicate that using the SOD mimic in
combination with candidate autoantigens may provide a form of
antigen-specific tolerance.
[0053] LPS-induced respiratory burst and cytokine production by
peritoneal macrophages is inhibited by the SOD mimic.
[0054] Macrophages are activated in the two-stage reactions of
priming and triggering (Meltzer, J. Immunol. 127:179-183 (1981)).
In order to assess the inhibitory effect of the SOD mimic on this
process, peritoneal macrophages (PC) were cultured with LPS in the
presence or absence of the mimic. The supernatants were collected
and the PC (peritoneal macrophages) were washed and triggered with
PMA to measure their NADPH oxidase-mediated respiratory burst and
superoxide production. FIG. 4A shows that 3.4 .mu.M SOD mimic
results in a 75% reduction in superoxide production and increasing
the concentration of SOD mimic to 34 .mu.M did not significantly
further decrease superoxide production. Moreover, FIG. 4B shows
that TNF-.alpha. production by LPS-primed PC was inhibited 34% by
17 .mu.M mimic, while 34 .mu.M mimic resulted in a 51% inhibition.
These data clearly demonstrate that pre-incubation of LPS-primed
macrophages with SOD mimic inhibited both activation of NADPH
oxidase and TNF-.alpha. production. It should be noted that the SOD
mimic had been washed off prior to the assay and, therefore, was
not present in the extracellular space where superoxide geneation
is measured. Therefore, a decrease in superoxide production was not
due to the SOD mimic scavenging the extracellular superoxide but
rather to a reduction in oxidase-dependent superoxide. The fact
that superoxide production by activated macrophages (FIG. 4A) is
inhibited by 3.4 .mu.M SOD mimic, while inhibition of TNF-.alpha.
or IFN-.gamma. production requires higher SOD mimic concentration
(FIG. 4B, 2C), indicates that the oxidant concentration necessary
to activate the NADPH oxidase of macrophages is lower than the
oxidant concentration necessary to activate the signal transduction
pathways required for cytokine production. These results point to
the fascinating prospect that biological responses to oxidants are
not just "all-or-none", but instead are specific to the pathway
involved.
[0055] SOD mimic-treated NIT-1 insulinoma cells are protected from
alloxan and cytokine-mediated cytotoxicity.
[0056] Both alloxan and pro-inflammatory cytokines have been shown
to be cytotoxic to .beta.-cells in vitro. This series of
experiments was designed to determine if the SOD mimic could
protect islet cells from alloxan and cytokine-mediated cytotoxicity
using the well established NIT-1 insulinoma cell line. FIG. 5A
shows that incubation of NIT-1 cells with 10 mM alloxan induces 50%
apoptosis compared to 5% for control untreated or control plus SOD
mimic. However, NIT-1 cells exposed to alloxan and treated with the
SOD mimic show 70% viability.
[0057] FIG. 5B demonstrates the protective effect of the SOD mimic
on NIT-1 cells exposed to IL-1.beta. in culture. The addition of 10
ng /ml IL-1.beta. was cytotoxic to NIT-1 cells (.about.50% of the
cells were apoptotic) compared to control or control plus SOD
mimic. A clear protective effect was seen when NIT-1 cells exposed
to IL-1.beta. were treated with SOD mimic. The SOD mimic's
protective effect is consistent with other reports of antioxidant
proteins conferring resistance to immunological damage in
insulinoma cells (Grankvist et al, Biochem. J. 199:393-398 (1981),
Malaisse et al, Proc. Natl. Acad. Sci. USA 79:927-930 (1982),
Lenzen et al, Free Radic. Biol. Med. 20:463-466 (1996), Tiedge et
al, Diabetes 46:1733-1742 (1997), Benhamou et al, Diabetologia
41:1093-1100 (1998), Tiedge et al, Diabetes 47:1578-1585 (1998),
Tiedge et al, Diabetologia 42:849-855 (1999)).
EXAMPLE 2
Protection from Streptozotocin-induced Diabetes and Facilitation of
Islet Engraftment by SOD Mimetic Treatment
[0058] 1: Protection from streptozotocin-induced diabetes by in
vivo treatment with SOD mimetic.
[0059] Experiment: Diabetes was induced in C57B1/6 male mice with
160 mg/kg streptozotocin (SZ) intravenously. Recipients were either
otherwise untreated or were treated with daily intraperitoneal
bolus injections with 1 mg/kg or 10 mg/kg of the SOD mimetic on
days -1 through +5 relative to SZ treatment.
[0060] Results: 1 mg/kg of the mimetic demonstrated some protection
from SZ-induced diabetes. Results indicate that the 10 mg/kg dose
led to protection in 2/5 animals (versus {fraction (0/9)} in
untreated animals). (See FIG. 6.)
[0061] 2: Protection of islet transplants from
streptozotocin-induced diabetes by in vitro culture with SOD
mimetic.
[0062] Experiment: Syngeneic C57B1/6 islet grafts were pre-treated
in vitro with SOD mimetic (34 .mu.M) for 2 hours and then
transplanted in C57B/16 challenged with 160 mg/kg SZ as described
above.
[0063] Results: Pre-treatment of the islet graft prior to
transplant led to protection in 2/3 islet grafts. (See FIG. 7.)
[0064] 3: Facilitation of islet engraftment in spontaneously
diabetic NOD mice by in vitro pre-treatment with SOD mimetic.
[0065] Experiment: Recurrence of disease in autoimmune diabetic NOD
mice is so vigorous that islet transplants often fail to engraft
(i.e., grafts fail to restore even transient euglycemia). This
experiment determined whether initial inflammatory damage to
syngeneic NOD islet grafts could be attenuated by treating NOD
islets in vitro with a SOD mimetic prior to transplant. Syngeneic
NOD islet grafts were pre-treated in vitro with SOD mimetic (34
.mu.M) for 2 hours and then transplanted into spontaneously
diabetic (autoimmune) NOD recipient.
[0066] Results: The treated NOD islet graft restored euglycemia
within 24 hours relative to the untreated control NOD graft that
failed to engraft during the initial 5 day observation period. (See
FIG. 8)
EXAMPLE 3
Human Islet Isolation
[0067] Islets are obtained from the pancreas of cadaveric donors
(islets comprise approximately 1-2% of the pancreas). The donor
pancreas is harvested and preserved with UW (University of
Wisconsin solution, DuPont Pharma, Wilmington, Del.). An automated
method is used to isolate islets from the donor pancreas (Ricordi
et al, Diabetes 37:413-420 (1988), Tzakis et al, Lancet 336:402-405
(1990)). All procedures are performed under aseptic conditions in
Class II biohazard hoods or clean rooms with solutions comprised of
sterile components.
[0068] The pancreas is removed from the shipping container and
placed into a sterile tray containing 500 ml a cold preservation
solution. A sample of the preservation solution is taken for
microbiological analysis. This tray is placed in a larger tray and
maintained cold via a cold bath or using 1 L of cold sterile ice
(from 2 L of frozen sterile water); the organ is trimmed of fat and
non-pancreatic tissue and weighed. After cleaning, the pancreas is
dipped in betadine and antibiotics and the tray containing the
pancreas is removed from the ice for the distension procedure.
[0069] The pancreatic duct is cannulated with catheters and the
pancreas is distended with sterile filtered collagenase solution.
The collagenase solution consists of Liberase-HI (Roche Molecular,
Indianapolis, Ind.) dissolved in 15 ml Hank's Balanced Salt
Solution and diluted to a maximal total volume of 350 ml.
Liberase-HI has been specifically formulated for use in human islet
isolation procedures (Linetsky, Diabetes 46:1120-1123 (1997)).
[0070] The distended pancreas is placed into a sterile stainless
steel chamber (Ricordi et al, Diabetes 37:413-420 (1988), Tzakis et
al, Lancet 336:402-405 (1990)), additional collagenase solution is
added, and the collaaenase solution is recirculated and brought to
37.degree. C., as the chamber is mechanically agitated (Ricordi et
al, Diabetes 37:413-420 (1988), Tzakis et al, Lancet 336:402-405
(1990)). During this digestion procedure, samples are taken at
intervals to monitor the breakdown of the pancreas via microscopy.
The length of digestion varies but in general, once the temperature
has reached 37.degree. C. inside the chamber and most of the islets
are free of the surrounding acinar tissue (15-25 minutes) the
digestion process can be stopped.
[0071] Once free islets are detected, the recirculation cylinder
and the heating circuit are bypassed and the islet separation is
conducted in a system in which the temperature is progressively
decreased and the collagenase solution is diluted with solutions.
The digest containing the free islets is collected into sterile
containers at 4.degree. C. to prevent enzymatic overdigestion. An
aliquot of the digest (composed of endocrine and acinar tissue) is
taken for staining with dithizone; the percentage of free islets,
degree of islet fragmentation, and the condition of the acinar
tissue are noted. The tissue is centrifuged and recombined and the
supernatant removed. The pellets are collected and resuspended in
tubes containing UW and held on ice for 30 minutes before
proceeding with purification steps. UW allows acinar cells to
reestablish osmotic equilibrium, hence preventing cell swelling
This procedure is aimed at increasing the difference in density
between islets and acinar tissue, a key parameter for effective
purification based on difference in density (Robertson, Br. J.
Surg. 79:899-902 (1992)).
[0072] The separation of islets from exocrine tissue is performed
via density gradient centrifugation in a COBE 2991 blood cell
processor (Ricordi et al, Diabetes 37:413-420 (1988), Tzakis et al,
Lancet 336:402-405 (1990)2). The gradients are composed of
dissolved sugar gradients (Ficoll) dissolved in Eurocollins
solution, using defined protocols. After collection of the
resulting fractions from the COBE 2991, islet enriched fractions
(purity=islets Vs non islets.about.60-90%) are washed extensively
to remove Ficoll and resuspended in culture medium composed of CMRL
plus 10% FCS, antibiotics and L-glutamine.
[0073] The islet cell suspension may be cultured prior to
transplant. Groups of islets are placed in a 22.degree. C.
incubator (95% air, 5% CO.sub.2) in MCRL 1066 media supplemented
with 10% FCS, 1% HEPES, 1% glutamine, and 1% antibiotic solution
and filtered with a 0.2 m filter. After suspension in culture
media, and immediately prior to placement in the incubator,
representative aliquots of islets will be removed for bacteriology
and mycology assessment, for enumeration, and for assessment of
viability, endotoxin content, and functional capacity.
[0074] Prior to transplantation, the islet are collected from the
tissue culture flasks/bags and placed into tubes, a sample of the
culture media is taken for mycoplasma testing, and the suspension
is centrifuged. The islets are resuspended in transplant media (TX
media: HBSS, 2.5% human serum albumin, HAS) and centrifuged to wash
out cellular debris, tissue culture media (FCS) and, soluble
proteolytic activity. The islets are resuspended once more in TX
media, aliquots are removed for islet enumeration and microbiology,
and the cells are centrifuged again. A sample is taken from the
supernatant for microbiological analysis, and the islets are
suspended in 200 ml of TX media for transplant.
[0075] In order to determine the functional capacity of the
preparation, two aliquots of freshly isolated for cultured islets
will be incubated overnight at 370.degree. C. On the subsequent
morning, standard techniques for static incubation and assessment
of insulin release, DNA content, and insulin content will be
utilized to determine the functional capacity of the islets
(Ricordi et al, Diabetes 37:413-420 (1988), Tzakis et al, Lancet
336:402-405 (1990)). Briefly, samples will be washed twice in basal
media (RPMI 1640+10% FBS) containing 2.8 mM glucose, followed by a
2-hour incubation in basal medium and a further 2 washes. One
aliquot will then be incubated in basal medium and one in medilum
containing 16.7 mM glucose to assess glucose mediated insulin
release. At the end of the incubation period, media will be
collected and frozen at -20.degree. C. until they are assayed for
immunoreactive insulin. The islets will be washed twice in basal
media, and acid alcohol will be added for a period of 18 hours to
assess islet insulin content. Standard RIA procedures will be used
to determine insulin content.
EXAMPLE 4
Preservation of Human Islets After Isolation Using a
Metalloporphyrin-Based Superoxide Dismutase Mimic
[0076] Islet transplantation is an attractive alternative to
chronic insulin administration for the restoration of normoglycemia
in type I diabetes. However, that single cadaveric donors do not
provide sufficient numbers of islets to achieve insulin
independence in recipients erects a stumbling block. One reason for
the limited number of islets obtained after isolation could be due
to the loss of cells by apoptosis during and after isolation. In
this study, it was demonstrated that incubation of human islets,
from cadaveric donors (n=5), for 6 days in a free-radical
scavenging, metalloporphyrin-based superoxide dismutase mimic
Mn(III) tetrakis (N-ethylpyridium-2-yl) porphyrin (SOD mimic),
exhibited a 3-fold increase in bata cell mass compared to control
islets as measured by DNA content. The increase in beta cell mass
correlated with an increase in overall cell viability. Dithizone
staining throughout the 6 day incubation period revealed that all
preparations maintained at least 75% of islet mass. There was no
detectable loss of beta cell function in SOD mimic-treated islets
as measured by static glucose stimulated insulin release. The
ability of the SOD mimic to efficiently scavenge free radicals and
protect cells from oxidative stress and apoptosis warrants their
use for the preservation of beta cells during islet isolation
procedures. (See FIGS. 10 and 11. )
EXAMPLE 5
AEOL 10113 and MnTBAP (AEOL 10201) Improve the Survival of Cultured
Neurons in Serum-free Media
[0077] The ability of AEOL 10201 and AEOL 10113 to improve the
survival of normal and SOD2-deficient cerebrocortical neurons in
primary culture was studied. Neuronal cultures were prepared from
cerebral cortices of SOD2 knockout (homozygous -/-, heterozygous
-/+ or wild-type +/+ genotypes) mice of embryonic days 14-16.
Neuronal cultures were initially plated in serun containing minimum
essential medium (MEM with Earle's salts) in a low oxygen
environment (5% O.sub.2. 95% Argon) for 18 hours. The presence of
serum during this initial period promotes adherence of neurons to
the substrate and the low oxygen levels protect SOD2-deficient
neurons from ambient oxygen levels. Following this plating
procedure, culture media was replaced with serum-free MEM
containing vehicle or varying concentrations of AEOL compounds and
placed in a normal oxygen environment. Cultures were observed for
injury and supernatant media assayed for the release of lactate
dehydrogenase 2, 3 and 5 days following the addition of drugs. SOD2
-/- cultures showed accelerated cell death in serum-free conditions
and under ambient oxygen (FIG. 12A). Neuronal cultures from SOD2+/+
and wild-type (normal) mice died from serum-deprivation 5 days
following media change. AEOL 10201 and 10113 improved the survival
of SOD2-/- cultures on days 2, 3 and 5 (FIGS. 12B, 12C and FIG.
13A). AEOL 10201 and 10113 dramatically improved the survival of
wild-type (normal) and SOD2+/- cultures 5 days after media change
to serum-free conditions. These results indicate that AEOL 10113
and 10201 can substitute for the presence of protective factors in
serum that promote cell survival. They further indicate than
catalytic antioxidants can be used for maintaining cultured cells
in serum-free media.
EXAMPLE 6
Preservation of Human Islet Cell Function Mass
Experimental Details
Human Islets
[0078] Human pancreata were obtained from CORE (Center for Organ
Recovery and Education-Pittsburgh) and from NDRI (National Disease
Research Interchange -Philadelphia). Twelve pancreata failing the
standard criteria for the use of whole pancreas or islet
transplantation were processed (Table 1). To more comprehensively
test the compound, it was set up to utilize all available organs
without applying any exclusion criteria. The cold ischemia time was
9.+-.2 hr ranging from 5 to 15 hr. The age was 49.+-.4 years (range
17-68 years) and body mass index 26.+-.2 (values are mean.+-.SEM).
The final islet yield (IEQ/g) for the pancreatic preparations (n=9)
obtained with the semi-automated method, which consisted of purity
between 60-80%, was 4628.+-.749 IEQ/g range 1790 to 7631, median
4542.
[0079] The percentage of islets over whole preparation was
determined immediately after isolation, by dithizone (Sigma, St.
Louis, Mich.) staining (Latif et al, Transplantation. 45:827-830
(1988). This was 60-80% in the first group of nine islet
preparations and respectively 60%, 60%, and 30% in the last three
preparations. The yield obtained using the second series of
experiments (stationary digestion) was 1744.+-.676 IEQ/g.
1TABLE 1 Donor Characteristics Human Cold % islets/ Pancreas
Ischemia Body Mass whole (HP) Age Time Gender Index preparation
Study group 1 48 68 7 M 22.9 60-70 49 50 15 F 30.2 60-70 50 54 5 M
27.7 65-75 52 49 12 M 30.4 65-75 53 51 8 F 17.6 60-70 54 46 6 F
17.6 70-80 55 17 8 F 24.2 70-80 57 65 7 F 23.4 60-65 58 55 7 M 22.4
60-70 Study group 2 60 24 11 M 31 60-65 68 48 9 F 37 60-65 71 57 10
M 29 30-35
Culturing of Purified Human Islets in the Presence of SOD-mimic
[0080] The initial series of experiments involved the addition of
SOD-mimic after islet isolation, as a culture supplement.
Metalloporphyrin SOD-mimics AEOL 10113, AEOL 10150 were provided by
Incara Pharmaceuticals. The islets were isolated using the method
described by Ricordi et al (Ricordi et al, Diabetes 38 Suppl
1:140-142 (1989)) with minor modifications. Prior to purification,
the digest was incubated in cold UW solution for 45 minutes. The
islet-enriched fractions were purified using discontinuous
Euro-ficoll density gradients and processed in a COBE 2991 Cell
Separator (Gambro, Lakewood, Co.). The islets were cultured for 7
days (37.degree. C., 5% CO.sub.2) in CMRL-1066 culture medium
supplemented with 10% heat inactivated fetal calf serum, 100
units/m1 penicillin and 0.1 mg/ml streptomycin, and 2 mmol/1
L-glutamine (Life Technologies, Grand Island, N.Y.) with or without
SOD-mimic at the final concentration of 34 .mu.mol/1. Fresh culture
medium (with or without SOD-mimic) was replaced three times per
week and islet samples were taken for assessment of DNA content and
for functional studies at selected time points.
Determination of Islet Cell Mass and Viability
[0081] DNA content has been used as an indirect measure of cell
mass, since the clustered nature of the islets, together with the
non-endocrine contaminants, makes direct counting inappropriate
(Ling et al, J Clin Invest 98:2805-2812 (1996)). DNA content was
measured in samples of the islet preparations from both control and
SOD-treated groups, using a Pico Green dsDNA Quantitation Kit
following the manufacturer protocol (Molecular Probes, Eugene,
Oreg.). Islet viability was determined by simultaneous staining of
live and dead cells using a two-color fluorescence assay
(Calcein-AM and Propidium Iodide, Molecular Probes) (Lorezo et al,
Nature 36:756-760 (1994)). The percentage of viable and dead cells
was estimated in both control and SOD-treated groups.
Static Glucose Challenge and Insulin Content Measurement
[0082] Handpicked islets from control and SOD-treated groups were
subjected to static glucose challenge in Krebs-Ringer bicarbonate
buffer (KREBB) (pH 7.35) containing 10 mmol/l HEPES and 0.5% bovine
serum albumin (Sigma). After conditioning, the islets were
incubated in KRBB containing low (2.8 mmol/l) and high (20 mmol/l)
glucose concentrations for one hour. At the end of the glucose
challenge, insulin extraction was carried out to determine islet
insulin content (Pipeleers et al, Diabetes 40:908-919 (1991)). The
insulin levels were measured by ELISA (ALPCO, Windham).
Immunocytochemistry
[0083] Samples of the purified islet preparations were fixed in
Bouin's solution for one hour and then transferred to 10% buffered
formalin. Using standard procedures, the islet samples were stained
for immunoreactive pro-insulin, glucagon, CK19 (Scytek
Laboratories, Logan, Utah) and amylase (Biogenex, San Ramon,
Calif.) and the percentage of positive cells was counted in both
control and SOD-treated groups.
Perifusion Protocol of Islet Insulin Release
[0084] Whether addition of SOD-mimic to the islet medium induces
unregulated insulin release, this was determined by a perifusion
protocol in three different human islet preparations, carried out
under constant, physiologic glucose concentration. Groups of 100
handpicked islets (diameter 100-150 .mu.m) cultured under control
conditions for 4-7 days were perifused with KREBB containing 5.6
mmol/l glucose. During the 90-minute perifusion period, the
SOD-mimic was added between minute 30 and 60. The insulin
concentration of the elution samples was measured by ELISA (ALPCO).
Administration of SOD-mimic during islet isolation
[0085] In the second series of experiments, SOD-mimic was
administered during the isolation phase as well as during culture.
The pancreatic lobe was divided in two parts (body and tail), of
similar size (27.+-.3 grams) randomly assigned to control and
experimental conditions. During isolation SOD-mimic (concentration
34 .mu.mol/l) was delivered to the half of the pancreatic tissue
together with Liberase .TM., by intra-ductal injection. In the
control group, only Liberase .TM. was infused. The split organs are
not amenable to authomatic processing therfore stationary digestion
was carried out in parallel on the two organ segments. The samples
were digested for 30 minutes at 37.degree. C. with periodic
shaking. Cell dissociation was finalized by manual teasing of the
tissue. Cells and aggregates were filtered through a 500 .mu.m mesh
and collected in cold RPMI-1640 culture medium (Life Technologies)
containing 10% heat-inactivated fetal calf serum. Prior to COBE
processing, the pancreatic digests were incubated with cold UW
solution with or without SOD-mimic (34 .mu.mol/1) for 45 minutes.
Purification and culture were then continued as described in the
first series of experiments.
Islet Transplantation Under the Kidney Capsule of Diabetic Mice
[0086] Islets grafts of 200 to 1000 IEQ (islet equivalents) from
four different organs, were cultured in the presence of SOD-mimic
or kept in control conditions for at least two hours before
transplantation. Streptozotocin-induced diabetic (Sigma, 250 mg/kg
body weight) NOD-scid or Rag 1 mice (Jackson Laboratories, Bar
Harbor,) exhibiting non-fasting blood glucose levels of >300
mg/dl were used as recipients. Animals were anesthetized by I.P.
injection of Avertin (0.30-0.40 mg/gram body weight). Control or
SOD-treated islets were transplanted under the mouse kidney capsule
(Alexander et al, Diabetes 51:356-365 (2002)). Successful
engraftment was defined by reduction of glycemic levels to <200
mg/dl following transplantation. After 4 to 7 weeks
post-transplantation, normalized recipients underwent nephrectomy
to remove the islet graft. Return to hyperglycemia was interpreted
as indirect proof of islet graft function.
Statistical Methods
[0087] Statistical analysis was carried out by Student's t test,
Mann-Whitney nonparametric test, and Kaplan and Meier analysis. P
values <0.05 were deemed statistically significant.
Results
Presence of SOD-mimic Preserves Islet Cell Mass
[0088] Isolated islets from nine donor pancreata, were divided into
two groups and cultured either in CMRL-1066 alone or supplemented
with SOD-mimic. The results in FIG. 14 demonstrate that in all
experimental groups (n=9), a 3-fold mean increase in cell mass was
observed when compared to controls (p=0.02). It should be noted
that the difference in DNA content between control and experimental
groups was appreciable starting from day 2 (24 hours after
isolation) and steadily observed over the remaining culture period.
However, in the first 24 hours of culture, a similar decrease in
cell mass was observed in control and SOD-treated islet
preparations accounting for 20-40% of the initial cell mass. These
data demonstrate that addition of SOD-mimic reduces cell loss
significantly over time, although it appears that there is no
protective effect on those cells that die very early, within 24
hours following isolation.
Cell Viability
[0089] Double fluorescence viability was performed to determine
whether DNA content might be affected by the presence of dead
non-degraded cells in higher proportion within the
SOD-mimic-treated preparations. The results indicate that in both
control and SOD-treated aggregates, a similar number of viable and
dead cells was present, however, the islet number was higher in
SOD-treated than in the control group. At a later stage of the
culture period, control preparations showed higher dead cell
contents, but the difference did not reach statistical significance
(Table 2a).
Static Glucose Challenge and In vitro Islet Function
[0090] Islet glucose responsiveness was assessed by a static
glucose challenge method between days 3 and 5 after isolation
(Table 2b). Upon glucose stimulation insulin release, expressed as
absolute value or as a percentage of the insulin content and
stimulation indices were similar, regardless of the culture
treatment. Also basal insulin release did not differ between
groups.
[0091] Also studied was the effect of addition of SOD-mimic on
insulin release during a 90-minute perifusion protocol of isolated
human islets (n=3) under constant glucose concentration. The
results demonstrate that SOD-mimic supplementation does not affect
insulin secretion. Basal release was similar in all groups,
regardless of the addition of SOD-mimic to the medium.
2TABLE 2 Islet functional properties a) Viability Culture day 1-4
Culture day 5-10 Groups Viable % Dead % Viable % Dead % Control 80
.+-. 5 20 .+-. 5 92 .+-. 5 8 .+-. 5 SOD-mimic 79 .+-. 5 21 .+-. 5
86 .+-. 5 14 .+-. 5 Percentage of viable and dead cells present in
islet preparations (n = 9). Values are mean .+-. SEM. Differences
are not statistically significant. b) In vitro
glucose-responsiveness Stimulated insulin % Stimulation Groups
secretion .mu.U Insulin index Control 13 .+-. 2 3 .+-. 1 5.5 .+-.
1.5 SOD-mimic 14 .+-. 2 3 .+-. 0.5 5.8 .+-. 1 Stimulated insulin
secretion values are obtained by subtracting basal from 20 mmol/l
glucose-induced insulin release. Values are mean .+-. SEM of five
(n = 5) islet preparations.
[0092] Stimulated insulin secretion values are obtained by
subtracting basal from 20 mmol/l glucose-induced insulin release.
Values are mean.+-.SEM of five (n=5) islet preparations.
Characterization of the Total Cell Mass
[0093] To exclude the possibility that higher DNA contents in
SOD-treated preparations could be attributed to selective survival
of non-islet tissue, the islets preparations (n=5) were
characterized by immunocytochemistry. The relative proportion of
pro-insulin (beta cells), glucagon (alpha cells), amylase
(exocrine), and CK19 (ductal cells) was determined (FIG. 15A). In
all cases, cell composition was minimally different between
SOD-mimic-treated and control groups. Both groups showed a
reduction in amylase immuno-reactive cells that changed from values
as large as 20% at start, to negligible values after culture.
regardless of the culture treatment (p<0.01). When the
proportion of pro-insulin positive cells (% of beta cells) was
expressed as a fraction of DNA content, used as an indicator of
cell number, the results demonstrate that SOD-mimic treatment
accounts for a minimum 1.3 to a maximum of 4.5 fold increase in
beta cell mass (FIG. 15B). These data demonstrate that treatment of
islets with SOD increases overall beta cell mass without
appreciable dysregulation in islet function.
Early Administration of SOD-mimic Reduces Cell Loss
[0094] The aim of the three additional experiments was to determine
whether administration of SOD- mimic during the digestion might
play a more protective role on cell survival than its addition in
culture after isolation. To avoid the impact of donor variables on
the quality of the isolated islets, the technical approach of
splitting the organs in two parts has been adopted. After infusion
of Liberase.TM. solution with or without SOD-mimic, an arbitrary
incubation time of 30 minutes was maintained in all groups.
Importantly, the presence of SOD-mimic did not appear to affect the
digestion duration. Treated and control preparations for each donor
organ yielded islet fractions of similar purity. FIG. 16
demonstrates the relative variations in DNA content of control and
experimental islet preparations following isolation. The data
indicate that in all three cases, a significant (p=0.0001) increase
in islet cell mass was appreciable 24 hours post isolation in the
SOD-treated organs, and that the difference was maintained over
time. Treated and control islets were tested for viability and
functional capacity in vitro. In both cases more than 80% of viable
cells were counted among the islet preparation samples with no
difference associated with the treatment. The values of insulin
content, basal and stimulated insulin release per islet was also
not different between the two groups. This demonstrates that the
addition of the SOD-mimic during digestion resulted in greater
numbers of functional islets with respect to controls.
Normalization of Diabetic Mice
[0095] Diabetic recipient mice were randomly assigned to receive
islet transplants from control and SOD-mimic treated groups (FIG.
17). The first three recipients of each group received an islet
mass of 700 to 1000 IEQ. All six recipients normalized the glucose
levels within the first week of the post-transplant period. When
the number of transplanted islets was reduced to less than 400 IEQ
per graft, all recipients (n=9) of SOD-treated islets normalized,
while in the control group, three recipients of islet grafts
(respectively composed of 200, 220, and 400 IEQ) never corrected
glucose levels. In all successful transplants, animals maintained
normoglycemia for more than 30 days. In the cases in which the
graft was removed by nephrectomy, all mice return to hyperglycemic
state.
[0096] All documents cited above are hereby incorporated in their
entirety by reference.
* * * * *