Note: Descriptions are shown in the official language in which they were submitted.
CA 02487171 2004-11-24
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METHOD FOR THE PROTECTION OF ENDOTHELIAL AND EPITHELIAL CELLS
DURING CHEMOTHERAPY
Field of the invention
The invention relates to the field use of radiation therapy and/or
chemotherapy. More
specifically, the invention relates to a method for assuaging side effects
associated
with such treatment.
State of the art
Allogeneic stem cell transplantation (SCT) is a well established method for
the treat-
ment of hematological neoplasias and an increasing variety of other malignant
disor-
ders. SCT mainly consists of two sequential steps: The pretransplant
conditioning,
classically consisting of total body irradiation (TBI) and chemotherapy,
leading to
minimal residual disease and the immunosuppression of the recipient as the
first
step, and the transfer of allogeneic stem cells that should finally provide
the cure as
the second step. However, due to disparities in major (MHC) and minor (mHAg)
his-
tocompatibility antigens, severe inflammatory reactions, including acute graft-
versus-
host disease (GvHD), can occur in different phases post transplant. Based on
studies
by the inventors' and several other investigators2~3 it is widely accepted
that condi-
tioning contributes via non-specific inflammation to these transplant-related
compli-
cations (TRC). In addition, a direct toxicity of especially TBl has been demon-
strated 4°5 This has led to a variety of alternative conditioning
regimens currently un-
der investigation. In addition, new pre transplant therapies allow the
extension of
treatment protocols and the patients' selection. One compound of these novel
condi-
tioning concepts is fludarabine, a non-myeloablative immunosuppressant that
had
originally been used for the treatment of chronic lymphatic leukemia.6
Fludarabine in
combination with e.g. BCNU and melphalan, cyclophosphamide or other agents can
replace TBI or is used together with dose reduced TBI regimens.'~$ The
clinical data
obtained so far argue for comparably low side effects and a hematopoetic and
im-
mune cell specificity of fludarabine.9 However, the influence of this compound
on
1
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non-hematopoetic cells like endothelial and epithelial cells has not been
subject to
investigation yet.
Virtually all TRC are associated with endothelial dysfunction and
damage.~° The in-
ventors and others have shown that the endothelium is a target of pre-
transplant
conditioning in vitro and in vivo. Ionizing radiation induces programmed cell
death
(apoptosis) in endothelial cells.~~-'4 At the same time the endothelium is
activated in
terms of adhesion molecule expression leading to increased leukocyte-
endothelial
interactions as a prerequisite for inflammatory processes,~5,~s These effects
are sig-
nificantly enhanced by bacterial endotoxin (lipopolysaccharide, LPS) that
might
translocate through damaged mucosal barriers from the gastrointestinal tract.'
In
addition, LPS has been shown to increase the antigenicity of endothelial cells
to-
wards allogeneic CD8+ cytotoxic T lymphocytes.~$
Clinical results with fludarabine containing reduced intensity conditioning
(RIC) regi-
mens obtained so far show a clear downregulation of conditioning-related
toxicity
without affecting immune reconstitution.25 The incidence of acute GvHD in
patients
receiving RIC is comparable or even less than in those patients receiving the
classi-
cal conditioning regimen.26 However, reports on equally severe or even
increased
late effects like osteonecrosis,2' pulmonal complications,2$ and more cases of
chronic
GvHD29 clearly demonstrate the potential for serious side effects associated
with flu-
darabine treatment.
Summary of the invention
The invention is based on the discovery that fludarabine activates and damages
en-
dothelial and epithelial cells. The activation of the cells leads to damage in
the treat-
ment situation where fludarabine is used, e.g., when treating malignancies
using
SCT. The epithelial and endothelial cells can be protected from this
activation and
damage by treatment with defibrotide. This treatment may be concomitant or
defi-
brotide may be given before treatment with fludarabine or thereafter.
Abbreviations and definitions
SCT: Haematopoetic stem cell transplantation.
2
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Immunosuppressant: substance that down-regulates the immune response of a sub-
ject upon administration. Immunosuppressants are used in suppressing the
immune
system of patients undergoing stem cell therapy. Examples of
immunosuppressants
include fludarabine, cyclophosphamide, BCNU, cyclosporin, sirolimus,
tacrolimus and
melphalan. Preferred within the context of this application is fludarabine
(also known
as 2-fluoro-9-~i-D-arabinofuranosyladenine).
Protective oligodeoxyribonucleotide: shall mean, within the context of this
application,
both oligodeoxyribonucleotides as defined in US patent 5,646,268 and
polydeoxyri-
bonucleotides as defined in US 5,223,609, which are incorporated by reference
herein in their entirety.
US patent 5,646,268 discloses a process for producing an
oligodeoxyribonucleotide
having the following physico-chemical and chemical characteristics:
Molecular weight: 4000-10000
h: < 10
A + TIC + G* 1.100-1.455
A + G/C + T* 0.800 -1.160
Specific rotation : +30° - + 48°
*base molar ratio
h= hyperchromicity parameter
A process for producing such an oligodeoxyribonucleotide comprises:
precipitating 0.8M sodium acetate aqueous solutions of polydeoxyribonucleotide
so-
dium salts at 20° C by addition of an alkyl alcohol selected from the
group consisting
of ethyl, propyl and isopropyl alcohol.
US patent 5,223,609 discloses a defibrotide which fulfills certain
pharmacological
and therapeutical properties and is therefore particularly suitable, if the
nucleotide
fractions forming it are in stoichiometrical agreement with the following
polydeoxyri-
bonucleotidic formula of random sequence:
P1-5, (dAP)~2-~~, (dGp)~o-20, (dTp)~3-26~ (dCp)~o-20
wherein
P=phosphoric radical
3
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dAp=deoxyadenylic monomer
dGp=deoxyguanylic monomer
dTp=deoxythymidylic monomer
dCp=deoxycytidylic monomer
The Defibrotide corresponding to this formula moreover shows the following
chemico-physical properties: electrophoresis=homogeneous anodic mobility;
extinc-
tion coefficient, E ~ cm~~~~ at 260 ~1 nm=220 ~ 10; extinction ratio, E2so
1E26° =0.45 ~
0.04; coefficient of molar extinction (referred to phosphorous), ~(P)=7.750 ~
500; ro-
tatory power [a]p2°~ =53 ° ~ 6; reversible hyperchromicity,
indicated as % in native
DNA, h=15 ~ 5.
A preferred protective oligodeoxyribonucleotide is Defibrotide (CAS Registry
Number:
83712-60-1 ), a polynucleotide well known to the person skilled in the art,
which nor-
mally identifies a polydeoxyribonucleotide obtained by extraction (US
3,770,720 and
US 3,899,481 ) from animal and/or vegetable tissue; this
polydeoxyribonucleotide is
normally used in the form of a salt of an alkali metal, generally sodium, and
usually
has a molecular weight of approximately 45-50 kDa. Defibrotide is used
principally for
its antithrombotic activity (US 3,829,567) although it may be used in
different applica-
tions, such as, for example, the treatment of acute renal insufficiency (US
4,694,134)
and the treatment of acute myocardial ischaemia (US 4,693,995). United States
pat-
ents US 4,985,552 and US 5,223,609 describe a process for the production of
defi-
brotide which enables a product to be obtained which has constant and well
defined
physico-chemical characteristics and is also free from any undesired side-
effects.
Detailed description of the invention
The invention relates to a method for the treatment of a patient undergoing
treatment
with an immunosuppressant, comprising the step of administering an effective
dose
of a protective oligodeoxyribonucleotide to the patient. The treatment with an
immu-
nosuppressant preferably occurs during SCT. The immunosuppressant is
preferably
selected from the group comprising antimetabolites (e.g., 5-fluorouracil (5-
FU),
methotrexate (MTX), fludarabine, anti-microtubule agents (e.g., vincristine,
vinblas-
tine, taxanes (such as paclitaxel and docetaxel)), alkylating agents (e.g.,
cyclophas-
phamide, melphalan, bischloroethylnitrosurea (BCNU)), platinum agents (e.g.,
cis-
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CA 02487171 2004-11-24
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platin (also termed cDDP), carboplatin, oxaliplatin, JM-216, CI-973),
anthracyclines
(e.g., doxorubicin, daunorubicin), antibiotic agents including mitomycin-C,
topoi-
somerase inhibitors (e.g., etoposide, camptothecin), cyclosporin, tacrolimus,
siro-
limus, and other cytotoxic agents that act to suppress the immune system. A
review
of such agents that are frequently used in the therapy of malignancies may be
found
in Gonzales et al., Alergol. Immunol. Clin. 15, 161-181, 2000, which is
incorporated
herein by reference. Preferred immunosuppressants are nucleosides (i.e. the
gly-
cosides resulting from the removal of the phosphate group from a nucleotide),
as for
instance fludarabine which, by the way, is the preferred immunosuppressant for
the
purposes of the present invention.
The protective oligodeoxyribonucleotide may be administered concurrently,
simulta-
neously, or together with the immunosuppressant. A preferred combination is
the si-
multaneous gavage of defibrotide and fludarabine.
The step of administering the protective oligodeoxyribonucleotide preferably
occurs
concurrently, concomitantly, simultaneously, after or before the gavage of the
immu-
nosuppressant to the~patient.
In a preferred embodiment of the invention, the step of administering the
protective
oligodeoxyribonucleotide occurs after gavage of the immunosuppressant to the
pa-
tient. In a further preferred embodiment, the time delay between step of
administering
the protective and the gavage of the immunosuppressant to the patient is about
one
hour to about two weeks. The time delay between the step of administering the
pro-
tective and the gavage of the immunosuppressant to the patient is preferably
about
two days to about seven days.
In another preferred embodiment of the invention, the step of administering
the pro-
tective oligodeoxyribonucleotide occurs before gavage of the immunosuppressant
to
the patient. Preferably, the time difference between step of administering the
protec-
tive and the gavage of the immunosuppressant to the patient is about one hour
to
about two weeks. More preferably, the time difference between step of
administering
the protective and the gavage of the immunosuppressant to the patient is about
two
hours to about two days.
s
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The preferred protective oligodeoxyribonuc~eotide is defibrotide, however,
other sub-
stances as mentioned above as protective oligonucleotides may be used. The fol-
lowing embodiments define preferred doses for defibrotide; however, similar
doses
may be used when using a protective oligodeoxyribonucleotide which is not defi-
brotide. The optimal dose for any protective oligodeoxyribonucleotide will be
deter-
mined by the attending physician. The experiments described below show the pro-
tective effects of defibrotide. The effective dose determined in such
experiments may
be used as a guide for determining an effective dose for treatment.
The defibrotide is preferably administered orally or is injected
intravenously.
The preferred dose of of defibrotide is chosen so as to reach a blood level of
about
100 pg/mL to 0.1 pg/mL. More preferably, the dose of defibrotide is chosen so
as to
reach a blood level of about 10 ~g/mL to about 100 pg/mL. Most preferably, the
dose
of defibrotide is chosen so as to reach a blood level of about 100 pg/mL.
In a preferred embodiment of the invention, the dose of defibrotide
administered is
about 100 mg/kg body weight of the patient to about 0.01 mglkg body weight.
Pref-
erably, the dose of defibrotide administered is about 20 mg/kg body weight of
the pa-
tient to about 0.1 mg/kg body weight. More preferably, the dose of defibrotide
ad-
ministered is about 15 mg/kg body weight of the patient to about 1 mg/kg body
weight. More preferably, a daily dosage of about 12 mg to about 14 mg per Kg.
of
body weight of the patient is administered. Most preferably, the dose of
defibrotide
administered is about 12 mg/kg body weight of the patient.
Preferably, administration of a protective oligodeoxyribonucleotide according
to the
invention according to the invention is able to protect endothelial cells and
epithelial
cells from the effects of the immunosuppressant. The immunosuppressant
preferably
activates epithelial cells and endothelial cells and induces apoptosis
therein. Thus, in
a preferred embodiment, the protecting olideoxynucleotide protects epithelial
and/or
endothelial cells from apoptosis and/or activation by the immunosuppressant.
The
immunosuppressant is preferably fludarabine. The protective
oligodeoxyribonucleo-
tide is preferably defibrotide.
The activation includes enhanced expression of ICAM-1 and of MHC class I mole-
cules. The enhancement of expression is preferably substantial. Further
preferably,
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the immunosuppressant induces a pro-inflammatory activation of endothelial
cells
andlor of epithelial cells in a patient. The cells are preferably human
microvascular
endothelial cells (HMEC) and/or dermal and/or alveolar epithelial cells. The
damage
preferably occurs when the patient's endothelial and/or epithelial cells have
been ex-
posed to the immunosuppressant for about 1 hour to about 1 week or more. More
preferably, said damage occurs when said cells have been exposed for about 5
hours to about 72 hours. Even more preferably, the duration of such exposure
is
between 20 hours and 72 hours. Most preferably, the duration of such exposure
is
more than 48 hours.
The treatment with the immunosupressant preferably occurs during haematopoetic
stem cell transplantation. The haematopoetic stem cell transplantation is
preferably
allogeneic haematopoetic stem cell transplantation.
The invention also relates to a pharmaceutical composition comprising at least
a
protective oligodeoxynucleotide, for the treatment ~of a patient in need
thereof, which
patient is being treated with an immunosuppressant. The administration of said
pharmaceutical composition alleviates or protects from side effects caused by
the
immunosuppressant or by the immunosuppressant and a transplant. The transplant
is preferably a bone marrow or haematopoetic stem cell transplant. More
preferably,
the transplant is an allogeneic bone marrow or haematopoetic stem cell
transplant.
The side effects are preferably related to endothial and/or epithelial cells
and/or tis-
sues of the patient. Preferably, said side effects involve apoptosis of said
cells,
andlor activation of said cells. The activation preferably comprises enhanced
expres-
sion of MHC class I molecules and/or of intercellular adhesion molecule 1
(/CAM-1 ).
The side effects damages human microvascular endothelial cells (HMEC) as well
as,
preferably, dermal and alveolar epithelial cell lines after 48 hours of
culture, when
used in pharmacologically relevant concentrations (range: 10 pg/mL to 1
pg/mL).
The side effects generally include damages to target tissues of transplant
related
complications and stimulated allogeneic immune responses.
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The side effects preferably include significant upregulation of the
intercellular adhe-
sion molecule 1 (ICAM-1 ) and MHC class I molecules in endothelial cells
andlor pei-
thelial cells of the patient, particularly in alveolar endothelial cells. The
side effects
further include a a pro-inflammatory activation of microvascular endothelial
cells. The
side effects further preferably include enhanced lysis of such cells by by
allogeneic
MHC class I restricted cytotoxic T lymphocytes derived from the transplant.
Adminstration of the protective oligodeoxynucleotide preferably protects
against im-
munosuppressant-induced side effects, including apoptosis and alloactivation.
The pharmaceutical compositions comprising the immunosuppressant of the
present
invention can be formulated with techniques, excipients and vehicles of
conventional
and well known type, for the administration both orally and by injection,
particularly by
intravenous route. The dosages of active ingredient in the compositions
according to
the present invention ranges between 50 and 1500 mg for unitary dose, whereas
to
attain the desired results the daily administration of 10 to 40 mglkg is
suggested.
Methods for the preparation defibrotide may be found in US 4,985,552 and US
5,223,609, which patents are incorporated hereby in their entirety by
reference.
The invention also relates to a pharmaceutical composition containing a
therapeuti-
cally effective dose of an immunosuppressant and of a protective
oligodeoxyribonu-
cleotide. The immunosuppressant is preferably fludarabine. protective
oligodeoxyri-
bonucleotide is preferably defibrotide.
Brief description of the Figures
Fig 1: Fludarabine induces programmed cell death in human micorvascular en-
dothelial cells (HMEC). HMEC were either left untreated or incubated with 2-
fluoro-
9-(3-D-arabinofuranosyladenine (hereinafter referred to as F-Ara, the
metabolized
form of fludarabine) in descending concentrations for 48 hours and subjected
to flow
cytometric analysis (A) or microscopic DAPI stain analysis. A: Contour plots
of the
side scatter (SSC) image (x-axis) of propidium iodide (PI)-negative cells
plotted
against the forward scatter image (y-axis) as a parameter for cellular
granularity ver-
sus cell size. B: Quantitative fluorescence microscopy analysis of DAPI-
stained en-
dothelial cells. Results are given in % apoptotic HMEC (% apoptotic cells) ~
standard
8
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WO 03/101468 PCT/EP03/05753
deviation (out of n= 10 microscopic fields with an average of 70 cells per
field). Rep-
resentatives of at least five independent experiments are shown. *: p<0.001 of
un-
treated control versus F-Ara (10 pglmL) treated cells.
Fig 2: Defibrotide (D) inhibits F-Ara-induced apoptosis in HMEC, evidence for
an intracellular antagonism. F-Ara: 10 pg/mL. D: 100 pg/mL. Flow cytometric
analysis of the SSC-image of PI-negative cells. A: reproducible induction of
apoptosis
by F-Ara. B: Dose-dependent inhibition of F-Ara-induced apoptosis by D. C:
left plot:
incubation of HMEC with F-Ara for 1 hour, subsequent incubation with D for 48
hours
after washing. Right plot: incubation of HMEC with D for 1 hour, subsequent
incuba-
tion with F-Ara for 48 hours after washing. For experimental details see
legend to Fig
1 and Materials and Methods. Shown is one representative out of three
independent
experiments.
Fig 3: F-Ara induces apoptosis in keratinocytes and alveolar epithelial cells,
but not in gut or bronchial epithelial cells; protective effect of
Defibrotide. F-
Ara: 10 pg/mL. D: 100 pg/mL. Flow cytometric analysis of the SSC-image of PI-
negative cells (Fig 3 A) and DAPI-stain analysis of apoptotic cells (Fig 3 B).
Results
are given in mean % apoptotic cells ~ standard deviation out of three
different ex-
periments. HaCaT: human keratinocyte cell line. SW 480: gut epithelial cells
line. A
549: lung carcinoma cell line from the alveolar epithelium. SEAS-2B: bronchial
epithelial cell line. Primary bronchial epithelial cells have been derived
from a bron-
choscopic brush procedure. Fig 3 A: *: p=0.005 of F-Ara- versus F-Ara+D
treated
HaCaT cells. **: p=0.116 of F-Ara versus F-Ara+D treated A 549 cells. 0 : no
apopto-
sis induction. Fig 3 B: +: p=0.026 of F-Ara- versus F-Ara+D treated HaCaT
cells. ++;
p=0.001 of F-Ara versus F-Ara+D treated A 549 cells. For experimental details
see
legend to Fig 1 and Materials and Methods. Three representative experiments
are
summarized for each cell line.
Fig 4: Defibrotide (D) does not interfere with the anti-leukaemic and the anti-
PBMC effect of F-Ara. F-Ara: 10 pglmL. D: 100 pg/mL. A: Propidium iodide
staining
of primary acute myeloid leukemia (AML) cells derived from a patient in blast
crisis
(70 % blasts of total PBMC count). Results are given in mean % vitality of
three inde-
pendent experiments. *: p=0.008 of Control- versus F-Ara-treated AML cells. B:
Flow
9
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WO 03/101468 PCT/EP03/05753
cytometric analysis of the SSC-image of PI-negative PBMC. Shown is one repre-
sentative out of five independent experiments with different blood donors.
Fig 5: F-Ara induces ICAM-1 expression on HMEC, protective effect of Defi-
brotide (D). Flow cytometric analysis of ICAM-1 positive cells. HMEC were
either left
untreated or incubated with F-Ara (10 pg/mL, or descending concentrations in
B) in
the presence or absence of descending concentrations of D. A: Histogram plot
of
ICAM-1 expression from a representative experiment. Dotted line: Background
staining (nil control); thin line: ICAM-1 expression of untreated control
cells; thick line:
ICAM-1 expression of F-Ara-treated cells. B: Dose-dependent induction of ICAM-
1
expression by F-Ara. Summary of three independent experiments. Results are
given
as mean % ICAM-1 positive cells ~ standard deviation. *: p=0.075 of F-Ara-
versus
untreated control cells. C: Dose-dependent inhibition of F-Ara-induced ICAM-1
ex-
pression by Defibrotide (D). Results are given as mean % ICAM-1 positive cells
~
standard deviation. **: p=0.004 of F-Ara- versus F-Ara+D-treated HMEC.
Fig 6: F-Ara increases the allogenicity of HMEC for CD8-positive cytotoxic T-
lymphocytes (CTL), protective effect of Defibrotide. A: PBMC were stimulated
with irradiated HMEC in the presence of interleukin 2 (50 U/mL) for 7 days and
sub-
jected to a 5~Cr release assay with untreated (Control) and F-Ara (10 pg/mL)-
treated
HMEC (24 hour-incubation) as target cells. autolog. B-LCL: autologous
(effector)
EBV-transformed B-lymphoblastoid cells. K 562: target cells for natural killer
(NK)
cells. Results are given as % specific lysis as described in Materials and
Methods.
* ~ % specific lysis of F-Ara-treated HMEC fn the presence of anti-MHC class I
an-
tibody w6/32. E/T ratio: effector/target ratio. B: Downregulation of F-Ara-
induced allo-
genicity of HMEC towards CD8-positive CTL by Defibrotide (D). CD8-positive
PBMC
have been negatively selected (non-CD8+-cell-depleted) by magnetic bead separa-
tion. For experimental details see legend to Fig 6 A.
Fig 7: F-Ara decreases the allogenicity of HMEC for NK cells, enhancement of
lysis by blockade of MHC class I. NK cells have been negatively selected (non-
NK-
cell-depleted) by magnetic bead separation and stimulated with irradiated HMEC
in
the presence of IL-2 (50 U/mL) for 4 days and subsequently subjected to a 5'Cr
re-
lease assay as described for Fig 6. Table below the graph: Flow cytometric
analysis
to
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WO 03/101468 PCT/EP03/05753
of the effector cell population pre and post stimulation with HMEC. NK cells
were
characterized as CD3-/CD16+CD56+. * ~ % specific lysis of K 562 cells at E/T
ratio
of 20:1.
Table 1: Anti-endothelial CTL elicit a T~1-like phenotype.
Effector IFN~y ~ IL-4
PBMC 319 [176] 0
CD8+ 524 [174] 0
ELISA for the production of interferon gamma (IFN~y) and interleukin 4 (IL-4)
in the
supernatants of stimulated effector cells (7 days, irradiated HMEC, 50 UImL IL-
2).
PBMC were either left unseparated or negatively selected for CD8+ T cells as
given
for the experiments in Fig 6. Results are given as mean pg/mL cytokine ~
standard
deviation of 3 independent experiments.
Examples
METHODS
Cell culture and reagents
The human dermal microvascular endothelial cell line CDC/EU.-HMEC-1 (further
re-
ferred to as HMEC) was kindly provided by the centres for Disease Control and
Pre-
vention (Atlanta, Georgia, USA) and has been established as previously
described.'9
HMEC were cultured in MCDB131 medium, supplemented with 15% fetal calf serum
(FCS), 1 pg/mL hydrocortisone (Sigma, Deisenhofen, Germany), 10 nglmL
epidermal
growth factor (Collaborative Biochemical Products, Bedford, MA, USA) and
antibiot-
ics. All cell culture reagents have been purchased by Gibco BRL (Karlsruhe,
Ger-
many) unless stated otherwise. 2-Fluoroadenine 9-beta-D-arabinofuranoside (F-
Ara)
was obtained from Sigma, Deisenhofen, Germany, Defibrotide vials were obtained
from ProciclideTM, Crinos, Como, Italy.
Apoptosis assays
An established method for detecting apoptosis in human endothelial cells was
per-
formed as previously described2° using flow cytometry (FACScanTM and
CeIIQuestTM
software, Becton Dickinson, Heidelberg, Germany). Endothelial and epithelial
cells
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were either left untreated or incubated with F-Ara in descending
concentrations
(range: 10pg/mL to 0.1 pg/mL) in the presence or absence of Defibrotide for 48
hours. Afterwards, cells were washed in PBS/10% FCS and stained with the
necrosis
detecting dye propidium iodide (PI, 0.2 pg/mL, Sigma, Deisenhofen, Germany).
Apoptotic cells were identified by a PI-negative staining and by a
characteristic side
scatter image distinct from that of non-apoptotic cells. At least three
experiments per
cell type have been performed.
An alternative method for the detection of apoptosis used microscopic analysis
of
DNA fluorescence labeled cells. 1 x105/plate endothelial cells were seeded in
35mm
petri dishes (Nunc, Wiesbaden, Germany). These cells were treated as given
above
and subsequently fixed with Methanol/Acetone (1:1 ) for 2 minutes, washed once
in
PBS and stained with 4,6-Diamidino-2-phenylindole (DAPI) (0.5 pg/mL, Sigma, De-
isenhofen, Germany), dissolved in 20% Glycerin/PBS. Samples were mounted and
subjected to microscopic analysis. Nuclear condensation as revealed by DAPI
stain-
ing in the absence of trypan blue uptake is considered characteristic of
apoptosis as
opposed to necrosis.2~ The quantitative analysis included counting the number
of
apoptotic relative to all identifiable cells from at least 10 microscopic
fields, with an
average of 70 cells per field.
For the sake of the clarity of the manuscript DAPI stain results are only
displayed for
the experiments with endothelial cells and HaCaT as well as A 549 cells.
Cel! surface analyses
Cell surface expression of ICAM-1 (Becton Dickinson/Pharmingen, Heidelberg,
Ger-
many) and MHC class I (w6/32, hybridoma supernatant, ATCC, Manassas, VA, IJSA)
molecules on HMEC was assessed by the indirect immunofluorescence technique
and subsequent flow cytometry using the FACScanT"" flow cytometer and the Cell-
QuestTM analysis program (Becton Dickinson, Heidelberg, Germany). Endothelial
cells were treated as given and after incubation harvested with trypsin/EDTA
(Gibco),
washed once in cold PBS/ 10% FCS and incubated 1 hour on ice with 5 pg/mL of
anti-adhesion molecule MoAbs. Cells were washed again and incubated with a
goat
anti-mouse IgG-FITC conjugated antibody F(ab)2 fragment (Dako, Hamburg, Ger-
many) for 45 minutes on ice. Cells were then washed in PBS/ 10% FCS and sub-
jected to analysis. Viability of the cells was determined by concurrent
propidium io-
dide (0.2 pglmL, Sigma, Deisenhofen, Germany) staining. Omitting of the first
anti-
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WO 03/101468 PCT/EP03/05753
body served as a negative control to detect unspecific fluorescence. This
approach,
instead of using isotype control antibodies, was justified by previous
observations
that endothelial cells lack Fc receptors. Therefore, a non-specific binding of
anti-
bodies through their Fc portion could be excluded.
Allostimulation of peripheral blood cells with HMEC
Peripheral blood mononuclear cells (PBMC) were derived from heparinized (Novo
Nordisk, Mainz, Germany) blood of healthy human volunteers or buffy coats from
the
Bavarian Red Cross according to a standard protocol using Ficoll hypaque
(Pharma-
cia, Freiburg, Germany) density gradient centrifugation. Cells were then
stimulated in
a ratio of 1:1 and 2:1 with irradiated (20 Gy) HMEC for 7 days in the presence
of In-
terleukin 2 (50 U/mL) and 10% human AB serum (Sigma, Deisenhofen, Germany).
Alternatively, PBMC were selected for CD8+ T cells and natural killer (NK)
cells using
cell isolation kits according to the manufactuer's instructions (MACSTM,
Miltenyi Bio-
tech, Bergisch-Gladbach, Germany) based on the deletion of non CD8+ and non NK
cells, respectively. Stimulation of the selected cells was identical to that
of whole
PBMC cultures, except for NK cells which were stimulated for only 3 days.
Cytotoxicity assa y
T cell- or NK-cell mediated cytotoxicity was assessed according to a well
established
protocol,23 using a 4h 5'Cr radioisotope assay. HMEC that had either been left
un-
treated or incubated with F-Ara (10pg/mL) overnight were used as target cells,
to be
labeled 0.4 mCi Na25'Cr04 for 2 hours. After 3 washing steps, target cells
were ad-
justed to 104 ceIIs/mL and coincubated with PBMC, CD8+ or NK effector cells at
de-
scending effector to target ratios for another 4 hours. Supernatants were
transfered .
to dry scintillation plates and counted in a y-counter (all from Canberra
Packard,
Darmstadt, Germany). Autologous (effector) B-Lymphoblastoid cell lines (B-LCL)
and
K562 as NK sensitive cells were taken as additional control targets. The
percentage
of specific lysis was calculated as: [(experimental release - spontaneous re-
lease)/(maximal release - spontaneous release)] x 100. Spontaneous release in
all
experiments was always below 20%.
Enzyme linked immunosorbent assays (EL1SA)
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The ELISA for the detection of Interleukin 4 (IL-4, T~2 response) and
Interferon y
(IFN-y, T~1 response), IL-1 and IL-10 in the supernatants of allogeneic
effector T cells
(see below) were performed exactly according to the manufacturer°s kit
instructions
(R&D Systems, Minneapolis, MN, USA).
Statistical analysis
The significance of differences between experimental values was assessed by
means of the Student's t-test.
Example 1
F-Ara induces apoptosis (programmed cell death) in human microvascular en-
dothelial cells (HMEC)
In order to assess the influence of F-Ara on the viability of cultured human
endothe-
lial cells, HMEC were incubated with descending pharmacologically relevant
concen-
trations (10 pg/mL to 0.1 pg/mL) of 2-Fluoroadenine 9-beta-D-arabinofuranoside
as
the metabolized form of fludarabine. The median intracellular level of the
active (cy-
totoxic) fludarabine triphosphate in target cells is 20 pM, representing a
concentration
5.8 pg/mL (medac SCHERING, manufacturers°s instructions). After 48
hours of incu-
bation HMEC were subjected to apoptosis assays using the detection of cellular
granularity of propidium iodide negative cells (side scatter (SSC) image in
flow cy-
tometry) and microscopic analyses of DAPI-stained cells, respectively.
Independent
of the assays system, Fig 1 A and B clearly demonstrate that F-Ara causes
apoptosis
in HMEC in concentrations of 10 and 5 pg/mL, whereas 1 pg/mL was no longer ef-
fective. The critical threshold of the cytotoxicity of F-Ara was between 2 and
3 pg/mL.
Apoptosis by F-Ara was already detectable after 24h, though to a lesser extent
(data
not shown).
Example 2
Defibrotide protects HMEC from the F-Ara induced apoptosis
HMEC had either been left untreated or treated with F-Ara in the presence or
ab-
sence of varying concentrations of Defibrotide (100 pg/mL to 0.1 pg/mL) and as-
sessed for programmed cell death after 48 hours using flow cytometric analyses
of
the SSC image as described for Fig 1 A. Fig 2 A (mid contour plot) shows that
Defi-
brotide alone as a second control did not influence endothelial cell
viability. The
14
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apoptotic effect of F-Ara is reproduced in Fig 2 A (right contour plot),
whereas Fig 2 B
shows a dose-dependent protection of F-Ara induced cell death by Defibrotide.
In or-
der to exclude unspecific artifical extracellular interaction of F-Ara and
Defibrotide in
vitro HMEC were pretreated with Defibrotide for 1 hour and subsequently, after
3
washing steps, incubated with F-Ara for another 48 hours and vice versa. Fig 2
C
(right contour plot) reveales that pretreatment of HMEC for 1 hour was
sufficient to
protect cells from F-Ara induced apoptosis. Similarly, pretreatment of HMEC
with F-
Ara for 1 hour (Fig 2 C, left contour plot) and subsequent incubation with
Defibrotide
did not lead to endothelial programmed cell death.
Example 3
Effect of F-Ara on different epithelial cell lines, protective effect of
Defibrotide
Skin, the gastrointestinal tract (GIT) and most likely the lung are among the
primary
targets of GvHD. Therefore, it was reasonable to test the influence of F-Ara
on cell
lines derived from these organs. Cells from keratinocyte (HaCaT), GIT (SW
480),
alveolar (A549) and bronchial epithelial (BEAS-2B) cell lines as well as
primary bron-
chial epithelial cells were incubated with F-Ara (10 pg/mL) as given for Figs
1 and 2
and assayed in flow cytometric apoptosis analyses 48 hours post treatment. Fig
3 A
summarizes that gut and bronchial epithelial cells appeared to be resistant to
the
apoptotic stimuli of F-Ara, whereas keratinocytes (HaCaT) and alveolar
epithelial
cells (A549) showed signs of apoptosis, as determined by flow cytometry of the
SSC
image (34.0 [~1.0] % apoptotic cells for HaCaT and 42.9 [~26.7] % for A549,
respec-
tively). Again, the protective potential of Defibrotide (100 pg/mL) was
assessed. Ha-
CaT (4.3 [~3.0] %) and A 549 (5.4 [~2.9] %) cells were completely protected
from
programmed cell death after cotreatment with F-Ara and Defibrotide Fig. 3 A,
in-
serted bar graphs). To confirm these results, DAPI-stain apoptosis assays were
per-
formed for HaCAT (Fig 3 B, left columns) and A 549 cells (Fig. 3 B, right
columns).
As shown for endothelial cells, Defibrotide alone did not influence the number
of
apoptotic cells in either cell line (data not shown).
Example 4
Defibrotide does not interfere with the anti-leukaemic and anti-PBMC effect of
F-Ara
is
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Next to its desirable protective capacity for endothelial and epithelial cells
against F-
Ara induced apoptosis it was important.to investigate whether Defibrotide
would also
interfere with the anti-leukaemic properties of F-Ara. To address this
question, pri-
mary peripheral blood derived acute myeloid leukaemic (AML) cells with a blast
amount of 70 % were thawed, kept in culture for 24 hours and subsequently
treated
with F-Ara in the presence or absence of Defibrotide for another 48 hours. Fig
4 A
demonstrates that already almost 50 % of the cells died spontaneously of
necrotic
cell death. However, F-Ara induced cell death in up to 80 % of the cells. In
contrast to
its effect on endothelial and epithelial cells, Defibrotide was not able to
protect the
AML cells from the F-Ara mediated toxicity. It is of note that Fig 4 A
describes % vi-
tality of the cells, not % apoptotic cells, due to the fact that F-Ara
directly caused ne-
crosis, rather than apoptosis in AML cells. This could be observed after as
early as
24 hours of incubation. Still, Fig 4 A clearly shows that Defibrotide does not
interfere
with the desirable toxicity of F-Ara against leukaemic cells. We next asked
whether
Defibrotide might modulate the effect of F-Ara against normal haematopoetic
cells
and performed apoptosis assays (SSC-image) with PBMC from normal human blood
donors. As could be learned from a representative experiment depicted in Fig 4
B, F-
Ara induced apoptosis in 40.1 % of the cells as compared to 5.1 % apoptotic
cells in
the untreated control. Again, Defibrotide did not interfere with the apoptotic
activity of
F-Ara against PBMC (43.1 % apoptotic cells), suggesting that the
immunosuppress-
sive properties of F-Ara are not harmed by cotreatment with Defibrotide.
Example 5
F-Ara upregulates intercellular adhesion molecule 1 (ICAM-1) on HMEC with an-
tagonistic effects of Defibrotide
Based on previous observations that pretransplant conditioning not only
damages,
but also leads to proinflammatory activation of endothelial cells in terms of
adhesion
molecule induction,'5 we next investigated the expression of ICAM-1 under the
influ-
ence of F-Ara. As depicted in Fig 5 A and B, flow cytometric analyses
demonstrated
that F-Ara, after 24 hours of incubation, significantly enhances expression on
HMEC
in a dose-dependent manner similar to that observed for apoptosis induction.
Con-
centrations down to 1 pg/mL of F-Ara were effective in inducing ICAM-1. We
next
asked whether Defibrotide would also be functional as an antagonist of F-Ara
in this
experimental setting. HMEC were treated with F-Ara as given and incubated in
the
16
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WO 03/101468 PCT/EP03/05753
presence or absence of descending concentrations of Defibrotide. Fig 5 C summa-
rizes 3 independent experiments showing that Defibrotide in fact antagonized
the F-
Ara induced ICAM-1 expression in concentrations of 100 pg/mL and 10 pg/mL. It
is of
note that Defibrotide alone did not activate endothelial cells, the ICAM-1
expresssion
remained unchanged with every concentration tested (data not shown).
Since a proinflammatory activation of target cells is often associated with
increased
expression of major histocompatibility antigens (MHC) class I and II, we did
further
flow cytometric analyses for these antigens after incubation with F-Ara in
various
concentrations for 24 hours. Despite its well described immunosuppressive
proper-
ties, F-Ara surprisingly induced MHC class I molecules on HMEC dose-
dependently
(1.5 fold induction of mean fluorescence intensity at 10 pg/mL, 1.3 fold
induction at 5
pg/mL), whereas MHC class II remained unchanged (data not shown).
Example 6
F-Ara increases the antigenicity of endothelial cells towards allogeneic
periph-
eral blood cells, protection by Defibrotide
The induction of MHC class I molecules on HMEC by F-Ara prompted us to examine
whether F-Ara would also enhance the capacity of HMEC to stimulate
allocytotoxic
responses. Peripheral blood mononuclear cells (PBMC) as effectors were either
de-
rived from heparinized blood of healthy human volunteers of from buffy coat
prepara-
tions, stimulated with irradiated (20 Gy) HMEC in the presence of 50 UImL
interleukin
2 (IL-2) for 7 days and subsequently subjected to a standard S~Cr release
assay (for
details see Materials and Methods). At day -1 fresh HMEC as targets were
either left
unstimulated or incubated with F-Ara (10pg/mL) in the presence or absence of
an
anti-MHC class I neutralizing antibody (w6/32). Autologous effector Epstein-
Barr vi-
rus transformed B-lymphoblastoid cell lines (B-LCL) and K562 cells as
classical natu-
ral killer (NK) cell targets served as controls. Fig 6 A demonstrates that F-
Ara indeed
increased the antigenicity of HMEC towards allogeneic PBMC at all E/T ratios
tested.
The lack of specific lysis of K 562 and autologous effector B-LCL verified the
in-
volvement of MHC restricted cytotoxic T lymphocytes (CTL). In addition, lysis
of ei-
ther untreated or F-Ara treated HMEC could almost fully be blocked after
coincuba-
tion of these cells with the anti-MHC class I antibody w6/32 (Fig 6 A, * ). To
further
confirm that CD8+ CTL were responsible for the anti-endothelial cytotoxic
activity,
PBMC were selected for CD8+ and CD4+ T cells (non-CD8 and non-CD4-depleted
17
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PBMC, respectively) using magnetic bead separation with MACS' M bead kits.
Purity
of the preparations was > 93% in all cases with a complete absence of the
other cell
population (not shown). Separated T cells were stimulated with HMEC and IL-2
ex-
actly as described for unselected PBMC (see above). As shown in Fig 6 B, lysis
of F-
Ara-treated HMEC by CD8+ CTL was , again, significantly higher than that of
control
HMEC. Furthermore, pretreatment of target HMEC with F-Ara and Defibrotide (F-
Ara+D) downregulated specific lysis even below control levels, suggesting that
Defi-
brotide also protects endothelial cells against the lysis of allogeneic
effector lympho-
cytes. HMEC stimulated CD4+ T cells did not show any signs of cytotoxic
activity in
this experimental setting (data not shown). Flow cytometric analyses of F-Ara
versus
F-Ara+D treated HMEC resulted in a significant downregulation of MHC class I
mole-
cules by Defibrotide, suggesting that MHC class I expression is the critical
element in
regulating the cytotoxic response induced by F-Ara (data not shown).
Example 7
Anti-endothelial CTL display an T~1-like phenotype
To gain information about the nature of the anti-endothelial CTL, PBMC and
CD8+ T
cells were stimulated as given above, and supernatants were collected for the
as-
sessment of interferon gamma (IFN-~y) and interleukin 4 (IL-4) using ELISA
analyses.
As depicted in Tab 1, stimulation with HMEC and IL-2 obviously led to the
outgrowth
of T~1-like T cells as could be told from the unique expression of IFNjy,
whereas no
IL-4 was produced.
Example 8
F-Ara downregulates lysis of HMEC by allogeneic NK cells
Another interesting question was how F-Ara induced modulations of the MHC
class I
expression affects the cytolytic response of natural killer (NK) cells against
endothe-
lial cells. PBMC from healthy individuals were negatively selected for NK
cells (non-
NK cell depleted) and stimulated for 4 days with irradiated HMEC in the
presence of
IL-2, as it was described for the experiment in Fig 6B. At day 4, HMEC as
target cells
have either been left untreated or incubated with F-Ara (10 pg/mL) for 24
hours and
subjected to a standard 5'Cr release assay with the stimulated NK cells as
effectors.
Fig 7 demonstrates that F-Ara significantly downregulated the allogenicity of
HMEC
towards NK cells. As a positive control for NK cell activity, lysis of MHC
class I nega-
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CA 02487171 2004-11-24
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tive K 562 cells could be observed (Fig 7, *-). Pretreatment of F-Ara
stimulated
HMEC with the anti-MHC class I antibody w6/32 completely abrogated the effect
of
F-Ara and led to almost 100 % specific lysis of HMEC (Fig 7), suggesting that
MHC
class I on the surface of HMEC is, again, the critical switch for the
regulation of the
cytotoxic response of NK cells. The role of killer cell inhibitory receptors
(KIR) that
have been found to be negatively regulated by high expression levels of MHC
class I
molecules24 might be responsible for the the decreased cytolytic response of
NK
cells.
DISCUSSION
Clinical results with fludarabine containing reduced intensity conditioning
(RIC) regi-
mens obtained so far show a clear downregulation of conditioning-related
toxicity
without affecting immune reconstitution.~5 The incidence of acute GvHD in
patients
receiving RIC is comparable or even less than in those patients receiving the
classi-
cal conditioning regimen.26 However, reports on equally severe or even
increased
late effects like osteonecrosis,2' pulmonal complications,2$ and more cases of
chronic
GvHD arise.29 Despite its well documented immunosuppressive properties fludara-
bine, in our study, has turned out to activate and damage endothelial and
epithelial
cells. This observation might, at least in part, explain the undesired
clinical side ef-
fects described above, since osteonecrosis is an expression of endothelial
dysfunc-
tion, and fludarabine appears to be toxic for alveolar epithelial cells. It is
interesting to
note that the harmful effects of fludarabine on lung cells seem to be
compartment-
specific, as bronchial epithelial cells did not undergo apoptosis in response
to this
immunosuppressant. The fact that a keratinocyte cell line (HaCaT) was also
sensitive
to fludarabine suggest that it might also be involved in cutaneous disorders
post
SCT. As the pathogenesis of late complications is multifactorial and might
also be in-
fluenced by increasing age of the SCT patients and the use of peripheral stem
cells
further evaluation in clinical analyses of pulmonal and dermatological
complications
is needed.
Since in many pre-transplant protocols fludarabine is used in combination with
ioniz-
ing radiation it was important to test whether these two compounds would
cooperate
in affecting endothelial cells. Interestingly, we could not find any
enhancement of ra-
diation induced cell death by fludarabine or vice versa (data not shown). This
sug-
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gests differential mechanisms of how the apoptotic death signal is transfered
to en-
dothelial cells.
The precise mechanism how fludarabine induces apoptosis in endothelial and
epithelial cells remains to be elucidated. It is likely that fludarabine - as
a purin ana-
logue -integrates into the DNA and thus causes mutations that lead to gene
deletion
like reported previously.3° It has also been suggested that fludarabine
can cooperate
with cytochrome c and apoptosis protein-activating factor-1 (APAF-1 ) in
triggering the
apoptotic caspase pathway.3~
Fludarabine increases the allogenicity of endothelial cell targets for CD8+ T
cells. In
contrast, Fludarabine significantly downmodulates the endothelial lysis by
allogeneic
NK cells. The MHC class I expression seems to be critical for the regulation
of any of
these immune responses, since a blockade of class I fully abrogated CTL lysis
and
tremendously upregulated lysis by NK cells. These opposing effects of
fludarabine,
taken together with the clinical observation that fludarabine shows less acute
and
equal or even more chronic toxicity than the classical conditioning regimen
raises the
speculation that NK cells and CTL might be active in different phases of GvHD
pathophysiology, i.e. NK cells would primarily act in the earlier (suppressed
by fluda-
rabine), and CTL in the later phase (enhanced by fludarabine) post transplant.
With regard to the nature of the anti-endothelial CTL it is an interesting
question
whether these CTL are endothelial- or simply alto-specific. The existence of
endothe-
lial-specific effector lymphocytes has been described previously.32 In
contrast to the
CTL we characterized as displaying a Tc1-like phenotype, many of the CTL
clones
reported show little, if any, IFN-y and unusually express CD40 ligand at rest
what
might enhance cytolytic activity.33 But these data do not rule out the
existence of ad-
ditional allogeneic CTL with a specificity for non-hematopoetic targets.
Defibrotide is a well tolerated drug successfully used for the treatment of
veno-
occlusive disease as one major hepatic complication post SCT.~ In addition,
there is
an increasing number of pre-clinical and clinical reports showing its efficacy
in treat-
ing ischemia/reperfusion injury and atherosclerosis, as well as recurrent
thrombotic
thrombocytopenic purpura.3s-3~ pefibrotide is known to act directly on
endothelial
cells without further metabolism required3$ and could, therefore, be used in
our in vi-
tro studies. Defibrotide fully protected endothelial and epithelial cells from
fludarabine
mediated apoptosis. Additional experimentation is needed to assess the precise
mechanism of protection by which Defibrotide antagonizes fludarabine, but one
can
Zo
CA 02487171 2004-11-24
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imagine a role for Defibrotide in an inhibition of DNA integration of
fludarabine or the
aforementioned caspase activation. Besides its anti-apoptotic effects,
Defibrotide
was able to downregulate anti-endothelial CTL responses by regulating MHC
class I
expression. In contrast, Defibrotide did not affect the desirable anti-
leukemic effect of
fludarabine, as shown by the lack of protection of AML cells. Another
important ob-
servation was that Defibrotide could not block the fludarabine-mediated
apoptosis of
PBMC. This suggests that the immunosuppressant effect of fludarabine mandatory
for conditioning is not influenced by a co-treatment with defibrotide.
It is of note that Defibrotide was not protective against radiation induced
endothelial
cell damage, suggesting its effect to be specific for fludarabine mediated
cellular
changes (data not shown).
Based on these results and with respect to its little, if any, side effects,39
we conclude
from our study that Defibrotide is a good candidate used in combination with
fludara-
bine during conditioning prior to SCT, especially in patients at risk for VOD.
Studies
analyzing endothelial protection against further conditioning agents should
help to
clarify whether Defibrotide can be used as a broad protective agent.
21
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REFERENCES
1. Holler E, Kolb HJ, Moller A et al. Increased serum levels of tumor necrosis
factor
a precede major complications of bone marrow transplantation. Blood.
1990;75:1011-1016.
2. Antin JH, Ferrara JLM. Cytokine dysregulation and acute graft-versus-host
dis-
ease. Blood. 1992;80:2964-2968.
3. Ferrara JL, Levy R, Chao NJ. Pathophysiological mechanisms of acute graft-
vs.-
host disease. Biol Blood Marrow Transplant. 1999;5:347-356.
4. Xun CQ, Brown BA, Jennings CD, Henslee-Downey PJ, Thompson JS. Acute
graft-versus-host-like diseases induced by transplantation of human activated
natural killer cells into SCID mice. Transplantation. 1993;56:409-417.
5. Weiner RS, Bortin MM, Gale RP et al. Interstitial pneumonitis after bone
marrow
transplantation. Assessment of risk factors. Ann Intern Med. 1986;104:168-175.
6. Weiss MA. Novel treatment strategies in chronic lymphocytic leukemia. Curr
On-
col Rep. 2001;3:217-222.
7. Wasch R, Reisser S, Hahn J et al. Rapid achievement of complete donor
chimer-
ism and low regimen-related toxicity after reduced conditioning with
fludarabine,
carmustine, melphalan and allogeneic transplantation. Bone Marrow Transplant.
2000;26:243-250.
8. Carella AM, Champlin R, Slavin S, McSweeney P, Storb R. Mini-allografts:
ongo-
ing trials in humans. Bone Marrow Transplant. 2000;25:345-350.
9. Slavin S, Nagler A, Naparstek E et al. Nonmyeloablative stem cell
transplantation
and cell therapy as an alternative to conventional bone marror transplantation
with lethal cytoreduction for the treatment of malignant and nonmalignant hema-
tologic diseases. Blood. 1998;91:756-763.
10. Holler E, Kolb HJ, Hiller E et al. Microangiopathy in patients on
cyclosporine pro-
phylaxis who developed acute graft-versus-host disease after HLA-identical
bone
marrow transplantation. Blood. 1989;73:2018-2024.
11. Eissner G, Kohlhuber F, Grell M et al. Critical involvement of
transmembrane
TNF-a in endothelial programmed cell death mediated by ionizing radiation and
bacterial endotoxin. Blood. 1995;86:4184-4193.
12. Lindner H, Holler E., Ertl B et al. Peripheral blood mononuclear cells
induce pro-
grammed cell death in human endothelial cells and may prevent repair - role of
cytokines. Blood. 1997;89:1931-1938.
22
CA 02487171 2004-11-24
WO 03/101468 PCT/EP03/05753
13. Haimovitz-Friedman A, Balaban N, Mcloughlin M et al. Protein kinase C
mediates
basic fibroblast growth factor protection of endothelial cells against
radiation-
induced apoptosis. Cancer Res. 1994;54:2591-2597.
14. Fuks Z, Persaud RS, Alfieri A et al. Bacic fibroblast growth factor
protects endo-
thelial cells against radiation-induced programmed cell death in vitro and in
vivo.
Cancer Res. 1994;54:2582-2590.
15. Eissner G, Lindner H, Behrends U et al. Influence of bacterial endotoxin
on radia-
tion-induced activation of human endothelial cells in vitro and in vivo,
protective
role of IL-10. Transplantation. 1996;62:819-827.
16. Lindner H, Holler E, Gerbitz A, Johnson JP, Bornkamm GW, Eissner G.
Influence
of bacterial endotoxin on radiation-induced activation of human endothelial
cells in
vitro and in vivo: 2. IL-10 protects against transendothelial migration.
Transplan-
tation. 1997;64:1370-1973.
17. Beelen DW, Haralambie E, Brandt H et al. Evidence that sustained growth
sup-
pression of intestinal anaerobic bacteria reduces the risk od acute graft-
versus-
host disease after sibling marrow transplantation. Blood. 1992;80: 2668-2676.
18. Eissner G, Multhoff G, Holler E. Influence of bacterial endotoxin on the
allogenic-
ity of human endothelial cells. Bone Marrow Transplant. 1998;21:1286-1287.
19.Ades EW, Candal FJ, Swerlick RA et al. HMEC-1: establishment of an immortal-
ized human microvascular endothelial cell line. J Invest Dermatol. 1992;99:683-
690.
20.Cotter TG, Lennon SV, Glynn JM, Green DR. Microfilament-disrupting agents
prevent the formation of apoptotic bodies in tumor cells undergoing apoptosis.
Cancer Res. 1992;52:997-1005.
21. Lee A, Whyte MK, Haslett C. Inhibition of apoptosis and prolongation of
neutrophil
functional longevity by inflammatory mediators. J Leukoc Biol. 1993;54:283-
288.
22. Westphal JR, Tax WJ, Willems HW, Koene RA, Ruiter DJ, De-Waal RM. Acces-
sory function of endothelial cells in anti-CD3-induced T-cell proliferation:
syner-
gism with monocytes. Scand J Immunol. 1992;35:449-457.
23. MacDonald HR, Engers HD, Cerrottini JC, Brunner KT. Generation of
cytotoxic T
lymphocytes in vitro. J Exp Med. 1974;140:718-722.
24. Long E. Regulation of immune responses through inhibitory receptors. Annu
Rev
Immunol. 1999;17:875-904.
23
CA 02487171 2004-11-24
WO 03/101468 PCT/EP03/05753
25. Nagler A, Aker M, Or R et al. Low-intensity conditioning is sufficient to
ensure en-
graftment in matched unrelated bone marrow transplantation. Exp Hematol.
2001;29:362-370.
26. Michallet M, Bilger K, Garban F et al. Allogeneic hematopoietic stem-cell
tran-
plantation after nonmyeloablative preparative regimens: impact of
pretransplanta-
tion and posttransplantation factors on outcome. J Clin Oncol. 2001;19:3340-
3349.
27. Holler E, personal communication
28. Hildebrandt G, Bertz H, Mestan A et al. Analysis of pulmonary function
after allo-
geneic bone marrow trasplantation (BMT) or blood stem cell transplantation
(PBSCT) using conditioning regimens with total body irradiation (TBI) and con-
ventional intensity compared to regimens without TBI with reduced intensity
[ab-
stract]. Bone Marrow Transplant. 2001;27 (Suppl. 1 ):S216.
29. Bornhauser M, Thiede C, Schuler U et al. Dose-reduced conditioning for
alloge-
neic blood stem cell transplantation: durable engraftment without
antithymocyte
globulin. Bone Marrow Transplant. 2000;26:119-125.
30. Huang P, Siciliano MJ, Plunkett W. Gene deletion, a mechanism of induced
mu-
tation by arabinosyl nucleosides. Mutat Res. 1989;210:291-301.
31. Genini D, Budihardjo I, Plunkett W et al. Nucleotide requirements for the
in vitro
activation of the apoptosis protein-activating factor-1-mediated caspase
pathway.
J Biol Chem. 2000;275:29-34.
32. Biederman BC, Pober JS. human vascular endothelial cells favor clonal
expan-
sion of unusual alloreactive CTL. J Immunol. 1999;162:7022-7030.
33. Briscoe DM, Alexander AI, Lichtman AH. Interactions between T lymphocytes
and
endothelial cells in allograft rejection. Curr Op Immunol. 1998;10:525-531.
34. Pegram AA, Kennedy LD. Prevention and treatemnt of veno-occlusive disease.
Ann Pharmacother. 2001;35:935-942.
35. Rossini G, Pompilio G, Biglioli P et al. Protestant activity of
defibrotide in cardio-
plegia followed by idchemia/(repersusion injury in the isolated rat heart. J
Card
Su rg. 1999;14:334-341.
36. Rossini G, Berti F, Trento F et al. Defibrotide normalizes cardiovascular
function
hampered by established atherosclerosis in the rabbit. Thromb Res. 2000;97:29-
38.
24
CA 02487171 2004-11-24
WO 03/101468 PCT/EP03/05753
37. Pogliani EM, Perseghin P, Parma M, Pioltelli P, Corneo G. Defibrotide in
recurrent
thrombotic thrombocytopenic purpura. Clin Appl Thromb Hemost. 2000;6:69-70.
38.San T, Moini H, Emerk K, Bilsel S. Protective efFect of defibrotide on
perfusion in-
duced endothelial damage. Throm Res. 2000;99:335-341.
39.Chopra R, Eaton JD, Grassi A et al. Defibrotide for the treatment of
hepatic veno-
occlusive disease: results of the European compassionate-use study. Br J Hae-
matol. 2000;111:1122-1129.
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