U.S. patent application number 14/148483 was filed with the patent office on 2014-05-08 for compounds and methods for the treatment of inflammatory diseases of the cns.
This patent application is currently assigned to INDEX PHARMACEUTICALS AB. The applicant listed for this patent is Lars-Goran Axelsson, Lisa Charlotta Bandholtz, Alexander Gielen, Oliver Von Stein, Arezou Zargari. Invention is credited to Lars-Goran Axelsson, Lisa Charlotta Bandholtz, Alexander Gielen, Oliver Von Stein, Arezou Zargari.
Application Number | 20140128456 14/148483 |
Document ID | / |
Family ID | 42153438 |
Filed Date | 2014-05-08 |
United States Patent
Application |
20140128456 |
Kind Code |
A1 |
Bandholtz; Lisa Charlotta ;
et al. |
May 8, 2014 |
Compounds and methods for the treatment of inflammatory diseases of
the CNS
Abstract
Inflammatory diseases in the CNS can be treated or alleviated by
the administration of an oligonucleotide in an amount sufficient to
reduce the influx of mononuclear cells to the central nervous
system by down-regulating the expression of at least one cell
surface marker. For example multiple sclerosis can be treated or at
least alleviated, by the administration of an oligonucleotide in a
dose effective to inhibit or reduce the influx of mononuclear
and/or autoaggressive cells to the central nervous system. The
oligonucleotide can be used alone, or in combination with other
treatment strategies.
Inventors: |
Bandholtz; Lisa Charlotta;
(Stockholm, SE) ; Gielen; Alexander; (Bandhagen,
SE) ; Zargari; Arezou; (Solna, SE) ; Von
Stein; Oliver; (Upplands Vasby, SE) ; Axelsson;
Lars-Goran; (Tierp, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bandholtz; Lisa Charlotta
Gielen; Alexander
Zargari; Arezou
Von Stein; Oliver
Axelsson; Lars-Goran |
Stockholm
Bandhagen
Solna
Upplands Vasby
Tierp |
|
SE
SE
SE
SE
SE |
|
|
Assignee: |
INDEX PHARMACEUTICALS AB
Stockholm
SE
|
Family ID: |
42153438 |
Appl. No.: |
14/148483 |
Filed: |
January 6, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13127190 |
Jul 25, 2011 |
8637479 |
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PCT/SE2009/051247 |
Nov 4, 2009 |
|
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14148483 |
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61111287 |
Nov 4, 2008 |
|
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
C12N 15/11 20130101;
C12N 2310/313 20130101; C12N 2310/315 20130101; A61K 2039/55561
20130101; A61P 25/28 20180101; C12N 15/117 20130101; C12N 2310/17
20130101; A61P 25/00 20180101 |
Class at
Publication: |
514/44.R |
International
Class: |
C12N 15/11 20060101
C12N015/11 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 4, 2008 |
SE |
0802339-2 |
Claims
1. A method for the treatment, and/or alleviation of multiple
sclerosis, comprising administering an oligonucleotide to a patient
in need thereof in an amount effective to inhibit or reduce the
influx of mononuclear and/or autoaggressive cells to the central
nervous system by down-regulating the expression of at least one
specific cell surface marker, wherein the oligonucleotide is an
isolated and substantially purified oligonucleotide selected from
the group consisting of SEQ ID NO: 9 (IDX0150), and SEQ ID NO: 10
(IDX0980).
2. The method according to claim 1, wherein the oligonucleotide is
SEQ ID NO: 9 (IDX0150).
3. The method according to claim 1, wherein the oligonucleotide is
SEQ ID NO: 10 (IDX0980).
4. The method according to claim 1, wherein the cell surface marker
is at least one of CD49d, CXCR3 (CD183), CCR2 (CD192), and CCR5
(CD195).
5. The method of claim 4, wherein the oligonucleotide is SEQ ID NO:
9 (IDX0150).
6. The method of claim 4, wherein the cell surface marker is CD49d
and the oligonucleotide is SEQ ID NO: 9 (IDX0150).
7. The method according to claim 1, wherein the route of
administration is chosen from mucosal, subcutaneous, intramuscular,
intravenous and intraperitoneal administration.
8. The method according to claim 7, wherein the mucosal
administration is chosen from nasal, oral, gastric, ocular, rectal,
urogenital and vaginal administration.
9. The method according to claim 7, wherein the oligonucleotide is
SEQ ID NO: 9 (IDX0150).
10. The method according to claim 8, wherein the oligonucleotide is
SEQ ID NO: 9 (IDX0150).
11. A method for the treatment, and/or alleviation of multiple
sclerosis, wherein an oligonucleotide is administered to a patient
in need thereof in an amount effective to inhibit or reduce the
influx of mononuclear and/or autoaggressive cells to the central
nervous system by reducing the production of VEGF, wherein the
oligonucleotide is an isolated and substantially purified
oligonucleotide selected from the group consisting of SEQ ID NO: 9
(IDX0150), and SEQ ID NO: 10 (IDX0980).
12. The method according to claim 11, wherein the oligonucleotide
is SEQ ID NO: 9 (IDX0150).
13. The method according to claim 11, wherein the oligonucleotide
is SEQ ID NO: 10 (IDX0980).
14. The method according to claim 11, wherein the route of
administration is chosen from mucosal, subcutaneous, intramuscular,
intravenous and intraperitoneal administration.
15. The method according to claim 14, wherein the mucosal
administration is chosen from nasal, oral, gastric, ocular, rectal,
urogenital and vaginal administration.
16. The method according to claim 14, wherein the oligonucleotide
is SEQ ID NO: 9 (IDX0150).
17. The method according to claim 15, wherein the oligonucleotide
is SEQ ID NO: 9 (IDX0150).
Description
[0001] This application is a continuation application under 35
U.S.C. 120 of U.S. Ser. No. 13/127,190 filed Jul. 25, 2011, which
is a 371 of PCT/SE2009/051247 filed Nov. 4, 2009, which claims
priority under 35 U.S.C. 119 to U.S. No. 61/111,287 filed Nov. 4,
2008.
[0002] This application incorporates by reference the material in
the text file submitted herewith named
"Jan-6-2014-186890A_ST25.txt" created Jan. 6, 2014 and having a
size of 4064 bytes.
FIELD OF THE INVENTION
[0003] The present invention concerns the treatment and/or
alleviation of inflammatory diseases of the central nervous system
(CNS), such as but not limited to multiple sclerosis, and makes
available compounds and methods for this use.
BACKGROUND
[0004] Cell migration is central to many processes in the human
body, such as the immune response, but can also be a component of
chronic inflammation. The migration of mononuclear cells to the CNS
is believed to be one factor underlying the pathogenesis of
inflammatory diseases of the CNS.
[0005] One example is multiple sclerosis (MS) which is an
autoimmune disease that affects the CNS, i.e. the brain and spinal
cord. MS is characterized by weakness, tremors, and visual
impairments. Other symptoms are slurred speech and impaired
mobility, e.g. that the patients drag their feet, stumble, and
frequently drop objects. These symptoms may remain mild, come and
go, or become crippling--but they tend to get progressively worse
with age (Hafler, 2004). MS usually affects women more than men.
The disorder most commonly begins between ages 20 and 40, but can
strike at any age. The exact cause is not known, but MS is believed
to result from damage to the myelin sheath, the protective material
which surrounds nerve cells. It is a progressive disease, meaning
that the damage gets worse over time. Inflammation destroys the
myelin, leaving multiple areas of scar tissue (sclerosis). The
inflammation occurs when the body's own immune cells attack the
nervous system.
[0006] One important step in the pathogenesis of MS is the
migration of cells to the CNS, wherein self-reactive T-cells, and
B-cells together with monocytes mediate inflammation of the CNS,
thereby causing demyelination of axons. Chemokines and their
receptors are proposed to play a major role in the recruitment of
these leukocytes to CNS. Thus, a reduction of chemokine receptors
may be an effective strategy to prevent migration of destructive
cells to CNS. Important chemokine receptors described in MS
pathology are CCR5 (a receptor for chemotactic proteins such as
RANTES and MIP-1alpha), CXCR3 (a receptor for chemotactic proteins
such as IP-10 and MIG), and CCR2 (a receptor for chemotactic
proteins such as MCP1-3) (Trebst C et al., 2009).
[0007] Another step that can lead the lymphocytes to enter into the
parenchyma of the CNS is adhesion of CD49d (very late antigen,
VLA-4 expressed on T-cells and B-cells) to their receptors on
endothelial cells, and thereby transmigrate through the blood-brain
barrier. A reduction of CD49d can reduce the transmigration and
accumulation of immune cells in the CNS (Steinman L, 2009).
[0008] In healthy individuals, immune cells cannot pass through the
CNS capillaries and venules into the CNS tissue because the walls
of the capillaries in the CNS are different from those in the rest
of the body in that they have very closely packed cells which do
not allow the passage of immune cells. This special feature of the
CNS vascular system is referred to as the blood-brain barrier
(BBB). Vascular endothelial growth factor (VEGF) has been described
to induce breakdown of the BBB, which in turn can exacerbate the
inflammatory response in autoimmune disease of the CNS (e.g. MS)
(Proescholdt M A et al., 2002). A reduction of VEGF is an effective
strategy to prevent the increased vascular permeability of BBB and
thereby reduce the influx of destructive cells into CNS.
[0009] The inflammation causes nerve impulses to slow down or
become blocked, leading to the symptoms of MS. Repeated episodes,
or flare ups, of inflammation can occur along any area of the brain
and spinal cord.
[0010] Symptoms vary because the location and extent of each attack
varies. Usually episodes which last days, weeks, or even months,
alternate with periods of reduced or no symptoms (remission).
Recurrence (relapse) is common although non-stop progression
without periods of remission may also occur.
[0011] Patients diagnosed as having MS can expect one of four
clinical courses of disease, each of which might be mild, moderate,
or severe:
1. Relapsing-Remitting
[0012] Characteristics: People with this type of MS experience
clearly defined flare-ups (also called relapses, attacks, or
exacerbations). These are episodes of acute worsening of
neurological function. They are followed by periods of partial or
complete recovery (remissions) free of disease progression.
[0013] Frequency: Most common form of MS at time of initial
diagnosis. Approximately 85% of patients.
2. Primary-Progressive
[0014] Characteristics: People with this type of MS experience a
slow but nearly continuous worsening of their disease from the
onset, with no distinct relapses or remissions. However, there are
variations in rates of progression over time, occasional plateaus,
and temporary minor improvements.
[0015] Frequency: Relatively rare. Approximately 10% of
patients.
3. Secondary-Progressive
[0016] Characteristics: People with this type of MS experience an
initial period of relapsing-remitting MS, followed by a steadily
worsening disease course with or without occasional flare-ups,
minor recoveries (remissions), or plateaus.
[0017] Frequency: 50% of people with relapsing-remitting MS
developed this form of the disease within 10 years of their initial
diagnosis, before introduction of the "disease-modifying" drugs.
Long-term data are not yet available to demonstrate if this is
significantly delayed by treatment.
4. Progressive-Relapsing
[0018] Characteristics: People with this type of MS experience a
steadily worsening disease from the onset but also have clear acute
relapses (attacks or exacerbations), with or without recovery. In
contrast to relapsing-remitting MS, the periods between relapses
are characterized by continuing disease progression.
[0019] Frequency: Relatively rare. Approximately 5% of
patients.
[0020] There is no consensus within the scientific community as to
what triggers an attack. Patients with MS typically have a higher
number of immune cells than a healthy person, which suggests that
an immune response might play a role. The most common theories
point to a virus or genetic defect, or a combination of both. There
also appears to be a genetic link to the disease. MS is more likely
to occur in northern Europe, the northern United States, southern
Australia, and New Zealand than in other areas. Geographic studies
indicate there may be an environmental factor involved. People with
a family history of MS and those who live in a geographical area
with a higher incidence rate for MS have a higher risk of the
disease.
[0021] Medications such as interferon-beta, glatiramer acetate, and
mitoxantrone can reduce the frequency and severity of attacks in
people with relapsing-remitting MS and may reduce or delay future
disability. Interferon-beta and mitoxantrone may also slow the
progression of secondary progressive MS.
[0022] Treatment with interferon-beta or glatiramer acetate should
begin as soon as relapsing-remitting MS has been diagnosed. Most
specialists now agree that permanent damage to the nervous system
may occur early on, even while the symptoms are still quite mild.
Early treatment may help prevent or delay some of this damage.
[0023] Interferon beta treatment is however accompanied by several
adverse effects. The most frequent adverse effects are flu-like
symptoms: increased body temperature, feeling ill, fatigue,
headache, muscle pain, convulsion, dizziness, hair thinning, and
depression. Erythema, pain and hardness on the spot of injection
are also frequently observed. Interferon therapy causes
immunosuppression and can result in some infections manifesting in
unusual ways.
[0024] Also corticosteroids may be given during a relapse to reduce
inflammation and shorten the attack. The potent effect of
corticosteroids can result in serious side effects which mimic
Cushing's disease, a malfunction of the adrenal glands resulting in
an overproduction of cortisol. The list of potential side effects
is long and includes: increased appetite and weight gain; deposits
of fat in chest, face, upper back, and stomach; water and salt
retention leading to swelling and edema; high blood pressure;
diabetes; black and blue marks; slowed healing of wounds;
osteoporosis; cataracts; acne; muscle weakness; thinning of the
skin; increased susceptibility to infection; stomach ulcers;
increased sweating; mood swings; psychological problems such as
depression; and adrenal suppression.
[0025] In 2004, the FDA approved the use of a monoclonal antibody
(natalizumab, Tysabri.RTM., Biogen Idec Inc., Cambridge, Mass.,
USA, and Elan Pharmaceuticals, Inc., Dublin, Ireland) for the
treatment of patients with relapsing forms of MS (FDA News PO4-107,
Nov. 23, 2004).
[0026] While generally well tolerated, natalizumab is occasionally
associated with severe adverse effects. Antibody therapy in general
is costly, and there is a need for improvements inter alia with
regards to efficacy.
[0027] Currently, a number of other monoclonal antibodies are being
investigated for MS, including some that are already in use in
other conditions. These include ocrelizumab (Genentech/Hoffmann-La
Roche) daclizumab (Biogen Idec, Inc.), alemtuzumab (Campath.RTM.,
MabCampath.RTM., Bayer Schering, BTG, Genzyme, Millenium), and
rituximab (Rituxan.RTM., MabThera.RTM., Genentech, Hoffmann-La
Roche, Biogen Idec Inc.)
[0028] WO 2006/065751 concerns a CpG oligonucleotide prodrug that
includes a thermolabile substituent on at least one nucleotide
thereof. Therapeutic methods of using such thermolabile CpG
oligonucleotide prodrugs are described. The induction of cytokines,
in particular interferons, e.g. interferon-alpha, interferon-beta,
or interferon-gamma is disclosed.
[0029] WO 2006/027776 concerns methods for regulating an
AChE-associated biological pathway having a miRNA component, the
methods comprising subjecting the AChE-associated biological
pathway to an agent capable of regulating a function of the miRNA,
thereby regulating the AChE-associated biological pathway. Said
agents include modified polynucleotide sequences.
[0030] WO 2007/095316 relates generally to immunostimulatory
nucleic acids, compositions thereof and methods of using the
immunostimulatory nucleic acids. In particular the invention
relates to palindrome-containing immunostimulatory nucleic acids
and the use of these nucleic acids in treating disease.
[0031] WO 2004/016805 discloses a class of soft or semi-soft CpG
immunostimulatory oligonucleotides that are useful for stimulating
an immune response.
[0032] In summary, there is a need for improving existing therapies
for MS, with the aim of improving efficacy, as well as reducing
cost and adverse effects. There is also a need for developing new
treatment strategies for the battle against MS.
SUMMARY
[0033] The present inventors realized that the existing approaches
to treat or alleviate MS were insufficient in view of both the
results of treatment, the cost of treatment, and the occurrence of
adverse effects. The inventors set out to identify novel compounds
useful for the treatment and/or alleviation of MS, and to develop
methods of treatment having improved efficacy and reduced adverse
effects on the patients. Other problems underlying the invention,
as well as advantages associated with the invention, will become
evident to the skilled person upon study of the description,
examples, and claims, incorporated herein by reference.
[0034] The inventors surprisingly found that specific
oligonucleotide sequences were effective to inhibit or reduce the
influx of autoaggressive cells to the central nervous system by
down-regulating the expression of specific cell surface markers.
The inventions and embodiments are as set out in the enclosed
claims, incorporated herein by reference.
SHORT DESCRIPTION OF THE DRAWINGS
[0035] In the following detailed description, reference will be
made to the attached drawings in which;
[0036] FIGS. 1-3 are bar diagrams, showing normalized relative mRNA
expression of IFN-alpha (FIG. 1), IFN-beta (FIG. 2), and IL-10
(FIG. 3) in rat splenocytes stimulated with the inventive compounds
and cultured for 24 hours. Values were normalized to the mean RQ
value of the samples that were stimulated with medium only. Data
are shown as means.+-.SD of splenocytes derived from 6 spleens.
[0037] FIG. 4 is a bar diagram showing the expression of CD49d
(MFI) in splenocytes from DA rat (n=3). Splenocytes
(2.times.10.sup.6/ml) left untreated or stimulated (10 .mu.M) with
drugs. The inventive compounds were able to down-regulate CD49d
expressed on CD3 expressing cells after 48 h incubation as analyzed
by FACS.
[0038] FIG. 5 shows the expression of CD49d (MFI) in PBMC from DA
rat. PBMC (2.times.10.sup.6/ml) left untreated or stimulated (10
.mu.M) with drugs. The inventive compounds were able to
down-regulate CD49d expressed on CD3 expressing cells after 48 h
incubation as analyzed by FACS.
[0039] FIG. 6 is a graph, illustrating the mean clinical score of
MOG-induced EAE in DA rats (female) showing therapeutic effect of
oligonucleotides and vehicle (PBS). Twelve rats in each group were
all immunized with rat MOG in Incomplete Freund's adjuvant (IFA) at
day 0. IDX9052, IDX9054, IDX0980 and vehicle were administered
before the peak of the first attack (day 9 and day 15), and during
the peak of the first attack (day 20). Hundred fifty pg of drugs
were administered s.c. in a total volume of 100 .mu.l.
[0040] FIG. 7 is a graph, showing the incidence of MOG-induced EAE
in DA rats. Disease severity was reduced in rats treated with
IDX0980 and IDX9054 compared to PBS and IDX9052 treated groups.
[0041] FIG. 8 illustrates the mean clinical score of MOG-EAE in DA
rats showing therapeutic effect of oligonucleotides and vehicle
(PBS). 16 rats in each group were all immunized with rat MOG in
Incomplete Freund's adjuvant (IFA) at day 0. IDX9054 and vehicle
were administered before the peak of the first attack (day 9 and
day 15), and during the peak of the first attack (day 20). 150
.mu.g of drugs were administered in a total volume of 100 .mu.l s.c
and 40 .mu.l i.n. PBS treated group was only treated i.n.
[0042] FIG. 9 shows the mortality of MOG-EAE in DA rats. Reduced
mortality in both IDX0980 and IDX9054 treated groups as compared to
PBS and IDX9052 treated group. The rats either die of the disease
or were killed according to ethical regulations. Data are presented
at both day 25 and 35.
[0043] FIG. 10 shows the expression of CD49d on CD3 positive cells
isolated from RRMS patients (n=9). PBMC (2.times.10.sup.6/ml) left
untreated or stimulated (1, 10 and 25 .mu.M) with drugs. The
oligonucleotides were able to down regulate expression of CD49d on
CD3 positive T cells after 48 h incubation as analyzed by FACS.
Error bars indicate SEM, *P<0.05, **P<0.01,***P<0.001 as
analyzed by nonparametric T test, Wilcoxon matched pair test.
[0044] FIG. 11 shows the expression of CXCR3 (CD183) on CD3
positive cells isolated from RRMS patients. PBMC
(2.times.10.sup.6/ml) left untreated or stimulated (1, 10 and 25
.mu.M) with drugs. The inventive compound was able to down-regulate
the expression of CXCR3 on CD3 positive T cells after 48 h
incubation as analyzed by FACS.
[0045] FIG. 12 shows the expression of CXCR3 (CD183) on CD19
positive cells isolated from RRMS patients. PBMC
(2.times.10.sup.6/ml) left untreated or stimulated (1, 10 and 25
.mu.M) with drugs. The oligonucleotides were able to down-regulate
expression of CXCR3 on CD19 positive cells after 48 h incubation as
analyzed by FACS.
[0046] FIG. 13 shows the expression of CXCR3 (CD183) on CD14
positive cells isolated from RRMS patients. PBMC
(2.times.10.sup.6/ml) left untreated or stimulated (1, 10 and 25
.mu.M) with drugs. The oligonucleotides were able to down-regulate
expression of CXCR3 on CD14 positive cells after 48 h incubation as
analyzed by FACS.
[0047] FIG. 14 shows the expression of CCR5 (CD195) on CD14
positive cells isolated from RRMS patients. PBMC
(2.times.10.sup.6/ml) left untreated or stimulated (1, 10 and 25
.mu.M) with drugs. The oligonucleotides were able to down-regulate
expression of CCR5 on CD14 positive cells after 48 h incubation as
analyzed by FACS.
[0048] FIG. 15 shows the expression of CCR2 (CD192) on CD14
positive cells isolated from RRMS patients in MFI (A) or % (B),
respectively. PBMC (2.times.10.sup.6/ml) left untreated or
stimulated (1, 10 and 25 .mu.M) with drugs. The oligonucleotides
were able to down-regulate expression of CCR2 on CD14 positive
cells after 48 h incubation as analyzed by FACS.
[0049] FIG. 16 Shows migration of cells towards MCP-1 and RANTES.
PBMCs 0.5.times.10.sup.6 (A) or 0.250.times.10.sup.6(B) from two
RRMS patients were incubated with the inventive compounds IDX9045,
IDX9054, IDX0980 (1, 10 and 25 .mu.M) or left untreated for 48 h.
Cells were then used in QCM migration assay in order to analyze
migration towards MCP-1 and RANTES. Both experiments showed reduced
migration of cells treated with inventive compounds compared to
untreated cells.
[0050] FIG. 17 shows the expression of VEGF in supernatant from
RRMS patients (n=6-11). PBMC (2.times.10.sup.6/ml) left untreated
or stimulated (1, 10 and 25 .mu.M) with drugs. The oligonucleotides
were able to down-regulate VEGF in supernatant from the stimulated
cells after 48 h incubation as analyzed by CBA. Error bars indicate
SEM, *P<0.05, **P<0.01, ***P<0.001 as analyzed by
nonparametric T test, Wilcoxon matched pair test.
[0051] FIG. 18 shows the induction of IFN-beta in PBMC isolated
from RRMS patients. PBMCs (2.times.10.sup.6/ml) from different RRMS
patients (n=6) were stimulated with three different concentrations
(1, 10 and 25 .mu.M) of oligonucleotides. IFN-beta-production was
analyzed after 48 h incubations using IFN-beta ELISA kit.
DESCRIPTION
[0052] The following description is of the best mode presently
contemplated for carrying out the invention. This description is
not to be taken in a limiting sense, but is made solely for the
purpose of describing the general principles of the invention. The
scope of the invention should be determined with reference to the
claims.
[0053] Before the invention is described in detail, it is to be
understood that this invention is not limited to the particular
compounds described or process steps of the methods described as
such compounds and methods may vary. It is also to be understood
that the terminology used herein is for purposes of describing
particular embodiments only, and is not intended to be limiting. It
must be noted that, as used in the specification and the appended
claims, the singular forms "a," "an" and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a sequence" includes more than one such
sequence, and the like.
[0054] Further, the term "about" is used to indicate a deviation of
+/-2% of the given value, preferably +/-5% and most preferably
+/-10% of the numeric values, when applicable.
[0055] The term "influx" as used in the expression "the influx of
autoaggressive cells to the central nervous system" is intended to
mean the accumulation of autoaggressive cells in the central
nervous system, and includes the steps of migration, adhesion, and
transmigration of mononuclear cells, in particular T-cells,
B-cells, and monocytes.
[0056] The invention makes available specific novel nucleotides,
i.e. the isolated oligonucleotide sequences according to any one of
SEQ ID NO 1-8. See Table 1, which correlates the SEQ ID No:s, the
internal references and the sequences.
TABLE-US-00001 TABLE 1 Oligonucleotides SEQ ID NO Seq 5'-3' IDX-No
1 G*G*G*TCGCAGC*T*G*G IDX9045 2 G*G*G*GTCGTCTGC*G*G*G IDX9054 3
T*C*G*TCGTTCGGCCGATCG*T*C*C IDX9038 4 G*G*G*GTCGCAGCT*G*G*G IDX9004
5 G*G*G*GTCGTCTG*C*G*G IDX9052 6 T*C*G*TCGTTCTGCCATCGTC*G*T*T
IDX9022 7 G*A*T*CGTCCGTCGG*G*G*G IDX9058 8 G*G*G*GATCGTCCG*G*G*G
IDX9060 9 G*G*A*ACAGTTCGTCCAT*G*G*C IDX0150 10
G*G*GGGACGATCGTCG*G*G*G*G*G IDX0980 *= phosphorothioate
modification
[0057] The above sequences SEQ ID NO 1-8 have been designed by the
inventors, and are to the best knowledge of the inventors, not
previously known. SEQ ID NO 10, although known for medical use (see
below), is to the best knowledge of the inventors not previously
known for use in the treatment of MS.
[0058] SEQ ID NO 9 (IDX0150) is known from U.S. Pat. No. 6,498,147,
and has been successfully tested in phase II clinical trials for
the treatment of inflammatory bowel disease (Kappaproct.RTM., Index
Pharmaceutical AB, Solna, Sweden). SEQ ID NO 10 (IDX0980) was
originally used, as it is known to be a strong immunomodulatory
olignucleotide in human (Kerkmann et al., 2005; Wikstrom et al.,
2007).
[0059] The present inventors make available an isolated and
substantially purified oligonucleotide chosen among SEQ ID NO
1-8.
[0060] According to one embodiment, at least one nucleotide in such
oligonucleotides has a phosphate backbone modification. Preferably
said phosphate backbone modification is a phosphorothioate or
phosphorodithioate modification.
[0061] The inventors also make available pharmaceutical
compositions comprising an oligonucleotide according to any one of
SEQ ID NO 1-8. Said pharmaceutical compositions further preferably
comprise a pharmacologically compatible and physiologically
acceptable excipient or carrier, chosen from saline, liposomes,
surfactants, mucoadhesive compounds, enzyme inhibitors, bile salts,
absorption enhancers, cyclodextrins, or a combination thereof.
[0062] Another embodiment of the invention is the use of an
isolated and substantially purified oligonucleotide according to
any one of SEQ ID NO 1-8 for the manufacture of a pharmaceutical
composition for the treatment, and/or alleviation of multiple
sclerosis.
[0063] Another embodiment is the use of an isolated and
substantially purified oligonucleotide according to SEQ ID NO 9
[IDX0150] or SEQ ID NO 10 [IDX0980] for the manufacture of a
pharmaceutical composition for the treatment, and/or alleviation of
multiple sclerosis, in particular relapsing-remitting multiple
sclerosis.
[0064] Without wishing to be bound to any specific theory, the
inventors contemplate that the effect of the inventive compounds at
least in part is accountable to their capability to inhibit or
reduce the influx of autoaggressive cells to the central nervous
system by down-regulating the expression of at least one cell
surface marker. Consequently, one embodiment of the invention
involves the administration of an oligonucleotide according to SEQ
ID NO 1-8 or 9 -10 in an amount effective to inhibit or reduce the
influx of autoaggressive cells to the central nervous system by
down-regulating the expression of at least one cell surface
marker.
[0065] It is contemplated that the oligonucleotides and the methods
of their use is also generally applicable to the treatment or
alleviation of an inflammatory disease of the central nervous
system wherein said oligonucleotide is administered in an amount
effective to inhibit or reduce the influx of mononuclear cells to
the central nervous system by down-regulating the expression of at
least one cell surface marker.
[0066] Preferably said at least one cell surface marker is chosen
among CD49d, CXCR3 (CD183), CCR2 (CD192), and CCR5 (CD195).
According to one embodiment, said oligonucleotide is chosen among
SEQ ID NO 1 [IDX9045], SEQ ID NO 2; [IDX9054]; SEQ ID NO 7
[IDX9058); SEQ ID NO 3 [IDX9038]. Preferably said oligonucleotide
is SEQ ID NO 1 [IDX9045]. According to another embodiment, said at
least one cell surface marker is CD49d and the oligonucleotide is
chosen from SEQ ID NO 3 [IDX9038] or SEQ ID NO 7 [IDX9058].
[0067] The inventors however also contemplate, again without
wishing to be bound to a specific theory, that the effect is at
least in part accountable to the inhibition or reduction of the
influx of autoaggressive cells to the central nervous system by
reducing the production of VEGF.
[0068] Consequently, according to another embodiment of the
invention, the oligonucleotide is chosen among SEQ ID NO 1
[IDX9045] and SEQ ID NO 10 [IDX0980].
[0069] The invention also makes available a method for the
treatment, and/or alleviation of multiple sclerosis, wherein an
oligonucleotide is administered in an amount effective to inhibit
or reduce the influx of autoaggressive cells to the central nervous
system by down-regulating the expression of specific cell surface
markers.
[0070] Preferably a pharmaceutical composition comprising an
oligonucleotide according to any one of SEQ ID NO 1-8, 9 and 10 is
administered to a patient.
[0071] Preferably, the route of administration is chosen from
mucosal, subcutaneous, intramuscular, intravenous and
intraperitoneal administration. Preferably the mucosal
administration is chosen from nasal, oral, gastric, ocular, rectal,
urogenital and vaginal administration.
[0072] In the above method, the cell surface marker is at least one
of CD49d, CXCR3 (CD183), CCR2 (CD192), and CCR5 (CD195), and the
oligonucleotide is chosen among SEQ ID NO 1 [IDX9045], SEQ ID NO 2;
[IDX9054]; SEQ ID NO 7 [IDX9058); SEQ ID NO 3 [IDX9038].
[0073] According to a preferred embodiment, the cell surface marker
is CD49d and the oligonucleotide is chosen from SEQ ID NO 3
[IDX9038] or SEQ ID NO 7 [IDX9058].
[0074] According to another embodiment of a method for the
treatment, and/or alleviation of multiple sclerosis, said
oligonucleotide is administered in an amount effective to inhibit
or reduce the influx of autoaggressive cells to the central nervous
system by reducing the production of VEGF. In this embodiment, the
oligonucleotide is preferably chosen among SEQ ID NO 1 [IDX9045]
and SEQ ID NO 10 [IDX0980].
[0075] According to an embodiment, the oligonucleotide is
administered in an amount of about 1 to about 2000 .mu.g per kg
body weight, preferably about 1 to about 1000 .mu.g per kg body
weight. Most preferably the oligonucleotide is administered in an
amount of about 1 to 500 .mu.g per kg body weight.
[0076] In a method according to the invention, the route of
administration is chosen from mucosal, subcutaneous, intramuscular,
intravenous and intraperitoneal administration. According to an
embodiment of the method, the mucosal administration is chosen from
nasal, oral, gastric, ocular, rectal, urogenital and vaginal
administration.
[0077] Nasal administration constitutes one embodiment of the
method according to the invention. There are several methods and
devices available for nasal administration; single or multi-dosing
of both liquid and powder formulations, with either topical or
systemic action. Using appropriate devices or administration
techniques, it is possible to target the olfactory bulb region for
delivery to the CNS. The present invention is not limited to
particular methods or devices for administering the
oligonucleotides to the nasal mucous membrane. The initial animal
studies have shown that simple instillation by pipette works
satisfactorily, although for human use, devices for reliable single
or multi dose of administration would be preferred.
[0078] According to another embodiment of the invention, the
oligonucleotides are administered to the mucous membrane of the
colon through rectal instillation, e.g. in the form of an aqueous
enema comprising the oligonucleotides suspended in a suitable
buffer.
[0079] According to another embodiment of the invention, the
oligonucleotides are administered to the mucous membrane of the
lungs or the airways through inhalation of an aerosol, comprising
the oligonucleotides suspended in a suitable buffer, or by
performing a lavage, also comprising the oligonucleotides suspended
in a suitable buffer.
[0080] According to yet another embodiment of the invention, the
oligonucleotides are administered to the mucous membrane of the
urogenital tract, such as the urethra, the vagina etc through
application of a solution, a buffer, a gel, salve, paste or the
like, comprising the oligonucleotides suspended in a suitable
vehicle.
[0081] A particular embodiment involves the use of an
oligonucleotide according to the invention for use in conjunction
with the administration of Tysabri, an antibody directed to CD49d,
a cell surface marker on T-cells. There are indications that the
oligonucleotides according to the invention can down regulate
CD49d, which might reduce transmigration of T-cells through the
blood-brain barrier. The inventors thus make available a
combination therapy involving the use of oligonucleotide compounds
together with an anti-CD49d antibody. This is contemplated to be
able to reduce antibody consumption, and thereby reduce the cost,
side-effects and risks associated with the said antibody therapy.
Consequently, in this embodiment, said compound is administered
sufficiently before the administration of an antibody in order to
allow the down-regulation of the specific cell surface molecules
towards which the antibody is directed.
[0082] A skilled person is well aware of the fact that there are
several approaches to the treatment of MS. Naturally new approaches
are constantly being developed, and it is conceived that the
oligonucleotides, their use and methods of treatment according to
the present invention, will find utility also in combination with
future treatments. The inventors presently believe that the
inventive oligonucleotides, their use and methods of treatment
would be useful as a stand-alone therapy for MS. It cannot however
be excluded that the inventive oligonucleotides will have utility
in combination with existing or future anti-MS treatments.
[0083] The oligonucleotide is administered in a therapeutically
effective dose. The definition of a "therapeutically effective
dose" is dependent on the disease and treatment setting, a
"therapeutically effective dose" being a dose which alone or in
combination with other treatments results in a measurable
improvement of the patient's condition. A skilled person can
determine a therapeutically effective dose either empirically, or
based on laboratory experiments, performed without undue burden.
The treating physician can also determine a suitable dose, based on
his/her experience and considering the nature and severity of the
disease, as well as the patient's condition.
[0084] Another embodiment is the administration of the
oligonucleotide in two or three or more separate doses, separated
in time by about 12, about 24 hours.
[0085] The invention finds utility in the treatment of MS, as
supported by the in vivo and in vitro data presented in the
experimental section and illustrated in the attached figures.
[0086] The embodiments of the invention have many advantages. So
far, the administration of an oligonucleotide in the doses defined
by the inventors has not elicited any noticeable side-effects.
Further, the mucosal administration is easy, fast, and painless,
and surprisingly results in a systemic effect. It is held that this
effect, either alone, or in combination with existing and future
anti-MS treatments, offers a promising approach to fight this
disease as well as related diseases.
EXAMPLES
Example 1
The Effect of Oligonucleotides on Rat Splenocytes
[0087] The present inventors set out to find and validate candidate
compounds that would be beneficial for the treatment and/or
alleviation of MS. The compounds used in these studies are based on
oligodeoxynucleotides. Before specific oligonucleotides could be
tested for their beneficial effect in in vitro and in vivo studies,
an assay was developed which enabled the inventors to test whether
an oligonucleotide has an immunomodulatory effect in rat. For this
purpose, the inventors used a rat splenocyte-based assay, where
splenocytes were incubated for a specified time with a selection of
inventive compounds. After the incubation, mRNA expression for
several immunological relevant markers was analyzed (IFN-alpha,
IFN-beta, IFN-gamma, IL-6, IL-10, TNF-alpha, VEGF-A, CCL-2, CCL-3,
CCL-4, CCL-5, CXCL-1, CXCL-2, CXCL-10 and TGF-beta1), which served
as a base to identify compounds that are showing immunomodulatory
effects in rat and enabled the selection of compounds to be used in
further rat studies.
Materials and Methods
[0088] Oligodeoxynucleotides: In the present study, 6 different
oligonucleotides were used for stimulation experiments using rat
derived splenocytes. All oligonucleotides were synthesized by
Biomers.net (Ulm, Germany). Upon arrival, all oligonucleotide were
diluted with sterile water in a series of different dilutions. The
optical density (OD) A260/280 was determined in at least five or
more samples of each dilution using a spectrophotometer
(SmartSpec.TM. 3000, Biorad, Hercules, Calif.). The average
concentration of all readings, for all dilutions, was calculated in
order to determine the concentration of the stock. These stock
solutions were all stored at -20.degree. C. The different working
solutions used in the experiments: 1 .mu.M and 10 .mu.M were
prepared by diluting the oligonucleotide stock solutions further in
distilled water (Invitrogen, Carlsbad, Calif.). Repeated
thawing/freezing cycles were minimized to limit any degradation of
the compounds.
[0089] PCR primers: Gene-specific primers were designed by using
Applied Biosystems Primer Express software (Table 2; Foster City,
Calif.). Amplification/detection of contaminating genomic DNA was
avoided by constructing one of the primers over an exon/intron
boundary. Primer oligonucleotides were ordered from MWG Biotech
(Ebersberg, Germany).
[0090] Rat splenocyte preparation: Six spleens derived from female
DA rats were used in the study. The spleens were not pooled but
dealt with individually to assess the degree of variability. Cell
suspensions were prepared under sterile conditions by using a 70
.mu.m nylon cell strainer (Becton Dickinson, Franklin Lakes, N.J.,
USA). Cells were then washed twice in complete RPMI 1640 (RPMI 1640
containing 5% heat inactivated FCS (Invitrogen), 2 mM L-glutamine
(Sigma-Aldrich), 100 U/ml penicillin and 100 .mu.g/ml streptomycin
(Invitrogen) at 1200 rpm for 7-10 minutes at 4.degree. C. The
supernatant was decanted and cells were resuspended in 1 ml red
blood cell lysing buffer (Sigma-Aldrich) and incubated further for
two minutes at room temperature. Another 5 ml of complete medium
was added before centrifugation, which was performed as previously
described. After decanting the supernatant, the pellet was
resuspended in complete medium and cell numbers were determined
with 0.4% Trypan blue exclusion (Sigma-Aldrich) using a Nikon
Eclipse TE2000-S microscope (Nikon, Tokyo, Japan).
[0091] In vitro stimulation: Cells were plated out in a 96-well
V-bottom plate in complete RPMI 1640 medium at a concentration of
10.times.10.sup.6 cells/ml, corresponding to 5.times.10.sup.5 cells
per well. Directly after plating the cells, an oligonucleotide
diluted in RPMI 1640 medium were added so that the final
concentration of the added oligonucleotide reached 1 .mu.M and 10
.mu.M respectively. Incubations were performed in duplicates. Cells
were incubated in a humidified cell culture incubator (Thermo
Scientific, Waltham, Mass.) with 5% CO.sub.2 in air at 37.degree.
C. for 24 hours. After the incubation period cell suspensions were
pooled and added to 1 ml ice cold PBS and thereafter washed at 1200
rpm for 10 minutes at 4.degree. C. Finally supernatant was removed
and the cell pellet subsequently dissolved and lysed in 350 .mu.l
RLT buffer with 1% of .beta.-mercaptoethanol added. The lysed cell
suspensions were frozen at -20.degree. C. until further
processing.
[0092] RT-PCR: Total RNA was extracted (Qiagen total RNA extraction
kit, Qiagen, Hilden, Germany). Samples were incubated with 27kU of
DNase for 30 minutes at 37.degree. C. in order to avoid
amplification/detection of contaminating genomic DNA. After the RNA
was eluted in 40 .mu.l RNase-free water, 5 .mu.l of RNA eluate was
taken for determination of the RNA concentration by
spectrophotometry. Reverse transcription was performed with 0.15-1
.mu.g of total RNA, random hexamers (0.1 .mu.g; Invitrogen), and
superscript reverse transcriptase (200 U; Invitrogen) following
manufacturers guidelines. The resulting cDNA was diluted with
sterile deionised water to form cDNA stock solutions. Amplification
was performed using the Applied Biosystems 7500 Real time PCR
System using SYBR-Green I (Applied Biosystems) with a two-step PCR
protocol (95.degree. C. for 10 minutes, followed by 40 cycles of
95.degree. C. for 15 seconds and 60.degree. C. for 1 minute). In
preliminary experiments, the primer pairs had been tested using a
conventional PCR protocol. The PCR products were run in an agarose
gel and were in all cases confined to a single band of the expected
size. All primers used are listed in Table 2.
[0093] Semi-quantitative assessment of mRNA levels was performed
using the .DELTA..DELTA.Ct method with amplification of mRNA and
actin-gamma, a house-keeping gene, in separate tubes. All samples
were run in duplicates. Real-time PCR data (individual .DELTA.Ct
and RQ values) were calculated and analysed with 7500 Real time PCR
System SDS Software. The individual values were then exported to
Excel. For each particular gene the average RQ values from the
duplicate samples were normalised against the average RQ value of
the samples that were stimulated with medium only. The mean values
and standard deviation of the six individual splenocyte populations
were calculated accordingly.
TABLE-US-00002 TABLE 2 Realtime PCR primer sequences Gene
Oligonucleotide sequence IFN-alpha .quadrature.Forward
5'-CCTCTTCACATCAAAGGAGTCATCT Reverse 5'-ACAGGCTTGCAGACCACTCA
IFN-beta .quadrature.Forward 5'-GCGTTCCTGCTGTGCTTCTC Reverse
5'-TGCTAGTGCTTTGTCGGAACTG IFN-gamma .quadrature.Forward
5'-TCGCCAAGTTCGAGGTGAA Reverse 5'-TAGATTCTGGTGACAGCTGGTGAA IL-6
Forward 5'-CACCCACAACAGACCAGTATATACCA Reverse
5'-TGCCATTGCACAACTCTTTTCT IL-10 Forward 5'-TGCGACGCTGTCATCGAT
Reverse 5'-GACACCTTTGTCTTGGAGCTTATTAA TNF-alpha .quadrature.Forward
5'-TGATCGGTCCCAACAAGGA Reverse 5'-TGCTTGGTGGTTTGCTACGA CXCL-1
Forward 5'-TCACTTCAAGAACATCCAGAGTTTG Reverse
5'-GTGGCTATGACTTCGGTTTGG CXCL-2 Forward
5'-AAGATACTGAACAAAGGCAAGGCTAA Reverse 5'-TTGATTCTGCCCGTTGAGGTA
CXCL-10 Forward 5'-CATGTTGAGATCATTGCCACAA Reverse
3'-CCGCTTTCAATAAGCTCTTGATG CCL-2 Forward 5'-CCAATGAGTCGGCTGGAGAA
Reverse 3'-GAGCTTGGTGACAAATACTACAGCTT CCL-3 Forward
5'-AGCCGGGTGTCATTTTCCT Reverse 3'-TTGGACCCAGGTCTCTTTGG CCL-4
Forward 5'-GCACCAATAGGCTCTGACCCT Reverse 3'-TTGGTCAGAAATACCACAGCTGG
CCL-5 Forward 5'-CCTTGCAGTCGTCTTTGTCACT Reverse
3'-GATGTATTCTTGAACCCACTTCTTCTC VEGF-A Forward
5'-GGGCTGCTGCAATGATGAA Reverse 3'-TTGATCCGCATGATCTGCAT TGF-beta1
Forward 5'-ACGTGGAAATCAATGGGATCA Reverse 3'-GGAAGGGTCGGTTCATGTCA
Reverse 5'-CCGACGATGGAAGGAAACAC
Results
[0094] Initially, the focus of the study was on the cytokines
IFN-alpha, IFN-beta and IL-10, due to their suggested protective
effects in MS and therefore graphs for only these factors are
included.
[0095] Both IFN-alpha (FIG. 1) and IFN-beta mRNA (FIG. 2) were
induced by several oligonucleotides and especially IDX9052,
IDX9054, IDX9060 and IDX0980 induced high levels of mRNA for these
genes. Oligonucleotides that were not capable of inducing
IFN-alpha/beta include IDX9022 and IDX9045.
[0096] In contrast to the type 1 interferons, IL-10, a potent Th-2
related cytokine was induced at much lower levels. IDX9060 and
IDX0980 are among the oligonucleotides that induce the highest
levels of IL-10 as shown in FIG. 3. Values were normalized to the
mean RQ value of the samples that were stimulated with medium only.
Data are shown as means.+-.SD of splenocytes derived from 6
spleens.
[0097] The patterns of IFN-gamma, CXCL-10 and VEGF-A were nearly
similar compared to the pattern of the type I interferons and were
moderately to strongly induced by several oligonucleotides (data
not shown). Most oligonucleotides induced only limited amounts of
IL-6 mRNA and the difference between the tested compounds was
relatively small (data not shown).
[0098] TNF-alpha and TGF-beta mRNA were hardly affected by any of
the tested oligonucleotides as none of these molecules could change
its expression significantly. Interestingly, however was the
observation that IDX9022 and IDX9045 gave a conspicuous down
regulation of TGF-beta (data not shown).
[0099] If one attempts to summarize the data on the remaining
chemokines, then it becomes clear that there are two groups: one
group consisting of genes that respond upon treatment with an
oligonucleotide and one group consisting of genes that do not
respond or even show a down-regulation after culture with an
oligonucleotide. To the first group belong MIP-1alpha, MCP1 and
CCL-5, whereas CCL-4, MIP-2 and CINC belong to the latter group. In
neither of the groups a clear overlap between the genes could be
observed.
[0100] In conclusion, it has become evident that the inventors'
candidate drugs are able to stimulate rat cells and thereby change
expression levels of several immunological relevant marker genes.
These results provided the basis for focusing on specific
oligonucleotides in further studies, including in a rat EAE
model.
Example 2
Reduction of CD49d Expression on Rat Cells in Vitro
[0101] The inventors set out to investigate if the inventive
compounds can reduce CD49d (an important molecule on surface of
lymphocytes that interacts with receptors on endothelial cells) and
thereby reducing the transmigration of the cells into the CNS, i.e.
a critical step in MS pathogenesis. The inventors used splenocytes
or blood from rat for studying expression of CD49d upon stimulation
with the inventive compounds.
Material and Methods
[0102] Splenocytes (n=3) or PBMC (n=1) from DA rats were incubated
at 37.degree. C. in a volume of 500 .mu.l of complete RPMI-medium
(containing 10% FCS, 1% PenStrep, 2 mM L-glutamine, 10 mM HEPES and
1 mM Sodium Pyruvate) in 48-well plates at a concentration of
2.times.10.sup.6 cells/ml and treated with 10 .mu.M of IDX9022,
IDX9058, IDX9038, IDX0150, IDX9054 and IDX9045.
[0103] After 48 hours, 200 .mu.l of the cell suspension was spun
down in 96-well plates, resuspended in 100 .mu.l of 2% FCS (in PBS)
and incubated with fluorochrome conjugated anti-CD3 and anti-CD49d
antibodies (Becton Dickinson, San Jose, Calif.) for 30 min at
4.degree. C. The cells were then washed twice in pure PBS and
subsequently analyzed by FACS using a FACSArray bioanalyzer (BD)
for surface antigen expression analysis.
Results
[0104] Splenocytes from 3 pooled DA rats showed down-regulation of
CD49d upon treatment with inventive compounds (FIG. 4). This
down-regulation was most pronounced with IDX0150, IDX9045 and
IDX9054, 48 h after incubation. PBMC from 1 DA rat showed
down-regulation of CD49d upon treatment with inventive compounds
(FIG. 5). This down-regulation was most pronounced with IDX0150 and
IDX9038, 48 h after incubation.
[0105] A decrease in CD49d expression was observed in splenocytes
and PBMC treated with the inventive compounds. These properties of
the inventive compounds could reduce the process of transmigration
and there by influx of cells into CNS, either as stand alone or as
a combinatory treatment with antibody therapy.
Example 3
Animal Studies
[0106] The inventors commissioned animal studies to confirm their
hypothesis and to test different candidate compounds. Two studies
were performed with MOG-induced Experimental Autoimmune
Encephalomyelitis (EAE) in DA rats.
[0107] MOG-induced EAE in DA rats is a well characterised
experimental model with high reproducibility sharing many features
with its human counterpart multiple sclerosis, and as such is an
appropriate model for therapeutic testing (Gold et al., 2006,
Friese et al., 2006). Following immunization with MOG in IFA,
animals become progressively paralysed from the tail, through the
back legs to the front legs due to progressive degeneration of
myelin caused by infiltrating inflammatory immune cells into the
spinal cord and brain. Pathogenesis is chronic relapsing, with
animals partially recovering and then relapsing with heightened
disease, and is a result of both monocytes and T cell (type 1
cytokines) and B cell (antibody) activities.
[0108] The rat was chosen as a rodent species since it is a widely
accepted relapsing/remitting experimental model. The selected
strain has documented susceptibility to CNS inflammation. In
general, 50-80% of immunized rats develop relapsing disease after a
titrated immunization. The remaining ones develop either late
chronic disease or die during the first attack.
[0109] Test animals: DA-rats (female, 180-250 g, 10-14 weeks of
age) from B & K, Sollentuna, Sweden were used. The animals were
allowed to acclimatize for at least 7 days before test starting. In
this period the animals were observed daily to ascertain their
fitness for the study. Animals showing signs of ill-health, any
abnormalities or bodyweight range extremes were replaced before the
study.
[0110] The groups were distributed within and between barriers in a
manner which allowed equalisation of environmental influences
across the study. The test animals were identified by an ear tag.
The cage card stated number of the experiment, and the group code
alone.
[0111] Housing: The animals were housed four per cage, unless
reduced by mortality or isolation. The cages were conventional
makrolon plastic cages with stainless steel lids. Aspen chips were
used as flooring material. Filtered, not recirculated air was
supplied. The temperature was maintained within the range
20-23.degree. C. and the relative humidity within the range 40-60%.
Both temperature and relative humidity were monitored daily.
Lighting conditions were 12 hours light and 12 hours dark
(07.00-19.00).
[0112] Diet: The test animals were given conventional rodent diet
(R 36 (irradiated) from Lactamin, Sweden) ad lib. Before delivery,
each batch of diet was analyzed by the supplier for various
nutritional components and chemical and microbiological
contaminants. The suppliers' analytical certificates were
scrutinized and approved before any batch of diet was released for
use.
[0113] This diet contained no added antibiotic or other
chemotherapeutic or prophylactic agent. The test animals were given
water ad lib (municipal drinking water) via polyethylene or
polycarbonate pipes with sipper tubes.
[0114] Immunization procedure: The rats were immunized (day=0) by
injecting i.d. in the base of the tail 0.1 ml of an emulsion
composed of 30 .mu.g rat MOG pH3 (5.2 mg/ml stock) in Incomplete
Freund's Adjuvant (Sigma, St. Louis, USA). This procedure is known
to result, starting approximately from day 10, in the appearance of
a progressive paralysis, arising from the tail and progressively
ascending up to the forelimbs.
[0115] Duration of treatment: The test compounds and control were
administered twice before (i.e. day 11 and day 15) the peak of the
first attack, and the third treatment at day 20 (during the peak of
the first attack).
[0116] Test substances: In the first study, two proprietary
oligonucleotides were used, IDX9052 (SEQ ID NO 5) and IDX9054 (SEQ
ID NO 2), together with a publicly available oligonucleotide,
IDX0980 (SEQ ID NO 10). In the second study, one proprietary
oligonucleotide was used; IDX9054 (SEQ ID NO 2). The sequence of
the test substances is given in Table 1.
[0117] The test compounds in the first study (IDX9052, IDX9054 and
IDX0980) were administered s.c. in a total volume of 100 .mu.l in
the neck. This dose was administered three times. The test compound
in the second study (IDX9054) was administered s.c in a total
volume of 100 .mu.l in the neck or i.n in a total volume of 40
.mu.l on the nose. The drugs were administered three times either
s.c or i.n. In both studies, PBS was used as both vehicle and
control (blank). The dose administered was 150 .mu.g per
immunization in all studies.
[0118] Study design: The first study involved four groups of 12
animals each. All the groups were immunized with rat MOG in IFA,
according to the immunization protocol. All compounds were
immunized three times. [0119] Group 1: control group: vehicle alone
PBS by s.c. route [0120] Group 2: dosed with 150 .mu.g of IDX9052
by s.c. route [0121] Group 3: dosed with 150 .mu.g of IDX9054 by
s.c. route [0122] Group 4: dosed with 150 .mu.g of IDX0980 by s.c.
route
[0123] The number of animals per group was the minimum number
allowing an accurate assessment of observed pharmacological
effects.
[0124] The second study involved three groups of 16 animals each.
All the groups were immunized with rat MOG in IFA, according to the
immunization protocol. All compounds were immunized three times.
[0125] Group 1: dosed with 150 .mu.g of IDX9054 by s.c route [0126]
Group 2: control group: vehicle alone PBS by i.n. route [0127]
Group 3: dosed with 150 .mu.g of IDX9054 by i.n route
[0128] Clinical observations: The animals were visually inspected
at least twice daily for evidence of reaction to treatment or
ill-health. Starting from day 5 the animals were examined
individually for the presence of paralysis by means of a clinical
score as follows: [0129] 0=no sign of disease [0130] 1=tail
weakness or tail paralysis [0131] 2=hind leg paraparesis or
hemiparesis [0132] 3=hind leg paralysis or hemiparalysis [0133]
4=complete paralysis (tetraparaplegy) [0134] 5=moribund state or
death
[0135] Ataxia was routinely assessed. A disease remission was
defined as an improvement in disease score for at least 2 days
consecutively. A relapse was defined as an increase in the clinical
deficit of at least 2 points that lasted for at least 2 days.
[0136] The results of the clinical scores were expressed as the
mean (.+-.SEM) score within each group. The effects of the test
substances were compared with that of the vehicle-treated control
group. Differences of clinical score values among groups were
analyzed by Kruskal-Wallis test followed, in case of significance,
by the pair wise Wilcoxon test, at each measurement time.
[0137] Observation of the animals took place in a quiet room.
Clinical signs were monitored daily in each group of treatment in a
blind fashion by a technician who was unaware of the treatments.
The body weight of the animals was also monitored daily.
[0138] Animals considered to be in pain, distress or in moribund
condition were examined by the staff veterinarian or authorized
personnel and, if necessary, humanely sacrificed to minimize undue
pain or suffering.
Results
[0139] Animal status: In the first study all animals tolerated s.c
treatment with vehicle or IDX9052, IDX9054 and IDX0980 without any
adverse behavioral or physical effects as assessed daily. In the
second study, all animals tolerated i.n and s.c. treatment with
vehicle or IDX9054 without any adverse behavioral or physical
effects as assessed daily, except from one animal in the PBS i.n
control group that died immediately following a treatment, an event
not considered to be natural. One animal in the same group
developed arthritis and was scarified according to ethical
guidelines.
[0140] At disease debut there was the usual associated drop in
weight which continued thereafter in all studies. Due to the
variable disease incidence and mortality between groups,
statistical analysis of differences between groups is not
informative. However, both IDX9054 and IDX0980-treated groups lost
less weight than PBS control group in the first study and in the
second study IDX9054 i.n and s.c--treated groups lost less weight
than PBS i.n control group.
[0141] Clinical disease course: In study number one all rat groups
were assessed daily for clinical signs of disease. They began to
develop EAE from day 10 (FIG. 6). Four animals in the IDX0980
treated group and three animals in the IDX9054 treated groups never
developed disease, but otherwise all other groups reached 100%
clinical disease incidence by day 16 (FIG. 7). There was a
significant difference between incidence in the IDX0980 treated
group and the control group (p=0.0285). All animals are included in
the presented analysis. In the second study, rats began to develop
EAE from day 11 (FIG. 8). Five animals in both the s.c and i.n
IDX9054 treated groups never developed disease, but the PBS i.n
group reached 100% clinical disease incidence by day 26. There was
a significant difference between incidence in the IDX9054 s.c and
i.n--treated groups compared to the i.n control group (p=0.02).
[0142] Disease course developed with a relapsing-remitting course
which progressively worsened over time. As not all animals in a
group developed disease at exactly the same time, and their disease
courses were thus not completely in-phase with each other, the
standard deviations of the mean values presented are variable (Not
shown).
[0143] In the first study, there was a significant difference in
onset of EAE in the IDX9054 treated group (p=0.0318), being
slightly delayed compared to the control group. While there was no
statistically significant difference in either mean disease
severity, cumulative score or mean maximum score between groups,
there was a definite tendency that both IDX0980- and
IDX9054-treated groups had reduced weight loss, reduced disease
severity and lower mortality rates than both other groups (FIG. 9).
In the second study, there was no statistically significant
difference in either onset of EAE mean disease severity, cumulative
score or mean maximum score between groups, but there was a
definite tendency that both i.n- and s.c-IDX9054-treated groups had
reduced weight loss, reduced disease severity and lower mortality
rates than the control PBS i.n group. The cumulative score and mean
maximal score for the i.n-IDX9054-treated group was marginally less
than in the s.c-IDX9054-treated group.
[0144] This MOG-EAE model is a severe disease model, and rats
either died or were sacrificed due to ethical regulations in each
group, being assigned a maximum score of 5 or 4, respectively,
thereafter in all presented analyses. The mortality was generally
high, but reflects the natural variation we routinely experience in
the model.
[0145] A summary of all clinical data is presented in Tables 3-4,
and a summary of the statistical analysis is presented below each
table.
TABLE-US-00003 TABLE 3 Summary of pre-clinical data from the first
EAE study Mean Inci- Cumu- Mean Mortality Weight dence lative
cumulative % (no./ Change GROUP n (%) Score Score (SD) total) gram
0980 s.c 12 67 543 45 (37) 50.0 (6/12) +8.25 9054 s.c 12 75 604 50
(38) 58.3 (7/12) +3.88 PBS s.c 12 100 793 66 (24) 66.6 (8/12) -2.67
9052 s.c 12 100 762 64 (24) 66.6 (8/12) -4.16
P-Values for Pre-Clinical Study Number One
IDX0980 vs PBS
[0146] CUMULATIVE SCORE p=0.2128 [0147] MAX SCORE p=0.2444 [0148]
DISEASE DURATION p=0.3008 [0149] DAY OF ONSET p=0.2873 [0150]
INCIDENCE p=0.0285*
IDX9054 vs PBS
[0150] [0151] CUMULATIVE SCORE p=0.3548 [0152] MAX SCORE p=0.4490
[0153] DISEASE DURATION p=0.1018 [0154] DAY OF ONSET p=0.0318*
[0155] INCIDENCE p=0.0641
IDX9052 vs PBS
[0155] [0156] CUMULATIVE SCORE p=0.6858 [0157] MAX SCORE p=0.6806
[0158] DISEASE DURATION p=0.3907 [0159] DAY OF ONSET p=0.6123
[0160] INCIDENCE p=-(both 100%)
TABLE-US-00004 [0160] TABLE 4 Summary of pre-clinical data from the
second EAE study Mean Inci- Cumu- Mean Mortality Weight dence
lative cumulative % (no./ Change GROUP n (%) Score Score (SD)
total) gram PBS i.n 16 100 867 62 (25) 50.0 (7/14).sup. -8.71
IDX-9054 16 68 672 42 (39) 21 (5/16) +11.44 i.n IDX-9054 16 68 726
45 (39) 21 (5/16) +6.3 s.c
P-Values for Pre-Clinical Study Number Two
IDX9054 i.n vs PBS i.n
[0161] CUMULATIVE SCORE p=0.20 [0162] MAX SCORE p=0.055 [0163]
DISEASE DURATION p=0.29 [0164] DAY OF ONSET p=0.17 [0165] INCIDENCE
p=0.02*
IDX9054 s.c vs PBS i.n
[0165] [0166] CUMULATIVE SCORE p=0.21 [0167] MAX SCORE p=0.12
[0168] DISEASE DURATION p=0.24 [0169] DAY OF ONSET p=0.16 [0170]
INCIDENCE p=0.02*
[0171] MOG-induced EAE in DA rats is a well characterized
experimental model with high reproducibility. It shares many
features with its human counterpart multiple sclerosis, and as such
is an appropriate model for therapeutic testing (Gold et al., 2006,
Friese et al., 2006).
[0172] Following immunization with MOG in IFA, animals become
progressively paralyzed from the tail, through the back legs to the
front legs due to progressive degeneration of myelin caused by
infiltrating inflammatory immune cells into the spinal cord and
brain. Pathogenesis is chronic relapsing, with animals partially
recovering and then relapsing with heightened disease, and is a
result of both monocytes and T cell (type 1 cytokines) and B cell
(antibody) activities.
[0173] The test substances are oligonucleotides with proven
immunostimulatory activity in vitro. Their specific
immunostimulatory profile differs depending on the sequence of the
oligonucleotide, which makes comparative analyses of their effects
in vivo on ongoing inflammatory responses of therapeutic interest.
The purpose of the two EAE-animal studies was to investigate the
effect of IDX9052, IDX9054 and IDX0980 in a model of
relapsing/remitting MOG-induced EAE in DA rats.
[0174] Treatment with all test oligonucleotides, or with vehicle
alone, did not cause any adverse physiological or behavioral
effects in recipient rats. Treatment was initiated at just prior to
the first bout of disease, in order to have first allowed disease
to develop normally but then to dampen the ongoing proinflammatory
cascade. Additional administrations were timed to coincide with
height of disease and start of the clinical disease phase. Recovery
from this first period of EAE was more pronounced in both IDX0980-
and IDX9054-treated groups, and development of subsequent clinical
bouts was less pronounced in the IDX0980-treated group. All
measured parameters were reduced in both IDX0980- and
IDX9054-treated groups compared to vehicle-treated groups.
[0175] SEQ ID NO 5 (IDX9052) was a strong inducer of IFN alpha/beta
in rat splenocytes but showed no reduced severity of disease in the
EAE rat model. Thus, the reduced severity of disease observed in
the EAE rat model upon treatment with IDX9054 and IDX0980 can not
be correlated to only IFN beta production. Furthermore, the results
obtained with SEQ ID NO 2 (IDX9054) indicate that this compound has
a therapeutic effect, both s.c. and i.n. in MOG-EAE in DA rats.
Example 4
Reduction of CD49d in Vitro in Patient Samples
[0176] To investigate if the inventive compounds can reduce CD49d
expression the inventors used PBMC isolated from RRMS patients for
studying expression of CD49d upon stimulation with candidate
compounds.
Material and Methods
[0177] PBMC from RRMS patients (n=9) was obtained from BD CPT
vacutainer (Becton Dickinson). The cells were immediately incubated
at 37.degree. C. in a volume of 500 .mu.l of complete RPMI-medium
(containing 10% FCS, 1% PenStrep, 2 mM L-glutamine,10 mM HEPES and
1 mM Sodium Pyruvate) in 48-well plates at a concentration of
2.times.10.sup.6 cells/ml and treated with 1, 10 and 25 .mu.M of
each of the oligonucleotide compounds (Table 1). Cells incubated
with oligonucleotides were harvested after 48 h, washed in PBS and
re-suspended in PBS supplemented with 2% FCS. The cells were
stained with two different sets of fluorochrome conjugated antibody
mixtures; (1) anti-CD3 APC, anti-CD49d PE and (2) anti-CD19 PE Cy7,
anti-CD49d APC for 30 min at 4.degree. C. The antibodies were
purchased from Becton Dickinson. After staining, the cells were
washed in PBS and subsequently analyzed by FACS using a FACSArray
bioanalyzer (Becton Dickinson).
Results
[0178] PBMC isolated from RRMS patients showed down-regulation of
CD49d on T cells in a dose dependent manner upon stimulation with
the oligonucleotides (FIG. 10).
[0179] A decrease in CD49d expression on cells was observed in PBMC
isolated from RRMS patients treated with the inventive compounds.
These properties of the inventive compounds could reduce the
transmigration and thereby the influx of cells into CNS.
Example 5
Reduction of Chemokine Receptors in Vitro in Patient Samples
[0180] The influx of blood mononuclear cells (e.g. T-cells,
B-cells, monocytes) in CNS plays a crucial role in the pathogenesis
of MS. Blocking or reducing the influx would therefore be
beneficial for the treatment of MS. Chemokine receptors like CCR5
(CD195), CCR2 (CD192), and CXCR3 (CD183) are expressed on
mononuclear cells and are involved in recruitment of the cells to
the site of inflammation. To investigate if the inventive compounds
can reduce expression of said chemokine receptors, the inventors
stimulated PBMC isolated from RRMS patients with the inventive
compounds.
Material and Methods
[0181] PBMCs from RRMS patients (n=3) were isolated using BD CPT
vacutainer. The cells were immediately incubated at 37.degree. C.
in a volume of 500 .mu.l of complete RPMI-medium (containing 10%
FCS, 1% PenStrep, 2 mM L-glutamine, 10 mM HEPES and 1 mM Sodium
Pyruvate) in 48-well plates at a concentration of 2.times.10.sup.6
cells/ml and treated with 1, 10 and 25 .mu.M of each inventive
compounds. Cells incubated with the oligonucleotides were harvested
after 48 h, washed in PBS and re-suspended in PBS supplemented with
2% FCS. The cells were stained with three different sets of
fluorochrome conjugated antibody mixtures; (1) anti-CD3 PE-Cy-7,
anti-CCR5 APC-Cy7, anti-CCR2 Alexa Fluor 647, anti-CXCR3-PE, (2)
anti-CD19 PE-Cy-7, anti-CCR5 APC-Cy7, anti-CCR2 Alexa Fluor 647,
anti-CXCR3 PE and (3) anti-CD14 PE-Cy-7, anti-CCR5 APC-Cy7,
anti-CCR2 Alexa Fluor 647, anti-CXCR3 PE, for 30 min at 4.degree.
C. The antibodies were purchased from Becton Dickinson. After
washing in PBS, the cells were analyzed using a FACSarray flow
cytometer (Becton Dickinson).
Results
[0182] PBMC isolated from RRMS patients (n=3) showed
down-regulation of CXCR3 on T cells (CD3 positive) after
stimulation with inventive compounds, especially by IDX9045 (FIG.
11). CCR5 was also down-regulated on T cells after oligonucleotide
treatment, especially by IDX9022 (data not shown).
[0183] PBMC isolated from RRMS patients (n=3) showed
down-regulation of CXCR3 on CD19 positive cells. The
oligonucleotides that showed the most potent down-regulatory effect
of this receptor were IDX9038, IDX9054, IDX9058, IDX9045, IDX9004,
and IDX0980 (FIG. 12).
[0184] PBMC isolated from RRMS patients (n=3) showed
down-regulation of CXCR3, CCR5 and CCR2 on CD14 positive cells. The
oligonucleotides showing the most potent down-regulatory effect of
CXCR3, CCR5 and CCR2 are shown in FIGS. 13, 14 and 15A and B,
respectively.
[0185] A decrease in chemokine receptor (CCR5, CXCR3, CCR2)
expression on T, B and monocytes was observed in PBMC isolated from
RRMS patients treated with the inventive compounds. These
properties of the inventive compounds could play important role in
reduction of the migration of these cells towards the CNS.
Example 6
Reduction of Chemotaxis of Leukocytes Isolated from RRMS
Patients
[0186] MCP-1 and RANTES are potent chemokines for the recruitment
of blood mononuclear cells, in particular monocytes, T-cells, and
B-cells to the site of inflammation. Chemotaxis of blood
mononuclear cells towards MCP-1 and RANTES is mediated mainly
through CCR2, and CCR5 receptors, respectively. The aim of this
study was to demonstrate that the decreased expression of CCR2, and
CCR5 by inventive compounds as shown in example 5, can indeed
reduce the migration of mononuclear cells.
Material and Methods
[0187] Chemotaxis of blood mononuclear cells was investigated using
the QCM.TM. colorimetric chemotaxis assay (Millipore, Temecula,
Calif.) according to the manufacturer's instructions. Briefly,
PBMCs were isolated from RRMS patients using BD CPT vacutainer and
treated with 1, 10 and 25 .mu.M of inventive compounds as described
in example 5. After 48 h the cells were washed and transferred
(3.times.10.sup.5 cells in 250 .mu.l medium) to the top inserts of
24-well cell migration plate assembly having a pore size of 3
.mu.m. 300 .mu.l of medium containing chemo-attractants MCP-1 (10
ng/mL) and RANTES (10 ng/mL) was then added to the lower chamber.
The cells were then allowed to migrate through the filter towards
the chemoattractant for 16 h at 37.degree. C. in a humidified cell
culture incubator (Thermo Scientific) with 5% CO.sub.2 in air.
Thereafter, the cells from the lower chamber, i.e. migrated cells,
were detected by incubation with the cell viability stain WST-1 for
1 h followed by quantification by measuring the absorbance at 450
nm using a microplate reader (Tecan, Mannedorf, Switzerland).
Results
[0188] PBMCs isolated from two different RRMS patients treated with
inventive compounds (IDX9045, IDX9054, IDX0980) showed less
migration than untreated cells towards the chemo-attractants in a
functional migration assay (FIG. 16A-B).
[0189] This indicates that the reduction of cell migration is due
to a lower expression of the receptors. There is reason to predict
that these results also reflect an in vivo scenario, consequently
leading to less chemotaxis of cells into the central nervous
system.
Example 7
Reduction of VEGF in Vitro in Patient Samples
[0190] The active lesions in MS are characterized by blood-brain
barrier (BBB) breakdown, suggesting that altered vessel
permeability is involved in the pathogenesis of the disease. In
patients with MS, it has been reported that VEGF induces blood
vessel permeability thereby increases the influx of the
autoaggressive cells into CNS (Proescholdt M A et al., 2002). To
investigate if the inventive compounds can reduce production of
VEGF, the inventors used PBMC isolated from RRMS patients for
studying production of VEGF upon stimulation with the inventive
compounds.
Material and Methods
[0191] PBMCs were isolated from RRMS patients (n=6-11) using BD CPT
vacutainer. The cells were immediately incubated at 37.degree. C.
in a volume of 500 .mu.l of complete RPMI-medium in 48-well plates
at a concentration of 2.times.10.sup.6 cells/ml and treated with 1,
10 and 25 .mu.M of each inventive compounds. After 48 h the
supernatants were analyzed for presence of VEGF using cytometric
bead array (CBA, Becton Dickinson).
Results
[0192] PBMC isolated from RRMS patients showed significant VEGF
reduction in cell supernatants after stimulation with IDX9038,
IDX9045, IDX9004, and IDX0980 (FIG. 17), however, IDX9022, IDX9058,
IDX9054, IDX9060, IDX0150 and IDX9052 did not reduce VEGF in the
cell supernatant (data not shown).
[0193] A reduction of VEGF was observed in the cell supernatant
from cells treated with the inventive compounds. This property of
the inventive compounds is believed to reduce vascular permeability
of the BBB and thereby prevent the infiltration of immune cells
into CNS.
Example 8
Induction of IFN-Beta in Vitro in Patient Samples
[0194] The inventors used blood from RRMS patients to test
different candidate compounds in vitro for induction of
IFN-beta.
Material and Methods
[0195] PBMCs were isolated from RRMS patients (n=6) using BD CPT
vacutainer. The cells were immediately incubated at 37.degree. C.
in a volume of 500 .mu.l of complete RPMI-medium in 48-well plates
at a conc. of 2.times.10.sup.6 cells/ml and treated with 1, 10 and
25 .mu.M of each of oligonucleotide compounds. After 48 h the
supernatants were analyzed for IFN-beta production using an
IFN-beta ELISA kit (Invitrogen).
Results
[0196] PBMC isolated from RRMS patients showed significant IFN-beta
production in cell supernatants after stimulation with IDX9058,
IDX9045, IDX9004, IDX9054, IDX9060 and IDX0980 after 48 h (FIG.
18). IDX9022, IDX9038 and IDX 9052 did not induce significant
IFN-beta production in supernatant (data not shown). IDX0150 showed
no IFN-beta production at all (data not shown).
[0197] An increased IFN-beta production was observed in cell
supernatant from PBMC isolated from RRMS patients treated with the
inventive compounds. This property of the inventive compounds to
elicit IFN-beta production could contribute to less inflammation
due to the known beneficial effects of this cytokine, as currently
used in RRMS therapy.
[0198] In general, the results of the experiments performed by the
inventors indicate that the oligonucleotides can be effective in an
in vivo situation where an inhibition or reduction of mononuclear
cells to the central nervous system could be beneficial for the
treatment of inflammatory diseases of the CNS. This beneficial
effect of the inventive compounds can be mediated through
down-regulating the expression of at least one cell surface marker
or VEGF.
[0199] Although the invention has been described with regard to its
preferred embodiments, which constitute the best mode presently
known to the inventor, it should be understood that various changes
and modifications as would be obvious to one having the ordinary
skill in this art may be made without departing from the scope of
the invention as set forth in the claims appended hereto.
REFERENCES
[0200] FDA News P04-107, Nov. 23, 2004. [0201] Friese, M A et al.,
The value of animal models for drug development in multiple
sclerosis, Brain, 2006; 129 (Pt 8):1940-52. [0202] Gold et al.,
Understanding pathogenesis and therapy of multiple sclerosis via
animal models: 70 years of merits and culprits in experimental
autoimmune encephalomyelitis research, Brain, 2006; 129 (Pt
8):1953-71. [0203] Hafler, D A, Multiple sclerosis. J Clin Invest,
2004. [0204] Kerkmann, M. et al., Spontaneous formation of nucleic
acid-based nanoparticles is responsible for high interferon-alpha
induction by CpG-A in plasmacytoid dendritic cells, J Biol Chem,
2005; 280(9):8086-93. [0205] Wikstrom, F H et al.,
Structure-dependent modulation of alpha interferon production by
porcine circovirus 2 oligodeoxyribonucleotide and CpG DNAs in
porcine peripheral blood mononuclear cells., J Virol. 2007;
81(10):4919-27. [0206] Proescholdt M A. et al, Vascular endothelial
growth factor is expressed in multiple sclerosis plaques and can
induce inflammatory lesions in experimental allergic
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61(10):914-25 [0207] Trebst C, Ransohoff R M. Investigating
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Sequence CWU 1
1
10113DNAArtificial SequenceSynthesized 1gggtcgcagc tgg
13215DNAArtificial SequenceSynthesized 2ggggtcgtct gcggg
15321DNAArtificial SequenceSynthesized 3tcgtcgttcg gccgatcgtc c
21415DNAArtificial SequenceSynthesized 4ggggtcgcag ctggg
15514DNAArtificial SequenceSynthesized 5ggggtcgtct gcgg
14622DNAArtificial SequenceSynthesized 6tcgtcgttct gccatcgtcg tt
22716DNAArtificial SequenceSynthesized 7gatcgtccgt cggggg
16815DNAArtificial SequenceSynthesized 8ggggatcgtc cgggg
15919DNAArtificial SequenceSynthesized 9ggaacagttc gtccatggc
191020DNAArtificial SequenceSynthesized 10gggggacgat cgtcgggggg
20
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