U.S. patent application number 12/632727 was filed with the patent office on 2010-12-09 for immunomodulatory polymeric antigens for treating inflammatory pathologies.
Invention is credited to Neil Thomas BLACKBURN, Larry Chris BLASZCZAK, Kathleen Ann TAYLOR.
Application Number | 20100310470 12/632727 |
Document ID | / |
Family ID | 27807987 |
Filed Date | 2010-12-09 |
United States Patent
Application |
20100310470 |
Kind Code |
A1 |
TAYLOR; Kathleen Ann ; et
al. |
December 9, 2010 |
IMMUNOMODULATORY POLYMERIC ANTIGENS FOR TREATING INFLAMMATORY
PATHOLOGIES
Abstract
Provided are natural and synthetic immunomodulatory polymeric
antigens (SPAs), compositions containing SPAs, and methods of using
these natural and synthetic SPAs and compositions to prevent or
treat inflammatory pathologies.
Inventors: |
TAYLOR; Kathleen Ann;
(Fishers, IN) ; BLASZCZAK; Larry Chris;
(Indianapolis, IN) ; BLACKBURN; Neil Thomas;
(Indianapolis, IN) |
Correspondence
Address: |
TechLaw LLP
10755 Scripps Poway Parkway, Suite 465
San Diego
CA
92131
US
|
Family ID: |
27807987 |
Appl. No.: |
12/632727 |
Filed: |
December 7, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10506312 |
Sep 1, 2004 |
7629313 |
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PCT/US03/05575 |
Mar 7, 2003 |
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12632727 |
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60363065 |
Mar 8, 2002 |
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60365211 |
Mar 15, 2002 |
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Current U.S.
Class: |
424/9.8 ;
424/93.7; 424/93.71; 435/375; 514/54 |
Current CPC
Class: |
A61P 17/00 20180101;
C12N 5/0639 20130101; C12N 5/064 20130101; A61K 2039/5154 20130101;
A61P 37/00 20180101; A61P 41/00 20180101; A61K 31/726 20130101;
A61K 38/14 20130101; A61P 29/00 20180101 |
Class at
Publication: |
424/9.8 ;
435/375; 424/93.7; 514/54; 424/93.71 |
International
Class: |
A61K 49/00 20060101
A61K049/00; C12N 5/02 20060101 C12N005/02; A61K 35/12 20060101
A61K035/12; A61K 31/715 20060101 A61K031/715; A61K 35/26 20060101
A61K035/26 |
Claims
1. A method of inhibiting the maturation of an antigen presenting
cell, comprising contacting in vitro said antigen presenting cell
and an effective amount of a compound selected from the group
consisting of CP1, Compound 15, and mixtures thereof, for a time
and under conditions effective to inhibit maturation of said
antigen presenting cell.
2. The method of claim 1, wherein said antigen presenting cell is a
dendritic cell.
3. The method of claim 1, wherein inhibition of maturation of said
antigen presenting cell is accompanied by a reduction in the level
of expression of one or more surface markers selected from the
group consisting of CD80, CD86, and MHC II by said antigen
presenting cell.
4. The method of claim 1, wherein inhibition of maturation of said
antigen presenting cell is accompanied by a reduction in the level
of expression of one or more cytokines selected from the group
consisting of IL6, IL12, interferon alpha, and interferon gamma by
said antigen presenting cell.
5. A method of inhibiting the maturation of an antigen presenting
cell in a mammal, comprising administering to a mammal other than a
rat or a mouse an effective amount of a compound selected from the
group consisting of CP1, Compound 15, and mixtures thereof, and
inhibiting maturation of said antigen presenting cell.
6. The method of claim 5, wherein said antigen presenting cell is a
dendritic cell.
7. The method of claim 5, wherein inhibition of maturation of said
antigen presenting cell is accompanied by a reduction in the level
of expression of one or more surface markers selected from the
group consisting of CD80, CD86, and MHC II by said antigen
presenting cell.
8. The method of claim 5, wherein inhibition of maturation of said
antigen presenting cell is accompanied by a reduction in the level
of expression of one or more cytokines selected from the group
consisting of IL6, IL12, interferon alpha, and interferon gamma by
said antigen presenting cell.
9. A method of inhibiting an inflammatory response in a mammal in
need thereof other than a rat or a mouse, comprising: (a) isolating
peripheral blood mononuclear cells, or a monocyte-containing
fraction thereof, from said mammal; (b) contacting in vitro said
isolated peripheral blood mononuclear cells or monocytes and a
composition containing an effective amount of cytokines that
differentiate monocytes to immature dendritic cells for a time and
under conditions effective to generate immature monocyte-derived
dendritic cells; (c) contacting in vitro said immature
monocyte-derived dendritic cells and an effective amount of a
compound selected from the group consisting of CP1, Compound 15,
and a mixture thereof for a time and under conditions effective to
prevent maturation of said immature monocyte-derived dendritic
cells; and (d) administering said immature monocyte-derived
dendritic cells to said mammal, reducing the ability of dendritic
cells of said mammal to drive cognate interactions with T cells and
inhibiting said inflammatory response in said mammal.
10. The method of claim 9, wherein said cytokine composition of
step (b) comprises granulocyte-macrophage colony-stimulating factor
and IL4.
11. The method of claim 9, wherein said inflammatory response is
selected from the group consisting of abscesses and post-surgical
adhesions, sepsis; rheumatoid arthritis; myesthenia gravis;
inflammatory bowel disease; colitis; systemic lupus erythematosis;
multiple sclerosis; coronary artery disease; diabetes; hepatic
fibrosis; psoriasis; eczema; acute respiratory distress syndrome;
acute inflammatory pancreatitis; endoscopic retrograde
cholangiopancreatography-induced pancreatitis; burns; atherogenesis
of coronary, cerebral, and peripheral arteries; appendicitis;
cholecystitis; diverticulitis; visceral fibrotic disorders; wound
healing; skin scarring disorders; granulomatous disorders; asthma;
pyoderma gangrenosum; Sweet's syndrome; Behcet's disease; primary
sclerosing cholangitis; and cell, tissue, or organ
transplantation.
12. A method of inhibiting an inflammatory response in a mammal in
need thereof other than a rat or a mouse, comprising: administering
to said mammal an effective amount of a compound selected from the
group consisting of CP1, Compound 15, and mixtures thereof,
preventing dendritic cells or other antigen presenting cells of
said mammal from maturing and rendering them incapable of
stimulating T cell activation, thereby inhibiting said inflammatory
response in said mammal.
13. The method of claim 12, wherein said antigen presenting cells
are B cells or macrophages.
14. The method of claim 12, wherein said inflammatory response is
selected from the group consisting of abscesses and post-surgical
adhesions, sepsis; rheumatoid arthritis; myesthenia gravis;
inflammatory bowel disease; colitis; systemic lupus erythematosis;
multiple sclerosis; coronary artery disease; diabetes; hepatic
fibrosis; psoriasis; eczema; acute respiratory distress syndrome;
acute inflammatory pancreatitis; endoscopic retrograde
cholangiopancreatography-induced pancreatitis; burns; atherogenesis
of coronary, cerebral, and peripheral arteries; appendicitis;
cholecystitis; diverticulitis; visceral fibrotic disorders; wound
healing; skin scarring disorders; granulomatous disorders; asthma;
pyoderma gangrenosum; Sweet's syndrome; Behcet's disease; primary
sclerosing cholangitis; and cell, tissue, or organ
transplantation.
15. A method of inhibiting an inflammatory response in a mammal in
need thereof other than a rat or a mouse, comprising: (a) isolating
peripheral blood mononuclear cells, or a monocyte-containing
fraction thereof, from said mammal; (b) contacting in vitro said
isolated peripheral blood mononuclear cells or monocytes and a
composition containing an effective amount of cytokines that
differentiate monocytes to immature dendritic cells for a time and
under conditions effective to generate immature monocyte-derived
dendritic cells; (c) contacting in vitro said immature
monocyte-derived dendritic cells and an effective amount of a
compound selected from the group consisting of CP1, Compound 15,
and mixtures thereof for a time and under conditions effective to
prevent maturation of said immature monocyte-derived dendritic
cells; (d) contacting in vitro said immature dendritic cells and
naive T cells to generate T regulatory cells; and (e) administering
said T regulatory cells that suppress T effector cells to said
mammal, thereby suppressing said inflammatory response.
16. The method of claim 15, further comprising contacting said T
regulatory cells and IL2 for a time and under conditions effective
to exparid the number of said T regulatory cells.
17. The method of claim 15, wherein said inflammatory response is
selected from the group consisting of abscesses and post-surgical
adhesions, sepsis; rheumatoid arthritis; myesthenia gravis;
inflammatory bowel disease; colitis; systemic lupus erythematosis;
multiple sclerosis; coronary artery disease; diabetes; hepatic
fibrosis; psoriasis; eczema; acute respiratory distress syndrome;
acute inflammatory pancreatitis; endoscopic retrograde
cholangiopancreatography-induced pancreatitis; burns; atherogenesis
of coronary, cerebral, and peripheral arteries; appendicitis;
cholecystitis; diverticulitis; visceral fibrotic disorders; wound
healing; skin scarring disorders; granulomatous disorders; asthma;
pyoderma gangrenosum; Sweet's syndrome; Behcet's disease; primary
sclerosing cholangitis; and cell, tissue, or organ
transplantation.
18. A method of inhibiting an inflammatory response in a mammal in
need thereof other than a rat or a mouse, comprising: administering
to said mammal an effective amount of a compound selected from the
group consisting of CP1, Compound 15, and mixtures thereof,
generating T regulatory cells that suppress T effector cells and
that inhibit said inflammatory response.
19. The method of claim 18, wherein generation of said T regulatory
cells is associated with a lack of maturation of dendritic cells or
other antigen presenting cells.
20. The method of claim 19, wherein said antigen presenting cells
are B cells or macrophages.
21. The method of claim 18, wherein said inflammatory response is
selected from the group consisting of abscesses and postsurgical
adhesions, sepsis; rheumatoid arthritis; myesthenia gravis;
inflammatory bowel disease; colitis; systemic lupus erythematosis;
multiple sclerosis; coronary artery disease; diabetes; hepatic
fibrosis; psoriasis; eczema; acute respiratory distress syndrome;
acute inflammatory pancreatitis; endoscopic retrograde
cholangiopancreatography-induced pancreatitis; burns; atherogenesis
of coronary, cerebral, and peripheral arteries; appendicitis;
cholecystitis; diverticulitis; visceral fibrotic disorders; wound
healing; skin scarring disorders; granulomatous disorders; asthma;
pyoderma gangrenosum; Sweet's syndrome; Behcet's disease; primary
sclerosing cholangitis; and cell, tissue, or organ
transplantation.
22. The method of claim 15, wherein expression of both IL10 and
IL19 by said T regulatory cells is upregulated.
23. The method of claim 22, wherein said T regulatory cells are a
subset of CD3+ T cells.
24. The method of any claim 15, wherein expression of IL17 in said
T effector cells is down-regulated.
25. The method of claim 24, wherein said T effector cells are a
subset of CD3+ T cells.
26. A method of measuring the immunological activity of CP1 or
Compound 15 in a mammal, comprising: administering CP1 or Compound
15 to said mammal; administering Candin to said mammal; and
measuring the inhibition of delayed type hypersensitivity skin
lesions elicited by said Candin, wherein a reduction in lesion size
in said mammal compared to lesion size in an untreated control
mammal that has not received CP1 or Compound 15 indicates that said
compounds are effective in inhibiting a localized inflammatory
response.
27. The method of claim 26, wherein said immunological activity is
the activity of T regulatory cells.
28. The method of claim 26, wherein said immunological activity is
associated with inhibition of cognate interactions between antigen
presenting cells and naive T cells.
29. The method of claim 28, wherein said antigen presenting cells
are dendritic cells.
30. The method of claim 28, wherein said antigen presenting cells
are B cells or macrophages.
31-33. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. patent
application Ser. No. 10/506,312 filed Sep. 1, 2004, which is a U.S.
National Phase application based on International Patent
Application No. PCT/US2003/05575 filed Mar. 7, 2003, now U.S. Pat.
No. 7,629,313 issued Dec. 8, 2009, which claims priority to U.S.
provisional application Ser. No. 60/363,065 filed Mar. 8, 2002 and
to U.S. provisional application No. 60/365,211 filed Mar. 15, 2002.
The aforementioned applications are hereby incorporated herein by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the field of immunology,
and more particularly to immunomodulation. The present invention
provides novel methods for preventing and treating inflammatory
pathologies employing natural or synthetic polymeric antigens (N/S
PAs) possessing immunomodulatory properties in these methods. The
present invention also provides a process for preparing novel
synthetic SPAs that can be used to induce the activity of T
regulatory cells and the expression of interleukin 10 (IL10) in
humans and other animals, affording protection against, and/or
treatment for, a wide variety of inflammation-based
pathologies.
[0004] 2. Description of Related Art
[0005] Microbial antigens are the most powerful immunomodulators
known. Among the most common examples are lipopolysaccharide (LPS)
from Gram negative bacteria, and bacterial cell wall glycopeptides,
also known as murein or peptidoglycan (PG), from both Gram negative
and Gram positive bacteria. Bacterial PG is well established as a
potent inflammatory agent (Wahl et al. (1986) J. Exp. Med.
165:884). Many microbial antigens, including PG, are thought to
exert their pro-inflammatory effects by activating one of the
mammalian cell surface receptors known as Toll-like receptors
(TLRs). The TLR then triggers an intracellular signaling pathway
through transcription factor NF-.kappa.B, which in turn induces
expression of genes coding for inflammatory mediators (chemokines
and certain cytokines). PG itself is thought to activate through
TLR2 (Hallman et al. (2001) Pediatr. Res. 50:315).
[0006] Recently, cDNA array technology has brought even higher
resolution to our understanding of pro-inflammatory mediator
induction by PG (Wang et al. (2000) J. Biol. Chem. 275:20260). The
most highly activated genes are those expressing chemokines (IL-8
and MIP-1.beta.), and the second most highly activated genes are
those expressing cytokines (TNF-.alpha., ILL and IL6). Regardless
of mechanistic detail, the downstream effect of bacterial PG on the
host is a potent inflammatory response. In fact, PG has long been
used for induction of arthritis in animal models (Cromartie et al.
(1977) J. Exp. Med. 146:1585). Partially purified PG from the
bacterium Streptococcus pyogenes is now commercially available for
such purpose (Lee Laboratories, Atlanta, Ga.). Fragments of PG,
known collectively as muropeptides, also exhibit inflammatory
effects in animals, and these effects are dependent on muropeptide
structure (Tuomanen et al. (1993) J. Clin. Invest. 92:297). Even
the very smallest fragments of PG, designated muramyl dipeptide
(MDP), and glucosaminyl MDP (GMDP), as well as their derivatives,
exhibit inflammatory effects in animals (Kohashi et al. (1980)
Infect. Immun. 29:70).
[0007] Kasper and Tzianabos have demonstrated that certain
polysaccharides purified from the surface of bacterial cells
exhibit protective effects in vivo when tested in models of
inflammation such as the formation of intraabdominal abscesses,
intraabdominal sepsis, and post-surgical adhesions (U.S. Pat. Nos.
5,679,654 and 5,700,787; PCT International Publications WO
96/07427, WO 00/59515, and WO 02/45708). These investigators have
demonstrated that when purified from whole capsule, certain
polysaccharides derived from Bacteroides fragilis, Staphylococcus
aureus, and Streptococcus pneumoniae have unique characteristics
that set them apart from many polysaccharide antigens. The former
molecules are high molecular weight, helical, and zwitterionic in
nature (Wang et al. (2000) Proc. Natl. Acad. Sci. USA
97:13478-13481, and references 5-9 therein). Most bacterial
polysaccharides are neutral or negatively charged, and are
considered to be T cell-independent antigens (Abbas et al. (2000)
Cellular and Molecular Immunobiology, W.B. Saunders, Philadelphia).
Kasper and Tzianabos suggest that the zwitterionic nature of these
polysaccharides plays a role in the interaction of these molecules
with CD4+ T cells (Tzianabos et al. (1993) Science 262: 416-419;
Tzianabos et al. (2001) Proc. Natl. Acad. Sci. USA 98:9365-9370).
More recent work by this group suggests that some of these
molecules may interact with antigen presenting cells (APCs) via
their zwitterionic characteristics and further, that stimulation of
CD4+ T cells by these polysaccharide antigens is dependent on MHC
II-bearing APCs (Kalka-Moll et al. (2002) J. Immunol.
169:6149-6153). It has yet to be determined precisely how these
interactions between zwitterionic polysaccharides and APCs may
stimulate CD4+ T cells. These investigators have shown that
zwitterionic polysaccharides activate CD4+ T cells in vitro as
evidenced by the stimulation of proliferation and the production of
the cytokines IL2, INF7, and IL10, and that the protection is
adoptively transferred by polysaccharide-stimulated T cells in vivo
(PCT International Publication WO 00/59515; Kalka-Moll et al.
(2000) J. Immunol. 164:719-724; Tzianabos et al. (2000) J. Biol.
Chem. 275:6733-6738). In earlier studies by this group, stimulation
of CD4+ cells did not necessarily depend on the presence of APCs,
and the mitogenic properties of these molecules on T cells derived
from rat and mouse species was different: rat splenocytes
proliferated in response to CP1 treatment, while mouse splencocytes
did not (Tzianabos et al. (1995) J. Clin. Invest. 96:2727-2731;
Brubaker et al. (1999) J. Immunol. 162:2235-2242).
[0008] Overall, however, their observations led this group to
hypothesize that the activation of CD4+ T cells by these
polysaccharides leads to the production of cytokines such as IL2 or
IL10 that protect against inflammatory responses (PCT International
Publication WO 00/59515; Kalka-Moll et al. (2000) J. Immunol.
164:719-724; Tzianabos et al. (2000) J. Biol. Chem. 275:6733-6738;
Tzianabos et al. (1999) J. Immunol. 163: 893-897). It remains
unclear, however, exactly how these molecules activate T cells or
how they exert their protective effects. Further complicating an
understanding of these polysaccharides, this group has reported
other studies indicating that the same zwitterionic polysaccharides
can induce the formation of abscesses in the same in vivo model
where protective effects of these molecules have been observed
(Tzianabos et al. (1993) Science 262: 416-419; Tzianabos et al.
(1994) Infect. Immun. 62:3590-3593). Therefore, from this body of
literature, it is difficult to ascertain the mechanism whereby
these zwitterionic polysaccharides act as modulators of the immune
system.
[0009] Another group of investigators has described
immunomodulatory effects of the exopolysaccharide (capsule-like) of
Paenibacillus jamilae, a gram positive bacillus isolated from olive
mill wastewaters (Ruiz-Bravo et al. (2001) Clin. Diag. Lab.
Immunol. 8:706-710). Although the authors do not disclose the
structural features of this polysaccharide, their results are
similar to the work of Kasper and Tzianabos, summarized above. The
molecule, referred to as CP-7, stimulates the proliferation of
lymphocytes in culture, as well as significant expression of
IFN.gamma. and GMCSF. Further, this group reports that this
compound renders mice resistant to Listeria monocytogenes
infection. The investigators suggest that the mechanism may be
through the stimulation of a Th1 response, which is in direct
contrast to the invention disclosed herein.
[0010] In view of the confusing and sometimes contradictory effects
reported in the literature for various immunomodulatory
polysaccharides, there exists a need in the art for an
understanding of the mechanism of action of protective,
anti-inflammatory immunomodulatory molecules, including
polysaccharides, as well as a need for additional therapeutic
molecules that modulate the immune response in both a safe and
effective manner. Such insight and additional molecules will
facilitate the development of even more effective anti-inflammatory
strategies and therapeutics.
SUMMARY OF THE INVENTION
[0011] Accordingly, in view of the need in the art for an
understanding of the mechanism(s) by which immunomodulatory
polysaccharide antigens induce protection against inflammation, as
well as the need for additional molecules that can be used to
modulate the immune response in humans and animals for
anti-inflammatory therapeutic purposes, the present inventors have
investigated the properties and effects of an immunomodulatory
bacterial polysaccharide and a novel synthetic peptidoglycan on
immune system function. They have discovered that the bacterial
polysaccharide derived from the capsule of Streptococcus
pneumoniae, referred to as CP1, as well as the novel synthetic
peptidoglycan (PG) Compound 15 disclosed herein, which is a
synthetic polymeric antigen, protect against the induction of
inflammation in models of intraabdominal abscesses and
post-surgical adhesions. They have also surprisingly discovered
that when human peripheral blood mononuclear cells (PBMCs) are
treated in vitro with an SPA as disclosed herein, the response is
most notably the expression of IL10. Only minimal and early
expression of IL2, IFN-.gamma., or TNF-.alpha. is observed. The
stimulation of an anti-inflammatory response by the synthetic
peptidoglycan polymer disclosed herein is completely novel and
unexpected in view of the current body of evidence discussed above:
while natural peptidoglycans are inflammatory, the presently
disclosed synthetic peptidoglycan is anti-inflammatory. The
inventors' surprising discovery of the in vitro anti-inflammatory
activity of an SPA contrasts markedly with previously published
observations on the activity of purified bacterial surface
polysaccharides, and prompted them to test the activity of this SPA
in an animal model of inflammation. The inventors observed that
this SPA exhibits protective therapeutic effects in this animal
model of inflammation-based pathology.
[0012] Accordingly, in one aspect, the present invention provides a
synthetic polymeric antigen having the structure shown in Formula
I:
##STR00001## [0013] wherein n is an integral in the range of from
about 375 to about 75, or a pharmaceutically acceptable salt
thereof.
[0014] In another aspect, the present invention provides a
composition, comprising the synthetic polymeric antigen or
pharmaceutically acceptable salt thereof of Formula I, and a
buffer, carrier, diluent, or excipient.
[0015] In another aspect, the present invention provides a
pharmaceutical composition, comprising the synthetic polymeric
antigen or pharmaceutically acceptable salt thereof of Formula I,
and a pharmaceutically acceptable buffer, carrier, diluent, or
excipient.
[0016] In another aspect, the present invention provides a method
of inhibiting the maturation of an antigen presenting cell,
comprising contacting in vitro said antigen presenting cell and an
effective amount of a compound selected from the group consisting
of CP1, Compound 15, and mixtures thereof, for a time and under
conditions effective to inhibit maturation of said antigen
presenting cell.
[0017] In another aspect, the present invention provides a method
of inhibiting the maturation of an antigen presenting cell in a
mammal, comprising administering to a mammal other than a rat or a
mouse an effective amount of a compound selected from the group
consisting of CP1, Compound 15, and mixtures thereof, and
inhibiting maturation of said antigen presenting cell. [0018] In
another aspect, the present invention provides a method of
inhibiting an inflammatory response in a mammal in need thereof
other than a rat or a mouse, comprising: [0019] (a) isolating
peripheral blood mononuclear cells, or a monocyte-containing
fraction thereof, from said mammal; [0020] (b) contacting in vitro
said isolated peripheral blood mononuclear cells or monocytes and a
composition containing an effective amount of cytokines that
differentiate monocytes to immature dendritic cells for a time and
under conditions effective to generate immature monocyte-derived
dendritic cells; [0021] (c) contacting in vitro said immature
monocyte-derived dendritic cells and an effective amount of a
compound selected from the group consisting of CP1, Compound 15,
and a mixture thereof for a time and under conditions effective to
prevent maturation of said immature monocyte-derived dendritic
cells; and [0022] (d) administering said immature monocyte-derived
dendritic cells to said mammal, reducing the ability of dendritic
cells of said mammal to drive cognate interactions with T cells and
inhibiting said inflammatory response in said mammal.
[0023] In this and the other ex vivo methods disclosed herein,
administration of treated cells can be performed intravenously,
intraperitoneally, or via intercardiac route.
[0024] Inflammatory responses that can be treated via the foregoing
and following methods include abscesses and post-surgical
adhesions, sepsis; rheumatoid arthritis; myesthenia gravis;
inflammatory bowel disease; colitis; systemic lupus erythematosis;
multiple sclerosis; coronary artery disease; diabetes; hepatic
fibrosis; psoriasis; eczema; acute respiratory distress syndrome;
acute inflammatory pancreatitis; endoscopic retrograde
cholangiopancreatography-induced pancreatitis; burns; atherogenesis
of coronary, cerebral, and peripheral arteries; appendicitis;
cholecystitis; diverticulitis; visceral fibrotic disorders; wound
healing; skin scarring disorders; granulomatous disorders; asthma;
pyoderma gangrenosum; Sweet's syndrome; Behcet's disease; primary
sclerosing cholangitis; and cell, tissue, or organ
transplantation.
[0025] In yet another aspect, the present invention provides a
method of inhibiting an inflammatory response in a mammal in need
thereof other than a rat or a mouse, comprising: [0026]
administering to said mammal an effective amount of a compound
selected from the group consisting of CP1, Compound 15, and
mixtures thereof, preventing dendritic cells or other antigen
presenting cells of said mammal from maturing and rendering them
incapable of stimulating T cell activation, [0027] thereby
inhibiting said inflammatory response in said mammal.
[0028] In another aspect, the present invention provides a method
of inhibiting an inflammatory response in a mammal in need thereof
other than a rat or a mouse, comprising: [0029] (a) isolating
peripheral blood mononuclear cells, or a monocyte-containing
fraction thereof, from said mammal; [0030] (b) contacting in vitro
said isolated peripheral blood mononuclear cells or monocytes and a
composition containing an effective amount of cytokines that
differentiate monocytes to immature dendritic cells for a time and
under conditions effective to generate immature monocyte-derived
dendritic cells; [0031] (c) contacting in vitro said immature
monocyte-derived dendritic cells and an effective amount of a
compound selected from the group consisting of CP1, Compound 15,
and mixtures thereof for a time and under conditions effective to
prevent maturation of said immature monocyte-derived dendritic
cells; [0032] (d) contacting in vitro said immature dendritic cells
and naive T cells to generate T regulatory cells; and [0033] (e)
administering said T regulatory cells that suppress T effector
cells to said mammal, [0034] thereby suppressing said inflammatory
response.
[0035] In a further aspect, the present invention provides a method
of inhibiting an inflammatory response in a mammal in need thereof
other than a rat or a mouse, comprising: [0036] administering to
said mammal an effective amount of a compound selected from the
group consisting of CP1, Compound 15, and mixtures thereof, [0037]
generating T regulatory cells that suppress T effector cells and
that inhibit said inflammatory response.
[0038] In another aspect, the present invention provides a method
of measuring the immunological activity of CP1 or Compound 15 in a
mammal, comprising: [0039] administering CP1 or Compound 15 to said
mammal; [0040] administering Candin to said mammal; and [0041]
measuring the inhibition of delayed type hypersensitivity skin
lesions elicited by said Candin, [0042] wherein a reduction in
lesion size in said mammal compared to lesion size in an untreated
control mammal that has not received CP1 or Compound 15 indicates
that said compounds are effective in inhibiting a localized
inflammatory response.
[0043] Further scope of the applicability of the present invention
will become apparent from the detailed description provided below.
However, it should be understood that the detailed description and
specific examples, while indicating preferred embodiments of the
present invention, are given by way of illustration only since
various changes and modifications within the spirit and scope of
the invention will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] The above and other aspects, features, and advantages of the
present invention will be better understood from the following
detailed description taken in conjunction with the accompanying
drawings, all of which are given by way of illustration only, and
are not limitative of the present invention, in which:
[0045] FIG. 1 is a schematic showing the normal events that occur
when interactions between dendritic cells and T cells lead to
inflammation or adaptive immunity
[0046] FIG. 2 is a schematic showing the T regulatory cell
hypothesis of the present invention.
[0047] FIG. 3 shows the cytokine profile from human peripheral
blood mononuclear cells (PBMCs) treated with CP1 or Compound 15
(PG). Human PBMCs in culture are treated with CP1 at 6.0
micrograms/ml (panel A) or PG at 0.6 micrograms/ml (panel B), and
the expression of cytokines is measured over the course of eight
days. Results are normalized against untreated media controls. Data
are expressed as the average of triplicate wells 3.+-.the standard
error of the concentration of cytokines represented. The results
show that the primary response to treatment with these molecules is
the expression of IL10.
[0048] FIG. 4 shows Confocal microscope images of human iDCs
treated with either FITC-Dextran (FITC-Dx, 40 kDa in size) or
Oregon-green labeled Compound 15 (OG-PG, approx. 150 kDa in size)
for two minutes. After incubation with the polymers, the cells are
washed extensively to remove any external polymer and the
internalized material followed at two-minute intervals.
Localization of polymer in endocytic vacuoles can be seen using
either compound, and fluorescence is visualized in the photographs
as white punctate material within the dark field of the cells.
[0049] FIG. 5 shows flow cytometric analysis of uptake of either
FITC-Dextran (panel A) or Oregon-green labeled Compound 15 (panel
B) by human DC at 37.degree. C. or 0.degree. C., respectively. Each
histogram shows the mean fluorescence intensity of fluorescent
signal versus cell number at the time intervals indicated. The
results show that the uptake of each molecule is similar, and that
this uptake is inhibited when the cells are metabolically inactive
at 0.degree. C.
[0050] FIG. 6 shows that neither CP1 nor PG induces PBMCs to divide
in culture. Isolated PBMCs are incubated with 50 .mu.g/ml CP1 ( ),
100 .mu.g/ml pg (.largecircle.), 25 .mu.g/ml PHA (.gradient.), or
left untreated () for the number of days indicated. Radioactive
thymidine [.sup.3H]-Thy is added to cultures 18 h prior to each
time point and the amount of radiolabel incorporated by the cells
is measured by scintillation counting. Radioactivity is measured as
counts per minute.
[0051] FIG. 7 shows that CP1 induces an increase in the percent of
CD4+CD25+ T regulatory cells in human PBMCs in a dose-dependent
manner. Isolated PBMCs are incubated with CP1 at 0.6 micrograms/ml
(closed triangle) or 6.0 micrograms/ml (closed square) for the
number of days indicated. Untreated PBMCs (closed circle) are
included as a measure of the base line number of CD4+CD25+ cells
present in the culture.
[0052] FIG. 8 shows that CP1 and synthetic PG Compound 15 inhibit
anti-CD3 antibody-mediated proliferation of human PBMCs. PBMCs are
pre-incubated for 24 hours with 50 mg/ml of CP1 or 100 mg/ml of PG
Compound 15 prior to incubation on tissue culture plates coated
with varying concentrations of anti-CD3 antibody for 48 hours
(panel A) or 72 hours (panel B). Cell proliferation is evaluated
using a .sup.3H-Thymidine incorporation assay followed by liquid
scintillation counting.
DETAILED DESCRIPTION OF THE INVENTION
[0053] The following detailed description of the invention is
provided to aid those skilled in the in practicing the present
invention. Even so, the following detailed description should not
be construed to unduly limit the present invention as modifications
and variations in the embodiments discussed herein can be made by
those of ordinary skill in the art without departing from the
spirit or scope of the present inventive discovery.
[0054] The contents of each of the references cited herein are
herein incorporated by reference in their entirety.
DEFINITIONS
[0055] As used herein, unless indicated otherwise, the following
abbreviations shall be understood to have the following
meanings:
TABLE-US-00001 Abbreviation Reagent or Fragment h or hr hour(s)
min. minute(s)
[0056] As used above, and throughout the description of the
invention, the following terms, unless otherwise indicated, shall
be understood to have the following meanings:
[0057] "Biomarker" means a marker of a specific activity that
correlates with the administration of a drug. Non-limiting examples
of biomarkers include a cell surface receptor, a soluble mediator,
an mRNA message, or an in vivo response that is modulated and that
can be measured.
[0058] "CP1" means a capsular polysaccharide obtainable from
Streptococcus pneumoniae serotype 1. CP1 is used in the methods
disclosed herein in a form isolated and purified as described
below.
[0059] "Effective amount" refers to an amount of a compound or
composition of the present invention effective to produce the
desired or indicated immunologic or therapeutic effect.
[0060] "IL10" is an endogenous mediator that shifts equilibrium
away from inflammation. Directed, endogenous generation of IL10
maximizes efficacy and minimizes toxic effects.
[0061] "Immune cell" means any cell capable of responding or
mounting a response within the entirety of the host immune system.
Generally these cells are referred to as "white blood cells" but
are not necessarily limited to this category. Examples of immune
cells include T and B cells, monocytes, macrophages, natural killer
cells, dendritic cells, antigen presenting cells, and
polymorphonuclear leukocytes.
[0062] "Modulate" means either an increase or a decrease in a
selected parameter.
[0063] The terms "patient" or "subject" refer to mammals including
humans and other primates, and companion, zoo, and farm animals,
including, but not limited to, cats, dogs, rodents, horses, cows,
sheep, pigs, goats, etc.
[0064] "Non-immune cell" means a cell that is not normally involved
in immune responses but that may have the capacity to be modulated
by products of the immune system.
[0065] "SPA''means "synthetic polymeric antigen." Compound 15
disclosed herein, which is a synthetic peptidoglycan (PG), is a
particular SPA. SPAs can be produced by total synthesis.
[0066] "T regulatory cells" or "T.sub.regs" refers to a unique
lineage of immunoregulatory T cells that potently suppress
inflammatory effector T cells in vitro and in vivo. T.sub.regs are
characterized by expression of certain cell surface markers
including, for example, CD4 and CD25 (CD4+/CD25+).
[0067] Natural and Synthetic Polymeric Antigens Useful as
Immunomodulators
[0068] The structures of CP1, synthetic PG (Compound 15), and a
generalized synthetic polymeric antigen (SPA) are shown below:
##STR00002##
[0069] For the present SPAs, "R" represents a substituted
amine.
[0070] For comparison, the structure of natural peptidoglycan
is:
##STR00003## ##STR00004##
[0071] CP1 is a homopolymer of the indicated repeat unit (or one of
its sequence isomers). It exists as a distribution of molecular
weights centered around 270 kilodaltons as judged by size exclusion
chromatography (dextran as standard). The material is isolated as a
hygroscopic white powder that is soluble in water or saline.
[0072] SPAs are homopolymers of the indicated general repeat unit
structure. These polymers resemble bacterial cell wall
peptidoglycans, but are accessed through chemo-enzymatic total
synthesis from N-acetylglucosamine.
[0073] Synthetic PG is an example of an SPA. It is a homopolymer of
the indicated repeat unit, existing as a distribution of molecular
weights centered around 150 kilodaltons. The polymer is a
hygroscopic white powder that is soluble in water or saline.
[0074] Natural peptidoglycan in the bacterial cell wall is a single
covalently closed macromolecule that precisely defines the shape of
a bacterial cell throughout the cell cycle. It is composed of a
rigid axis of parallel polymeric peptidoglycan glycan strands
wherein the repeat unit is .beta.[1,4]-inked
N-acetylglucosaminyl-.beta.[1,4]-N-acetylmuramylpentapeptide. The
glycan strand is helical in shape with about four repeat units per
complete turn of the helix. The more flexible pentapeptide axes
extend N to C from the lactyl carboxyls of the muramic acid
residues. The peptide is generally H.sub.2N-Ala-D-iso-Glu(or
iso-Gln)-Lys(or diaminopim-elate, DAP)-D-Ala-D-Ala-COOH (SEQ ID
NO:1). The peptides may be crosslinked between Lys(or DAP) from a
donor strand to the carbonyl of the penultimate D-Ala of an
acceptor strand. Although the diagram shows complete crosslinking
for clarity, the actual degree of crosslinking in a living cell
varies with genus and is always less than 100%.
[0075] In comparison, synthetic PG Compound 15 dislcosed herein is
linear, i.e., there is no crosslinking in the peptides. In
addition, in amino acid position 2, GABA replaces the naturally
occurring D-iso-Glu (D-iso-Gln) residues.
[0076] As described below in the method of preparation of CP1
(Example 2), the starting material for the preparation of CP1 as
used herein is obtainable from the American Type Culture Collection
(Manassas, Va.) as crude capsular material from Streptococcus
pneumoniae (type 1), originally prepared for production of the
Pneumovax vaccine (Merck Pharmaceuticals). As described above, CP1
as used in the present studies is highly purified, and consists
solely of the CP1 polysaccharide, without the addition of any other
stimulatory antigens or adjuvants. In contrast, Pneumovax vaccine
contains more than 20 capsular type antigens, and is formulated
with an adjuvant. The vaccine is designed to stimulate adaptive
immunity and does so effectively. As such, the immune response to
this vaccine is completely opposite from that observed when using
the present isolated, purified CP1 (or Compound 15).
[0077] The present inventors have discovered that the bacterial
polysaccharide derived from the capsule of Streptococcus pneumoniae
(CP1), as well as the synthetic PG antigen Compound 15 disclosed
herein, protect against the induction of inflammation in models of
intraabdominal abscesses and post-surgical adhesions. As
demonstrated in the examples presented below, investigations into
the mechanism of protection induced by these molecules reveal that
they appear to inhibit the maturation of dendritic cells, the most
powerful antigen presenting cells (APCs) in the immune cell
repertoire. Immature APCs are unable to activate T cells due to the
their inability to signal T cells through co-stimulation. Treatment
of human PBMCs with either molecule fails to stimulate activation
or proliferation of T cells. This is completely unexpected in view
of the literature on both zwitterionic polysaccharides and
naturally occurring peptidoglycans, discussed earlier. Both of
these classes of molecules have been reported to be mitogens for T
cell activation (PCT International Publication WO 00/59515;
Kalka-Moll et al. (2000) J. Immunol. 164:719-724; Tzianabos et al.
(2000) J. Biol. Chem. 275:6733-6738; Levinson et al. (1983) Infect.
Immun. 39:290-296). Furthermore, CP1 and synthetic PG fail to
stimulate Toll-like receptors in reporter cells in vitro, or to
stimulate the expression of inflammatory cytokines in PBMC
cultures, events that would be expected if maturation of APCs
occurs through stimulation of TLR2 or other TLRs (Schwander et al.
(1999) J. Biol. Chem. 274:17406-17409; Medzhitov et al. (2001) Nat.
Rev. Immunol. 6: 135-145) with subsequent activation of T cells
through the expected cognate interactions between the two cells
types in the presence of antigen. The present inventors also
observe an increase in the number of CD4+CD25+ cells present in
PBMC cultures following treatment with CP1, suggesting that
treatment with this molecule creates a population of immature APCs
that drive the stimulation of T regulatory cells within the
culture. This hypothesis is further supported by functional
observations of suppression of proliferation of T cells in PBMC
cultures stimulated with anti-CD3 antibodies following treatment
with the natural or synthetic polymeric antigens. Finally, the
inventors have also surprisingly discovered that when human PBMCs
are treated in vitro with CP1 or synthetic PG Compound 15 as
disclosed herein, the response is most notably the expression of
IL10. Negligible expression of IL2, IFN-.gamma., TNF-.alpha., IL6,
or IL12 is observed. These results are in direct contrast to the
body of literature on the recognition of bacterial polysaccharides
by the immune system. Furthermore, the stimulation of an
anti-inflammatory response by the synthetic peptidoglycan polymer
disclosed herein is completely novel and unexpected in view of the
current body of evidence regarding natural peptidoglycans,
discussed above, indicating that bacterial peptidoglycan is a
potent inflammatory agent. Thus, while natural peptidoglycans are
inflammatory, the presently disclosed synthetic peptidoglycan
Compound 15 is anti-inflammatory. The inventors' surprising
discovery of the in vitro anti-inflammatory activity of this
synthetic peptidoglycan contrasts markedly with previously
published observations on the activity of purified bacterial
peptidoglycans, and prompted them to test the activity of this SPA,
as well as CP1, in animal models of inflammation. As demonstrated
below, the inventors observe that both this synthetic peptidoglycan
as well as CP1 exhibit protective therapeutic effects in this
animal model of inflammation-based pathology.
[0078] Immunomodulatory Activities of Natural and Synthetic
Polymeric Antigens (N/S PAs)
[0079] The N/S PAs of the present invention induce peripheral blood
mononuclear cells (PBMCs) from animals and humans to secrete IL10.
IL10 is a type II cytokine with pleomorphic effects (Moore et al.
(2001) Annu. Rev. Immunol. 19:683-765). It has been shown to have
potent anti-inflammatory activity, down-modulating inflammatory
responses of T effector cells (Morel et al. (2002) Immunol.
106:229-236), dendritic cells (Martin et al. (2003) Immunity
18:155-167), and other antigen presenting cells (Williams et al.
(2002) J. Leuko. Biol. 1.72:800-809). IL10 is produced by a variety
of cell types, including T cells, dendritic cells, monocytes (Moore
et al. (2001) Annu. Rev. Immunol. 19:683-765), and a specialized
sub-set of T cells known as T regulatory (Treg) cells (Suri-Payor
et al (2001) J. Autoimmun. 16:115-123). In many ways, this cytokine
functions to help maintain a dynamic balance within the immune
system. IL10 acts to tamp down unchecked inflammatory responses
that could otherwise be deleterious to the host (Moore et al.
(2001) Annu. Rev. Immunol. 19:683-765).
[0080] Interactions of Natural and Synthetic Polymeric Antigens
with Dendritic Cells
[0081] Most microbial antigens signal the immune system through
highly conserved structural motifs referred to as
pathogen-associated microbial patterns (PAMPs) (Medzhitov (2001)
Nat. Rev. Immunol. 135-145). PAMPs interact with Toll-like
receptors (TLRs) present on a variety of antigen presenting cells
to initiate a signaling cascade that results in the expression of
pro-inflammatory cytokines such as IL12 and IL6, and a variety of
chemokines (Janeway et al. (2002) Annu. Rev. Immunol. 20:197-216).
Activation of antigen presenting cells through TLRs, in particular
dendritic cells, leads to a maturation process that is
characterized by increased expression of surface MHC II molecules
and co-stimulatory molecules such as CD80 and CD86 (Chakraborty et
al. (2000) Clin. Immunol. 94:88-98). This cascade is designed to
marshal early defenders of the innate immune system to respond
immediately to invasion, and forms the basis for the link to
long-standing adaptive immunity through antigen presentation to T
cells (Keller (2001) Immunol. Lett. 78:113-122). Since CP1 is
derived from bacterial capsule and synthetic peptidoglycan Compound
15 is patterned after natural bacterial cell wall-derived
peptidoglycan, one might expect that these polymers would possess
PAMPs that could signal through TLRs. Indeed, natural peptidoglycan
has been shown to be a ligand for TLR2 (Schwandner et al. (1999) J.
Biol. Chem. 274:17406-17409). As surprisingly discovered by the
present inventors, N/S PAs do not appear to activate TLR2 or any
other TLR tested in either human or rodent cells. This is further
evidenced by the lack of expression of IL12, IL6, or other
pro-inflammatory cytokines in PBMC cultures stimulated with N/S
PAs. In addition, human monocyte-derived dendritic cells are not
driven to maturation by stimulation with N/S PAs. Following
treatment with N/S PAs, immature dendritic cells do not demonstrate
the characteristic upregulation in MHC 11, CD80, or CD86 on their
surface, despite the fact that these cells are considered to be the
most potent of antigen presenting cells and avidly internalize
these molecules and concentrate them in endocytic vacuoles.
[0082] Bacterial lipopolysaccharide (LPS) is a powerful TLR4
agonist (Beulter (2002) Curr. Top Microbiol. Immunol.
270:109-120.), and is commonly used as a maturation signal for
immature dendritic cells (Ardavin et al. (2001) Trends Immunol.
22:691-700). LPS specifically upregulates co-stimulatory molecules
such as CD80 and CD86 on dendritic cells (Michelsen et al. (2001)
J. Biol. Chem. 276:25680-25686). These surface molecules are
essential for signaling T cells to elaborate effector functions
such as inflammatory responses. When immature dendritic cells are
co-cultured with N/S PAs and LPS, CD80 and CD86 are not
upregulated, suggesting that N/S PAs inhibit the maturation of
dendritic cells.
Dendritic Cells
[0083] Dendritic cells (DCs) are a family of professional antigen
presenting cells that are found in virtually every organ. Dendritic
cell subtypes have been well defined, and it has been demonstrated
that these cell types evolve through several levels of
differentiation and maturation throughout their life span (Jonuleit
et al. (2001) Trends in Immunol. 22:394-400). Immature dendritic
cells are characterized by low expression of MHC II molecules, as
well as limited expression of the co-stimulatory molecules CD80 and
CD86. The expression of these surface molecules is dramatically
upregulated in response to inflammatory stimuli such as IFN.gamma.
or ligation of TLR. Functionally, immature DCs in the periphery are
especially adept at the capture and processing of antigens.
Maturing DCs downregulate these activities, and significantly
upregulate their ability to stimulate naive T cells through the
presentation of antigen via MHCII and co-stimulation through
CD80/86 (Banchereau et al (2000) Annu. Rev. Immunol. 18:767-811).
Summarized in FIG. 1.
[0084] In the absence of inflammation, most peripheral DCs are in
an immature state, and it is thought that these cells play a major
role in maintenance of peripheral T cell tolerance (recognition of
self), induction of T cell anergy, and protection against
autoimmunity (Jonuleit et al. (2001) Trends in Immunol.
22:394-400).
[0085] The present inventors have observed that treatment of
immature dendritic cells with CP1 or synthetic polymeric antigen
Compound 15 inhibits their ability to mature, despite the presence
of a potent inflammatory stimulus (LPS). The consequences for
immune regulation through immature or semi-mature (low CD80 and
CD86 expression) dendritic cells are only beginning to be fully
appreciated (Lutz et al. (2002) Trends Immunol. 23:445-449). It has
been suggested that the induction of adaptive immunity versus
tolerance or suppression of inflammation may be determined by the
ratio of immature or semi-mature DCs to fully mature DCs in the
periphery (Jonuleit et al. (2001) Trends in Immunol. 22:394-400;
Garza et al. (2000) J. Exp. Med. 191:2021-2028). Chemotherapeutic
maintenance of an immature DC population through treatment with N/S
PAs may inhibit the cognate interactions between T cells and DCs,
thus preventing the clonal expansion of antigen-specific effector T
cells in response to inflammatory stimuli. In view of the entire
body of evidence presented herein, however, it is more likely that
the immature DCs generated by N/PA treatment induce a T regulatory
cell population that directly inhibits the activity of inflammatory
effector T cells, thus affording protection against inflammatory
pathologies. Evidence is mounting in the literature that immature
DCs induce T regulatory cells in vivo, and further, T regulatory
cells have been induced by immature DCs that specifically protect
animals from influenza virus infection and prevent rejection in
models of transplantation (Jonuleit et al. (2001) Trends in
Immunol. 22:394-400; Dhodapkar et al. (2001) J. Exp. Med.
193:233-238; Thomson et al. (1999) Transplant. Proc. 31:2738-2739).
In these studies, immature DCs were expanded ex vivo and then
administered to animals. N/S PAs could provide a unique therapy in
which autologous or immunologically compatible DCs are rendered
chronically immature through ex vivo treatment and then
reintroduced into patients to stimulate T regulatory activity.
[0086] T Regulatory Cells
[0087] Recent studies from several laboratories have demonstrated
that the immature dendritic cell is a critical component in the
generation of T regulatory cells (Tregs) (Jonuleit et al. (2001)
Trend Immunol. 22:394-400). T regulatory cells function to maintain
peripheral tolerance, protect against autoimmunity, and participate
in modulating inflammation to allow for appropriate responses to
microbial invasion or tissue damage while protecting the host from
deleterious bystander effects (Maloy et al. (2001) Nat. Immunol.
2:816-822).
[0088] The most intensely studied Treg phenotype is characterized
by the constitutive expression of the surface markers CD4 and CD25
(Shevach (2002) Nat. Rev. Immunol. 2:389-400). T regulatory cells
with this phenotype have been identified both in vitro and in vivo
in both rodents (Taylor et al. (2001) J. Exp. Med. 193:1311-1317)
and man (Jonuleit et al. (2001) J. Exp. Med. 193:1285-1294).
CD4+CD25+ T cells naturally occur in the peripheral circulation at
a frequency of approximately 2-10% (Shevach (2002) Nat. Rev.
Immunol. 2:389-400). During co-culture of CD4+CD25- target cells
with CD4+CD25+ T regulatory cells, the T regulatory cells inhibit
the proliferation of CD4+CD25- target cells despite the presence of
potent proliferative signals such as antiCD3 antibodies or
allogeneic APCs (Pasare et al. (2003) Science 299:1033-1036). To
date, there have been no reports describing a definitive chemical
means to generate T regulatory cells in vivo. Early studies
reported in the literature indicated that CD4+CD25+ Treg cells
expressed some IL10 in vitro (Shevach (2002) Nat. Rev. Immunol.
2:389-400). Furthermore, in inflammatory models, CD4+CD25+ cells
were unable to inhibit inflammation in IL10 knockout animals
(Shevach (2002) Nat. Rev. Immunol. 2:389-400). These studies led to
the widely held belief that the mechanism of T regulatory
anti-inflammatory activity is via the expression of IL10. Elegant
studies performed in several laboratories (Jonuleit et al. (2001)
J. Exp. Med. 193:1285-1294; Levings et al. (2001) J. Exp. Med.
193:1295-1302; Dieckman et al. (2001) J. Exp. Med. 193:1303-1310)
have shown that while CD4+CD25+ T cells do indeed express IL10
and/or other cytokines, the mechanism by which they suppress
inflammatory T cells is dependent on cell-cell contact. In the
initial interactions between CD4+CD25+ T cells and their targets,
cytokine expression does not play a role. Recently, this seemingly
paradoxical set of observations was clarified by the work of
Diekman et al. ((2002) J. Exp. Med. 196:247-253). This group has
also shown that CD4+CD25+ T cells interact with inflammatory T
cells through cell-cell contact. Although the exact nature of the
signals transduced by this contact is not yet known, these workers
demonstrated that one important consequence of contact is that the
target cells, i.e., CD4+CD25- T cells, become anergized, and begin
to express high levels of IL10. Since T regulatory cells are
relatively rare in the context of the entirety of the immune
system, this provides a mechanism to amplify the anti-inflammatory
effect, and explains the body of data indicating a role for IL10 in
systemic anti-inflammation mediated by CD4+CD25+ T cells.
[0089] Human PBMC cultures treated with N/S PAs do not respond by
proliferation when compared to control cultures treated with
polyclonal mitogens such as phytohaemagglutinin (PHA) or
superantigens such as Staphylococcus aureus enterotoxin A (SEA).
N/S PAs do, however, stimulate an increase in the percentage of
CD4+CD25+ cells present in the culture. Furthermore, when
N/PA-treated PBMC cultures are stimulated with .alpha.CD3
antibodies, there is a marked suppression in the proliferative
capacity of the culture compared to that of untreated controls.
Microarray analysis further reveals that PBMC cultures treated with
N/S PAs and .alpha.CD3 antibodies selectively upregulate the
expression of IL10 and IL19 (an IL10 paralogue) messages in the
CD3+ T cell population while downregulating several inflammatory
cytokine messages such as IL17 and TNFI.beta..
[0090] Taken together, the data disclosed herein suggest that N/S
PAs inhibit the maturation of dendritic cells. Immature dendritic
cells have a unique capacity to drive the generation of T
regulatory cells. Treg cells may then participate in the inhibition
of inflammatory responses through cell-cell signaling as well as
through the stimulation of IL10 expression from anergized T cells
at the sites of inflammation.
[0091] IL10
[0092] The concept of using recombinant IL10 as an
immunotherapeutic is widely accepted (Madsen (2002) Gastroenterol.
123:2140-2144; Barnes (2001) Curr. Opin. Allergy Clin. Immunol.
1:555-560; Bremeanu et al (2001) Int. Rev. Immunol. 20:301-331; St.
Clair (2000) Curr. Dir. Autoimmun. 2:126-149). There are numerous
animal models of inflammation in which IL10 has been shown to be
efficacious, e.g., inflammatory bowel disease (IBD), Crohn's
disease, rheumatoid arthritis, autoimmune diabetes, and allergic
disease (Madsen (2002) Gastroenterol. 123:2140-2144; Barnes (2001)
Curr. Opin. Allergy Clin. Immunol. 1:555-560; Bremeanu et al (2001)
Int. Rev. Immunol. 20:301-331; St. Clair (2000) Curr. Dir.
Autoimmun. 2:126-149). Clinical trials using recombinant IL10 for
the treatment of inflammatory bowel disease have, however, met with
mixed results. Requirements for repeated high dose regimens, as
well as some resulting toxicity, have hampered the success of these
efforts. Harnessing an individual's immune system to selectively
produce endogenous IL10 via T regulatory activity may provide a
better route to immunotherapy. Expression of endogenous IL10,
modulated by the host within the entirety of the immune system, may
provide the appropriate context to achieve efficacy without the
requirement for repeated dosing or the problems of cytokine
toxicity. Furthermore, the selective enhancement of a cell
population may prove to be the ideal delivery system for such a
potent cytokine. Inherent in the immune cell repertoire is the
ability to traffic within the body to sites of inflammation. An
immune cell population that has been given a specific trafficking
signal via a N/PA-tolerized dendritic cell may populate specific
sites and locally induce IL10 expression. This therapeutic approach
would avoid the problems associated with systemic administration of
potent cytokines and better mimic the naturally localized action of
this immune mediator.
[0093] Intra-Abdominal Abscesses
[0094] The formation of intra-abdominal abscesses is the
consequence of contamination of the peritoneal cavity with colonic
bacteria. This usually occurs during trauma or surgical
interventions. Bacteria stimulate a vigorous inflammatory response,
resulting in the recruitment of macrophages, polymorphonuclear
leukocytes (PMNs), and lymphocytes, and the release of a variety of
inflammatory mediators such as IL113, TNF.alpha., TNF.beta., IL17,
as well as a number of chemokines (Whal. et al. (1986) J. Exp. Med.
163:884-891; Tzianabos et al. (2002) Curr. Opin. Micro. 5:92-95).
One possible outcome of this response is the encapsulation of
invading bacteria by a variety of immune cells interlaced with
deposits of fibrin. Once formed, the abscess is relatively
resistant to antibiotic therapy, and patients often require
surgical intervention to drain the abscess. Although prophylactic
antibiotics are given to patients at risk, these interventions are
not fully successful. A method to prevent the initial formation of
an abscess by modulation of the host response through T regulatory
cell activity and the expression of IL10 represents a better form
of therapy that could become a standard of care for at risk
surgical procedures.
[0095] Post-Surgical Adhesions
[0096] Post-surgical adhesions are a significant complication of
abdominal, gynecologic, orthopedic, and cardiothoracic surgeries.
In the abdomen and pelvic cavity, adhesions are associated with
considerable morbidity and can be fatal. In pre-clinical models,
exogenously administered IL10 has been shown to limit the formation
of adhesions (Laan. et al. (1999). J. Immunol. 162:2347-2352; Chung
et al. (2002). J. Exp. Med. 195:1471-1476). Current therapies in
human medicine are, however, designed to interrupt the formation of
adhesions after surgical insult. These products involve the
introduction of gels or barrier products into the surgical site.
These devices have met with only limited success due to enhanced
infection rates, lack of efficacy, and relatively low rates of use
within the medical community. Better methods to prevent the
formation of adhesions are urgently needed.
[0097] Like abscess formation, current evidence suggests that the
formation of adhesions also involves activation of inflammatory
processes, most notably the consistent expression of the
inflammatory mediator, IL17, and the deposition of fibrin and other
matrix proteins. Together, these processes define a unique
intersection between the immune system and pathways of
fibrinogenesis and wound repair. Due to the unique nature of N/S
PAs and the potential to manipulate the structures of synthetic PAs
and thus their modulating activity, N/S PAs could prove to be
useful tools to explore these interactions in greater detail.
Specifically, N/S PAs may be useful in the identification and
development of biomarkers that are indicative of specific immune or
fibrinogenic responses.
[0098] Delayed Type Hypersensitivity Assay for Use as a Clinical
Study Biomarker
[0099] In view of the observations that N/S PAs elicit their
protective effects through the response of a T regulatory
population to inflammatory stimuli, there is a need to develop a
specific assay to measure this activity for clinical studies. Early
phase clinical trials typically employ healthy volunteers for
safety and dose response assessment, a scenario that does not
necessarily include the induction or measurement of a specific
inflammatory pathology. It is therefore necessary to develop a
surrogate biomarker for the activity of these compounds. Delayed
Type Hypersensitivity (DTH) reactions in the skin have been used
for decades to assess exposure to Mycobaterium tuberculosis (TB) in
humans, and more recently to determine the state of T cell
responsiveness in the face of immunocompromise (Anderson et al.
(1968) Immunology 15:405-409; Gray et al (1994) Curr. Opin.
Immunol. 6:425-437; Kuby et al. (2000) Immunology, W.H. Freeman and
Co.) Studies in the literature have demonstrated that the DTH
response is primarily mediated by T cells and that the inflammatory
activity can be adoptively transferred to naive animals by DTH T
cells alone (Elices et al. (1993) Clin. Exp. Rheumatol.
11:s77-s80). As disclosed herein, a Guinea pig model of DTH has
been developed to assess the ability of N/S PAs to limit the
localized inflammatory reaction in the skin. Direct measurements of
the DTH response can be readily observed and measured in humans and
Guinea pigs. Flares, wheals, and/or indurations can be observed and
readily measured quantitatively on the surface of the skin. The
antigen used to elicit inflammatory T cell activity in this assay,
derived from Candida albicans (Candin), is currently being used
clinically to measure immune competence in individuals undergoing
transplant therapies or suffering from AIDs. This antigen is also
considered to be safer for the general population than TB antigens.
When tested in this model, CP1 demonstrates significant efficacy in
preventing the characteristic skin lesions of DTH. Since it has
been reported in the literature that CD4+CD25+ T regulatory cells
are essential components of the memory and protective immunity to
C. albicans (Montagnoli et al. (2002) J. Immunol. 169:6298-6308),
these results provide further evidence that the protective effects
of N/S PAs are derived from T regulatory activity.
[0100] Mechanism of Action of Naturally Occurring and Synthetic
Polymeric Antigens: The T Regulatory Cell Hypothesis
[0101] The present inventors have conducted detailed investigations
into the mechanism(s) by which immunomodulatory molecules such as
CP1 and the synthetic polymeric antigen Compound 15 direct and
elicit anti-inflammatory effects in mammals, including the
induction of T regulatory cell populations. From these studies, the
following picture, summarized in FIG. 2, has emerged.
[0102] As depicted in FIG. 2, natural or synthetic immunomodulatory
polymeric antigens inhibit the maturation of dendritic cells.
Immature dendritic cells (iDCs) express low CD80 and CD86
co-stimulatory molecules. In this state, iDCs have the unique
ability to interact with naive T cells and induce the generation of
CD4+CD25+ T regulatory cells (pathway B). In the face of an
inflammatory response, T regulatory cells interact with T effector
cells through cell-cell dependent contact and inhibit the
proliferative capacity of these T inflammatory effector cells.
Further, contact between T regulatory cells and T effector cells
renders the effectors anergic and stimulates these cells to express
large amounts of IL10. Elicitation of IL10 expression in the former
inflammatory T cell effectors serves to amplify the suppressive
effects of direct T regulatory cell contact and broadens the
protection against an ongoing inflammatory process. The inhibition
of maturation of dendritic cells observed by the present
investigators could also inhibit the clonal expansion of T effector
cells through the lack of cognate interactions between these two
cell types (pathway A). However, the data presented herein more
compellingly support the hypothesis that T regulatory cells are
ultimately generated by the natural or synthetic polymeric antigens
of the present invention and afford protection against inflammatory
pathologies.
[0103] Pharmaceutical Compositions
[0104] The natural and synthetic immunomodulatory polymeric
antigens disclosed herein can be used to prevent or treat
inflammatory pathologies in humans and other mammals. Thus, in one
aspect, the present invention provides pharmaceutical compositions
for human and veterinary medical use comprising CP1 and the
synthetic PG of the present invention, together with one or more
pharmaceutically or physiologically acceptable carriers,
excipients, or diluents, and optionally, other therapeutic agents.
Thus, the present invention also relates to pharmaceutical
compositions of the presently described immunomodulating polymers
in combination with a antibacterial agent or other therapeutic
agent, and a pharmaceutically acceptable carrier, excipient, or
diluent.
[0105] The immunomodulatory polymers of the present invention can
be delivered separately with another anti-bacterial antibiotic
drug(s), or in the form of anti-bacterial antibiotic cocktails. An
anti-bacterial antibiotic cocktail is a mixture of a molecule of
the present invention and an anti-bacterial antibiotic drug and/or
supplementary potentiating agent. The use of antibiotics in the
treatment of bacterial infection is routine in the art. In this
embodiment, a common administration vehicle (e.g., tablet, implant,
injectable solution, etc.) can contain both a natural or synthetic
polymeric antigen and the anti-bacterial antibiotic drug and/or
supplementary potentiating agent. Alternatively, the anti-bacterial
antibiotic drug can be separately dosed.
[0106] Non-limiting examples of anti-bacterial antibiotic drugs
useful in the present invention include: penicillin G, penicillin
V, ampicillin, amoxicillin, bacampicillin, cyclacillin, epicillin,
hetacillin, pivampicillin, methicillin, nafcillin, oxacillin,
cloxacillin, dicloxacillin, flucloxacillin, carbenicillin,
ticarcillin, avlocillin, mezlocillin, piperacillin, amdinocillin,
cephalexin, cephradine, cefadoxil, cefaclor, cefazolin, cefuroxime
axetil, cefamandole, cefonicid, cefoxitin, cefotaxime, ceftizoxime,
cefinenoxine, ceftriaxone, moxalactarn, cefotetan, cefoperazone,
ceftazidme, imipenem, clavulanate, timentin, sulbactam, neomycin,
oritavancin, erythromycin, metronidazole, chloramphenicol,
clindamycin, lincomycin, vancomycin, trimethoprim-sulfamethoxazole,
aminoglycosides, quinolones, tetracyclines, and rifampin. Note
Goodman & Gilman's The Pharmacological Basis of Therapeutics,
Ninth Edition, Hardman et al., Eds., McGraw-Hill, New York, (1996)
in this regard. The precise amounts of the therapeutic agent used
in combination with the immunomodulatory polymers of the present
invention will depend upon a variety of factors, including the
polymer itself, the dose and dose timing selected, the mode of
administration, the nature of any surgery that may be contemplated,
and certain characteristics of the subject. Where local
administration is carried out, it will be understood that very
small amounts may be required (nanograms, or possibly picograms).
The precise amounts selected can be determined without undue
experimentation, particularly since a threshold amount will be any
amount that will favorably enhances the desired immune response. A
dose in the range of from about one picogram to about one milligram
may be efficacious, depending upon the mode of delivery; a dose in
the range of from about one nanogram to about one microgram may
also be useful.
[0107] Dosing, Treatment Regimen, and Administration
[0108] The compounds of the present invention can be administered
in an effective amount for inducing protection against a wide
variety of different inflammation-based pathologies, including
post-surgical adhesions and intra-abdominal abscesses associated
with bacterial infection. For such purposes, an effective amount is
that amount of a compound of the present invention that will, alone
or together with further doses or additional therapeutic compounds,
inhibit, ameliorate, or prevent the inflammation-based pathology.
The dose range can be from about one picogram/kilogram bodyweight
to about one milligram/kilogram bodyweight, or from about one
nanogram/kilogram bodyweight to about one microgram/kilogram
bodyweight. The absolute amount will depend upon a variety of
factors, including the nature of the inflammatory pathology to be
treated, whether the administration is in conjunction with elective
surgery or emergency surgery, concurrent treatment, the number of
doses, individual patient parameters including age, physical
condition, size and weight, and the severity of the
inflammation-based pathology, and can be determined by the medical
practitioner with no more than routine experimentation. It is
generally preferred that a maximum dose be used, that is, the
highest safe dose according to sound medical judgment. Multiple
doses of the pharmaceutical compositions of the invention are
contemplated.
[0109] Determination of the optimal amount of compound to be
administered to human or animal patients in need of prevention or
treatment of an inflammation-based pathology, as well as methods of
administering therapeutic or pharmaceutical compositions comprising
such compounds, is well within the skill of those in the
pharmaceutical, medical, and veterinary arts. Dosing of a human or
animal patient is dependent on the nature of inflammation-based
pathology, the patient's condition, body weight, general health,
sex, diet, time, duration, and route of administration, rates of
absorption, distribution, metabolism, and excretion of the
compound, combination with other drugs, severity of the
inflammation-based pathology, and the responsiveness of the disease
state being treated, and can readily be optimized to obtain the
desired level of effectiveness. The course of treatment can last
from several days to several weeks or several months, or until a
cure is effected or an acceptable diminution or prevention of the
disease state is achieved. Optimal dosing schedules can be
calculated from measurements of drug accumulation in the body of
the patient in conjunction with the effectiveness of the treatment.
Persons of ordinary skill can easily determine optimum dosages,
dosing methodologies, and repetition rates. Optimum dosages can
vary depending on the potency of the immunomodulatory polymeric
compound, and can generally be estimated based on ED.sub.50 values
found to be effective in in vitro and in vivo animal models.
Effective amounts of the present compounds for the treatment or
prevention of inflammation-based pathologies, delivery vehicles
containing these compounds, agonists, and treatment protocols, can
be determined by conventional means. For example, the medical or
veterinary practitioner can commence treatment with a low dose of
the compound in a subject or patient in need thereof, and then
increase the dosage, or systematically vary the dosage regimen,
monitor the effects thereof on the patient or subject, and adjust
the dosage or treatment regimen to maximize the desired therapeutic
effect. Further discussion of optimization of dosage and treatment
regimens can be found in Benet et al., in Goodman & Gilman's
The Pharmacological Basis of Therapeutics, Ninth Edition, Hardman
et al., Eds., McGraw-Hill, New York, (1996), Chapter 1, pp. 3-27,
and L. A. Bauer, in Pharmacotherapy, A Pathophysiologic Approach,
Fourth Edition, DiPiro et al., Eds., Appleton & Lange,
Stamford, Conn., (1999), Chapter 3, pp. 21-43, and the references
cited therein, to which the reader is referred.
[0110] In the context of the present invention, the terms
"treatment," "therapeutic use," or "treatment regimen" as used
herein are meant to encompass prophylactic, palliative, and
therapeutic modalities of administration of the immunomodulatory
polymers of the present invention, and include any and all uses of
the presently claimed compounds that remedy a disease state,
condition, symptom, sign, or disorder caused by an
inflammation-based pathology, or which prevents, hinders, retards,
or reverses the progression of symptoms, signs, conditions, or
disorders associated therewith. Thus, any prevention, amelioration,
alleviation, reversal, or complete elimination of an undesirable
disease state, symptom, condition, sign, or disorder associated
with an inflammation-based pathology is encompassed by the present
invention.
[0111] A particular treatment regimen can last for a period of time
which may vary depending upon the nature of the particular
inflammation-based pathology, its severity, and the overall
condition of the patient, and may involve administration of
compound-containing compositions from once to several times daily
for several days, weeks, months, or longer. Following treatment,
the patient is monitored for changes in his/her condition and for
alleviation of the symptoms, signs, or conditions of the disorder
or disease state. The dosage of the composition can either be
increased in the event the patient does not respond significantly
to current dosage levels, or the dose can be decreased if an
alleviation of the symptoms of the disorder or disease state is
observed, or if the disorder or disease state has been ablated.
[0112] An optimal dosing schedule is used to deliver a
therapeutically effective amount of the compounds of the present
invention. For the purposes of the present invention, the terms
"effective amount" or "therapeutically effective amount" with
respect to the compounds disclosed herein refers to an amount of
compound that is effective to achieve an intended purpose,
preferably without undesirable side effects such as toxicity,
irritation, or allergic response. Although individual patient needs
may vary, determination of optimal ranges for effective amounts of
pharmaceutical compositions is within the skill of the art.
Human-doses can be extrapolated from animal studies (A. S. Katocs,
Remington: The Science and Practice of Pharmacy, 19.sup.th Ed., A.
R. Gennaro, ed., Mack Publishing Co., Easton, Pa., (1995), Chapter
30). Generally, the dosage required to provide a therapeutically
effective amount of a pharmaceutical composition, which can be
adjusted by one skilled in the art, will vary depending on the age,
health, physical condition, weight, type and extent of the disease
or disorder of the recipient, frequency of treatment, the nature of
concurrent therapy (if any), and the nature and scope of the
desired effect(s) (Nies et al., Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 9.sup.th Ed., Hardman et
al., eds., McGraw-Hill, New York, N.Y., 1996, Chapter 3).
[0113] Prophylactic modalities for high risk individuals are also
encompassed by the present invention. As used herein, the term
"high risk individual" is meant to refer to an individual for whom
it has been determined, via, e.g., individual or family history or
genetic testing, living or working environment or conditions, etc.,
that there is a significantly higher than normal probability of
being susceptible to an inflammation-based pathology or the onset
or recurrence of an associated disease or disorder. For example, a
patient could have a personal and/or family medical history that
includes frequent occurrences of a particular disease or disorder.
As another example, a patient could have had such a susceptibility
determined by genetic screening according to techniques known in
the art (see, e.g., U.S. Congress, Office of Technology Assessment,
Chapter 5 In: Genetic Monitoring and Screening in the Workplace,
OTA-BA-455, U.S. Government Printing Office, Washington, D.C.,
1990, pages 75-99). As part of a treatment regimen for a high risk
individual, the individual can be prophylactically treated to
prevent inflammation-based pathologies or the onset or recurrence
of the disease, disorder, sign, symptom, or condition. The term
"prophylactically effective amount" is meant to refer to an amount
of a pharmaceutical composition of the present invention that
produces an effect observed as the prevention of infection or
inflammation, or the onset or recurrence of a disease, symptom,
sign, condition, or disorder. Prophylactically effective amounts of
a pharmaceutical composition are typically determined by the effect
they have compared to the effect observed when a second
pharmaceutical composition lacking the active agent is administered
to a similarly situated individual.
[0114] For therapeutic use, the immunomodulatory compounds
disclosed herein can be administered to a patient suspected of
suffering from an inflammation-based pathology in an amount
effective to reduce the symptomology of the disease, symptom, sign,
condition, or disorder. One skilled in the art can determine
optimum dosages and treatment schedules for such treatment regimens
by routine methods.
[0115] It should be noted that the present invention encompasses
the use of both CP1 and Compound 15 in combination therapy with one
another.
[0116] In the case of surgery- or trauma-related abscesses and
adhesions, the methods of the present invention can be effectuated
by administering multiple doses over a three week period preceding
surgery, over a two week period preceding surgery, over a one week
period preceding surgery, when the first dose is administered only
24 hours preceding surgery, and even when given only after exposure
to bacteria. Further doses can be administered after surgery as
well. Any regimen that results in an enhanced immune response to
bacterial infection/contamination and subsequent abscess/adhesion
formation can be used, although optimal doses and dosing regimens
are those which would not only inhibit the development of abscess
and/or adhesion formation, but also would result in a complete
protection against abscess or adhesion formation by a particular
bacterial organism or a variety of bacterial organisms. Desired
time intervals for delivery of multiple doses of a particular
polymer can be determined by one of ordinary skill in the art
employing no more than routine experimentation.
[0117] Thus, the present invention is useful whenever it is
desirable to prevent bacterial abscess or adhesion formation in a
human or animal subject. This includes prophylactic treatment to
prevent such conditions in planned surgical procedures, as well as
in emergency situations. Elective surgeries include the following
intraabdominal surgeries: right hemicolectomy; left hemicolectomy;
sigmoid colectomy; subtotal colectomy; total colectomy;
laparoscopic or open cholecystectomy; gastrectomy; caesarian
section; etc. Emergency surgeries include those to correct the
following conditions: perforated ulcer (duodenal or gastric);
perforated diverticulitis; obstructive diverticulitis; acute
appendicitis; perforated appendicitis; blunt abdominal trauma;
penetrating abdominal trauma; second operation to drain abscess;
etc. The methods of the present invention are also useful in
nonintraabdominal surgeries such as cardiac surgeries and surgeries
to correct wound infections. The present methods are also useful in
connection with diseases that predispose a subject to abscess
formation such as pelvic inflammatory disease, inflammatory bowel
disease, urinary tract infections, and colon cancer. The present
methods are therefore useful with abscesses of virtually any tissue
or organ, including specifically, but not limited to, dermal
abscesses such as acne. Those of ordinary skill in the art to which
this invention pertains will readily recognize the range of
conditions and procedures in which the present invention is
applicable.
[0118] In another aspect, the present invention includes a method
for inducing protection against postoperative surgical adhesion
formation associated with many common types of surgery. The method
includes the step of administering to a subject in need of such
protection a pharmaceutical preparation containing an effective
amount for reducing postoperative surgical adhesion formation of
the immunomodulating polymer of the present invention. It is fully
expected that administration of one or more such polymers at a site
separate from the operative site will be effective in inducing
protection against postoperative surgical adhesion formation. This
is particularly surprising in view of previous observations, as
discussed above.
[0119] PCT International Publication WO 00/59515 teaches that local
administration of certain polymers into the surgical site is
effective for reducing the incidence of postoperative surgical
adhesions. In accordance with the present invention, an
immunomodulatory polymer can be effective when given subcutaneously
apart from the surgical site at which adhesions are likely to
form.
[0120] The presently disclosed compounds can be administered in an
effective amount for inducing protection against postoperative
surgical adhesion formation. An effective amount for inducing
protection against postoperative surgical adhesion formation as
used herein is that amount of immunomodulating polymer of the
present invention that will, alone or together with further doses
or additional therapeutic compounds, inhibit or prevent the
formation of postoperative surgical adhesion. It is believed that
doses ranging from about one picogram/kilogram bodyweight to about
one milligram/kilogram bodyweight, or from about one
nanogram/kilogram bodyweight to about one microgram/kilogram
bodyweight, will be effective, depending upon the mode of
administration. The absolute amount will depend upon a variety of
factors (including whether the administration is in conjunction
with elective surgery or emergency surgery, concurrent treatment,
number of doses, and individual patient parameters including age,
physical condition, size and weight), and can be determined via
routine experimentation. It is preferred generally that a maximum
dose be used, that is, the highest safe dose according to sound
medical judgment.
[0121] Multiple doses of the pharmaceutical compositions of the
present invention are contemplated for inducing protection against
postoperative surgical adhesion formation. Such multiple doses can
be administered over a three day period beginning on the day
preceding surgery. Further doses can be administered post surgery
as well. Any regimen that results in a reduced postoperative
surgical adhesion formation can be used, although optimum doses and
dosing regimens are those which would not only inhibit the
development of postoperative surgical adhesion formation, but would
also result in complete protection against postoperative surgical
adhesion formation. Desired time intervals for delivery of multiple
doses of one of the present immunomodulatory polymers can be
determined by one of ordinary skill in the art employing no more
than routine experimentation.
[0122] Thus, the methods disclosed herein are useful whenever it is
desirable to prevent postoperative surgical adhesion formation in a
human or animal subject. This includes prophylactic treatment to
prevent adhesion formation following planned surgical procedures,
as well as following emergency operations. Elective surgeries
include the following intraabdominal surgeries: right
hemicolectomy; left hemicolectomy; sigmoid colectomy; subtotal
colectomy; total colectomy; laparoscopic or open cholecystectomy;
gastrectomy; pancreatectomy; splenectomy; liver, pancreas, small
bowel, or kidney transplantation; lysis of adhesions; etc.
Emergency intraabdominal surgeries include those to correct the
following conditions: perforated ulcer (duodenal or gastric);
perforated diverticulitis; obstructive diverticulitis; bowel
obstruction; acute appendicitis; perforated appendicitis; blunt
abdominal trauma; penetrating abdominal trauma; second operation to
drain abscess; ruptured abdominal aortic aneurysm, etc. The methods
of the present invention are also useful in the case of
nonintraabdominal surgeries such as cardiac surgeries, open and
endoscopic orthopedic surgeries, neurosurgeries, gynecologic and
pelvic surgeries, and surgeries to correct wound infections. The
present methods are also useful in connection with diseases that
predispose a subject to spontaneous adhesion formation, such as
pelvic inflammatory disease, inflammatory bowel disease, urinary
tract infections, and colon cancer. The present methods are thus
useful with inflammatory processes involving virtually any tissue
or organ.
[0123] When administered to prevent postoperative surgical adhesion
formation, the compounds of the present invention can be
administered either distant from the operative site, including
systemically, or locally into the operative site at which it is
desirable to reduce the likelihood of postoperative surgical
adhesion formation. The compounds of the present invention can be
administered as an aqueous solution, as a crosslinked gel, or as
any temporal or physical combination of aqueous solution and
crosslinked gel forms.
[0124] The preparations of the present invention can be
administered "in conjunction with" infection, meaning close enough
in time with the surgery, trauma, or diseases that predispose the
host to abscess or adhesion formation so that a protective effect
against abscess or adhesion formation is obtained. The preparations
can be administered long before surgery in the case of elective
surgery (i.e., weeks or even months), preferably with booster
administrations closer in time to (and even after) the surgery.
Particularly in emergency situations, the preparations can be
administered immediately before (minutes to hours) and/or after the
trauma or surgery. It is important only that the preparation be
administered close enough in time to the surgery so as to enhance
the subject's immune response against bacterial
infection/contamination, thereby increasing the chances of a
successful host response and reducing the likelihood of abscess or
adhesion formation.
[0125] Those of ordinary skill in the art to which this invention
pertains will recognize that the present methods can be applied to
a wide range of diseases, symptoms, conditions, signs, disorders,
and procedures. Besides abscesses and adhesions, other inflammatory
processes and pathologies to which the compounds, compositions, and
methods of the present invention can be applied include: sepsis;
rheumatoid arthritis; myesthenia gravis; inflammatory bowel
disease; colitis; systemic lupus erythematosis; multiple sclerosis;
coronary artery disease; diabetes; hepatic fibrosis; psoriasis;
eczema; acute respiratory distress syndrome; acute inflammatory
pancreatitis; endoscopic retrograde
cholangiopancreatography-induced pancreatitis; burns; atherogenesis
of coronary, cerebral, and peripheral arteries; appendicitis;
cholecystitis; diverticulitis; visceral fibrotic disorders (liver,
lung, intestinal); wound healing; skin scarring disorders (keloids,
hidradenitis suppurativa); granulomatous disorders (sarcoidosis,
primary biliary cirrhosis); asthma; pyoderma gangrenosum; Sweet's
syndrome; Behcet's disease; primary sclerosing cholangitis; and
cell, tissue, or organ transplantation.
[0126] Formulations
[0127] The compounds of the present invention can be administered
in pharmaceutically or physiologically acceptable solutions that
can contain pharmaceutically or physiologically acceptable
concentrations of salts, buffering agents, preservatives,
compatible carriers, and optionally, other therapeutic ingredients.
The synthetic PG (Compound 15) of the present invention is soluble
up to ca. 20 mg/mL in water at neutral pH. Furthermore, aqueous
solutions of this compound can accommodate low (about 0.5 to about
5) weight percentages of glycerol, sucrose, and other such
pharmaceutically acceptable excipient materials. CP1 and the SPA
Compound 15 disclosed herein can thus be formulated in a variety of
standard pharmaceutically acceptable parenteral formulations.
[0128] The pharmaceutical compositions of the present invention can
contain an effective amount of CP1 or the presently disclosed SPA,
optionally included in a pharmaceutically or physiologically
acceptable carrier, excipient, or diluent. The term
"pharmaceutically or physiologically acceptable carrier, excipient,
or diluent" means one or more compatible solid or liquid fillers,
dilutants, or encapsulating substances that are suitable for
administration to a human or other animal. The term "carrier"
denotes an organic or inorganic ingredient, natural or synthetic,
with which the active ingredient is combined to facilitate the
application. The components of the pharmaceutical compositions also
are capable of being comingled with the polymers of the present
invention, and with each other, in a manner such that there is no
interaction that would substantially impair the desired
pharmaceutical efficiency of CP1 or the SPA.
[0129] Compositions suitable for parenteral administration
conveniently comprise sterile aqueous preparations, which can be
isotonic with the blood of the recipient. Among the acceptable
vehicles and solvents are water, Ringer's solution, and isotonic
sodium chloride solution. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose, any bland fixed oil can be employed, including synthetic
mono- or di-glycerides. In addition, fatty acids such as oleic acid
are useful in the preparation of injectables. Carrier formulations
suitable for subcutaneous, intramuscular, intraperitoneal,
intravenous, etc. administrations can be found in Remington: The
Science and Practice of Pharmacy, 19.sup.th Edition, A. R. Gennaro,
ed., Mack Publishing Co., Easton, Pa., (1995). CP1 and the SPA
polymer of the present invention can be delivered individually, or
in a mixture comprising the two polymers.
[0130] A variety of administration routes are available. The
particular mode selected will depend upon whether CP1 or the
present SPA is selected, the particular condition being treated,
and the dosage required for therapeutic efficacy. Generally
speaking, the methods of the present invention can be practiced
using any mode of administration that is medically acceptable,
meaning any mode that produces effective levels of an immune
response without causing clinically unacceptable adverse effects.
Preferred modes of administration are parenteral routes. The term
"parenteral" includes subcutaneous, intravenous, intramuscular, or
intraperitoneal injection, or infusion techniques.
[0131] The compositions can be conveniently presented in unit
dosage form or dosage unit form, and can be prepared by any of the
methods well known in the art of pharmacy. All methods include the
step of bringing CP1 or the SPA into association with a carrier
that constitutes one or more accessory ingredients. In general, the
compositions are prepared by uniformly and intimately bringing CP1
or the SPA into association with a liquid carrier, a finely divided
solid carrier, or both, and then, if necessary, shaping the
product. CP1 or the SPA can be stored lyophilized.
[0132] Other delivery systems can include time-release,
delayed-release, or sustained-release delivery systems. Such
systems can avoid repeated administrations of the anti-inflammatory
agent, increasing convenience to the subject and the physician.
Many types of release delivery systems are available and known to
those of ordinary skill in the art, including polymer-based systems
such as poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides.
[0133] Microcapsules of the foregoing polymers containing drugs are
described in, for example, U.S. Pat. No. 5,075,109. Delivery
systems also include non-polymer systems such as: lipids, including
sterols such as cholesterol, cholesterol esters, and fatty acids or
neutral fats such as mono-, di-, and tri-glycerides; hydrogel
release systems; silastic systems; peptide-based systems; wax
coatings; compressed tablets using conventional binders and
excipients; partially fused implants; and the like. Specific
examples include, but are not limited to: (a) erosional systems in
which an agent of the invention is contained in a form within a
matrix such as those described in U.S. Pat. Nos. 4,452,775,
4,675,189, and 5,736,152, and (b) diffusional systems in which an
active component permeates at a controlled rate from a polymer such
as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.
In addition, pump-based hardware delivery systems can be used, some
of which are adapted for implantation.
[0134] The foregoing descriptions provide a comprehensive overview
of the many aspects of the present invention. The following
examples illustrate various aspects thereof and are not intended,
nor should they be construed, to be limiting thereof in any
way.
Example 1
[0135] A general procedure for preparing SPA precursor 14 described
in PCT International Publication Number WO 01/79242. The synthetic
approach used herein is outlined in Scheme I and exemplified
below.
##STR00005## ##STR00006##
DEFINITIONS
[0136] DBU 1,8-diazabicyclo[5.4.0]undec-7-ene
HOBT N-hydroxybenzotriazole
[0137] EDCI 1-[3-(dimethyamino)propyl]-3-ethylcarbodiimide
hydrochloride Py.sup.+protonated pyridine Ac acetyl Ph phenyl
NHS N-hydroxysuccinimide
[0138] TLC thin-layer chromatography TFA trifluoroacetic acid THF
tetrahydrofuran DMF dimethyl formamide RP/HPLC reverse-phase HPLC
DMAP 4-dimethylaminopyridine
NH(TFA) NHC(O)CF.sub.3
General Experimental Conditions
[0139] Reactions are carried out with continuous stirring under a
positive pressure of nitrogen, except where noted. Reagents and
solvents are purchased and used without further purification,
except as noted. TLC is performed using 0.25 mm silica gel 60
plates from E. Merck with a 254 nm fluorescent indicator. Solvent
system specifications are expressed as percents or ratios by
volume. Plates are developed in a covered chamber and visualized by
ultraviolet light or by treatment with 5% phosphomolybdic acid in
ethanol, with ceric ammonium molybdate in aqueous sulfuric acid, or
in the cases of amino acid and peptide derivatives with ninhydrin
in acetic acid/n-butanol. All such visualization treatments are
followed by heating. Flash chromatography is carried out with
silica gel 60, 230-400 mesh (0.040-0.063 mm particle size)
purchased from EM Science or with commercial Biotage prepackaged
32-63 m.mu. KP-Sil cartridges. HPLC analyses and purifications are
performed using Waters X-Terra C8 columns with the specified
solvent system and flow rate.
[0140] NMR spectra are reported as chemical shifts in
parts-per-million (ppm) downfield from a tetramethylsilane internal
standard (0 ppm). .sup.11-1 NMR spectra are recorded in the solvent
indicated on a Bruker Avance spectrometer at 500.2 MHz, a Varian
Mercury spectrometer at 400.21 MHz, or a GE QE-300 spectrometer at
300.2 MHz. Electrospray mass spectra (ES/MS) are recorded on a
Micromass Platform LCZ spectrometer. High resolution mass spectra
are recorded on a Micromass QTOF mass spectrometer.
Peptide Starting Material
##STR00007##
[0142] A flask is charged with DMF (117 mL) and compound 1 (20 g,
58.4 mMol). K.sub.2CO.sub.3 (12.1 g, 87.5 mMol) and 1-bromohexane
(16.7 mL, 119 mMol) are added with vigorous stirring and the
mixture is heated to 45.degree. C. After 5 hr the reaction is
complete as evidenced by TLC analysis (25% ethyl acetate in
hexanes). The mixture is cooled to room temperature and diluted
with ethyl acetate. The solids are filtered under suction and the
filtrate is washed successively with water (1.times.), N HCl
(3.times.), and 0.5M pH 7 buffer (1.times.). The organic phase is
dried (MgSO.sub.4) and concentrated in vacuo to a thick oil which
solidifies on standing. The oil is taken up in dichloromethane and
crystallized from hexanes and dichloromethane. The crystals are
dried in vacuo to afford compound 2 (24.3 g, 97%). .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 6.51 (br s, 1H), 5.09 (d, 1H, J=7.5 Hz),
4.28 (m, 1H), 4.13 (t, 2H, J=6.8 Hz), 3.37 (m, 2H), 1.82 (m, 1H),
1.64 (m, 6H), 1.44 (s, 9H), 1.31 (m, 6H), 0.89 (t, 3H, J=7.0 Hz);
ES/MS m/z=427.1 [M+H].sup.+, 425.2 [M-H]
[0143] Trifluororacetic acid (62 mL, excess) is added to a stirred
solution of compound 2 (17.1 g, 13.1 mMol) in dichloromethane (200
mL) at 0.degree. C. The cooling bath is removed and the reaction
mixture is stirred 2 hr at ambient temperature. TLC analysis (50%
ethyl acetate in hexanes) indicates the absence of starting
material. The reaction mixture is transferred to a beaker and
layered with water. The pH is adjusted to 9 with aqueous NaOH. The
organic phase is separated, and the aqueous phase extracted
(2.times.) with dichloromethane. The organic phase is dried
(MgSO.sub.4), and concentrated in vacuo to afford compound 3 as a
thick oil (13.7 g, 95%), which is taken directly to the coupling
step. ES/MS m/z=327.2 [M+H].sup.+, 325.2 [M-H].sup.-
[0144] EDCI {1-[3-(dimethyamino)propyl]-3-ethylcarbodiimide
hydrochloride} (7.65 g, 39.9 mMol) is added to a stirred solution
of N-BOC-.gamma.-aminobutyric acid (7.95 g, 39.1 mMol) and NHS
(4.59 g, 39.9 mMol) in DMF (120 mL) at room temperature. Stirring
is continued overnight, at which time the reaction is judged to be
complete by TLC analysis (10% methanol in chloroform). The crude
compound 3 (12.8 g, 39.1 mMol) is added in DMF (75 mL), using 5 mL
DMF to aid the transfer, followed by diisopropylethyl amine (7.24
mL, 43.0 mMol). After 4 hr the reaction is complete, as judged by
TLC analysis (10% methanol in chloroform). The reaction mixture is
diluted with ethyl acetate, then washed with water (1.times.), N
HCl (2.times.), and water (1.times.). The combined aqueous extracts
are washed with a single portion of ethyl acetate. The combined
organic extracts are washed with brine, dried (Na.sub.2SO.sub.4),
and concentrated in vacuo. The residue is chromatographed (Biotage
65M; step gradient: 1:1 ethyl acetate:hexanes followed by 5% MeOH
in ethyl acetate). Appropriate fractions are combined and
concentrated in vacuo to afford pure compound 4 (17.7 g, 89%).
ES/MS m/z=512.1 [M+H].sup.+, 510.1 [M-H].sup.-
[0145] Trifluoroacetic acid (5.1 mL, excess) is added to a stirred
solution of compound 4 (1.69 g, 3.31 mMol) in dichloromethane (33
mL) at 0.degree. C. The cooling bath is removed and the mixture
stirred at ambient temperature for 30 min, at which time the
reaction is complete as judged by TLC analysis (10% methanol in
chloroform). The dichloromethane solution is transferred to a
beaker, layered with water, and the pH adjusted to 9 with aqueous
NaOH. The organic phase is drawn off and the aqueous phase is
extracted with dichloromethane. The combined organic extracts are
dried (Na.sub.2SO.sub.4) and concentrated in vacuo to afford
compound 5 as a thick oil (quantitative yield). ES/MS m/z=412.3
[M+H].sup.+, 410.2 [M-H].sup.-
Phytanol Phosphate Triethylammonium Salt Starting Material
##STR00008## ##STR00009##
[0147] Phytol 6 (5.0 g, 16.9 mMol) in ethanol (20 mL) is added to a
stirred slurry of Raney Ni (about 500 mg) in ethanol (10 mL). The
system is brought under a hydrogen atmosphere at balloon pressure
at room temperature and stirring is continued overnight. About 50
.mu.L of reaction mixture is removed, filtered through a syringe
filter, and concentrated under a nitrogen stream. .sup.1H nmr
analysis confirms the disappearance of the phytol vinylic hydrogen
absorption. The reaction mixture is filtered through celite and
concentrated in vacuo to a yellow oil. The oil is adsorbed on
silica gel 60 (10 g) and flash-chromatographed over silica gel 60
(10 g) using a gradient elution (hexanes to 10% ethyl acetate in
hexanes). Appropriate fractions are combined and concentrated in
vacuo to afford pure phytanol 7 (4.57 g, 90.5%) as a colorless oil.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.68 (m, 2H), 1.46 (m,
25H), 0.74 (m, 15H)
[0148] Bis(2,2,2-trichloroethyl)phosphorochloridate (18.7 g, 49.2
mMol) is added to a stirred solution of compound 7 (9.80 g, 32.8
mMol) and DMAP (802 mg, 6.56 mMol) in dichloromethane. After
cooling the solution to 0.degree. C., triethylamine (13.7 mL, 98.4
mMol) is added dropwise via syringe. The cooling bath is removed
and the reaction mixture is stirred overnight at ambient
temperature, at which point TLC analysis (10% ethyl acetate in
hexanes) indicates complete reaction. The mixture is then diluted
with dichloromethane, washed with N HCl (3.times.), and the aqueous
layer is back extracted with dichloromethane. The combined organic
extracts are dried (MgSO.sub.4) and concentrated to an oil. The oil
is chromatographed over silica gel 60 (50 g) using a gradient of
hexanes to 15% ethyl acetate in hexanes. Appropriate fractions are
combined and concentrated in vacuo to afford
bis(2,2,2-trichloroethyl)phytanyl phosphate 8 (20.2 g, 96%) as a
colorless oil. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 4.62 (m,
4H), 4.31 (m, 2H), 1.80 (m, 1H), 1.56 (m, 4H), 1.27 (m, 20H), 0.86
(m, 15H); ES/MS m/z=641.2 [M+H].sup.+, 662.2 [M+Na].sup.+
[0149] An HCl/THF solution is prepared by bubbling HCl(g) through
anhydrous THF at 0.degree. C. for 2 min. The HCl/THF solution (20
mL) is added portionwise to a stirred suspension of compound 8
(20.1 g, 31.3 mMol) and Zn.degree. dust (24.5 g, 375.6 g-atom) at
0.degree. C. in THF (300 mL). Gas evolution is evident upon each
addition. After 1 hr, the Zn.degree. forms small clods in the
reaction mixture, and TLC analysis (25% ethyl acetate in hexanes)
indicates complete reaction. The reaction mixture is filtered
thorough celite, concentrated to about 1/4 volume, and diluted with
ethyl acetate. The organic solution is washed with 1N HCl
(3.times.) and dried (MgSO.sub.4). After removal of the
MgSO.sub.4by suction filtration, triethylamine (4.6 mL, 32.9 mMol)
is added and the system is then concentrated to a colorless foam.
The product is co-distilled with dichloromethane and toluene and
then evacuated (<30 Torr) at room temperature overnight to yield
phytanyl phosphate triethylamine salt 9. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 8.74 (m, 1H), 5.06 (br, 1H), 4.04 (t, 2H, J=6.5
Hz), 3.28 (dt, 6H, J=9.7, 5.5 Hz), 1.68 (m, 1H), 1.52 (m, 2H), 1.38
(t, 9H, J=7.3 Hz), 1.24 (m, 12H), 1.10 (m, 9H), 0.86 (m, 15H);
ES/MS m/z=377.3 [M-H].sup.-
Lactol Intermediate
##STR00010##
[0151] The orthogonally-protected disaccharide monopeptide 10 (Saha
et al. 2001 Organic Lett. 3:3575) (12.0 g, 12.1 mMol) is added to a
stirred suspension of 10% Pd/C (6.0 g) in 0.23 M HCl in acetic acid
(120 mL). The reaction mixture is stirred under an atmosphere of
hydrogen (balloon pressure) at 25.degree. C. for 1.5 hr. Analysis
of the reaction mixture by TLC (5% MeOH/CHCl.sub.3) shows complete
consumption of starting material. The reaction mixture is filtered
through a pad of Celite, concentrated to about 1/4 volume and
diluted with methylene chloride. The organic solution is washed
with aqueous NaHCO.sub.3 (.times.3) and water (.times.2). The
aqueous extracts are combined and extracted with methylene
chloride. The combined organic layers are washed with brine, dried
with Na.sub.2SO.sub.4, and concentrated in vacuo to afford the
lactol product 10a as a white solid (10.2 g, 94% by mass).
Inspection of the .sup.1H NMR spectrum reveals the presence of an
unidentified related substance impurity at about 20%. The substance
is taken forward with the desired product and removed via
purification of a subsequent intermediate. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 8.22 (d, 1H, J=3.4 Hz), 7.93 (dt, 2H, J=6.3,
1.7 Hz), 7.70 (dt, 1H, J=7.4, 1.5 Hz), 7.61 (m, 2H), 7.40 (t, 1H,
J=6.1 Hz), 7.17 (m, 1H), 6.05 (d, 1H, J=9.7 Hz), 5.58 (d, 1H, J=3.4
Hz), 5.10 (m, 2H), 2.35 (s, 3H), 2.14 (s, 3H), 2.05 (s, 3H), 2.03
(s, 3H), 2.02 (s, 3H), 1.96 (s, 3H), 1.37 (m, 6H); ES/MS m/z=904.4
[M+H].sup.+, 902.3 [M-H].sup.-
Monopeptide Dibenzyl Phosphate Intermediate
##STR00011##
[0153] Compound 10a (13.7 g, 15.2 mMol) in anhydrous
dichloromethane (60 mL) is added rapidly via pressure-equalizing
dropping funnel to a vigorously stirred suspension of tetrazole
(4.0 g, 57.8 mMol) and dibenzyl N,N'-diethylphosphoramidite (10.4
mL, 29.5 mMol) in anhydrous dichloromethane (40 mL) under argon at
25.degree. C. The reaction mixture becomes homogeneous within a few
minutes. After 2 hr, TLC (5%
[0154] MeOH/CHCl.sub.3) shows a complete reaction. The mixture is
cooled to -78.degree. C., and 0.2M peracetic acid in methylene
chloride (190 mL) is added dropwise over 10 min with vigorous
stirring. After the addition is complete, the cooling bath is
removed and the mixture allowed to warm to room temperature over 2
hr. TLC (5% MeOH/CHCl.sub.3) shows complete reaction. The mixture
is diluted with methylene chloride and extracted: ice-cold
saturated Na.sub.2S.sub.2O.sub.3 (1.times.), N HCl (1.times.) and
water (1.times.). The combined aqueous extracts are back-extracted
once with methylene chloride. The combined methylene chloride
solutions are dried over MgSO.sub.4 and concentrated in vacuo to a
colorless oil. The crude product is adsorbed on
pyridine-deactivated silica gel and chromatographed over
pyridine-deactivated silica gel using a gradient elution
(chloroform to 5% methanol in chloroform). Evaporation of solvent
provides the pure monophosphate triester 11 (12.4 g, 70%) as a
colorless solid. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.88 (d,
2H, J=7.3 Hz), 7.67 (t, 1H, J=7.6 Hz), 7.57 (t, 2H, J=7.7 Hz), 7.33
(m, 10H), 7.18 (d, 1H, J=7.4 Hz), 5.98 (m, 2H), 5.14 (m, 2H), 5.05
(dd, 2H, J=8.2, 3.1 Hz), 5.00 (d, 2H, J=8.1 Hz) 4.53 (m, 2H), 4.37
(m, 3H), 4.29 (dd, 1H, J=12.3, 4.1 Hz), 4.02 (m, 8H), 3.61 (d, 1H,
J=8.8 Hz), 3.52 (dd, 1H, J=10.9, 8.6 Hz), 3.33 (t, 2H, J=5.8 Hz),
2.05 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.95 (s,
3H), 1.81 (s, 3H), 1.37 (d, 3H, J=6.4 Hz), 1.33 (d, 3H, J=7.3 Hz);
ES/MS m/z=1164.6 [M+H].sup.+, 1186.6 [M+Na].sup.+, 886.6
[glycosyl].sup.+, 1162.6 [M-H].sup.-
Disaccharide Tripeptide Dibenzyl Phosphate Intermediate
##STR00012##
[0156] DBU (1.30 mL, 8.57 mMol) is added dropwise to a solution of
monophosphate triester 11 (9.07 g, 7.79 mMol) in dichloromethane
(78 mL) under an argon atmosphere. After 15 min, TLC (10%
MeOH/CHCl.sub.3) shows complete consumption of the starting
material. The reaction solution is diluted with dichloromethane and
washed twice with 1N HCl. The organic layer is dried
(Na.sub.2SO.sub.4) and concentrated in vacuo, affording the acid
analog of compound 11 as a foam (6.73 g, 87%). ES/MS m/z=1018.6
[M+Na].sup.+, 718.5 [glycosyl].sup.+, 994.6 [M-H].sup.-
[0157] The acid analog (2.32 g, 2.33 mMol), peptide 5 (1.05 g, 2.56
mMol) and N-hydroxy-benzotriazole (315 mg, 2.33 mMol) are dissolved
in anhydrous DMF (23 mL) at 0.degree. C. EDCI (491 mg, 2.56 mMol)
is added and the reaction mixture is stored at -20.degree. C. for
48 hr. Analysis of the reaction mixture by TLC (15%
MeOH/CHCl.sub.3) reveals complete consumption of the starting
material. The reaction mixture is concentrated in vacuo,
redissolved in ethyl acetate, and washed sequentially with water
(2.times.), N HCl (2.times.), water and brine. The organic solution
is dried with MgSO.sub.4 and concentrated to a foam. The foam is
adsorbed on pyridine-deactivated silica gel and chromatographed
over pyridine-deactivated silica gel using a gradient of
choloroform to 4% methanol in chloroform. Evaporation of solvent
affords compound 12 (2.33 g, 72%) as a white solid.
[0158] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.34 (m, 10H),
6.74 (t, 1H, J=5.9 Hz), 6.45 (d, 1H, J=9.3 Hz), 5.97 (dd, 1H,
J=5.4, 3.2 Hz), 5.07 (m, 5H), 2.06 (s, 3H), 2.05 (s, 3H), 2.02 (s,
3H), 1.84 (s, 3H), 0.88 (t, 3H, J=7.0 Hz); ES/MS m/z=1389.8
[M+H].sup.+, 1412.8 [M+Na].sup.+, 1111.7 [glycosyl].sup.+, 1387.7
[M-H].sup.-
Disaccharide Tripeptide Phosphate Monopyridyl Salt Intermediate
##STR00013##
[0160] The disaccharide tripeptide dibenzyl phosphate 12 (7.25 g,
5.22 mMol) in methanol (30 mL) is added to a suspension of 10% Pd/C
(3.63 mg) in methanol (25 mL), cooled in an ice bath to aid in
degassing the reaction solution. The solution is then warmed to
room temperature and hydrogenated at baboon pressure for 2 hr. The
catalyst is removed by filtration through celite and the filtrate
is treated with pyridine (1.0 mL). The resulting mixture is
concentrated to a white solid, which is collected and dried under
high vacuum for 16 hr to afford compound 13 (6.32 g, 94%). .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 8.73 (m, 2H), 7.96 (m, 1H), 7.56
(m, 2H), 5.88 (m, 1H), 2.09 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H),
2.01 (s, 3H), 1.95 (s, 3H), 1.94 (s, 3H), 0.88 (t, 3H, J=6.8
Hz);
[0161] ES/MS m/z=1209.7 [M+H].sup.+, 1232.8 [M+Na].sup.+, 1111.8
[glycosyl].sup.+, 1207.6 [M-H].sup.-
[0162] Modified Lipid II Intermediate
##STR00014##
[0163] To a stirred solution of phytanol phosphate triethylamine
salt 9 (57 mg, 0.118 mMol) in dichloromethane (1.2 mL) at room
temperature is added carbonyl diimidazole (15 mg, 0.094 mMol). The
reaction mixture is stirred overnight. .sup.1H NMR analysis
indicates an 85:15 mixture of imidazolate derivative and starting
phosphate as evidenced by the chemical shifts of the protons on the
oxygen-bearing carbon (phosphate at 3.993 ppm, imidazolate at 3.818
ppm). The crude imidazolate is concentrated in vacuo, taken up in
THF to 0.7 mL/mequiv, and used as thus obtained.
[0164] The crude imidazolate solution (1.0 mL, 1.5 equiv) is added
to a solution of monopyridyl salt 13 (450 mg, 0.349 mMol) in DMF
(0.35 mL) and THF (1.5 mL) at room temperature. Vacuum dried
4,5-dicyanoimidazole (103 mg, 0.873 mMol) is added and the reaction
mixture stirred at room temperature for 18 hr. Imidazolate solution
(0.7 mL, 1.0 equiv) is added and stirring continued for 23 hr. The
reaction is complete as evidenced by RP/HPLC analysis:
[0165] Waters Xterra C8 analytical column--(4.6 mm.times.250
mm.times.5 .mu.m)
[0166] Gradient--1:1=50 mM aq. NH.sub.4HCO.sub.3/MeOH to MeOH
[0167] Rate--1 mL/min, .lamda.=214 nm, 2 .mu.L reaction mix
[0168] Chart--Starting Material @ 10 min, Product @ 20 min
[0169] The crude pyrophosphate product is taken on without further
manipulation.
[0170] Aqueous NaOH (1.0N, 4.4 mL) is added to the stirred reaction
mixture and stirring is continued while the reaction course is
monitored until starting material is consumed as evidenced by
RP/HPLC (vide supra). At completion, the reaction mixture is
diluted with 50 mM aqueous NH.sub.4HCO.sub.3, transferred to a
separatory funnel, and extracted with ether (3.times.). The aqueous
phase is lyophilized to a tan solid (1.15 g). The solid is taken up
in minimal 50 mM aq. NH.sub.4HCO.sub.3, passed through a 0.45 .mu.m
syringe filter, and chromatographed over a CG-71 resin column (2.2
cm.times.26 cm, 100 mL bed volume) using a nine column volume
gradient from 30% MeOH in 50 mM aq. NH.sub.4HCO.sub.3 to 100% MeOH.
Appropriate 15 mL fracions are pooled, concentrated in a rotary
evaporator, and lyophilized to afford pure modified lipid II 15 as
a white solid (270 mg, 61%). .sup.1H NMR (400 MHz,
CD.sub.3CN:D.sub.2O=2:1) .delta. 5.37 (dd, 1H, J=7.1, 3.2 Hz), 4.48
(d, 1H, J=8.3 Hz), 4.25 (q, 1H, J=6.7 Hz), 4.15 (m, 1H), 3.46 (m,
1H), 2.89 (t, 1H, J=7.6 Hz), 2.22 (t, 1H, J=8.1 Hz), 1.95 (m, 5H),
0.82 (t, 5H, J=6.4 Hz), 0.82 (t, 15H, J=6.4 Hz); ES/MS m/z=1221.9
[M+H].sup.+, 1219.9 [M-H].sup.-
##STR00015##
[0171] A 20 mM stock solution of 15 is prepared by dissolving the
white powder (370 mg) in water (14.5 mL). To water (79.7 mL) is
added PEG 8000 (28.8 mL as a 50% stock in water). To this solution
is added 0.5M sodium phosphate buffer at pH 7.0 (5.8 mL), 1M
aqueous magnesium chloride (3.6 mL). The resulting solution is
divided equally among three conical tubes, and to each is added
compound 15 stock solution (4.8 mL) with thorough mixing. The
polymerization reaction is initiated by addition of 123 .mu.M
Staphylococcus aureus MtgA enzyme stock solution (3.9 mL). The
reaction solutions are mixed well and allowed to stand undisturbed
for 24 hr.
[0172] As the polymer forms, it aggregates and settles to the
bottom of the tube. The supernatant is removed and centrifuged
(3500 rpm, 20 min) to recover any polymer that has been
adventitiously removed with the supernatant. The pellet is
dissolved in 0.2M aqueous HCl (5 mL) and taken on to the next step
in this form.
[0173] To each of the crude polymer suspensions that remains after
decanting the supernatant is added 5M aqueous HCl (2.times.100
.mu.L with mixing after each addition). The system becomes
homogeneous after addition of the acid. To the yellowish solutions
thus obtained are added the acidified pellet solutions from
processing of the original supernatants (vide supra). These aqueous
acidic solutions are incubated at 37.degree. C. overnight, after
which the tube contents are pooled to a final volume of about 30
mL. The solution is neutralized to pH 7-8 using about 1.2 mL of 5M
aqueous NaOH, at which point the homogeneous solution becomes
cloudy. The cloudy solution is centrifuged twice (3500 rpm, 20
min), the pellet being washed with water each time and then
discarded (final volume of retained supernatant=36 mL).
[0174] Aqueous 5M NaOH (3.6 mL) is added to bring the final
concentration to 0.5M. This solution is allowed to stand at room
temperature for 2 hr and is then neutralized to pH 6 with 5M
aqueous HCl. The solution is divided into eight aliquots (8.times.5
mL, 1.times.3 mL), each in a 50 mL conical tube. Nine volumes of
ethanol are added to each tube and the solutions are stored
overnight in the -20.degree. C. freezer. The tubes are centrifuged
(3500 rpm, 20 min) and the supernatants carefully removed. After
brief drying in vacuo, the pellets are dissolved in minimal aqueous
NaCl (100 mM) and pooled to a final volume of 16 mL. Nine volumes
of ethanol are again added and the precipitation process repeated.
Finally, a third round of precipitation is executed.
[0175] The final pellet is dissolved in water (40 mL), placed in an
Amicon Model 8050 stirred cell concentrator, and subjected to
concentration/dilution cycles until the effluent conductance is
near zero. The solution is then concentrated as much as possible,
filtered through a pre-washed Millipore Steriflip filter, and
lyophilized. The synthetic peptidoglycan 15 is thus isolated as a
white solid (144 mg, 66%).
[0176] Verification of Synthetic Peptidoglycan Structure
[0177] The structural identity of the synthetic peptidoglycan 15 is
determined by size exclusion chromatography, .sup.1H NMR
spectroscopy, enzymatic susceptibility and mass spectrometry. Size
exclusion chromatography (3.2 mm.times.30 mm Pharmacia Superose 6
column, 20 mM sodium phosphate buffer at pH=7) indicates the
midpoint of the size distribution to be about 150 kilodaltons based
on dextran as standard (range about 75 kD to about 375 kD). .sup.1H
NMR (400 MHz, D.sub.2O) .delta. 4.45 (br s, 1H), 4.32 (br s, 1H),
3.50 (br m, 13H), 2.90 (m, 2H), 2.26 (M, 2H), 1.95 (s, 3H), 1.89
(s, 3H), 1.75 (m, 3H), 1.62 (m, 3H), 1.31 (m, 6H).
[0178] Synthetic peptidoglycan 15 is rapidly degraded by lysozyme.
Bacterial cell wall glycan polymer, a substructure of
peptidoglycan, is the natural substrate for lysozyme. Therefore,
lysozyme susceptibility represents prima facie evidence for the
glycan substructure of 15. Finally, the lysozyme hydrolysis product
of 15,
N-acetylgulcos-aminyl-.beta.-[1,4]-N-acetylmuramyl-[Ala-GABA-Lys]-peptide-
, is confirmed by ES/MS m/z 781.6 [M+H].+-., 779.5 [M-H].sup.-.
Example 2
Stimulation of IL10 Expression in Human Peripheral Blood
Mononuclear Cells by Synthetic Bacterial Antigen
[0179] Since natural peptidoglycans and bacterial capsular antigens
have been shown to stimulate inflammatory cytokines in vitro and in
vivo, we sought to determine the cytokine profile elicited from
human peripheral blood mononuclear cells (PBMCs) exposed to either
CP1 or synthetic PG.
[0180] Preparation of Synthetic PG (Compound 15)
[0181] For this and all succeeding examples, Compound 15 is
prepared as described in Example 1.
[0182] Purification of Cp1 from Pneumococcal Polysaccharide Powder
(ATCC)
[0183] For this and all succeeding examples, CP1 is prepared as
follows.
[0184] One gram of pneumococcal polysaccharide powder (American
Type Culture Collection, Manassas, Va.; lot #2059900, 5 bottles) is
dissolved in pyrogen-free distilled water (ca. 25 mL) with
intermittent shaking over the course of 8 h. The mixture is
transferred to the refrigerator (4.degree. C.) and allowed to stand
overnight to complete the dissolution and then transferred to a
Teflon bottle. The total volume after quantitative transfer is ca.
50 mL. Aqueous NaOH (4N, 50 mL) is added and the mixture is heated
for 1 hr at 80.degree. C. with occasional swirling. After cooling
to room temperature, the solution is carefully neutralized with
glacial acetic acid (11.3 mL).
[0185] The solution is dialyzed (3.times., 6-8 kD molecular weight
cutoff) against MilliQ water. After dialysis, the solution is
filtered (sterile, pyrogen-free, 0.2 .mu.nylon) and the filtrate is
adjusted to 50 mM Tris-HCl, pH 8.0. The crude basic hydrolysis
product is purified by IEC (ion exchange chromatography; stationary
phase=Q-Sepharose Fast Flow, gradient elution mobile phase=50 mM
Tris-HCl, pH 8.0 to 50 mM Tris-HCl, pH 8.0 and 1.0M in NaCl). The
CP1 containing fractions (180 mL in total) are identified by
analytical SEC (size exclusion chromatography; 3.2 mm.times.30 mm
Pharmacia Superose 6 column, 20 mM sodium phosphate buffer at pH
8). The solution is concentrated to 100 mL.
[0186] Aqueous NaCl (0.5 M, 200 mL) is added (3.times. dilution)
and the solution is concentrated to 100 mL using an Amicon
concentrator (YM series membrane, 10 kD mw cutoff). The NaCl
dilution/concentration process is repeated. The resulting solution
is diluted with distilled water (200 mL) and concentrated (Amicon)
to 100 mL. This process is repeated five times. The final
concentration takes the solution to a volume of 30 mL at which
point the conductance of the effluent is <40 .mu.S/cm (1 mM
NaCl=110 .mu.S/cm) and the pH=6. After filtration (sterile,
pyrogen-free, 0.2 .mu.nylon) and rinsing, the final solution volume
is 55 mL. The concentration is ca. 10 mg/mL as determined by SEC.
The pure CP1 can be stored indefinitely in this aqueous solution
form at 4.degree. C.
[0187] The identity of the CP1 is verified from an aliquot by
.sup.1H nmr (Stroop et al. (2002) Carbohydr. Res. 337 335-344) and
determined to be free from traces of protein and endotoxin by
standard assay methodologies.
[0188] Human PBMCs are obtained from anonymous donors through the
Eli Lilly and Company donor program. Mononuclear cells are
separated by Ficoll-hypaque (Stem Cell Technologies, Vancouver,
Canada) sedimentation to eliminate red blood cells and
polymorphonuclear leukocytes. The mononuclear layer, consisting of
T, B, and mononuclear cells, is cultured in RPMI 1640 with 10%
fetal bovine serum (Gibco, BRL, Carlsbad Calif.). PBMCs
(2.times.10.sup.6 cells/well) are cultured with several
concentrations of Compound 15 or CP1 to determine the optimal
response. Although the response to Compound 15 or CP1 typically
varies among human donors, a concentration of 0.6 .mu.g/ml of
Compound 15 and 6.0 .mu.g/ml of CP1 gives reproducible and
consistent results and is therefore used in these experiments (FIG.
3). Following isolation, human PBMCs are treated with Compound 15
(0.6 .mu.g/ml) or CP1 (6.0 .mu.g/ml) and maintained in culture for
eight days. Supernatants are sampled daily and analyzed for
cytokine expression using a multiplex Enzyme Linked Immunosorbent
Assay (Luminex, Linco Research, St. Charles, Mo.; catalog no.
HCYTO-60K). The human multiplex cytokine kits employed in these
experiments measure IL1, IL2, IL4, IL6, IL8, IL10, TNFci, and INFy.
In additional experiments, a custom IL12 specific antibody bead
complex is added to further define the cytokine response (Luminex,
Linco Research, St. Charles, Mo.). In all assays, results are
normalized against untreated media controls. Data are expressed as
the average of triplicate wells.+-.the standard error of the
concentration of cytokines represented. The data represent typical
results from at least three experiments.
[0189] As shown in FIG. 3, in several experiments, the data reveal
that treatment of human PBMCs with Compound 15 or CP1 results in
only minimal expression of most inflammatory cytokines represented
in the kit. Surprisingly, the predominant response is the
expression of the anti-inflammatory cytokine IL10. The expression
of IL10 occurs late in the time course, detectable at day 5 and
continuing to rise at day 8 to concentrations of approximately 80
pg/ml (Compound 15) to 250 pg/ml (CP1). IL2 and INFy are only
barely detectable early in the time course, whereas the expression
of IL4, IL6, IL12 or TNF are not detected at any time point.
[0190] These results suggest that CP1 and synthetic PG selectively
induce the expression of IL10 in PBMC cell cultures, and that they
may be efficacious in animal models of inflammation.
Example 3
Interaction of CP1 and Synthetic PG With Toll-Like Receptor 2
(TLR2)
[0191] Toll-like receptors (TLRs) play a critical role in early
innate immunity to invading pathogens by sensing the presence
microorganisms within the body (Akira et al. (2001) Nature Immunol.
2:675-680.) These receptors recognize highly conserved structural
motifs only expressed by microbial pathogens, called
pathogen-associated microbial patterns (PAMPs) (Medzhitov (2001)
Nat. Rev. Immunol. 135-145). PAMPs include various bacterial cell
wall components such as lipopolysaccharides (LPS), peptidoglycan
and lipopeptides, as well as flagellin, bacterial DNA, and viral
double-stranded RNA. Stimulation of TLRs by PAMPs initiates a
signaling cascade leading to the activation of the transcription
factor NF-.kappa.B, which induces the secretion of pro-inflammatory
cytokines and effector cytokines that direct the adaptive immune
response (Janeway et al. (2002) Annu. Rev. Immunol. 20:197-216).
Since natural peptidoglycan is a PAMP that activates cells via
TLR-2 (Iwaki et al. (2002) J. Biol. Chem. 277:24315-24320), we
sought to determine if synthetic peptidoglycan (Compound 15) could
also activate NF-.kappa.B in vitro. In addition, since CP1 behaves
in vivo like Compound 15, we investigated its ability to activate
human TLR2 using an NF-.kappa.B-reporter assay in HEK293 cells.
[0192] These experiments involve transfecting HEK293 cells
(American Type Culture Collection, Manassas, Va.) with two plasmid
DNAs. The first plasmid, called pcDNA3.1/hygrow, contains the human
TLR-2 gene. The second plasmid, pNF-.kappa.B-luc (Stratagene, La
Jolla, Calif.), encodes the NF-.kappa.B gene linked to a luciferase
reporter gene whose product can be followed in vitro as a direct
measure of NF-.kappa.B-activation. To prepare the DNA for
transfection into the cells, Fugene6 (Roche, Base1 Switzerland)
transfecting reagent is diluted 1:6 in OPTI-MEM (Invitrogen,
Carlsbad, Calif.) growth medium. Next, 75 ng of pNF-.kappa.B-luc
and 300 ng of pcDNA3.1/hygrow DNA are added to the diluted Fugene6
and the mixture is incubated at 37.degree. C. for 30 minutes.
HEK293 cells at a concentration of 10.sup.6 cells/ml are added to
the DNA/Fugene6 mixture. After gentle mixing, the cell/DNA mixtures
are aliquoted into 96 well tissue culture plates at a concentration
of 10.sup.5 cells/well and incubated for 24 h at 37.degree. C. in a
5% CO.sub.2 environment. After incubation, varying concentrations
of test compounds are added to the cells and incubation is allowed
to continue for an additional 24 h. The amount of luciferase
activity resulting from incubation with the compounds is evaluated
by removing the growth media from the cells and replacing it with
100 .mu.l of RLB lysis solution (Promega, Madison, Wis.). Lysis is
completed by a single freeze/thaw cycle at -80.degree. C. The
luciferase activity of each cell culture is determined in a 25
.mu.l aliquot of cell lysate in a Victor Luminometer (Perkin Elmer
Life Sciences, Shelton, Conn.) according to the manufacturer's
instructions. A positive control for NF.kappa.B activation in
HEK293 cells is incubation of transfected cells with TNF.alpha.
(Pharmingen, Palo Alto, Calif.) at a concentration of 1 ng/ml.
[0193] Table 1 shows that, using varying concentrations of
commercially-available natural peptidoglycan isolated from
Staphylococcus aureus (Fluka, St. Louis, Mo.), up to 54.5-fold
induction of NF.kappa.B activity is observed compared with that of
unstimulated cultures. Another commercially available preparation
of peptidoglycan and polysaccharide mixture (PG/PS; Lee Labs Inc.,
Grayson, Ga.) stimulates up to a 33.7-fold induction of NF-.kappa.B
in HEK293 cells. The data in Table 1 show the lack of NF-.kappa.B
activation by either Compound 15 or CP1 at concentrations up to 500
.mu.g/ml.
TABLE-US-00002 TABLE 1 Luciferase Assay for Measurement of TLR2
Activity in HEK293 Cells Compound.sup.1: Staphylococcus aureus
Concentration peptidoglycan PG/PS Cpd 15 (.mu.g/ml) (Fluka) (Lee
Labs Inc) CP1 (PG) 500 48.0 33.7 0 0 250 51.8 27.0 0 0 125 54.5
15.2 0 0 62.5 50.7 8.8 0 0 31.2 48.6 5.3 0 0 15 37.7 3.0 0 0 7.5
34.9 2.6 0 0 3.7 31.8 1.9 0 0 1.8 24.7 1.6 0 0 0.93 20.7 1.5 0 0
0.46 17.8 1.0 0 0 .sup.1Positive stimulation control: cultures
incubated with 1 ng/ml TNF.alpha. yielded a 22.5-fold increase in
luciferase activity compared with unstimulated cultures.
[0194] These results demonstrate that unlike natural peptidoglycan
or bacterial capsular material (which are PAMPs), CP1 and synthetic
PG do not induce activation of NF.kappa.B through TLR2.
Example 4
Interaction of CP1 and Synthetic PG With Other Toll-Like Receptors
(TLRs)
[0195] Concurrently with the studies investigating the interaction
of CP1 and synthetic PG with TLR2, we also tested the interaction
of CP1 and synthetic PG with an expanded list of TLR constructs
using the same NF-.kappa.B-reporter assays described above in
Example 3 (Table 1). The results are shown in Table 2.
TABLE-US-00003 TABLE 2 Summary of TLR activation.sup.1 via
NF.kappa.B using N/S PAs. Compound Escherichia coli Cpd 15 PG/PS
Receptor LPS CP1 (PG) (Lee Labs) TLR2 + - - ++ TLR2/CD14 ++ - - ++
TLR4/CD14 +++ - - - TLR5 + - - - TLR7 +/- - - - TLR8 - - - -
.sup.1The relative positive activation of NF.kappa.B is indicated
by the number of "+" signs while a lack of activation is indicated
by a "-" sign.
[0196] As shown in Table 2, concentrations of either Compound 15 or
CP1 between 0.001-100 .mu.g/ml elicit no NF-.kappa.B-signaling with
any of the other TLR receptors. In all of these experiments, LPS
serves as a positive control for TLR4 activation and natural PG
serves as a positive control for TLR2 activation.
[0197] These experiments confirm the previous observation (Example
3) that neither Compound 15 nor CP1 activates TLR2, even in the
presence of a necessary adaptor molecule CD14 (Janeway et al.
(2002) Annu. Rev. Immunol. 20:197-216), and extends this
observation to five other TLRs.
Example 5
CP1 and Synthetic PG Do Not Stimulate Maturation of Human Dendritic
Cells (DCs)
[0198] DCs are often referred to as professional antigen presenting
cells and sentinels of the immune system (Banchereau et al. (2000)
Annu. Rev. Immuno. 1.18:767-811). They reside in almost all
peripheral tissues in an immature state (iDC), which allows them to
phagocytose (or engulf) antigens so they can be processed and
presented to the immune system, specifically to naive T cells
(Shortman et al. (2002) Nat. Rev. Immunol. 2:151-161). With their
cargo of processed antigens, the dendritic cells migrate via the
blood and lymphatic circulation to lymph nodes, spleen, and other
lymphoid tissues. During this journey, they mature, losing their
ability to take up and process antigen, and begin to display that
antigen on their surfaces. By the time they reach their
destinations, they have become potent stimulators of T cells and,
with their multitentacled (dendritic) shape, proceed to make
cell-cell contact with large numbers of T cells (Banchereau et al.
(2000) Annu. Rev. Immuno. 1.18:767-811).
[0199] Certain CD (cluster of differentiation) markers, which are
surface-exposed proteins and glycoproteins, can be used to track
the maturation state of the dendritic cells (Chakraborty et al.
(2000) Clin. Immunol. 94:88-98). Table 3 lists the commonly used CD
markers for this purpose and their relative expression levels on
monocytes, immature dendritic cells (iDC), and mature dendritic
cells (mDC) (Chakraborty et al. (2000) Clin. Immunol.
94:88-98).
TABLE-US-00004 TABLE 3 Cluster of Differentiation (CD) Markers used
to distinguish monocytes (MO), immature-(iDC) and mature-(mDC)
dendritic cells. Cell Surface Marker.sup.1: CD1a CD14 CD83 CD86
HLA-DR MO - ++ - - - iDC ++ - - - - mDC ++ - +++ +++ +++ .sup.1The
relative amount of each cell surface marker is indicated in the
table by the number of "+" signs while the absence of the cell
surface marker is indicated by a "-" sign
[0200] Labeling cells with fluorescently-conjugated anti-CD
antibodies permits analysis of dendritic cell maturation status via
determination of mean fluorescence intensity (MFI) of the marker on
the surface of a cell population. Flow cytometry is used to analyze
large cell samples for the presence of cell surface markers. In
vitro, iDC can be produced by isolating CD14(+) monocytes from
human blood and culturing these cells for four days with a cocktail
of two cytokines (Granulocyte-Macrophage Colony-Stimulating Factor
(GM-CSF) and Interleukin-4 (IL-4)). Since several bacterial
molecules, for example LPS (Matsunaga et al. (2002) Scand. J.
Immunol. 56:593-601) and peptidoglycan (Michelsen et al. (2001) J.
Biol. Chem. 276:25680-25686), can induce the differentiation of
iDCs to the mDC phenotype (as would occur during activation of the
innate immune system), we were interested in evaluating the potency
of CP1 and synthetic PG in maturing human monocyte-derived
dendritic cells.
[0201] Human PBMCs are obtained from anonymous donors through the
Eli Lilly and Company donor program. Mononuclear cells are
separated by Ficoll-hypaque (Stem Cell Technologies, Vancouver,
Canada) sedimentation to eliminate red blood cells and
polymorphonuclear leukocytes. The CD14(+) monocyte fraction is
isolated from PBMCs by incubation with CD14-conjugated magnetic
beads (Miltenyi Biotech Inc., Auburn, Calif.) followed by physical
separation in a magnetic field using an autoMACS apparatus
(Miltenyi Biotech, Inc., Auburn, Calif.). Once isolated, the
CD14(+) monocytes are incubated in complete DC media consisting of
RPMI 1640 containing 10% heat-inactivated Australian fetal bovine
serum (FBS), non essential amino acids, sodium pyruvate,
2-mercaptoethanol, penicillin-streptomycin (as 1.times.solutions
all from Gibco BRL, Carlsbad Calif.). In addition, some cultures
are induced to differentiate into iDCs using complete DC medium
containing 20 ng/ml IL-4 (Sigma, St. Louis, Mo.) and 40 ng/ml
GM-CSF (Pharmingen, Palo Alto, Calif.) for four days at 37.degree.
C. with 5% CO.sub.2. After the four day incubation, cells are
incubated with CP1, synthetic PG, or LPS for an additional 24 h
before being stained for CD marker analysis by flow cytometry. The
standard staining protocol for flow cytometry involves washing the
cells twice in Dulbecco's phosphate buffered saline (DPBS, Gibco
BRL, Carlsbad, Calif.) containing 2% heat inactivated FBS (Gibco
BLR, Carlsbad, Calif.) and 0.05% sodium azide (Sigma, St. Louis,
Mo.), hereafter referred to as "flow wash solution." After washing,
10.sup.5 cells/sample are resuspended in 100 .mu.l of flow wash
solution and 20 .mu.l of pre-diluted phycoerythrin-conjugated
primary anti-CD marker antibody (all antibodies used are from
Pharmingen, Palo Alto, Calif.) for 15 min on ice. A similarly
conjugated isotype control antibody is included in all analyses.
After incubation, cells are washed three times in flow wash
solution. After the final wash, cells are fixed by resuspension in
the flow wash solution containing 1% paraformaldehyde (Becton
Dickinson, Palo Alto, Calif.). Cell samples are stored at 4.degree.
C. and protected from light until analysis using an FC500 flow
cytometer (Beckman Coulter, Miami, Fla.). Once cells are correctly
gated for forward and side scatter profiles, mean fluorescent
intensity (the amount of marker on the cell surface) is evaluated
for 10,000 cells/sample.
[0202] The results of these experiments are summarized in Table
4.
TABLE-US-00005 TABLE 4 Flow cytometric analysis of monocyte-derived
dendritic cells after incubation with N/S PAs or LPS. Cell Surface
Marker.sup.1: Cell type CD1a CD14 CD83 CD86 HLA-DR MO 5.1 16.5 5.6
12.8 23.9 iDC 116.1 3.3 7.6 10.9 7.7 iDC + 122.7 3.3 7.6 11.4 8.7
CP1 iDC + 109.8 3.4 9.5 12.1 9.1 Cpd 15 iDC + 124.4 4.1 46.7 75.4
29.2 LPS .sup.1Numbers represent mean fluorescence intensity of
cell surface markers in 10,000 cells/sample.
[0203] As shown in Table 4, the panel of surface markers used in
this experiment confirms that the four day incubation of CD14(+)
monocytes with GM-CSF and IL-4 induces the differentiation of the
cells into immature dendritic cells (compare the results in Table 4
with the expected phenotype summarized in Table 3. As shown in
Table 4, these immature dendritic cells are functionally capable of
reaching a mature state since incubation of these cells with E.
coli LPS (the positive control for maturation) significantly
increases the staining of CD-83, -86 and HLA-DR on their cell
surfaces, which is the expected phenotype of a mature DC. The data
in Table 4 show that incubation with either CP1 or Compound 15
fails to change the staining profile from the iDC state, indicating
that neither compound is capable of effecting the maturation of
dendritic cells.
Example 6
Uptake of Synthetic PG by Immature Human Dendritic Cells (iDCs)
[0204] The inhibition of maturation of DCs induced by CP1 and
synthetic PG may be due to the inability of these cells to process
these molecules internally. Antigen uptake and processing
(degradation) are two fundamental properties of APCs (Banchereau et
al. (2000) Annu. Rev. Immunol. 18:767-811). DCs are the most potent
APCs of the immune system in part because of their powerful
capacity to endocytose or sample material from their environment
(Shortman et al. (2002) Nat. Rev. Immunol. 2:151-161). To determine
whether iDCs are capable of endocytosing high molecular weight
immunomodulatory polysaccharide antigens such as synthetic PG, we
prepared a fluorescent derivative of Compound 15 for use in uptake
studies employing confocal microscopy. This imaging technique can
be used to localize within cells fluorescent probes such as the
Oregon-green labeled Compound 15 disclosed herein. In these
experiments, we use as a control molecule fluorescently labeled
(FITC) dextran polymer. Dextran (40 kDa in size) is a macromolecule
commonly used for endocytosis experiments (Sallusto et al. (1995)
J. Exp. Med. 182:389-400). Since it is a high molecular weight
carbohydrate polymer, it is a useful comparator for Compound
15.
[0205] Oregon-green labeled Compound 15 is prepared as described in
PCT International Publication WO 01/79242. Briefly, Oregon-green
(Molecular Probes, Eugene, Oreg.)-conjugated Lipid II is included
in an MtgA-polymerization reaction at a ratio of 1:4 with unlabeled
Lipid II to produce a 25% Oregon-green labeled polymer. The
polymeric material is purified and treated as previously described.
For uptake studies, fluorescent Compound 15 at a final
concentration of 50 .mu.g/ml, or Lysine-fixable FITC-conjugated
dextran (40 kDa size, Molecular Probes, Eugene, Oreg.) at 1 mg/ml,
is incubated with human monocyte-derived iDC prepared as described
in Example 5 for two minutes at 37.degree. C. After incubation,
extracellular probe is removed by washing the cells four times in
ice cold complete DC medium (Example 5). Washed cells are then
incubated at 37.degree. C. and staining is stopped at two-minute
intervals by washing in 1% paraformaldehyde fix diluted in flow
wash solution (which also contains the metabolic poison sodium
azide; protocol described in Example 5). Glass slide samples are
prepared at each time interval and sealed with clear nail polish.
Samples are stored at -20.degree. C. and protected from light until
analysis on a Radiance 2100 confocal microscope (BioRad
Laboratories, Hercules, Calif.).
[0206] FIG. 4 shows black and white confocal images of human iDCs
treated with either FITC-Dextran (40 kDa in size) or Oregon-green
labeled Compound 15 (approx. 150 kDa in size) for two minutes.
After incubation with the polymers, the cells are washed
extensively to remove any external polymer and the internalized
material is followed at two-minute intervals.
[0207] Intracellular localization of either Compound 15 or Dextran
is visible as bright areas in the dark field of the cells after a
two-minute incubation with the polymers (FIG. 4). Furthermore, the
internalized polymers are not spread throughout the cytoplasm, but
are instead localized in discrete packets or vesicles, consistent
with their presence in endocytic vacuoles.
[0208] These results demonstrate that iDCs are capable of
endocytosing synthetic PG.
Example 7
Kinetics of Uptake of Synthetic PG by Immature Human Dendritic
Cells (iDCs)
[0209] Since there appears to be such robust uptake of Compound 15
by iDCs (FIG. 4), the fluorescent version of this molecule was used
in flow cytometry to visualize the kinetics of polymer uptake.
[0210] In these experiments, human monocyte-derived dendritic cells
are prepared as described in Example 5. Dendritic cells are
resuspended at 5.times.10.sup.5 cells/sample and incubated on ice
at 37.degree. C. At the start of each time course, cells are
incubated with either fluorescent Compound 15 at a final
concentration of 50 .mu.g/ml or Lysine-fixable FITC-conjugated
dextran (40 kDa size, Molecular Probes, Eugene, Oreg.) at 1 mg/ml.
At 0, 2, 10, 20, 30, 40, and 50 minutes after the start of
incubation, uptake is stopped by washing the cells with four washes
of ice cold flow wash buffer (Example 5). The washed cells are
fixed in paraformaldehyde also as described in Example 5. Stained,
fixed cells are stored at 4.degree. C. protected from light until
analysis using a FC500 flow cytometer (Beckman Coulter, Miami,
Fla.). Once cells are correctly gated for forward and side scatter
profiles, mean fluorescent intensity of the population is evaluated
for 10,000 cells/sample. FIG. 5 shows that over time, Compound 15
accumulates in the iDC cytoplasm. The same is true for the control
molecule FITC-Dextran. To control for non-specific adhesion of the
molecules to the cell surface (which could be read as a positive in
this assay), cells are also incubated with fluorescent polymers at
0.degree. C. At this temperature, the iDCs are viable yet unable to
endocytose material, i.e., they are metabolically inactive
(Sallusto et al. (1995) J. Exp. Med. 182:389-400). At this
temperature, signal from neither the control molecule
(FITC-dextran) nor Compound 15 increases over time (FIG. 5). This
indicates that the uptake seen at 37.degree. C. is a result of
cellular endocytosis.
[0211] These results demonstrate that iDCs are capable of rapidly
endocytosing fluorescently labeled Compound 15, and that the
inability of this molecule to mature DCs is not due to
recalcitrance to endocytic uptake thereof.
Example 8
CP1 and Synthetic PG Interfere With LPS-Induced Maturation of
iDCs
[0212] As shown above in Table 4 (Example 5), LPS at 50 .mu.g/ml is
capable of transforming iDCs to an mDC phenotype characterized by
an increase in co-stimulatory markers (CD83 and CD86) as well as
class II Major Histocompatibility (MHC) markers (HLA-DR)
(Chakraborty et al. (2000) Clin. Immunol. 94:88-98). We next
investigated whether CP1 and synthetic PG are capable of
interfering with the transformation of iDCs to mDCs. The results
are shown in Table 5.
TABLE-US-00006 TABLE 5 Flow cytometric analysis of monocyte-derived
dendritic cells matured with E. coli LPS in the presence of N/S PAs
Cell Surface Marker.sup.1: Cell type CD1a CD14 CD83 CD86 HLA-DR iDC
+ 126.4 4.1 46.7 75.4 29.2 LPS iDC + 132.2 4.3 51.1 49.0 31.8 LPS +
CP1 iDC + 120.2 4.1 52.6 59.2 31.9 LPS + Cpd 15 .sup.1Numbers
represent mean fluorescence intensity of 10,000 cells/sample.
[0213] In these experiments, CD14(+) monocytes are isolated from
human PBMCs and differentiated into iDCs as described in Example 5.
After differentiation, iDCs are incubated with either of two known
inducers of cell maturation: E. coli LPS (Matsunaga et al. (2002)
Scand. J. Immunol. 56:593-601) or a cytokine cocktail containing
Tumor Necrosis Factor-.alpha. (TNF-.alpha.),
Interleukin-1.beta.(IL-1.beta., Prostaglandin E.sub.2, and IL-6
(Dieckman et al. (2002) J. Exp. Med. 196:247-253) for 24 h. To some
induced cultures we also add 50 .mu.g/ml CP1 or 100 .mu.g/ml
Compound 15 at the same time we add either LPS or cytokines. After
incubation, the cells are evaluated for CD 1a, CD14, CD83, CD86,
and HLA-DR expression by flow cytometry as described in Example
5.
[0214] In the case of cytokine-matured iDCs, flow cytometry
confirms that maturation by incubation with the cytokine cocktail
occurs; however, incubation with CP1 or synthetic PG has no
influence on the matured phenotype as determined by surface marker
analysis (data not shown). In contrast to this, Table 5 shows that
both CP1 and synthetic PG are able to interfere with LPS-induced
maturation of iDCs. Specifically, surface expression of the
co-stimulatory marker CD86 is decreased in the presence of these
molecules, while the other markers tested are essentially
unchanged. Additional experiments also demonstrate that CD80,
another marker of co-stimulation, is also decreased (data not
shown).
[0215] The powerful capacity of DCs to activate T cells is linked
to their constitutive expression of both MHC and costimulatory
markers like the family B7 markers (i.e., CD80 and CD86)
(Banchereau et al. (2000) Annu. Rev. Immunol. 18:767-811). If these
molecules are decreased or absent from the DC cell surface, the DCs
are unable to participate in stimulatory cognate interactions with
T cells. Schwartz (1990) Science 248:1349-1356 was the first to
observe that presentation of antigen on MHC molecules in the
absence of costimulatory molecules induces T-cell anergy. Thus, DCs
can provide both stimulatory (by virtue of being APCs) and
downregulatory signals for immune reactions.
[0216] To understand fully the significance of the above findings,
it is important to understand the role of DCs in immune tolerance.
Tolerance is an essential property of the immune system whereby
self- or auto-antigens do not trigger an immune response (Belz et
al. (2002) Immunol. Cell Biol. 80:463-468). Others have shown that
when DCs undergo an incomplete maturation (low levels of CD80 and
or CD86), or have been treated with antibodies that block the B7
family of costimulatory markers (i.e., CD80 and CD86), these cells
can induce antigen-specific unresponsiveness in vitro and T cell
anergy in vivo (Lu et al. (1996) J. Immunol. 157:3577-3586; Gao et
al. (1999) Immunology 98:159-170). Immature DCs are now understood
to contribute to peripheral tolerance by inducing the
differentiation of human T regulatory cells (Jonuleit et al. (2000)
J. Exp. Med. 192:1213-1222), a group of T cells that display
regulatory functions in vitro and in vivo. Activated T regulatory
cells have also been shown to elicit the production of IL-10, an
anti-inflammatory cytokine, through autocrine expression or
induction in effector T cells (Dieckmann et al. (2002) J. Exp. Med.
196:247-253). Thus, the fact that Compound 15 and CP1 appear to
influence the expression of costimulatory markers on the DC surface
suggests a mechanism of action for these molecules in the induction
of toleragenic DCs. These anergic DCs could then induce T-cell
anergy directly or through the activity of a T regulatory cell
population.
Example 9
CP1 and Synthetic PG Are Not Polyclonal Mitogens and Do Not
Stimulate Proliferation of Lymphocytes in Human PBMC Cultures
[0217] Mitogens are substances that nonspecifically induce DNA
synthesis and cell division in lymphocytes. LPS is a B-cell
specific mitogen (Moller et al. (1973) J. Infect. Dis. 128:52-56),
while phytohaemagglutinin (PHA) specifically induces T cells to
divide (Boldt et al. (1975) J. Immunol. 114:1532-1536).
Peptidoglycan is another T cell mitogen (Levinson et al. (1983)
Infect. Immun. 39:290-296). We were therefore interested in
determining whether CP1 or Compound 15 could stimulate human
peripheral blood mononuclear lymphocytes (PBMCs) to divide in
culture, particularly since Compound 15 is a completely synthetic
peptidoglycan. Cell division is measured in these experiments by
uptake of radiolabeled nucleotide base into the DNA of the
proliferating cells. The radioactive counts per minute (cpm) of the
culture, measured by scintillation counting, are a direct measure
of cellular proliferation.
[0218] In this experiment, PBMCs are isolated from a healthy human
volunteer as described in Example 2. Isolated PBMCs are aliquoted
into round-bottomed 96-well tissue culture plates (Falcon Brand,
Becton Dickinson, Palo Alto, Calif.) at density of 10.sup.5
cells/well. Some cells are also incubated with 50 .mu.g/ml of CP1,
100 .mu.g/ml Compound 15, or 25 .mu.g/ml PHA (Sigma, St. Louis,
Mo.) as a positive control for T cell proliferation. Cells are
incubated at 37.degree. C. in a 5% CO.sub.2 atmosphere for up to
four days. At 30, 54, and 78 hours post inoculation, some cultures
are pulsed with 1 .mu.Ci/well of [.sup.3H]-thymidine (Specific
Activity 6.7 Ci/mmol; ICN Inc, Costa Mesa, Calif.) and returned to
37.degree. C. incubation for a further 18 hours before being
harvested onto filter plates (Packard Instruments, Shelton, Conn.)
using a Filtermate harvestor (Packard Instruments, Shelton, Conn.).
Filterplates are dried after harvesting, prior to the addition of
20 .mu.l/well of Microscint-O scintillation cocktail (Packard
Instruments, Shelton, Conn.). Scintillation counting is performed
with a MicroBeta TriLux liquid scintillation counter (Perkin Elmer,
Shelton, Conn.).
[0219] FIG. 6 shows the typical proliferation response of human
PBMCs to the polyclonal T cell activator PHA. The incorporation of
[.sup.3H]-thymidine into PHA-treated cells is close to 100,000
times that of untreated cells after two days exposure, and
proliferation rates increase up to four days. In contrast, neither
CP1-nor Compound 15-treated cells respond by DNA proliferation and
expansion (FIG. 6). Therefore, these molecules do not appear to
behave like polyclonal mitogens in human PBMC cultures.
Example 10
CP1 Stimulates an Increase in CD4+CD25+ T Cells
[0220] As CP1 and Compound 15 do not behave like mitogens (Example
9), we hypothesized that the lack of proliferation is due T
regulatory cell suppression. This experiment examines the
possibility that these compounds stimulate an increase in T
regulatory cell numbers as defined by the surface markers CD4 and
CD25.
[0221] In these experiments, human PBMCs are isolated and cultured
at a density of 10.sup.5 cells/well in 96-well tissue culture
plates as described in Example 2. Some cultures also receive 0.6 or
6.0 .mu.g/ml of CP1 immediately after being aliquoted into the
tissue culture plates. Cell cultures are incubated at 37.degree. C.
in a 5% CO.sub.2 atmosphere for up to six days. Each day during
culture, cell samples are removed and stained for the co-expression
of CD4 and CD25 on the cell surface by flow cytometry. Samples are
stained using the standard staining protocol outlined in Example 5.
Antibodies for human CD4, CD25, as well as an isotype control
antibody, are obtained from Pharmingen (Palo Alto, Calif.).
[0222] FIG. 7 shows the percentage of CD4+/CD25+ cells in
CP1-treated PBMC cultures sampled each day over the course of six
days. The percentage of CD4+/CD25+ cells in untreated PBMCs is
highest after two days of culture, and accounts for up to 2% of
total cells in the culture. Incubation of human PBMCs with 0.6 or
6.0 .mu.g/ml CP1 increases the percentage of CD4+/CD25+ cells to
4.5% and 9.0%, respectively, in these cultures (FIG. 7).
[0223] These data suggest that treatment of human PBMCs with a
polysaccharide immunomodulator such as CP1 induces an increase in
the number of cells possessing a T regulatory phenotype.
Example 11
CP1 and Synthetic PG Suppress the .alpha.CD3 Antibody-Induced
Proliferation of Lymphocytes in Human PBMCs
[0224] When an antigen (Ag) is presented to a naive T cell in the
context of MHCII on the surface of an antigen presenting cell
(APC), there is engagement of the MHC-Ag complex with the T cell
receptor (TCR)/CD3 complex on the surface of the T cell (Weiss et
al. (1986) Annu. Rev. Immunol. 4:593-619). This interaction,
together with an amplification signal generated by CD28-B7 (CD80,
CD86) interaction on these two cell types leads to T cell
activation, cytokine stimulation, and cell division (Weiss et al.
(1986) Annu. Rev. Immunol. 4:593-619. In the absence of Ag or APC,
T lymphocytes can become activated and proliferate in vitro by
incubation with plate-bound anti-CD antibodies (van Lier et al.
(1989) Immunol. 68:45-50). Mimicking the activation by antigens,
the binding of CD3 antibodies to T cells results in the activation
of tyrosine kinase, a rise in the intracellular calcium
concentration, generation of diacylglycerol, and activation of
protein kinase C. Both calcium and protein kinase C serve as
intracellular messengers for the induction of gene activation (van
Lier et al. (1989) Immunol. 68:45-50). As shown in FIG. 6 of
Example 9, anti-CD3 antibody-mediated T cell proliferation is also
measured by the incorporation of [.sup.3H]-thymidine into the DNA
of dividing cells.
[0225] Since proliferation of PBMCs is not observed following
treatment with either CP1 or Compound 15 (FIG. 6), we hypothesized
that these molecules may stimulate T regulatory cells as suggested
by the results shown in FIG. 7. The present experiment is performed
to investigate whether these molecules induce suppression of
lymphocyte proliferation.
[0226] In these experiments, human PBMCs are isolated and cultured
as described in Example 2 and plated at 10.sup.6 cell/ml in T-25
tissue culture flasks (Corning Inc., Corning, N.Y.) for 24 h at
37.degree. C. in a 5% CO.sub.2 atmosphere. Cultures are exposed to
either CP1 at 50 .mu.g/ml or PG at 100 .mu.g/ml during this period.
One day prior to the incubation of cells on antibody coated plates,
anti-human CD3 antibody (Clone UCHT1, Pharmingen, Palo Alto,
Calif.) or an isotype-matched control antibody (Pharmingen, Palo
Alto, Calif.) is diluted in Dulbecco's phosphate buffered saline
(DPBS) (Gibco, BRL, Carlsbad, Calif.), and the wells of a 96-well
tissue culture plate are coated with 100 .mu.l aliquots of diluted
antibody. Plates are coated overnight at 4.degree. C. and washed
three times in DPBS before use. Human PBMCs, exposed to CP1,
Compound 15, or not exposed to either compound, are plated into
antibody-coated wells at a density of 10.sup.5 cells/well. Tissue
culture plates are incubated at 37.degree. C. in a 5% CO.sub.2
atmosphere for 30 or 54 hours before 1 .mu.Ci/well of
[.sup.3H]-thymidine (Specific Activity 6.7 Ci/mmol; ICN Inc, Costa
Mesa, Calif.) is added to each well. Cells are then returned to
37.degree. C. incubation for an additional 18 h before the cells
are harvested as described in Example 9. The liquid scintillation
counting procedure is also as described Example 9. The data for
this experiment are presented both as raw counts per minute (cpm)
of radioactivity and as a stimulation index (SI), which is the
ratio of the cpm of cells in .alpha.CD3 antibody-coated wells to
the cpm of cells in isotype (control) antibody-coated wells.
[0227] FIG. 8 shows that either 48 or 72 hours exposure to
.alpha.CD3 antibody causes human PBMCs to proliferate as shown by
the uptake of [.sup.3H]-thymidine (FIG. 8, triangles). Furthermore,
the amount of proliferation is directly correlated to the amount of
.alpha.CD3 antibody in the well, with the highest proliferation
seen in cells exposed to 0.4 .mu.g/ml .alpha.CD3 antibody. FIG. 8
also shows that pre-incubation of human PBMCs with either 50
.mu.g/ml of CP1 or 100 .mu.g/ml PG for 24 h prior to incubation
with .alpha.CD3 antibody causes a decrease in the amount subsequent
proliferation (FIG. 8, diamonds for CP1 and squares for PG Compound
15).
[0228] These results demonstrate that N/S PAs inhibit
anti-CD3-induced lymphocyte proliferation.
Example 12
Micro-Array Analysis of Human CD3+ Cells Following treatment with
N/S PAs and .alpha.CD3 Antibody
[0229] The results demonstrating cytokine expression shown in FIG.
3 are corroborated and extended by measurement of cytokine
modulation using microarray technology.
[0230] PBMCs are isolated as described in Example 2 and added to
6-well tissue culture plates in a medium containing RPMI with 10%
fetal bovine serum (Gibco BRL, Carlsbad, Calif.), 50 .mu.M
(3-mercaptoethanol, and 500 .mu.g/ml penicillin/streptomycin
(complete medium). T cell density is 2.5.times.10.sup.6 cells per
well. Either 50 .mu.g/ml CP-1, 100 .mu.g/ml Compound 15, or
complete medium is added to each well of the appropriate plate.
Incubation is at 37.degree. C. for 24 hours. Simultaneously, 6-well
tissue culture plates are treated with either 0.2 .mu.g/ml
.alpha.CD3 in sterile Phosphate-Buffered Saline (PBS, Gibco BRL,
Carlsbad, Calif.), 5 ml/well, or an equal volume of sterile PBS.
The uninoculated plates are incubated overnight at 4.degree. C.
Following incubation, cells treated with CP1 orsynthetic PG, or
untreated control cells, are gently resuspended and added to plates
that have either been coated with .alpha.CD3 or not, and incubation
is continued at 37.degree. C. for an additional 48 hours.
[0231] PBMCs are then processed with a Pan T Cell Isolation Kit
(Miltenyi Biotec, cat. #130-053-001; Auburn, Calif.) in substantial
accordance with the manufacturer's instructions. This kit is a
magnetic labeling system designed to isolate untouched T cells from
peripheral blood. Non-T cells are removed by magnetic separation
from unlabeled CD3.sup.+cells using an autoMACS (Miltenyi Biotec
Inc, Auburn, Calif.). The isolated T cells are stored at
-80.degree. C.
[0232] Total RNA is isolated from the cells using Trizol (GibcoBRL,
Carlsbad, Calif.) followed by chloroform extraction and subsequent
alcoholic precipitation following procedures specified by the
manufacturer. The RNA is quantitated spectrophoto-metrically, and
its integrity assessed by gel analysis. All RNA preparations are
stored at -80.degree. C. until needed.
[0233] Total RNA serves as the template for the synthesis of
biotin-labeled cDNA. This labeled cDNA is subsequently used as a
probe for commercially available directed microarrays.
Specifically, a GEArray Q Series Human Common Cytokine Kit, cat. #
HS-003N (SuperArray Bioscience Corporation, Frederick, Md.) is
employed. Probe synthesis and microarray processing are performed
as suggested by the manufacturer. A Typhoon 8600 Imager (Amersham
Pharmacia Biotech, Piscataway, N.J.) is used in chemiluminescent
mode to capture and store images that are then analyzed using
ImageQuant software (Amersham Pharmacia Biotech, Piscataway, N.J.).
Data are exported to Microsoft Excel, and image intensity is
corrected for background and normalized between experiments using
GEArray Analyzer software (SuperArray Bioscience Corporation,
Frederick, Md.).
[0234] Analysis of the data reveals a cytokine modulation pattern
that is consistent with that seen using the multiplex Enzyme Linked
Immunosorbent Assay as shown in FIG. 3. Table 6 shows that cells
exposed to CP1 consistently demonstrate an up-regulation of IL10
and IL19, which is a homolog of IL10, and a down-regulation of
IL17. IL17 is thought to be expressed mainly by activated T cells,
and functions to initiate and maintain an inflammatory response.
Cells that are exposed to .alpha.CD3 are activated and therefore
show an up-regulation of IL17, TNF-.beta., and other cytokines
known to participate in the inflammatory process. Anti-CD3-treated
cells also show decreases in both IL10 and IL19. When either CP1 or
Compound 15 is added to cells that are subsequently exposed to
.alpha.CD3, there is a dramatic increase in IL10 levels,
accompanied by concomitant decreases in IL17 and TGF-.beta..
TABLE-US-00007 TABLE 6 Cytokine Response in T Cells Exposed to
Various Stimuli Stimulus Up-Regulation* Down-Regulation* CP-1 (50
.mu.g/ml) IL19 IL17 .alpha.-CD3 (0.2 .mu.g/ml) IL17 IL10 TNF-.beta.
IL19 .alpha.-CD3 (0.2 .mu.g/ml) + IL10** IL17 CP-1 (50 .mu.g/ml)
IL19 TNF-.beta. *Change from untreated cells of at least 5-fold
**Also seen with Compound 15 @ 100 .mu.g/ml
[0235] The up-regulation of IL10 expression in CD3+ T cells induced
by both CP1 and Compound 15 in these microarray experiments
corroborates the results observed in Example 2, and in animal
models, and suggests that this cytokine can be used as a biological
marker to monitor the biological/immunological activity of both CP1
and synthetic PG in vitro and in vivo. The data also suggest that
directed microarrays can be used to monitor not only the biological
activity of the present compounds, but also the biological activity
of derivative compounds to determine the effects of structural
differences on immunodulatory potency.
Example 13
N/S PAs Protect Against the Formation of Intra-Abdominal
Abscesses
[0236] Since CP1 and synthetic PG induce T regulatory cells with
suppressive function in vitro as well as the late production of
IL10 from human PBMCs (Examples 10 and 11, and Example 2,
respectively), we were interested in assessing the ability of these
polymer antigens to protect animals against the inflammatory
formation of abscesses in vivo. A rat intra-abdominal abscess model
is used to address this question.
[0237] The rat model of abscess formation employed in these studies
is a modification of that described by Onderdonk et al. ((1977) J.
Infect. Dis. 136:82-87) and Tzianabos et al. ((1993) Science
262:416-419). Male Lewis rats (Charles River Laboratories,
Wilmington, Mass.), weighing between 135-175 grams, are used for
all experiments. Rats are housed in microisolator cages and given
chow (Ralston Purina, St. Louis, Mo.) and water ad libitum. Upon
arrival, animals are allowed to acclimate for 24 hours.
Intra-abdominal abscesses are induced by a single intraperitoneal
injection of prepared inoculum containing Bacteroides fragilis
(ATCC 23745; American Type Culture Collection, Manassas, Va.)
(10.sup.8 colony forming units per animal) mixed at a 1:6 dilution
with an adjuvant solution containing sterile rat cecal contents. B.
fragilis is maintained at -80.degree. C. in brain heart infusion
broth. Cultures are grown anaerobically in brain heart infusion
broth to log phase and diluted for use with rat sterile cecal
contents (rSCC). rSCC is prepared from rat cecal pellets that are
solubilized in brain heart infusion broth, autoclaved, and then
filtered. Animals are euthanized at six days post-inoculation and
assessed for abscess formation. Animals with one or more fully
formed abscesses are scored as positive. Animals with no abscesses
yield a negative score. Individuals scoring the results are blinded
to the identity of the experimental groups.
[0238] Animals (10 rats/group) are dosed subcutaneously with three
doses of Compound 15 or CP1 at twenty four hour intervals the day
before, the day of, and the day after challenge with B.
fragilis/rSCC (Tzianabos et al. J. Clin. Invest. 96:2727 (1995)).
Challenge with the inoculum is carried out by the intraperitoneal
route. Animals are administered log dilutions of Compound 15 or CP1
at 100, 10, and 1 .mu.g (X3)/animal. Results are expressed as the
percent protection (number of animals with no abscesses/treatment
group), and statistical significance is calculated using the
Fishers Exact Probability Test.
[0239] As shown in Table 7, both CP1 and Compound 15 produce
considerable protection against the formation of abscesses at both
the 100 .mu.g and 10 .mu.g doses when compared to that of saline
controls. Protection is assessed as the complete absence of
abscesses as compared to control animals with one or more abscess.
Protected animals show no deleterious effects of antigen
administration, with few, if any, signs of fever or lethargy, which
are common symptoms of inflammation. Nor do these animals display
symptoms of sepsis.
TABLE-US-00008 TABLE 7 Activity of N/S PAs in the Rat Abscess Model
Animals with % of Animals % Treatment Group Abscesses/group with
Abscesses Protection CP1 100 .mu.g .times. 3 SC 1/8 12.5 87.5 CP1
10 .mu.g .times. 3 SC 2/8 25 75 CP1 1.0 .mu.g .times. 3 SC 3/8 37.5
62.5 Saline 0.1 ml .times. 3 SC 6/8 75 25 Cpd 15 100 .mu.g .times.
3 SC 1/8 12.5 87.5 Cpd 15 10 .mu.g .times. 3 SC 1/8 12.5 87.5 Cpd
15 1.0 .mu.g .times. 3 SC 2/8 25 75 Saline 0.1 ml .times. 3 SC 6/8
75 25
[0240] Taken together with the data shown in Examples 2-12, these
data suggest that protection against the inflammatory processes
required for the formation of abscesses in response to bacterial
challenge in this model is inhibited by the presence of immature
dendritic cells, which can directly inhibit T cell activation or
induce the generation of a T regulatory population. Direct
inhibition of inflammatory cells by T regulatory cell contact can
further stimulate the expression of IL-10. In total, one or more of
these events may orchestrate the inhibition of inflammation seen in
the in vivo abscess model.
Example 14
N/S PAs Reduce the Incidence and Severity of Post-Surgical
Adhesions
[0241] Exogenous IL10 has been shown to limit the formation of
post-surgical adhesions (Holschneider et al. (1997) J. Surg.
Research 70:138-143). Further, T regulatory cells have potent
anti-inflammatory activity and have been shown to limit
inflammation in in vivo models (Maloy et al. (2001) Nat. Immunol.
2:816-822; Shevach (2002) Nat. Rev. Immunol. 2389-400). T
regulatory cells have also been shown to elicit the production of
IL10 from their target inflammatory T cells (Diekman et al. (2002)
J. Exp. Med. 196:247-253). As variously shown in Examples 2, 10,
11, 12, and 13, above, CP1 and Compound 15 stimulate the production
of IL10 from PBMCs, an increase in T regulatory cell numbers and
function in vitro, and afford protection from the formation of
abscesses in vivo. Since the inflammatory responses that lead to
fibrin deposition and the formation of abscesses is similar to the
pathologies involved in adhesion formation, we hypothesized that
treatment with CP1 or Compound 15 in an adhesion model would
likewise stimulate the activity of T regulatory cells and
ultimately the endogenous production of IL10 that may result in
reduction in the formation of post-surgical adhesions.
[0242] To test this hypothesis, male Lewis rats (Charles River
Laboratories, Wilmington, Mass.) are dosed subcutaneously with
three injections of Compound 15 or CP1 at twenty four hour
intervals the day before, the day of, and the day after surgical
induction of adhesions (Tzianabos et al. (1995) J. Clin. Invest.
96:2727-2731). Rats are administered log dilutions of each compound
at 100 jag, 10 .mu.g, and 1 .mu.g (X3) in 0.2 ml saline/animal.
Control groups are administered saline in 0.2 ml volumes at the
same dosing schedule. Peritoneal adhesions are induced following
the methods of Kennedy et al. ((1996) Surgery 120:866-871) and
Tzianabos et al. (PCT International Publication WO 00/59515) with
minor modifications. Briefly, rats are anesthetized with 2-5%
isoflurane in oxygen to a surgical plane of anesthesia. A one to
two cm midline incision is made into the abdominal cavity to expose
the cecum. The cecum is aseptically removed from the peritoneal
cavity and abraded with surgical gauze to induce visible
microhemorrhages. The cecum is then re-inserted into the peritoneal
cavity. The left and right lateral abdominal walls are inverted
aseptically and also abraded in the manner described above.
Following this procedure, 0.2-0.3 ml of rat sterile cecal contents
(rSCC), prepared as described in Example 13, are added to the
peritoneal cavity as an inflammatory adjuvant (Onderdonk et al.
(1982) J. Clin. Invest. 69:9-14). The peritoneum is closed with 3-0
silk followed by skin closure with tissue adhesive (3M Animal Care
Products, St. Paul, Minn.). Animals are sacrificed one week
following surgical manipulation and evaluated for the formation of
adhesions. Adhesions are scored on a scale of 0-5 using the method
described by Kennedy et al ((1996) Surgery 120:866-871): 0=no
adhesions; 1=thin filmy adhesion; 2=more than one thin adhesion;
3=thick adhesion with focal point; 4=thick adhesion with planar
attachment; and 5=very thick vascularized adhesions or more than
one planar adhesion. This scoring system approximates the system
used in human medicine, enumerates adhesions present, and indicates
the severity of the adhesion pathology; higher scores indicate
greater severity in inflammation and adhesion formation. The
results are shown in Table 8.
TABLE-US-00009 TABLE 8 Activity of N/S PAs in the Rat Adhesion
Model Range of Adhesion Mean Scores/Individual Adhesion Treatment
Group Scores Score Median CP1 100 .mu.g .times. 3 SC 0-4 1.8 2.0
(0, 1, 2, 24) CP1 10 .mu.g .times. 3 SC 1-4 2.4 3.0 (1, 1, 3, 3, 4)
CP1 1.0 .mu.g .times. 3 SC 1-4 2.6 3.0 (1, 1, 3, 3, 4) Saline 0.1
ml .times. 3 SC 3-4 4 4 (3, 3, 4, 5, 5) Cpd 15 100 .mu.g .times. 3
SC 0-4 1.6 2 (0, 0, 2, 2, 4) Cpd 15 10 .mu.g .times. 3 SC 0-4 2.2
3.0 (0, 1, 3, 3, 4) Cpd 15 1.0 .mu.g .times. 3 SC 0-4 2.2 3 (0, 1,
3, 3, 4) Cpd 15 0.1 .mu.g .times. 3 SC 1-4 3 3 (1, 3, 3, 4, 4)
Saline 0.1 ml .times. 3 SC 3-4 3.6 4 (3, 3, 4, 4, 4)
[0243] The data shown in Table 8 demonstrate that adhesion
formation in rats treated with 100 .mu.g of Compound 15 or CP1 is
significantly limited (median score=2.0) when compared to that in
saline controls (median score=4.0). These data demonstrate that
these polysaccharide antigens effectively protect rats from the
formation of severe surgically induced adhesions, and suggest that
these polymers induce an anti-inflammatory effect in vivo.
Example 15
N/S PAs Inhibit Delayed Type Hypersensitivity Reactions in a Guinea
Pig Model
[0244] Clinical evaluation of the safety and efficacy of immune
modulators such as CP1 and Compound 15 requires a convenient
biomarker. This is necessary because safety and dose determination
are usually determined in healthy volunteers, where a defined
inflammatory process is not measured. Furthermore, such a biomarker
would be useful in later stage trials as abscesses and/or adhesions
cannot be readily observed and graded for therapeutic efficacy in a
non-invasive manner following therapy with immune modulators.
Consequently, we developed a delayed type hypersensitivity (DTH)
animal model (Gray et al. (1994) Curr. Opin. Immunol. 6:425-437).
This assay can also be used in humans as a biomarker for clinical
efficacy studies using the present immune modulators. Clinically,
DTH skin tests are of significant value in the overall assessment
of immunocompetence in humans (Gray et al. (1994) Curr. Opin.
Immunol. 6:425-437; Kuby et al. (2000) Immunology, W.H. Freeman and
Co). Such tests including the administration of candin as described
below are commonly used to test immuno-competence in AIDS
patients.
[0245] A Guinea pig model is used to demonstrate the utility of a
DTH response as a biomarker. A localized DTH response in an animal
model represents an important source of information with regard to
T cell function. Direct measurements of the DTH response can be
readily observed and measured in humans and animals. Flares,
wheals, and/or indurations can be observed and readily measured
quantitatively on the surface of the skin.
[0246] For this purpose, female Hartley Guinea pigs (Charles River
Laboratories, Wilmington, Mass.) weighing 250-299 grams are used
for all DTH experiments. Guinea pigs are housed in microisolator
cages and given chow (Ralston Purina, St. Louis, Mo.) and water ad
libitum. Upon arrival, the animals are allowed to acclimate for 24
hours. Hair is then clipped from the back of the animal in an area
approximately 2.times.2 inches. The area is scrubbed with
povidone-iodine (H&P Industries/Triad Medical Inc., Mukwonago,
Wis.) followed by an alcohol scrub. Next, the animal is sensitized
to Candida albicans antigens by injecting a 0.2 ml saline
suspension of Candida albicans A26 (ATCC 90234) intradermally on
the dorsal side of the neck region. Cultures of Candida albicans
A26 are maintained at -80.degree. C. in a glycerol and lactose
freezing solution, and are grown aerobically on Sabourauds and
dextrose agar slants (DIFCO, Detroit, Mich.) at 35.degree. C. for
24 hours. Cultures are then suspended in sterile saline and
adjusted spectrophotometrically to a predetermined optical density
equivalent to approximately 2.0.times.10.sup.7 cells/ml before
use.
[0247] Three days following sensitization, the animals are treated
with the immunomodulator CP1 formulated in sterile water for
injection (Abbott Laboratories, North Chicago, Ill.) at 100, 10 and
1.0 ng per 0.2 ml. The animals are injected subcutaneously on the
dorsal side of the neck with 0.2 ml. A third group of animals dosed
with the water vehicle serves as the positive control group.
[0248] Four days following sensitization, the animals are shaved
and scrubbed as described above. Four equally spaced areas in the
shaved region are injected intradermally with 0.1 ml of Candin
(Allermed Laboratories, Inc., San Diego, Calif.), which serves as a
recall antigen for T cells that have been previously sensitized to
C. albicans. The animals are observed daily over three days for
erythema, wheals, and indurations at these four sites. Two traverse
(vertical and horizontal) diameters of the flares are recorded for
each site. These are averaged and a mean of the flare area
(mm.sup.2) is calculated. Treated animals are compared to untreated
controls in order to assess therapeutic efficacy. The results are
shown in Table 9.
TABLE-US-00010 TABLE 9 The Activity of CP1 in a Model of Delayed
Type Hypersensitivity (DTH) Area of Flare (mm.sup.2) H.sub.2O CP1
CP1 CP1 Control 1.0 ng .times. 1 (SC) 10 ng .times. 1 (SC) 100 ng
.times. 1 (SC) 76.26 .+-. 5.52 63.34 .+-. 9.64 50.92 .+-. 4.26
43.99 .+-. 5.56
[0249] The data shown in Table 9 demonstrate a significant
reduction in the flare area in animals treated with CP1 as compared
to that of control animals.
[0250] These findings demonstrate that a DTH skin assay is an
appropriate biomarker for clinical use and evaluation of
polysaccharide immunomodulators such as CP1 and synthetic PG
Compound 15.
[0251] The invention being thus described, it is obvious that the
same can be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of the present
invention, and all such modifications as would be obvious to one
skilled in the art are intended to be included within the scope of
the following claims.
Sequence CWU 1
1
115PRTArtificialsynthetic construct 1Ala Xaa Xaa Xaa Xaa1 5
* * * * *