U.S. patent application number 13/470072 was filed with the patent office on 2012-08-30 for methods for modulating macrophage proliferation in ocular disease using polyamine analogs.
This patent application is currently assigned to PATHOLOGICA LLC. Invention is credited to Michael McGrath.
Application Number | 20120219970 13/470072 |
Document ID | / |
Family ID | 36927925 |
Filed Date | 2012-08-30 |
United States Patent
Application |
20120219970 |
Kind Code |
A1 |
McGrath; Michael |
August 30, 2012 |
METHODS FOR MODULATING MACROPHAGE PROLIFERATION IN OCULAR DISEASE
USING POLYAMINE ANALOGS
Abstract
Methods for modulating macrophage proliferation in an individual
afflicted with or at risk for an ocular disease such as ARMD are
provided. The methods employ a polyamine analog, or salt or
protected derivative thereof. Macrophage proliferation has been
implicated in a number of serious disorders, including ARMD. The
invention also provides methods for aiding diagnosis and monitoring
therapy of an ocular disease such as ARMD.
Inventors: |
McGrath; Michael;
(Burlingame, CA) |
Assignee: |
PATHOLOGICA LLC
Burlingame
CA
|
Family ID: |
36927925 |
Appl. No.: |
13/470072 |
Filed: |
May 11, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11064781 |
Feb 22, 2005 |
8198334 |
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13470072 |
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09179383 |
Oct 26, 1998 |
7087648 |
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11064781 |
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60063317 |
Oct 27, 1997 |
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60063318 |
Oct 27, 1997 |
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Current U.S.
Class: |
435/7.24 ;
435/39; 435/7.92 |
Current CPC
Class: |
A61K 31/131 20130101;
A61P 25/28 20180101; G01N 2333/523 20130101; G01N 33/6896 20130101;
A61K 31/13 20130101; A61P 43/00 20180101; G01N 2800/164 20130101;
A61P 27/02 20180101; A61K 31/155 20130101 |
Class at
Publication: |
435/7.24 ;
435/39; 435/7.92 |
International
Class: |
G01N 33/566 20060101
G01N033/566; C12Q 1/06 20060101 C12Q001/06 |
Goverment Interests
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] This invention was made in part during work supported by a
federal grant from the National Institutes of Health, Grant No. CA
96230-03. The government may have certain rights in the invention.
Claims
1. A method of determining whether an individual is afflicted with,
or at risk for, a macrophage-associated ocular disease comprising:
detecting a level of activated or proliferating macrophages in a
biological sample from the individual, wherein detection of
activated or proliferating macrophages at a level greater than a
normal level indicates the individual is afflicted with, or is at
risk for, a macrophage-associated ocular disease.
2. The method of claim 1, wherein the disease is selected from the
group consisting of macrophage-associated retinal disease,
age-related macular degeneration (ARMD), vitreoretinopathy, and
diabetic retinopathy.
3. The method of claim 1, wherein the disease is non-exudative
ARMD.
4. The method of claim 1, wherein detecting the level of activated
or proliferating macrophages comprises detecting the level of a
biomarker for activated or proliferating macrophages.
5. The method of claim 1, wherein detecting the level of activated
or proliferating macrophages comprises detecting the level of
CD14+/CD16+ expression, CD16+/PCNA+ expression, CCR2 expression,
CD16+ expression, PCNA expression, or systemic Monocyte
Chemoattractant Protein (MCP-1).
6. A method of monitoring therapeutic treatment of a
macrophage-associated ocular disease comprising: detecting the
presence of activated or proliferating macrophages, or systemic
MCP-1 in a biological sample from a subject before and after
administration of a therapeutic agent, wherein a decrease in
activated or proliferating macrophages or systemic MCP-1 after
administration of the therapeutic agent is indicative of a
therapeutic response in the subject.
7. The method of claim 6, wherein the therapeutic agent is selected
from the group consisting of a polyamine analog, a salt of a
polyamine analog, a protected derivative of a polyamine analog,
MGBG, or a combination thereof.
8. The method of claim 6, wherein the disease is selected from the
group consisting of macrophage-associated retinal disease,
age-related macular degeneration (ARMD), vitreoretinopathy, and
diabetic retinopathy.
9. The method of claim 6, wherein said disease is non-exudative
ARMD.
10. A method for determining severity of a macrophage-associated
ocular disease comprising: detecting a level of activated or
proliferating macrophages or systemic MCP-1 in a biological sample
from a subject, wherein the level of activated or proliferating
macrophages or systemic MCP-1 corresponds to severity of the
macrophage-associated ocular disease in the subject.
11. The method of claim 10, wherein detecting the level of
activated or proliferating macrophages comprises detecting the
level of a biomarker for activated or proliferating
macrophages.
12. The method of claim 10, wherein detecting the level of
activated or proliferating macrophages comprises detecting the
level of CD14+/CD16+ expression, CD16+/PCNA+ expression, CCR2
expression, CD16+ expression, or PCNA expression.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
patent application Ser. No. 11/064,781, filed Feb. 22, 2005, which
is a continuation-in-part of U.S. patent application Ser. No.
09/179,383, filed Oct. 26, 1998, which claims the benefit of U.S.
Provisional Application Ser. Nos. 60/063,317 and 60/063,318, both
filed Oct. 27, 1997, all of which are incorporated by reference
herein.
TECHNICAL FIELD
[0003] This invention relates to diseases associated with
proliferating macrophages, particularly as it relates to
macrophage-associated ocular diseases, particularly macrophage
associated retinal diseases such as vitreoretinopathy, diabetic
retinopathy, and age-related macular degeneration (ARMD). More
specifically, it relates to the use of polyamine analogs or salts
or protected derivatives thereof to modulate macrophage
proliferation, particularly in individuals afflicted with or at
risk for a macrophage-associated retinal disease such as ARMD. The
invention also relates to methods of aiding diagnosis, monitoring
therapy, and delaying development of macrophage proliferation
disorders such as ARMD that entail detection and/or modulation of
macrophage proliferation.
BACKGROUND
[0004] Ocular diseases, such as degenerative cell proliferative
diseases exemplified by age-related macular degeneration (ARMD),
present a major health issue today. ARMD alone affects more than
1.75 million people in the U.S. It has been estimated that the
number will increase to about 3 million by 2020 due to the rapid
growing of the aging U.S. population. (see, e.g., Arch Ophthalmol,
(2004) 122:564). ARMD is the principal cause of registered legal
blindness and other visual disability among individuals over 60
years old in many parts of the world, including U.S., Western
Europe, Australia, and Japan. (Ambati, et al., Surv. Ophthalmol.,
48:257 May-June 2003; Zarbin, Arch. Ophthalmol., 2004,
122:598-614)
[0005] Clinical hallmarks of ARMD include drusen, hyperplasia or
the retinal pigment epithelium (RPE), geographic atrophy, and
choroidal neovascularization (CNV). Drusen are localized deposits
of extracellular material found between the basement membrane of
the RPE and Bruch's membrane. Drusen are characterized
morphologically as either "soft", with fuzzy, indistinct edges, or
"hard", with discrete, well-demarcated edges. Typically, drusen are
clustered in the central macula, and exhibit a varied and complex
morphology as determined by fundoscopic examination. It is well
established in the art that the size, number and confluency of
drusen are significant determinants for risk of developing ARMD.
For a discussion of drusen as biomarkers of immune-mediated
processes at the RPE-Bruch's membrane interface in ARMD, see
Hageman et al. (2001) Retinal Eye Res. 20:705-732. In general, the
precise pathogenic mechanisms that lead to ARMD are not well
understood (for a review, see Zarbin, (2004), supra).
[0006] ARMD is generally characterized into two forms. The
exudative or "wet" form of ARMD is characterized by CNV growth
under the RPE and retina with subsequent hemorrhage, exudative
retinal detachment, disciform scarring, and retinal atrophy, and
can also be accompanied by serous or hemorrhagic pigment epithelial
detachment. In the non-exudative or "dry" form of ARMD the
accumulation of drusen is thought to cause atrophy of the macula,
leading to vision loss. Wet ARMD accounts for about 75% of cases
with several central vision loss. About 18% of people aged 65 to 74
years, and about 30% of people older than 74 years, have early
ARMD, characterized by the presence of soft drusen or drusen with
RPE degeneration or hyperpigmentation. (see, e.g., Zarbin (2004)
supra)
[0007] There have been a number of reports assessing
immune/inflammatory mechanisms in the formation of drusen in ARMD.
One group has suggested that impaired macrophage recruitment may
allow accumulation of C5a and IgG in the eye, which in turn induces
vascular endothelial growth factor (VEGF) production by RPE,
possibly mediating development of CNV, the primary cause of visual
loss in the exudative or "wet" form of ARMD. Ambati et al. Nat.
Med. 2003 November; 9(11):1390-7. Epub 2003 Oct. 19. Macrophages
and foreign body giant calls have been reported near the Bruch's
membrane where drusen is found (van der Schaft et al. Br J.
Ophthalmol. 1993 October; 77(10):657-61; Lopez et al. Am J.
Ophthalmol. 1991 112:647-56; Killlingsworth et al. Eye. 1990 4 (Pt
4):613-21).
[0008] Others have hypothesized that macrophages and other
inflammatory cells may be involved in CNV, which is part of the
symptom in ARMD. For example, Oh et al. (Invest Ophthalmol Vis Sci
1999 40:1891-1898) suggested that IL-1.beta. and TNF-.alpha.
secreted by macrophages may promote, at least in part, angiogenesis
in CNV membranes by stimulating VEGF production in RPE cells.
Tsutsumi et al. (J Leukoc Biol. 2003 74:25-32) reported that mice
that lack CCR2, the receptor for MCP-1, the number of infiltrating
macrophage and the area of CNV were significantly reduced. Cousins
et al. (Arch Ophthalmol. 2004 122:1013-8) evaluated the activation
state of macrophage function in patients with age-related macular
degeneration (AMD) by quantifying the production of the
proinflammatory and angiogenic factor tumor necrosis factor alpha
(TNF-.alpha.) and by correlating its expression with dry and wet
AMD. This group reported that although wide variability in
TNF-.alpha. expression by blood monocytes was observed among
different patients, those patients with monocytes that expressed
the greatest amount of TNF-.alpha. demonstrated higher prevalence
of CNV.
[0009] Macrophages have also been reported to be associated with
the Bruch's membrane in ARMD. Killingsworth, et al., Eye, 1990, 4:
613-621. Weller et al. (1991) Eur J. Ophthalmol. 1:161-6 reported
that posttraumatic proliferative retinopathy was apparently
characterized by a severe initial inflammatory reaction as
evidenced by the presence of numerous macrophages. Capeans et al.
(Retina. 1998; 18(6):546-50) reported that monocyte chemotactic
protein-1 (MCP-1) present at significantly higher levels in the
vitreous of eyes with vitreoretinal disorders than the vitreous of
control eyes, and hypothesized that MCP-1 may be involved in the
recruitment of macrophages and monocytes into the vitreous of eyes
with proliferative vitreoretinal disorders. Other groups have
reported that macrophages are activated in rabbits during the
inflammatory phase of the development of proliferative
vitreoretinopathy (PVR). Hui et al. Graefes Arch Clin Exp
Ophthalmol. 1999 July, 237(7): 601-5; Chen et al. Ocul Immunol
Inflamm. 2002 March, 10(1): 27-39; Martin et al. Ophthalmic Res.
2003 July-August, 35(4): 232-8.
[0010] Other immune mechanisms have also been implicated in ARMD.
For example, auto-antibodies with specificity for retinal tissue
have been detected in ocular pathologies, including ARMD (Penfold
et al. Graefes Arch Clin Exp Ophthalmol. 1985 223:69-76).
C-reactive protein, a serum protein associated with inflammation,
is elevated in subjects with ARMD. (Seddon et al. (2004) JAMA
291:704-10) At the tissue level, confirmation of inflammatory cell
infiltrates has been documented for early, intermediate, and late
stage disease. (Penfold et al. (1985) Graefes Arch Clin Exp
Ophthalmol. 223:69-76) Immunohistochemical analysis of drusen has
demonstrated the presence of immunologic antigens, to include
complement components C5, C5b-9, immunoglobulin, and HLA-DR.
(Russell et al. (2000) Am J Ophthalmol. 129:205-14) For a review of
the immunological and etiological aspects of macular degeneration,
see Penfold et al. (2001) Retinal Eye Res 20:382-414.
[0011] More recently, the role of macrophages in ARMD has been
examined using transgenic mice deficient for either monocyte
chemoattractant protein (MCP-1), or its cognate chemokine receptor,
CCR-2. (Ambati et al. (2003) Nat. Med. 9:1390-7). These transgenic
animals developed ARMD abnormalities that include the
characteristic display of RPE drusen, accumulation of lipofuscin,
photoreceptor atrophy, and CNV. Ambati et al. hypothesized that
impaired macrophage recruitment may allow accumulation of C5a and
IgG, which induces vascular endothelial growth factor (VEGF)
production by RPE, possibly mediating development of CNV.
[0012] Diagnosis and prognosis of ARMD has primarily focused on
assessing drusen (e.g., total drusen area or the size of drusen),
which has been identified as the most important conventional risk
factors for ARMD progression (Latkany (2004)
medscape.com/viewarticle/494566). Other conventional techniques in
screening and diagnosis include fluorescence angiography (FA),
optical coherence tomography (OCT), spectral OCT, and scanning
laser ophthalmoscope with OCT (SLO-OCT). Therapies for ARMD are
largely in the experimental stage and focus on treatment of wet
ARMD. Exemplary therapies include those directed toward inhibition
of neovascularization such as laser photocoagulation, photodynamic
therapy (which may be accompanied by administration of a
light-activated drug such as VISUDYNE.RTM.), transpupillary
thermotherapy, microcurrent stimulation, and administration of
antiangiogenic agents, radiation therapy, and surgery. A review of
convention therapies is provided by Lois et al. (2004) Comp
Ophthalmol Update 5:143-161.
[0013] There is a need for methods of indicating development and/or
progression of these ocular diseases such as ARMD, and for
treatment of such diseases. The present invention addresses these
needs.
ADDITIONAL LITERATURE
Ocular Diseases, Including ARMD
[0014] Additional literature which may be of interest relating to
ocular diseases, and particularly ARMD, includes: Penfold et al.
(1987) Graefes Arch Clin Exp Ophthalmol 225:70-6; Killingsworth et
al. (1990) Eye 4:613-621; Nishimura et al. (1990) Ophthalmologica
200:39-44; Weller et al. (1991) Exp Eye Res. 53(2):275-81); Otani
et al. (1999) Invest Ophthalmol Vis Sci 40:1912-1920; Grossniklaus
et al. (2000) Mol Vis 8:119-26; Spandau et al. (2000) Invest
Ophthalmol Vis Sci 41:S836; Van der Schaft et al. (2001) Invest
Ophthalmol Vis Sci 33:3493; Grossiklaus et al. (2002) Mol. Vis
8:119-226.
Macrophages
[0015] Macrophages are terminally differentiated cells generally
incapable of further cell division. Macrophage proliferation has
been implicated in certain serious proliferative diseases such as
lymphoma, cardiovascular disease, and nephrosclerosis. U.S. Pat.
No. 5,639,600. Gabrielian et al. reported the role of macrophage
infiltration in traumatic proliferative vitreoretinopathy. ((1994)
Curr. Eye. Res. 13: 1-9). McGrath et al. disclosed the involvement
of clonally expanded macrophages in the induction of cancerous
tumor growth and AIDS dementia. U.S. Pat. Nos. 5,639,600 and
5,580,715; see also Pulliam et al. (1997) Lancet 349:692-695;
McGrath et al. (1995) J. Acquired Imm. Def. Syn. Hum. Retro. 8:
379-385; Shiramizu et al. (1994) Cancer Res. 54:2069-2072.
Polyamine Analogs and Anti-Proliferative Activity
[0016] Certain anionic oligomers have antiproliferative activity.
In particular water soluble polyureas and polyamides with a
molecular weight of less than 10,000 inhibit smooth muscle cell
proliferation in culture and in vivo, and have been suggested for
treatment of atherosclerosis (U.S. Pat. No. 5,460,807; see also
U.S. Pat. No. 5,516,807 (relating to use of bis-ethyl norspermine
in vascular proliferative disorders)). Certain triazoles are
antiproliferatives; in particular amino 1, 2, 3 triazoles inhibit
labeled thymidine incorporation into intact pig skin, inhibit
keratinocyte proliferation, and have been suggested for use in
treatment of psoriasis, a chronic skin disease characterized by
epidermal hyperproliferation (U.S. Pat. No. 4,847,257). Derivatives
of valproic acid decrease neuro-2a cell proliferation in vitro, and
have been suggested for use in prevention and treatment of
neurodegenerative disorders such as Alzheimer's disease to inhibit
pathologic neural cell growth (U.S. Pat. No. 5,672,746).
[0017] The level of polyamines is intimately related to cell
proliferation. Cellular levels of polyamines are carefully
regulated by opposing synthetic and catabolic pathways. Compounds
that are able to lower polyamine levels are proposed for use in the
treatment of rapidly proliferating host cells such as cancer and
psoriasis. A key polyamine catabolizing enzyme spermidine-spermine
N1-acetyltransferase (SSAT) is among the few genes known to be
inducible by the natural polyamines. Certain polyamine analogs
exaggerate this response. 1,11-diethylnorspermine (DENSPM)
increases SSAT mRNA levels in human melanoma cells up to 20-fold,
with an increase in immunodetectable SSAT protein by 300-fold. By
comparison, natural polyamine spermine is far less effective,
increasing SSAT mRNA by .about.3-fold and immunodetectable protein
by .about.7-fold. Fogel-Petrovic et al. (1996) Biochemistry
35:14435. Polyamine analogs also induce Z-DNA structure in vitro.
This property correlates inversely with the effects on
cis-diaminedichloroplatinum (II) (CDDP) cytotoxicity in human brain
tumor cells. Basu et al. (1996) Anticancer Res. 16:39.
[0018] U.S. Pat. No. 5,498,522 outlines the use of SSAT, or other
determinants related to SSAT induction such as SSAT co-factor
acetylCoA, and the SSAT products N1-acetylspermine and
N1-acetylspermidine, as a prognostic indicator or tumor response
marker. Measurement of these determinants is proposed as a
prognostic indicia and tumor response marker to evaluate the
clinical effectiveness of anticancer agents comprising polyamine
analogs. Hibasami et al. [(1989) Cancer Res. 49:2065] prepared
methylglyoxal-bis(cyclopentylamidinohydrazone) (MGBCP) as an
inhibitor of the natural polyamine synthetic pathway. MGBCP
inhibits S-adenosylmethionine decarboxylase, spermine synthase, and
spermine synthetase, competing with S-adenosylmethionine,
spermidine, and putrescine, respectively. MGBCP depleted spermidine
and spermine in leukemic ascites cells, and prolonged survival time
of mice bearing P388 leukemia.
[0019] U.S. Pat. No. 5,541,230 (Basu et al.) indicates that
spermine derivatives decrease growth in a number of human tumor
cell lines, and propose their use in cancer chemotherapy. Bergeron
et al. (Cancer Chemother. Pharmacol.) showed that the polyamine
analogs 1,14-bis(ethylamino)-5,10-diazatetradecaone (BE-4-4-4), and
1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4; see U.S.
Pat. No. 5,541,230)) directly affects growth, survival, and cell
cycle progression in human brain tumor cell lines. For other
publications relating to the synthesis and use of certain
polyamines, the reader is referred to EP 277,635, EP 162,413, EP
399,519, JP 85/6348, and U.S. Pat. No. 5,679,682; and to Bellevue
et al. (1996) Bioorg. Med. Chem. Lett. 6:2765, and Porter et al.
(1992) Falk Symposium 62:201; Marton and Pegg (1995) Ann Rev.
Pharmacol. Toxicol. 35:55-91.
SUMMARY OF THE INVENTION
[0020] The present invention provides methods for modulating
proliferating macrophages using a composition comprising a
polyamine analog or salt or protected derivative thereof,
preferably in an individual afflicted with or at risk for a disease
with which macrophage proliferation is associated, particularly a
macrophage-associated ocular disease, more particularly a
macrophage-associated retinopathy, wherein the composition is
administered in amount sufficient to modulate proliferating
macrophages in the individual.
[0021] In another aspect, the invention features a method for
increasing phagocytic activity of macrophages in an individual
having or at risk of a macrophage-associated ocular disease (e.g.,
a macrophage-associated retinal disease (particularly
macrophage-associated retinopathy, e.g., macrophage-associated
retinal disease, e.g., ARMD, particularly dry ARMD) by
administering to the individual a composition comprising a compound
selected from a polyamine analog, a salt of a polyamine analog, and
a protected derivative of a polyamine analog, in an amount
sufficient to enhance macrophage phagocytosis in the individual;
wherein said administering provides for an increase in phagocytic
activity of macrophages in the individual (e.g., an increase in
phagocytic activity of a population containing macrophages, e.g.,
blood macrophages, e.g., as found in a population of peripheral
blood monocytes) in the individual).
[0022] In one aspect of the invention, the invention provides a
method of aiding diagnosis of a macrophage-associated ocular
disease (e.g., a macrophage-associated retinal disease
(particularly macrophage-associated retinopathy, e.g.,
macrophage-associated retinal disease, e.g., ARMD, particularly dry
ARMD) in an individual by detecting in a biological sample for the
individual the level of proliferating macrophages (e.g., by
assessing a level of cells having markers of proliferating
macrophages, e.g., CD14+/CD16+ cells, CD16+/PCNA+ cells) and/or by
assessing a level of systemic MCP-1. An elevated level of
proliferating macrophages, or elevated level of systemic MCP-1,
relative to a normal level of proliferating macrophages or systemic
MCP-1 (i.e., a level of such markers in an non-diseased, preferably
age-matched control) indicates an increased risk of, or the
presence of, a macrophage-associated ocular disease. In a related
embodiment, sensitivity of proliferating macrophages to a polyamine
analog is tested, which sensitivity is indicative of the presence
of a macrophage-associated disease. Where an increased risk of a
macrophage-associated ocular disease, such as ARMD, is identified
using such a diagnostic test, the patient can be identified for
diet modification, increased frequency of follow-up visits to a
clinician, and more frequent tests to assess progression toward
disease.
[0023] In a further aspect of the invention, the invention provides
a method of monitoring therapy of a macrophage-associated ocular
disease (e.g., a macrophage-associated retinal disease) in an
individual comprising detecting the level of proliferating
macrophages (and/or a level of systemic MCP-1) in a biological
sample from said individual.
[0024] In another aspect of the invention, the invention provides a
method of modulating macrophage proliferation in an individual
afflicted with or at risk for a macrophage-associated ocular
disease (e.g., a macrophage-associated retinal disease, more
particularly a vitreoretinopathy, with the "dry" form of ARMD being
of particular interest) comprising administering to the individual
a composition comprising a compound selected from the group
consisting of a polyamine analog, a salt of a polyamine analog, and
a protected derivative of a polyamine analog, wherein all nitrogen
atoms of said polyamine analog are secondary, tertiary, or
quartenary amino groups, and where the composition is administered
in an amount sufficient to modulate macrophage proliferation in the
individual.
[0025] In another aspect of the invention, the invention provides a
method of decreasing a level or accumulation of drusen in a subject
comprising administering to the individual a composition comprising
a compound selected from the group consisting of a polyamine
analog, a salt of a polyamine analog, and a protected derivative of
a polyamine analog, wherein all nitrogen atoms of said polyamine
analog are secondary, tertiary, or quartenary amino groups, and
where the composition is administered in an amount sufficient to
modulate macrophage proliferation in the individual.
[0026] In one aspect of the invention, the invention provides a
method of delaying development of a macrophage-associated ocular
disease (e.g., a macrophage-associated retinal disease, more
particularly a vitreoretinopathy, with the "dry" form of ARMD being
of particular interest) in an individual comprising administering
to the individual an effective amount of an agent that modulates
macrophage proliferation. In a related embodiment, delaying
development is assessed by stabilization of, reduction of, drusen
(e.g., as assessed by drusen score).
[0027] These and other features as well as advantages of the
invention will be readily apparent to the ordinarily skilled
artisan upon reading the instant specification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 is a graph showing the relationship between blood
macrophage (CD14/16+) and immune activation (mean HLA-DR level) in
ARMD patient samples. It shows that the higher the level of
macrophages in the blood (% CD14+/16+) the higher the level of
activation (higher HLA-DR expression/cell) in ARMD patient
blood.
[0029] FIG. 2 is a graph showing that higher levels of activated
blood monocytes are also associated with higher drusen scores. in
ARMD patients.
[0030] FIG. 3A is a graph showing MCP-1 plasma levels in subjects
with wet ARMD (5 patients), dry ARMD (7 patients), and
controls.
[0031] FIG. 3B is a graph showing the relationship between the
level of CCR2 expression (which indicates cells are regulated by
the MCP-1) and the percentage of peripheral blood monocytes that
are CD14+/CD16+ in ARMD patient blood samples. In ARMD patients,
the higher the percentage of activated blood macrophages, the lower
the level of CCR2 expression.
[0032] FIG. 4A is a graph showing the relationship between the
retinal drusen of ARMD patients and blood monocyte expression of
the MCP-1 receptor, CCR2. The data in this figure illustrates that
CCR2 expression is down regulated in direct proportion to the
degree of retinal drusen (i.e., CCR2 expression levels and drusen
score are inversely correlated, with a decrease in CCR2 expression
being associated with an increased drusen score).
[0033] FIG. 4B is a graph showing phagocytic activity of peripheral
blood monocytes in control subjects, subjects with early ARMD
(drusen score of 1-2), and in subjects with late ARMD (drusen score
of 3-4).
[0034] FIG. 4C is a schematic showing a hypothesis of the disease
mechanism underlying ARMD pathogenesis. (MO=macrophage)
[0035] FIG. 5 is a graph showing sensitivity of CD14+/PCNA+ cells
to the polyamine analog SL-11047 ("SL47") in ARMD patients ("MD
patients"), and age-matched non-ARMD patients (both "non-disease"
and "disease" controls).
[0036] FIG. 6 is a bar graph showing that ARMD blood cells
spontaneously produce high levels of MCP-1 ("MCP-1 pre") and that
exposure to SL-11047 markedly decreases MCP-1 production ("MCP-1
post").
[0037] FIG. 7 is a graph showing the effects of polyamine analog
treatment on the phagocytic activity of peripheral blood monocytes
in a patient prior to and following 4 cycles of polyamine analog
therapy.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The invention is based on the discovery that polyamine
analogs are particularly effective in modulating macrophage
proliferation. Further, the invention is based on the discovery
that certain proliferative diseases are associated with, and may be
supported by, proliferation macrophages. Among these cellular
proliferative diseases are ocular diseases, particularly
retinopathies such as vitreoretinopathy, age-related macular
degeneration (ARMD), and the like. The invention is further based
on the discovery that polyamine analogs can be used in the
treatment of non-exudate or "dry" ARMD. In addition, the invention
is based on the discovery that systemic MCP-1, as well as markers
of macrophage activation (e.g., CCR2 expression levels, CD14/CD16
expression levels, CD14/PCNA expression levels) are indicative of
ARMD disease severity, based on their correlation with drusen, the
conventional ARMD disease severity marker.
[0039] For example, it was discovered ARMD patients have elevated,
abnormal levels of macrophage proliferation in peripheral blood. In
addition, the level of activated macrophages in blood parallels the
level of drusen formation in the retina of both wet and dry ARMD
patients. Without wishing to be bound by a particular theory,
macrophage proliferation is associated with, and may contribute to,
ocular diseases, particularly retinal diseases such as ARMD
(including wet and dry ARMD), proliferative vitreoretinopathy
(PVR), and proliferative diabetic retinopathy (PDR).
[0040] The invention is further based on the observation that the
level of activated macrophages in the peripheral blood of patients
parallels the level of cellular activation of the macrophages. In
addition, the invention is based on the discovery that the
polyamine analog SL-11047 can kill activated macrophage cells
isolated from ARMD patients. Controlling unwanted and harmful
macrophage proliferation and activation is thus a crucial aspect of
developing new, effective treatment modalities for
macrophage-associated disorders, including these
macrophage-associated ocular diseases.
[0041] Accordingly, the invention provides methods for modulating
macrophage proliferation, which are useful for controlling,
palliating, and/or delaying development of macrophage-associated
diseases, with macrophage-associated ocular diseases, particularly
macrophage-associated retinal diseases (including, but not limited
to, ARMD, vitreoretinopathy, and diabetic retinopathy) being of
particular interest. The invention also provides methods of
modulating macrophage proliferation in individuals afflicted with
or at risk for a macrophage-associated ocular disease, particularly
a macrophage-associated ocular disease such as ARMD. The invention
also provides methods of aiding diagnosis and/or monitoring therapy
of such diseases which entail measuring the presence of
proliferating macrophages.
[0042] As discussed below, exemplary agents for modulation of
macrophages are polyamine analogs especially
1,11-bis(ethyl)norspermine; 1,8-bis(ethyl)spermidine (BES);
1,12-bis(ethyl)spermine (BESm; DESPM (N1, N12-diethylspermine);
1,11-bis(ethylamino)-4,8-diazaundecane (BE-3-3-3);
1,14-bis(ethylamino)-5,10-diazatetradecane (BE-4-4-4)
(Diethylhomospermine, N1, N14-diethylhomospermine; DEHOP or
DEHSPM); diethyl-norspermine (DENOP);
1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4);
N-ethyl-N'-(2-(3'-ethylamino-propylamino
methyl)-cis-cyclopropylmethyl)-propane 1,3-diamine
tetrahydrochloride (SL-11037);
N-ethyl-N'-(2-(3'-ethylamino-propylamino
methyl)-trans-cyclobutylmethyl)-propane 1,3-diamine
tetrahydrochloride (SL-11038);
N-ethyl-N'-(2-(3'-ethylamino-propylamino
methyl)-trans-cyclopropylmethyl)-propane 1,3-diamine
tetrahydrochloride (SL-11044; and
N,N'-bis(3-ethylaminopropyl)-cis-but-2-ene-1,4-diamine
tetrahydrochloride (SL-11047).
DEFINITIONS
[0043] As used herein, the terms "macrophage" and "monocyte" are
used interchangeably, as it is understood that in the art the term
"monocyte" is often used to describe a circulating mononuclear cell
that expresses the CD14 cell surface marker, and when in a tissue
this cell is also classified as a macrophage.
[0044] A "proliferating macrophage" is a term understood in the art
and as used herein denotes a macrophage which is dividing. Normally
a macrophage is a terminally differentiated cell incapable of
further division. For purposes of this invention, a "proliferating
macrophage" is capable of further division or is in a portion of
the cell cycle not considered to be terminal or end stage.
Preferably, the proliferation is clonal, i.e., is derived from a
single cell. Methods of detecting proliferating macrophage(s) is
discussed below.
[0045] As used herein, detecting the "presence of proliferating
macrophages" generally means detecting the level of proliferating
macrophages. It is understood that an absolute or even relative
level need not be determined; an observation of detectable
proliferating macrophages is sufficient.
[0046] An "ocular disease" refers to a disease, disorder, or
abnormality that relates to the state of the eye, particularly the
ability of the eye to perceive images. "Ocular disease" encompasses
retinal diseases, including proliferative diseases of the eye.
Exemplary ocular diseases include ARMD (including exduative ("wet")
and non-exudative ("dry")), vitreoretinopathy, diabetic
retinopathy, and the like. The terms "disorder" and "disease" are
used interchangeably herein.
[0047] "Proliferative retinopathy" as used herein refers to an
ocular disease involving growth, displacement, hypertrophy or
hyperplasia of cellular or acellular components of the retina
and/or vitreous including, where such components include vascular
cells; blood vessels; glial cells; fibrocytes; macrophages;
inflammatory cells; retinal pigment epithelial cells; astrocytes or
sensory retinal cells including photoreceptors (rods and cones);
bipolar cells; and ganglion cells. Drusen are products of
proliferative retinopathy, as drusen can be regarded as growths in
Bruch's membrane, which is an acellular layer beneath retinal
pigment epithelial cells. Further, drusen can cause displacement of
retinal pigment epithelial cells, and may also facilitate
hyperplasia, hypertrophy or hypotrophy of retinal pigment
epithelial cells. "Proliferative retinopathy" includes, but is not
necessarily limited to, ARMD (both wet and dry), proliferative
diabetic retinopathy (PDR), traumatic proliferative retinopathy,
and proliferative vitreoretinopathy (PVR).
[0048] A "macrophage-associated" disease, disorder or indication is
a disease, disorder or indication that is associated with an
elevated, or abnormal, level of macrophage proliferation as
compared to control sample(s). Such disorders include, but are not
limited to, ocular diseases having a proliferating macrophage
component (e.g., retinal disease (e.g., ARMD, vitreoretinopathy,
diabetic retinopathy, and the like)). Macrophage-associated
retinopathies are of particular interest. Macrophage-associated
ocular disease can generally be characterized as having a high
frequency of activated macrophages in tissues compared to a
frequency of such cells in non-disease controls, where activated
macrophages can be identified by CD16 expression, elevated HLA-DR,
elevated MCP-1 production, decreased CCR2 expression, and elevated
PCNA (relative non-disease macrophages).
[0049] An individual "afflicted with" an ocular disease (e.g., a
retinal disease (e.g., ARMD, vitreoretinopathy, and the like) means
that the individual has been diagnosed as having, or is suspected
as having, an ocular diseases such as ARMD, vitreoretinopathy, and
diabetic retinopathy, etc.
[0050] By a "polyamine", a term well-understood in the art, is
meant any of a group of aliphatic, straight-chain amines derived
biosynthetically from amino acids; polyamines are reviewed in
Marton et al. (1995) Ann. Rev. Pharm. Toxicol. 35:55-91. By
"polyamine" is generally meant a naturally-occurring polyamine or
natural polyamine, which are naturally produced in eukaryotic
cells. Examples of polyamines include putrescine, spermidine,
spermine and cadaverine.
[0051] By "polyamine analog" is meant an organic cation which
shares structure with, but is non-identical to, naturally-occurring
polyamines such as spermine and/or spermidine and their precursor,
diamine putrescine. Polyamine analogs can be branched or
un-branched, or incorporate cyclic moieties. Examples of polyamine
analogs include, without limitation, N1, N14-diethylhomo-spermine
(DEHSPM) and N1, N12-diethylspermine (DESPM). See, for example, WO
98/17624 and U.S. Pat. No. 5,541,230. U.S. Pat. Nos. 5,037,846 and
5,242,947 disclose polyamines comprising primary amino groups.
Especially preferred are polyamine analogs wherein all nitrogen
atoms of said polyamine analogs are independently secondary,
tertiary, or quartenary amino groups.
[0052] An "alkyl" is a cyclic, branched, or straight chain chemical
group containing carbon and hydrogen, such as methyl, butyl,
t-butyl, pentyl, cyclopropyl, and octyl. Alkyl groups can be either
unsubstituted or substituted with one or more substituents, e.g.,
halogen, alkoxy, acyloxy, amino, hydroxyl, mercapto, carboxy,
benzyl. Alkyl groups can be saturated or unsaturated (e.g.,
containing --C.dbd.C-- or --C.sup.oC-- subunits), at one or several
positions. Unless otherwise specified, alkyl groups will comprise 1
to 8 carbon atoms, preferably 1 to 6, and more preferably 1 to 4
carbon atoms. "Cycloalkyl" refers to cyclic alkyl groups only, such
as cyclopropyl, cyclobutyl, cyclopentyl, etc. "n-alkyl" refers to a
linear (i.e., straight-chain) alkyl group only, while "branched
alkyl" refers to branched alkyl groups to the exclusion of cyclic
and linear alkyl groups. "Alkenyl" refers to a cyclic, branched, or
straight chain chemical group containing carbon and hydrogen where
at least one bond is monounsaturated, such as ethenyl,
cyclopentenyl, or 1,3-butadienyl. Alkenyl groups can be substituted
as indicated for alkyl groups. Alkenyl groups can be designated as
cyclic, linear (n-alkenyl) or branched in an analogous fashion to
the preceding designations for alkyl. An "aryl" is an unsaturated
aromatic carbocyclic group having a single ring (e.g., phenyl), or
multiple condensed rings (e.g., naphthyl), which can optionally be
unsubstituted or substituted with amino, hydroxyl, alkyl, alkoxy,
chloro, halo, mercapto and other substituents.
[0053] A "stereoisomer" is defined as any optical isomer of a
compound, including enantiomers and diastereomers. Unless otherwise
indicated, structural formula of compounds are intended to embrace
all possible stereoisomers.
[0054] A "salt" is defined as a compound formed by the replacement
of one or more hydrogen atoms with elements or groups, which is
composed of anions and cations, which usually ionizes in water; a
salt is formed, for instance, by neutralization of an acid by a
base. A polyamine analog salt can comprise, for example, chloride
ions.
[0055] "Protected derivative" is used to refer to a compound
protected with a protecting group. "Protecting group" refers to a
chemical group that exhibits the following characteristics: 1)
reacts selectively with the desired functionality in good yield
(preferably at least 80%, more preferably at least 90%, more
preferably at least 95%, still more preferably at least 99%) to
give a protected substrate that is stable to the projected
reactions for which protection is desired; 2) is selectively
removable from the protected substrate to yield the desired
functionality; and 3) is removable in good yield (preferably at
least 80%, more preferably at least 90%, more preferably at least
95%, still more preferably at least 99%) by reagents compatible
with the other functional group(s) present or generated in such
projected reactions. Examples of suitable protecting groups can be
found in Greene et al. (1991) Protective Groups in Organic
Synthesis, 2nd Ed. (John Wiley & Sons, Inc., New York).
Exemplary protecting groups for the amino functionality include,
but are not limited to, mesitylenesulfonyl (MesSO.sub.2),
benzyloxycarbonyl (CBz), t-butyloxycarbonyl (Boc),
t-butyldimethylsilyl (TBDIMS), 9-fluorenylmethyloxycarbonyl (Fmoc),
or suitable photolabile protecting groups such as
6-nitroveratryloxy carbonyl (Nvoc).
[0056] An "individual" is a vertebrate, preferably a mammal, more
preferably a human. Mammals include, but are not limited to, farm
animals, sport animals, rodents, primates, and pets. A
"non-HIV-infected individual" is an individual who has not been
infected by HIV. An "HIV-infected" individual may or may not yet
display clinical manifestations of infection. HIV and methods of
detecting HIV infection are well known in the art and need not be
discussed herein.
[0057] As used herein, "biological sample" encompasses a variety of
sample types obtained from an individual and can be used in a
diagnostic or monitoring assay. The definition encompasses blood
and other liquid samples of biological origin, solid tissue samples
such as a biopsy specimen or tissue cultures or cells derived
therefrom, and the progeny thereof. The definition also includes
samples that have been manipulated in any way after their
procurement, such as by treatment with reagents, solubilization, or
enrichment for certain components, such as proteins or
polynucleotides. The term "biological sample" encompasses a
clinical sample, and also includes cells in culture, cell
supernatants, cell lysates, serum, plasma, biological fluid, and
tissue samples. Generally, the sample will be, or be derived from,
peripheral blood. Preferably, the blood will have been enriched for
a macrophage fraction, by using, for example, glass or plastic
adherence. As used herein, reference to a "blood sample" is meant
to encompass whole blood as well as fractions derived from whole
blood, with samples containing immune cells, particularly
peripheral blood monocytes, being of particular interest. Other
biological samples of interest include, but are not limited to,
ocular samples obtained from eye tissue or fluids, e.g., vitreal
fluid, aqueous fluid, and retinal or choroid biopsies.
[0058] As used herein, the term "biomarker" refers to a molecule
(e.g., protein, nucleic acid, or chemical compound (e.g.,
substrate, metabolite, catabolite, and the like), whether large or
small, which, when present in a biological sample of an individual,
is indicative of the presence of a particular physiological trait
in the individual. For example, PCNA is biomarker for the presence
of proliferating macrophages in a biological sample. In another
example of biomarker, the molecule Monocyte Chemoattractant Protein
MCP-1 is a biomarker for the presence of activated macrophages.
[0059] As used herein, "aiding diagnosis" means that these methods
assist in making a clinical determination regarding the
classification, or nature, of the ocular disease, and may or may
not be conclusive with respect to the definitive diagnosis. The
method of aiding diagnosis of an ocular disease, particularly a
retinal disease, can comprise the step of detecting the level of
proliferating macrophages in a biological sample from the
individual and determining whether the proliferating macrophage
level is abnormal (e.g., elevated) relative to a level associated
with an unaffected individual. In general, abnormal levels of
proliferating macrophages can indicate the individual is
susceptible or sensitive to development of a proliferative
retinopathy, particularly ARMD, PVR, or PDR. Determining whether an
ocular disease (e.g., retinal disease) may or may not be associated
with proliferating macrophages, particularly clonal macrophage
proliferation, and making this classification can assist in
developing and recommending treatment strategies as well as
evaluating prognosis. For example, where an individual has
increased proliferating macrophages further diagnostic tests (e.g.,
OCT, FA, and the like) may be indicated, and/or frequency of
follow-up visits.
[0060] "Development" of an ocular disease, such as a retinal
disease, herein means initial manifestations and/or ensuing
progression of the disease. Development of disease can be
detectable and assessed using any suitable standard clinical
techniques, such as visual observation of the size of drusen, the
presence of bleeding in the diseased eye, the structure of the
retina, loss or degradation of vision or the visual field, and the
like. Clinical signs and symptoms for various ocular diseases,
including retinopathies, are well known in the art. As used herein,
development also refers to disease progression that may be
undetectable by use of conventional signs and symptoms. For
purposes of this invention, progression refers to the biological
course of the disease state. "Development" includes occurrence,
recurrence, and onset. As used herein "onset" or "occurrence" of a
neurological disorder includes initial onset and/or recurrence. As
used herein, "delaying" development of disease means to defer,
hinder, slow, retard, stabilize, and/or postpone development of the
disease. This delay can be of varying lengths of time, depending on
the history of the disorder and/or the medical profile of the
individual being treated. As is evident to one skilled in the art,
a sufficient or significant delay can, in effect, encompass
prevention, in that the individual does not develop detectable
disease. A method that "delays" development of disease is a method
that reduces the extent of the disease in a given time frame, when
compared to not using the method. Such comparisons are typically
based on clinical studies, using a statistically significant number
of subjects, although this knowledge can be based upon anecdotal
evidence. "Delaying development" can mean that the extent and/or
undesirable clinical manifestations are lessened and/or time course
of the progression is slowed or lengthened, as compared to not
administering the agent. Thus the term also includes, but is not
limited to, alleviation of symptoms, diminishment of extent of
disease, stabilized (i.e., not worsening) state of disease, delay
or slowing of disease progression, and remission (whether partial
or total) whether detectable or undetectable.
[0061] As used herein, an "effective amount" (e.g., of an agent) is
an amount (of the agent) that produces a desired and/or beneficial
result. An effective amount can be administered in one or more
administrations. For purposes of this invention, an effective
amount is an amount sufficient to produce modulation of macrophage
proliferation. An "amount (of a polyamine analog) sufficient to
modulate macrophage proliferation" preferably is able to alter the
rate of proliferation of macrophages by at least 25%, preferably at
least 50%, more preferably at least 75%, and even more preferably
at least 90%.
[0062] Such modulation may have desirable concomitant effects, such
as to palliate, ameliorate, stabilize, reverse, slow or delay
progression of disease, delay or even prevent onset of disease.
[0063] As used herein, the term "agent" means a biological or
chemical compound such as a simple or complex organic or inorganic
molecule, a peptide, a protein or an oligonucleotide. A vast array
of compounds can be synthesized, for example oligomers, such as
oligopeptides and oligonucleotides, and synthetic organic compounds
based on various core structures, and these are also included in
the term "agent". In addition, various natural sources can provide
compounds, such as plant or animal extracts, and the like. Agents
include, but are not limited to, polyamine analogs. Agents can be
administered alone or in various combinations.
[0064] "Modulating" proliferating macrophages as used means a
change in a level of proliferating macrophages of at least 25%,
preferably at least 50%, more preferably at least 75%, and even
more preferably at least 90%. A decrease in proliferating
macrophages can result from, for example, a decrease that results
following administration of an agent, such as a polyamine analog,
that interferes with natural polyamine interaction with DNA
(including, but not limited to, interfering with a polyamine
biosynthetic pathway, interfering with the intracellular
concentration of spermidine, competitors, inhibitors of DNA
interaction by a natural polyamine, interfering with polyamine
metabolism, etc.). Generally, for purposes of this invention,
"modulating" a level of proliferating macrophages means that the
qualitative or quantitative level of proliferating macrophages
(e.g., as detected using a biomarker such as PCNA), and further
which may be expressed as a portion of total macrophages (e.g.,
percent CD16+/CD14+ or percent PCNA-CD14 positive compared to total
CD14 positive) is decreased when compared to the level in that
individual when no agent is administered.
[0065] A "target" of a polyamine or polyamine analog is an entity
which interacts, either directly or indirectly, with the polyamine
or polyamine analog(s). Examples of targets are DNA, RNA, and/or
membranes.
METHODS OF THE INVENTION
[0066] The invention contemplates both methods of treatment (e.g.,
using polyamine analogs, and other agents that inhibit macrophage
proliferation and/or disrupt a polyamine biosynthetic pathway)
which methods have as a therapeutic endpoint modulation of
proliferation macrophages (e.g., a decrease in proliferating
macrophages (e.g., a decrease in the percentage of PBMCs that are
proliferating macrophages) and/or a decrease in a rate of
proliferation of such macrophages). The invention also contemplates
methods of diagnosis of a macrophage-associated ocular disease,
particularly a macrophage-associated retinal disease, by assessing
particular biomarkers that are indicators of proliferating
macrophages. These methods are described in more detail below.
Methods of Modulating Proliferating Macrophages
[0067] The invention provides methods for modulating proliferating
macrophages in an individual afflicted with or at risk for a
macrophage-associated ocular disease, particularly a
macrophage-associated retinal disease, comprising administering a
polyamine analog, a salt of a polyamine analog, or a protected
derivative of a polyamine analog, in an amount sufficient to
modulate macrophage proliferation in the individual (i.e., an
effective amount). Alternatively, a composition comprising a
polyamine analog, or a protected derivative of a polyamine analog
is administered in an amount sufficient to modulate proliferating
macrophages (i.e., an effective amount). Examples of
macrophage-associated ocular diseases have been described above,
and include, but are not limited to, macrophage-associated retinal
diseases. Exemplary diseases of interest includes, but are not
limited to, ARMD (wet and dry), vitreoretinopathy, and diabetic
retinopathy. Treatment of vitreoretinopathy and dry ARMD are of
particular interest. Polyamine analogs are discussed below.
[0068] The invention further provides methods for decreasing
drusen, or preventing further accumulation of drusen, in a in an
individual afflicted with or at risk for a macrophage-associated
ocular disease, particularly a macrophage-associated retinal
disease, comprising administering a polyamine analog, a salt of a
polyamine analog, or a protected derivative of a polyamine analog,
in an amount sufficient to modulate macrophage proliferation in the
individual (i.e., an effective amount). Alternatively, a
composition comprising a polyamine analog, or a protected
derivative of a polyamine analog is administered in an amount
sufficient to modulate proliferating macrophages (i.e., an
effective amount). Reducing drusen, or at least preventing further
accumulation of drusen, can provide for prevention or a delay in
development of a macrophage-associated ocular disease. Drusen can
be assessed according to conventional techniques, and therapy can
be assessed by reduction of drusen score, or maintenance of a
drusen score (e.g., a drusen score indicative of early ARMD of
1-2). Of particular interest is maintenance of a drusen score that
is indicative of less severe disease according to conventional
criteria.
[0069] For purposes of this invention, an individual suitable for
administration of a polyamine analog (or an agent which modulates
macrophage proliferation) is one who has been diagnosed as or
suspected of having an ocular disease, particularly a retinal
disorder such as ARMD, vitreoretinopathy, or diabetic retinopathy,
or who is adjudged to be at high risk for developing such a
disorder. As is evident to one skilled in the art, these methods
can apply to those individuals not displaying any symptoms. An "at
risk" or "high risk" individual is an individual who has a discrete
and significant risk of developing an ocular disease, particularly
a retinal disease. An "at risk" or "high risk" individual may or
may not have detectable disease, and may or may not have displayed
detectable disease prior to receiving the method(s) described
herein. "High risk" (or "at risk") denotes that an individual has
one or more so-called risk factors, which are measurable parameters
that correlate with development of disease. An individual having
one or more of these risk factors has a higher probability of
developing disease than an individual without these risk factor(s).
These risk factors include, but are not limited to, drusen score,
genetic (i.e., hereditary) considerations (including family history
and genetic markers), and presence or absence of appropriate
chemical markers and exposure to environments, conditions, or
factors which would increase the possibility of acquiring a
particular disease. Retroviral infections, especially retroviral
insertions into particular genetic loci (such as fur or PDGF), may
also be considered a risk factor. A high risk individual has one,
preferably two, more preferably three, risk factors. However, it is
understood that having only one risk factor can often indicate high
risk.
[0070] Despite the advances in research and development in the area
of ocular diseases, such as retinal diseases, only certain risk
factors have been found so far to play a role in the disease
development. Such factors can be aging, gender, genetic, nutrition
or stress-related. Because all risk factors for developing an
ocular disease are not known, and the interplay among these factors
(in terms of overall risk) are not fully understood, it is clear to
one skilled in the art that individuals suitable for administration
of an agent for purposes of this invention can have clinical
features in common, and that individuals not falling clearly in the
categories described above can nonetheless be considered suitable
candidates for administration of an agent. For example, an
individual who has a family history of ARMD could be considered at
risk for developing ARMD herself, even though no obvious symptom
has been observed. In this context, administration of an agent to
such an individual could result in delay of occurrence of disease,
even to the extent that the individual does not develop ARMD within
his or her lifetime (or develops it later than would have been
expected). Another example is an individual who is being treated
using other modes of therapy, and who is showing clinical
responsiveness to the therapy (i.e., stabilization or remission).
Such an individual may be adjudged as at "high risk" even though
the initial course of therapy is not yet completed, due to
projection of clinical progress by the clinician, and can be a
suitable candidate for receiving an agent before completion of the
initial therapy. The clinician, as one skilled in the art, has
discretion to determine whether treatment using an agent may be
indicated.
[0071] In another embodiment, the invention provides methods for
modulating macrophage proliferation in an individual (who is
generally afflicted with or at risk of for a macrophage associated
disease) comprising administering a composition comprising an
effective amount of an agent that interferes with polyamine
interaction with proliferating macrophage target, such as DNA, RNA,
and/or membranes. An agent that interferes with polyamine
interaction with a proliferating macrophage target(s) is one which
interferes with any aspect of natural polyamine synthesis and/or
metabolism, intracellular concentration regulation, and/or function
(i.e., interaction with DNA).
Diagnostic Methods and Kits
[0072] The present invention further provides methods for aiding in
the diagnosis of an individual having or at risk of having, or
monitoring therapy in individuals having, a macrophage-associated
ocular disease, particularly a retinal disease such as ARMD,
vitreoretinopathy, and diabetic retinopathy.
[0073] In one embodiment, these methods involve detecting the
presence of proliferating macrophages in a biological sample from
the individual, e.g., by detecting a biomarker of a proliferating
macrophage (e.g., CD14+/CD16+ cells or CD14+/PCNA+ cells, e.g., as
a percentage of peripheral blood monocytes (PBMCs)), where the
presence of a level of proliferating macrophages present in the
sample that is significantly greater than a level of a non-disease
control is an indicator of risk or the presence of a
macrophage-associated ocular disease. In another embodiment, the
diagnostic methods involve detecting a level of CCR2 expression in
PBMCs, where a decreased level of CCR2 expression relative to a
level found in non-diseased controls is indicative of proliferating
macrophages, which in turn is an indicator of risk or the presence
of a macrophage-associated ocular disease. In still another
embodiment, the diagnostic methods involve detecting systemic MCP-1
levels (levels of MCP-1 in the peripheral blood, i.e., other than
in the eye per se), where systemic MCP-1 levels elevated relative
to a systemic MCP-1 level in a non-disease control is indicative of
proliferating macrophages in the individual, which in turn is an
indicator of risk or the presence of a macrophage-associated ocular
disease. Throughout, reference to an elevated (or decreased) level
relative to a non-disease control is generally meant to encompass a
different that is at least 1 standard deviation above (or below) an
average value for normal, age-matched non-disease controls.
[0074] In those individuals considered at high or significant risk
of developing ARMD or other ocular disease, detection of
proliferating macrophages, CCR2 levels (e.g., as expressed on the
surface of monocytes), and/or MCP-1 levels in a biological sample
(e.g., a biological sample containing peripheral blood monocytes
(PBMCs) may also assist in alerting the individual and/or the
clinician of possible precursor disease. Thus, the invention also
includes methods of monitoring an individual at risk or high risk
of developing a macrophage-associated ocular disease, particularly
a macrophage-associated retinal disease, comprising detection of
proliferating macrophages, CCR2 levels, and/or MCP-1 levels in a
biological sample from that individual (e.g., in a blood sample,
e.g., a blood sample containing PBMCs). In one embodiment, the
individual is "afflicted with" (e.g., diagnosed as having,
suffering from and/or displaying one or more clinical symptoms of)
a particular disease, disorder or indication, or at "risk" for
(e.g., having a genetic predisposition for, or family history of,
or being environmentally exposed to factors which increase the
probability of acquiring) a particular disease, disorder, or
indication.
[0075] In another embodiment, the invention provides methods of
monitoring therapy of an ocular disease, particularly a retinal
disease, comprising detecting the presence of (e.g., the level of)
proliferating macrophages, CCR2 levels (e.g., as expressed on the
surface of monocytes), and/or MCP-1 levels in a biological sample.
As the level of macrophage proliferation is associated with these
conditions, monitoring these levels may in turn indicate initial
responsiveness and efficacy, as well as the appropriate dosage of
the therapy. It is understood that monitoring therapy or an
individual at (high) risk means that biological sample(s) are
obtained at different times, for example, during application of
therapy, and are compared, either with each other, a control,
and/or a desired value. In one embodiment, monitoring therapy
includes the step of detecting macrophage proliferation.
[0076] Detection of proliferating macrophage(s), macrophage markers
(e.g., CD14, CD16, PCNA, HLA-DR, CCR2, and the like) and MCP-1
levels can be achieved using any of several techniques. In some
embodiments of the invention involving detection of macrophage
proliferation, proliferation is measured in relation to circulating
macrophages, and is performed on a leukocyte preparation from
peripheral blood. In other embodiments of the invention,
proliferation is measured in relation to tissue-fixed macrophages,
typically performed on tissue sections.
[0077] Proliferating macrophages may be detected, for example, by
assaying cell proliferative markers, such as PCNA. These markers
are distinct from those that identify only "activated" macrophages
(as opposed to proliferating macrophages), such as CD69 and CD25.
The cellular subset representing macrophages may be identified by
detection of certain cell specific markers, such as CD14, CD68,
CD16, or nonspecific esterase. Detection of these cell-type and/or
proliferative markers use methods standard in the art, such as
staining techniques and FACS sorting and analysis. These methods
are further described in Example 1. Further, it is possible that
these proliferating macrophages could be distinguished based on
other characteristics, such as cell density (as measured in
PERCOLL.TM. gradients, for example). These determinations may be
established empirically using standard techniques in the art.
[0078] Methods for assessing CCR2 levels can be performed according
to methods known in the art. In one embodiment a CCR2 expression
level is determined by contacting an anti-CCR2 antibody with a
blood sample, usually a blood-derived sample, containing peripheral
blood monocytes.
[0079] Methods for assessing MCP-1 levels are also well known in
the art for example, MCP-1 can be detected using an anti-MCP-1
antibody in an ELISA using a blood sample, usually a blood-derived
sample, containing peripheral blood monocytes.
[0080] For the purpose of aiding in the diagnosis of or predicting
an ocular disease, particularly a retinal disease, the level of
proliferating macrophages, CCR2 expression, and/or MCP-1 in a
sample is generally compared with a mean or median level in samples
taken from healthy individuals, matched where necessary for sex and
age. A level of proliferating macrophages, or a level of CCR2
expressing cells, can be calculated as the absolute number of
proliferating macrophages or CCR2+ cells obtained from a blood
sample (or detected by immunohistopathology of a tissue section).
More usually, the level is calculated as a percentage of total
macrophages in the sample, identifiable by cell markers or
morphological characteristics, since this normalizes for
differences in the number of macrophage-like cells recovered in the
sample. Similarly, MCP-1 levels in a biological sample can be
determined either quantitatively or qualitatively and compared to
an MCP-1 level associated with unaffected (healthy)
individuals.
[0081] As with many clinical tests, a finding of greater than about
two, usually at least about three, standard deviations above the
average associated with a normal (non-diseased, preferably
age-matched) value is statistically significant and indicates an
abnormality. A finding of about one or about two standard
deviations, above the average is reason for concern, although, as
described herein, can be an indicator of a less severe form of
disease (e.g., a disease severity associated with a drusen score of
1-2, rather than a relative increased disease severity associated
with a drusen score of 3-4). In combination with other indicators,
an elevated level of a marker of a macrophage-associated ocular
disease as described herein can aid in diagnosis of, for example,
ARMD, or some other condition associated with macrophage
proliferation. In general, markers of macrophage activation, as
well as systemic MCP-1 production, can serve as markers of a
macrophage-associated ocular disease, with levels of such markers
serving as markers of disease severity.
[0082] For example, peripheral blood leukocytes stained and counted
for PCNA/CD14 or CD16/CD14 cells are indicative of the presence of
proliferating macrophages, which in turn is consistent with
macrophage-associated ocular disease, particularly a
macrophage-associated retinal disease, in the level of such cells
is elevated relative to a non-disease, age-matched control. In
addition, a level of proliferating macrophages is also indicative
of the severity of a macrophage-associated ocular disease in the
subject, with higher levels of proliferating macrophages (e.g., a
greater percentage of proliferating macrophages of total PBMCs)
being associated with increased disease severity (e.g., a higher
drusen score).
[0083] In another example, a decreased level of peripheral blood
leukocytes stained and counted for CCR2 expression compared to
healthy individuals is consistent with a macrophage-associated
ocular disease in the individual if the percentage of positively
stained cells is significantly less than that of a percentage of
CCR2+ cells from a non-disease, age-matched control. Decreased CCR2
expression levels are also indicative of the severity of a
macrophage-associated ocular disease in the subject, with lower
levels of CCR2 expression (e.g., CCR2-expressing cells as a
percentage of total PBMCs, or a mean CCR2 expression level of total
PBMCs) being associated with increased disease severity (e.g., a
higher drusen score).
[0084] In another example, a decreased level of peripheral blood
leukocytes stained and counted for intensity of HLA-DR expression
on macrophages compared to healthy individuals is consistent with a
macrophage-associated ocular disease in the individual if the
percentage of positively stained macrophages is significantly
greater than that level found in a non-disease, age-matched
control. The higher the intensity of HLA-DR staining, the greater
the severity of disease. Elevated HLA-DR expression levels are also
indicative of the severity of a macrophage-associated ocular
disease in the subject, with higher levels of HLA-DR expression
(e.g., HLA-DR-expressing macrophages as a percentage of total PBMCs
or macrophages, or a mean HLA-DR expression level of total PBMCs)
being associated with increased disease severity (e.g., a higher
drusen score).
[0085] In another example, an elevated level of MCP-1 in blood of a
subject (systemic MCP-1, as opposed to MCP-1 levels in the eye or
eye tissue per se) compared to a non-disease, age-matched control
is consistent with a macrophage-associated ocular disease in the
individual if the MCP-1 levels are significantly greater than a
systemic MCP-1 level of non-disease, age-matched controls. MCP-1
levels are also indicative of the severity of a
macrophage-associated ocular disease in the subject, with higher
MCP-1 levels being associated with increased disease severity
(e.g., a higher drusen score).
[0086] In another example, sensitivity of MCP-1 production and/or
macrophages from an individual to a polyamine analog can also be
indicative of a macrophage-associated ocular disease in the
individual. A decrease of MCP-1 production by such macrophages, or
inhibition of growth of such macrophages, in the presence of a
polyamine analog (e.g., in culture) is another indicator that the
individual from whom the macrophages were obtained has or is at
risk of a macrophage-associated ocular disease. A decrease in MCP-1
production of at least 25%, 50%, 75%, 85% or more, or effective
growth inhibition (including killing) of at least 25%, 50%, 75%,
85% or more of macrophages in a sample, in the presence of a
polyamine analog as compared to MCP-1 production or cell growth in
the absence of the polyamine analog is indicative of sensitivity of
the macrophage to the polyamine analog.
[0087] The differential diagnosis will include any condition
associated with macrophage proliferation as a causative or
consequential effect, with the ultimate diagnosis being the
responsibility of the managing physician or clinician, and can be
assessed in the context of other clinical signs or symptoms, if
present.
[0088] For the purpose of monitoring the effect of a macrophage
proliferation inhibitor, the level of proliferating macrophages in
a treated sample is generally compared with the level in an
untreated sample. For the general screening of proliferation
inhibitors, peripheral blood leukocytes are isolated from an
individual affected with a disease associated with proliferating
macrophages. Samples of the cells are treated with the candidate
compound, and the effect is compared with cells not treated. When
administered to a patient, the effect of a macrophage proliferation
inhibitor is determined by comparing the level of proliferating
macrophages before and during treatment, with a downward trend
generally being consistent with a positive effect.
[0089] In another embodiment, the invention provides methods of
delaying development of a macrophage-associated ocular disease,
particularly a macrophage-associated retinal disease. These methods
comprise administration of an effective amount of an agent which
modulates macrophage proliferation to the individual. Such agents,
which include polyamine analogs (including stereoisomers, their
salts, and protected derivatives thereof), are described below. The
invention also includes methods of treatment or palliation of these
disorders using an agent(s) which modulates macrophage
proliferation.
Kits
[0090] The invention also provides kits using in the diagnostic
methods of the invention. In general the kits include detection
means for detecting one or more biomarkers for a
macrophage-associated ocular disease, particularly
macrophage-associated retinal disease, in a biological sample,
usually a blood sample, from a subject. Such biomarkers include
biomarkers for proliferating macrophages (e.g., PCNA, and the like
as discussed herein), activated macrophage (e.g., CD14, CD16, PCNA,
CCR2, HLA-DR ("dr")), and MCP-1.
Agents for Modulating Macrophage Proliferation
[0091] In some embodiments of the invention, macrophage
proliferation is accomplished by using a polyamine analog
(including stereoisomers, salts, and protected derivatives
thereof). In other embodiments, any agent which modulates
macrophage proliferation may be used. With respect to polyamine
analogs, it is understood that the discussion also applies to
stereoisomers, salts and protected derivatives thereof.
Polyamine Analogs
[0092] The polyamine analogs used in the present invention include
compounds of the structures 1, 2, 3, 4, and 5, and the
corresponding stereoisomers, salts, and protected derivatives
thereof:
##STR00001##
[0093] where R.sub.1, R.sub.2, R.sub.4, R.sub.6 and R.sub.7 are
independently selected from the group consisting of hydrogen, alkyl
and aryl, and where R.sub.3 and R.sub.5 are alkyl groups;
##STR00002##
[0094] where R.sub.1, R.sub.2, R.sub.4, R.sub.6, R.sub.8, and
R.sub.9 are independently selected from the group consisting of
hydrogen, alkyl and aryl, and where R.sub.3, R.sub.5 and R.sub.7
are alkyl groups;
##STR00003##
[0095] where R.sub.1, R.sub.2, R.sub.4, R.sub.6, R.sub.8, R.sub.10
and R.sub.11 are independently selected from the group consisting
of hydrogen, alkyl and aryl, and where R.sub.3, R.sub.5, R.sub.7
and R.sub.9 are alkyl groups;
##STR00004##
[0096] where R.sub.1 and R.sub.5 are independently selected from
the group consisting of methyl, ethyl, n-propyl, and isopropyl;
[0097] where R.sub.2, R.sub.3, and R.sub.4 are independently
selected from the group consisting of C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.3-C.sub.6 cycloalkyl,
C.sub.1-C.sub.6 alkyl-C.sub.3-C.sub.6 cycloalkyl-C.sub.1-C.sub.6
alkyl, C.sub.3-C.sub.10 aryl, and C.sub.1-C.sub.6
alkyl-C.sub.3-C.sub.10 aryl-C.sub.1-C.sub.6 alkyl;
[0098] and where R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are
independently selected from the group consisting of H, methyl, and
ethyl;
##STR00005##
[0099] where R.sub.1 and R.sub.6 are independently selected from
the group consisting of methyl, ethyl, n-propyl, and isopropyl;
[0100] where R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are
independently selected from the group consisting of C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.3-C.sub.6 cycloalkyl,
C.sub.1-C.sub.6 alkyl-C.sub.3-C.sub.6 cycloalkyl-C.sub.1-C.sub.6
alkyl, C.sub.3-C.sub.10 aryl, and C.sub.1-C.sub.6
alkyl-C.sub.3-C.sub.10 aryl-C.sub.1-C.sub.6 alkyl;
[0101] and where R.sub.7, R.sub.8, R.sub.9, R.sub.10 and R.sub.11
are independently selected from the group consisting of H, methyl,
and ethyl.
[0102] Preferably, the polyamine analogs will include compounds of
the structures 2 and 3, where R.sub.3, R.sub.5, R.sub.7 and R.sub.9
are independently (CH.sub.2).sub.x groups, where x is an integer
from 2 to 6, and further where R.sub.4, R.sub.6 and R8 are hydrogen
atoms.
[0103] More preferably, the polyamine analogs will include
compounds of the structures 2 and 3, where R3, R5, R7 and R9 are
independently (CH2)x groups, where x is an integer from 2 to 6, and
where R4, R6 and R8 are hydrogen atoms, and where R1 and R10 are
alkyl groups, and further where R2 and R11 are hydrogen atoms.
[0104] Most preferably, the polyamine analogs will include
compounds of the structures 2 and 3, where R3, R5, R7 and R9 are
independently (CH.sub.2).sub.x groups, where x is an integer from 2
to 6, and where R.sub.4, R.sub.6 and R.sub.8 are hydrogen atoms,
and where R.sub.1 and R.sub.10 are alkyl groups, and where R.sub.2
and R.sub.11 are hydrogen atoms, and further where the polyamine
analogs have a molecular weight less than 500.
[0105] Additional preferred compounds also include compounds of the
structure 4,
[0106] where R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are H;
[0107] where R.sub.1 and R.sub.5 are ethyl;
[0108] where R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are H and
R.sub.1 and R.sub.5 are ethyl;
[0109] and/or where R.sub.2 and R.sub.4 are independently selected
from the group consisting of C.sub.1-C.sub.6 alkyl and R.sub.3 is
selected from the group consisting of C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.3-C.sub.6 cycloalkyl,
C.sub.1-C.sub.6 alkyl-C.sub.3-C.sub.6 cycloalkyl-C.sub.1-C.sub.6
alkyl, C.sub.3-C.sub.10 aryl, and C.sub.1-C.sub.6
alkyl-C.sub.3-C.sub.10 aryl-C.sub.1-C.sub.6 alkyl.
[0110] Additional polyamine analogs useful in the present invention
include compounds of the formula 6, and the corresponding
stereoisomers, salts, and protected derivatives thereof:
##STR00006##
[0111] where R.sub.4 is C.sub.2-C.sub.6 n-alkenyl, C.sub.3-C.sub.6
cycloalkyl, C.sub.3-C.sub.6 cycloalkenyl, or C.sub.3-C.sub.6 aryl;
R.sub.3 and R.sub.5 are independently chosen from a single bond,
C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.6 alkenyl;
[0112] R.sub.2 and R.sub.6 are independently chosen from
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkenyl, C.sub.3-C.sub.6
cycloalkyl, C.sub.3-C.sub.6 cycloalkenyl, or C.sub.3-C.sub.6
aryl;
[0113] R.sub.1 and R.sub.7 are independently chosen from H,
C.sub.1-C.sub.6 alkyl, or C.sub.2-C.sub.6 alkenyl; and
[0114] R.sub.8, R.sub.9, R.sub.10, and R.sub.11 are H.
[0115] In preferred embodiments of the compounds of formula 6,
R.sub.1 and R.sub.7 are independently chosen from C.sub.1-C.sub.6
alkyl or C.sub.2-C.sub.6 alkenyl.
[0116] Additional polyamine analogs useful in the present invention
include compounds of the formula 7, and the corresponding
stereoisomers, salts, and protected derivatives thereof:
##STR00007##
[0117] where R.sub.4 is C.sub.1-C.sub.6 n-alkyl or C.sub.1-C.sub.6
branched alkyl;
[0118] R.sub.3 and R.sub.5 are independently chosen from a single
bond or C.sub.1-C.sub.6 alkyl;
[0119] R.sub.2 and R.sub.6 are independently chosen from
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkenyl, C.sub.3-C.sub.6
cycloalkyl, C.sub.3-C.sub.6 cycloalkenyl, or C.sub.3-C.sub.6
aryl;
[0120] R.sub.1 and R.sub.7 are independently chosen from H,
C.sub.1-C.sub.6 alkyl, or C.sub.2-C.sub.6 alkenyl; and
[0121] R.sub.8, R.sub.9, R.sub.10, and R.sub.11 are H.
[0122] In preferred embodiments of the compounds of formula 7,
R.sub.1 and R.sub.7 are independently chosen from C.sub.1-C.sub.6
alkyl or C.sub.2-C.sub.6 alkenyl, R.sub.4 is C.sub.1-C.sub.6
saturated n-alkyl or C.sub.1-C.sub.6 saturated branched alkyl, and
R.sub.3 and R.sub.5 are independently chosen from a single bond or
C.sub.1-C.sub.6 saturated n-alkyl.
[0123] When compounds of formulas 1-7 contain terminal primary
amino groups (that is, in compounds of formula 1, when R.sub.1 and
R.sub.2 are both H, and/or R.sub.6 and R.sub.7 are both H; in
compounds of formula 2, when R.sub.1 and R.sub.2 are both H, and/or
R.sub.8 and R.sub.9 are both H; in compounds of formula 3, when
R.sub.1 and R.sub.2 are both H, and/or R.sub.10 and R.sub.11 are
both H; in compounds of formula 4, when R.sub.1 and R.sub.6 are
both H, and/or R.sub.5 and R.sub.9 are both H; in compounds of
formula 5, when R.sub.1 and R.sub.7 are both H, and/or R.sub.6 and
R.sub.11 are both H; in compounds of formula 6, when R.sub.1 and
R.sub.8 are both H, and/or R.sub.7 and R.sub.1, are both H; in
compounds of formula 7, when R.sub.1 and R.sub.8 are both H, and/or
R.sub.7 and R.sub.11, are both H), the diseases treated with such
compounds include all diseases disclosed herein except Alzheimer's
disease.
[0124] Preferably, all the nitrogens of the polyamine analog are
independently secondary, tertiary, or quarternary amino groups.
[0125] Among polyamine analogs preferred for use in this invention
are those compounds with a demonstrated ability to modulate
naturally occurring polyamine levels in cells. Without intending to
be limited by theory, possible mechanisms include competition in
the polyamine synthesis pathway; upregulation of polyamine
catabolizers such as SSAT; affecting polyamine metabolism.
[0126] Of special interest are the following polyamine analogs:
[0127] 1,11-bis(ethyl)norspermine
(1,1'-bis(ethylamino)-4,8-diazaundecane; BE-3-3-3) [0128]
1,8-bis(ethyl)spermidine (BES) [0129] 1,12-bis(ethyl)spermine
(BESm; DESPM N.sup.1,N.sup.12-diethylspermine; SunPharm); [0130]
1,14-bis(ethylamino)-5,10-diazatetradecane (BE-4-4-4)
(Diethylhomospermine, N.sup.1, N.sup.14-diethylhomospermine; DEHOP
or DEHSPM; SunPharm) [0131] diethyl-norspermine (DENOP; SunPharm)
[0132] 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4)
[0133] N-ethyl-N'-(2-(3'-ethylamino-propylamino
methyl)-cis-cyclopropylmethyl)-propane 1,3-diamine
tetrahydrochloride (SL-11037), provided by S'LIL, Madison, Wis.
[0134] N-ethyl-N'-(2-(3'-ethylamino-propylamino
methyl)-trans-cyclobutylmethyl)-propane 1,3-diamine
tetrahydrochloride (SL-11038), S'LIL [0135]
N-ethyl-N'-(2-(3'-ethylamino-propylamino
methyl)-trans-cyclopropylmethyl)-propane 1,3-diamine
tetrahydrochloride (SL-11044), S'LIL. [0136]
N,N'-bis(3-ethylaminopropyl)-cis-but-2-ene-1,4-diamine
tetrahydrochloride (SL-11047), S'LIL [0137] The structures of
SL-11037, SL-11038, SL-11044, and SL-11047 are diagrammed
below:
##STR00008##
[0138] Besides the polyamine analogs listed above, stereoisomers,
salts or protected derivatives thereof, may be used.
Methods of Use of Polyamine Analogs
[0139] The invention also comprises methods of using an effective
amount of any of the polyamine analogs listed above, or
stereoisomers, salts or protected derivatives thereof (or a
composition comprising an effective amount of any of the polyamine
analogs listed above, or stereoisomers, salts or protected
derivatives thereof) in modulating macrophage proliferation (or in
treating or delaying development of macrophage-associated diseases,
such as macrophage-associated ocular diseases, particularly
macrophage-associated retinal diseases). The invention also
comprises any polyamine analog listed above, or stereoisomers,
salts or protected derivatives thereof, for use in preparing
compositions (i.e., medicaments) useful for treating
macrophage-associated diseases, such as macrophage-associated
ocular diseases, particularly macrophage-associated retinal
diseases).
[0140] Any polyamine analog listed above, or stereoisomers, salts
or protected derivatives thereof (or a composition comprising an
effective amount of any polyamine analog listed above, or
stereoisomers, salts or protected derivatives thereof) may be used
in vitro or in vivo. In vitro, a suitable biological sample (such
as a blood sample, which may or may not be enriched for the
macrophage population) is contacted with the composition(s). In
vivo, a composition of the invention is generally administered
according to the manufacturer's/supplier's instructions. Generally,
polyamine analogs are administered by subcutaneous or intravenous
injection. They may also be administered orally.
In Vivo Administration
[0141] The amount of a polyamine analog (or stereoisomers, salts or
protected derivatives thereof) administered will depend on several
variables, such as the particular analog (or sterioisomer, salt or
protective derivative) used, the time course of administration, the
condition of the individual, the desired objective, the extent of
disease, how many doses will be administered, and whether any other
substances are being administered. Generally, the amount used will
be as recommended by the manufacturer and/or based on empirical
studies. In the case of polyamine analogs (or stereoisomer, salt,
or protected derivative thereof), the amount will generally be
between about 1 to about 300 mg/m.sup.2/day, possibly between about
15 to about 150 mg/m.sup.2/day. Administration is generally
intermittant, meaning that analog (or stereoisomer, salt, or
protected derivative thereof) is administered per a period of at
least one to two days and then not administered for a period of at
least one to two days, with the cycle repeated as indicated. In one
embodiment, the polyamine analog (or stereoisomer, salt, or
derivative thereof) for 6 days every three weeks.
[0142] Routes of administration will generally depend on the nature
of the particular polyamine analog (or stereoisomer, salt or
protective derivative) used, and may be, for example, oral or by
injection (intraocular (e.g., intravitreal), subcutaneous or
intravenous). Other routes of administration include topical
administration (to the eye, e.g., using eye drops),
subconjunctival, perioocular, subtenon, retrobublar
(retro-orbital), or by iontophoretic delivery to the eye.
Formulations
[0143] Preferably, a polyamine analog (or stereoisomer, salt or
protected derivative), or other suitable agent that interferes with
the polyamine synthetic pathway, polyamine metabolism, and/or the
intracellular concentration maintenance of spermine) is
administered in a suitable pharmaceutical excipient. Pharmaceutical
excipients are known in the art and are set forth in Remington's'
Pharmaceutical Sciences, 18th edition, Mack Publishing (1990).
[0144] The polyamine analog may also be associated with another
substance that facilitates agent delivery to macrophages, or
increases specificity of the agent to macrophages. For example, an
agent(s) may be associated into liposomes. Liposomes are known in
the art. The liposomes in turn may be conjugated with targeting
substance(s), such as IgGFc receptors. Substances that increase
macrophage phagocytosis such as zymosan or tetrachlorodecaoxygen
(TCDO) and/or activation such as MCSF, GMCSF or IL-3 may be used to
increase uptake of anti-proliferative agent(s). In one embodiment,
the polyamine analog is provided as a sustained release formulation
which can be placed, for example, in the eye or adjacent a vascular
bed that leads to the eye.
[0145] A polyamine analog (or stereoisomer, salt or protected
derivative) may be administered alone, or in conjunction with other
substances and/or therapies, depending on the context of
administration (i.e., desired end result, condition of the
individual, and indications). "In conjunction with" means that an
agent is administered prior to, concurrently, or after other
substance or therapy. Examples of substances that might be
administered in conjunction with an agent include, but are not
limited to, antibiotics (e.g., agents to treat or prevent
bacterial, viral or fungal infection), anti-inflammatory agents,
anti-angiogenic agents, and the like. For example, a polyamine
analog (or a stereoisomer, salt or protected derivative thereof)
can be administered in conjunction with mitoguazone
dihydrochloride.
[0146] The mechanistic effectiveness of various polyamine analogs
and enzyme inhibitors can be determined at least in part by their
ability to deplete intracellular polyamine pools. Kramer et al.
[(1995) Biochem. Pharmacol. 50:1433] describe the use of
4-fluoro-L-ornithine to monitor metabolic flux through the
polyamine biosynthetic pathway. It was determined that the
metabolic flux indicated by the rate of appearance of fluorinated
polyamines, reflected the proliferation status of the cells. U.S.
Pat. No. 5,498,522 outlines the use of SSAT as a prognostic
indicator or tumor response marker. Either SSAT enzyme activity,
SSAT enzyme protein, or mRNA transcripts can be measured directly,
or other determinants related to SSAT induction can be measured,
such as SSAT co-factor acetylCoA, and the SSAT products
N1-acetylspermine and N1-acetylspermidine. To further determine the
effect of a polyamine analog's administration, an individual may be
monitored for disease (or precursor disease) progression as well as
biochemical and/or genetic markers of disease (or precursor
disease). With respect to disease progression, multiple rating
scales (i.e., indices of clinical function) have been established
and are known in the art for various macrophage proliferative
disorders such as AD and lymphomas. For macrophage-associated
neurological disorders, cognitive functions can be tested and, in
some cases, imaging modalities such as MRI may be used.
Other Agents for Modulating Macrophage Proliferation
[0147] Besides the polyamine analogs described above, suitable
agents for use in modulating macrophages in the context of
macrophage-associated ocular diseases, particularly
macrophage-associated retinal diseases, include general
anti-proliferative agents (i.e., proliferation-modulating agents),
which agents inhibit proliferation of macrophages. These include,
but are not limited to, daunomycin, mitomycin C, daunrorubicin,
doxorubicin, 5-FU, cytocine arabinoside, colchicine, cytochalasin
B, bleomycin, vincristin, vinblastine, methotrexate, cis platinum,
ricin, abrin, diphtheria toxin, and saporin.
[0148] Other suitable agents would be those which inhibit, or
interfere with, the polyamine synthetic pathway, or those which
affect the metabolism of polyamines. Other suitable agents are
those which affect the closely regulated intracellular
concentration of spermidine. An example of such an agent is MGBG
(mitoguazone dihydrochloride; XYRKAMINE.RTM.; Ilex, Tex.) which
inhibits S-adenosylmethionine decarboxylase which in turn is
required for the production of polyamines. Any agent that
interferes with polyamine interactions with proliferating
macrophage target, such as DNA, RNA, and/or membranes would
likewise be suitable. Another type of useful agent is one that
interferes with polyamine interactions with DNA. Such an agent(s)
could exert this function, for example, by any of the effects above
(i.e., interfering with the polyamine synthetic pathway and/or
metabolism, disturbing the concentration of intracellular spermine,
competitors, etc.) as well as affecting polyamine function in terms
of interacting with DNA. It is understood that, with respect to
these and any other agent described herein, toxicology
considerations also must be taken into account when determining
whether, and/or in what amount, an agent is to be used.
[0149] Administration and other considerations have been described
above. Contents of all references and publications cited herein are
hereby incorporated in their entirety.
[0150] The following examples are provided to illustrate but not to
limit the invention.
EXAMPLES
Example 1
Immune Cell Activation in Wet and Dry ARMD
[0151] The purpose of this example was to determine levels of
immune activation on peripheral blood monocytes and T-cells in
subjects with ARMD. Further, the purpose was to determine whether
levels of peripheral activation were correlated with clinically
determined levels of drusen progression. Immune cell activation
markers of choice included HLA-DR, CD16 (FC gamma III), and
CD38.
Methods and Materials:
[0152] Subjects.
[0153] The study included 32 subjects with ARMD (9 dry, 23 wet),
and from 10 age-matched control subjects. Macular drusen were
graded from 1 to 4 on basis of size, number, soft vs. hard &
associated pigment abnormalities. The grades of macular drusen were
not disclosed to the laboratory assessing macrophage abnormalities.
Fundus photographs (see Results) illustrate the different grades.
Table I provides the drusen scores in the right and left eyes of
each of the ARMD subjects in the study. Unless specifically
indicated, all subsequent data for ARMD patients uses average
drusen values from both wet and dry ARMD patients.
TABLE-US-00001 TABLE I ARMD Cohort subject Age Drusen score right
eye Drusen score left eye 1 84 4 4 2 88 end stage FV scar 4 3 83 4
4 4 83 end stage FV scar 3 5 84 end stage FV scar 4 6 87 scar 1 7
87 2 end stage scar 8 82 end stage FV scar 2 9 73 1 1 10 85 end
stage FV scar 4 11 84 3 3 12 78 4 4 13 80 2 2 14 90 3 3 15 77 end
stage FV scar end stage FV scar 16 2 2 17 end stage FV scar 5 18 82
1 SRN, 1 19 84 4 4 20 77 21 66 SRN 1 22 80 4 SRN, 4 23 77 3 SRN, 3
24 86 2 2 25 82 3 3 26 88 2 SRN, 3 27 83 2 2 28 81 SRN, 2 FV scar
29 4 4 30 3 3 31 1 1 32 2 2
[0154] Evaluation of Monocyte Activity by Flow Cytometry.
[0155] Heparinized blood was collected from the 32 ARMD and 10
age-matched control subjects. Phenotypic analysis of peripheral
blood monocytes was accomplished by flow cytometry. Plasma levels
of macrophage chemo-attractant protein (MCP-1) were determined by
Enzyme-linked immunosorbent assay (ELISA).100 microliters of whole
blood was stained with anti-CD 14-FITC, anti-CD 16-PE (Dako
Corporation, Carpenteria, Calif.; FITC=fluorescein isothiocyanate;
PE=phycoerythrin), or anti-HLA-DR-PE (Becton Dickinson, San Jose,
Calif.) or anti-CCR2-PE. Monocyte activation was also assessed
based on a phagocytosis assay (Molecular Probes, Eugene, Oreg.).
T-cell evaluation was determined staining with anti-CD4-FITC,
anti-CD38-PE, and anti-CD38-PerCP. Negative control antibody
(Isotype IgG-FITC, Isotype IgG-PE, (DAKO Corp.), and IgG-Per-CP
(Becton Dickinson), was included for each data set. Staining was
accomplished by incubation for 20 minutes at room temperature. Red
blood cell lysis was accomplished by addition of 2 ml of FACSLYSE
(Becton Dickinson), followed by centrifugation at 400.times.g for
five minutes. Supernatants were discarded and cell pellets fixed by
addition of 1 ml of fixing solution (1% paraformaldehyde, 0.1%
sodium azide, in phosphate-buffered saline, 0.01M, pH7.4). Flow
cytometry analysis was performed on a FACSCAN flow cytometer driven
by CELLQUEST software (Becton Dickinson). Statistical analysis of
selected populations by un-paired t-tests, Newman-Keuls Multiple
Comparisons, and Spearman correlations, were performed by GraphPad
Prizm software (San Diego, Calif.).
Results
[0156] The results of analysis of macrophage activation studies as
related to ARMD classification are summarized in Table II.
TABLE-US-00002 TABLE II Immune Parameters of ARMD All ARMD Dry ARMD
Wet ARMD Controls Parameter n = 32 n = 9 n = 23 n = 10 HLA-DR 795
+/ 1276.sup. 886 +/- 318 760 +/- 256 662 +/- 261 % CD14/CD16 24 +/
7* .sup. 29 +/ 8** .sup. 22 +/- 6 17 +/- 7 MCP-1 466 +/- 295 570
+/- 371*** 320 +/- 91 215 +/- 82 (n = 12) (n = 7) (n = 5) (n = 7) %
CD4/CD38 46 +/- 14 53 +/- 7 (n = 15) (n = 7) % CD8/CD38 37 +/-
11*** 27 +/- 7 (n = 15) (n = 7) *p < 0.003 vs controls **p <
0.011 vs wet ***p < 0.04 vs controls
[0157] FIG. 1, which is a graph of the relationship between blood
macrophage (CD14/16+) and immune activation (CD14/dr level) in ARMD
patient samples, shows that the higher the level of macrophages in
the blood (% CD14+/16+) the higher the level of activation (higher
DR expression/cell) in ARMD patient blood. As shown in FIG. 2, the
higher level of blood monocyte activation (as reflected by
increasing levels of cell surface HLA-DR concentrations), the
higher level of drusen in the eyes of ARMD patients (more severe
disease). Therefore, blood macrophage activation is directly
correlated to the severity of retinal disease.
[0158] Plasma levels of MCP-1 were highest for subjects with dry
ARMD, compared to controls (p<0.05) (FIG. 3A). The activation
marker CD16 was markedly elevated on CD14 monocytes for subjects
with dry ARMD compared to subjects with wet ARMD (p<0.01), and
compared to normal controls (p<0.001). FIG. 3B shows that
activated macrophages (CD14+/CD16+) and expression of the MCP-1
receptor, CCR2, expression are inversely related. That is, the
greater the percentage of activated macrophages, the lower the CCR2
expression levels.
[0159] FIG. 4A shows the relationship between drusen and blood
monocyte expression of the MCP-1 receptor, CCR2. The figures
demonstrates that down-regulation of expression of the MCP-1
receptor, CCR2, is directly proportional to the severity of ARMD as
measured by drusen score. Further, these data indicate that CCR2
expression and/or MCP-1 levels can be used assess severity of
disease, as well as response to therapy.
[0160] The results of the experiments show that blood levels of
macrophages (CD14+/16+ cells, which include proliferating
macrophages (ProMacs)) in ARMD patients parallels the level of
cellular activation (level of DR on CD14+ cells). In addition,
levels of activated macrophages and ProMacs in the blood parallels
levels of drusen in the retina of ARMD patients.
[0161] The results also show that there are two parallel markers of
disease activity (related to retinal drusen levels) present on
macrophages. CD16 and HLA-DR levels on macrophages increases with
severity of ARMD. Systemic MCP-1 also increases with severity of
disease. In addition, the CCR2 levels of these same macrophages are
low relative to non-disease macrophages, indicating down-regulation
of the CCR2 receptor by the endogenously produced MCP-1 ligand.
These findings indicate that MCP-1 and markers of macrophage
activation are indicative of ARMD disease severity, based on their
correlation with drusen, the conventional ARMD disease severity
marker.
[0162] The results above indicate that persistently activated
macrophages, which includes a population of proliferating
macrophages, are elevated in ARMD. Without being held to theory,
one possible mechanism for disease involves drusen accumulation in
the RPE, which in turn can lead to disease progression. This drusen
accumulation may result from reduced ability of macrophages present
in the eye to phagocytose, which in turn allows for drusen
accumulation To test this hypothesis, CD14+ cells from ARMD
patients (early and late ARMD, where early ARMD was defined by a
drusen score of 1-2, and late ARMD defined as a drusen score of
3-4) and age-matched controls were assayed for phagocytic ability.
FIG. 4B shows that the CD14+ cells of patients with late ARMD have
significantly decreased phagocytic ability, consistent with this
model of ARMD progression.
[0163] Conclusions:
[0164] Data presented in this study support a mechanistic model of
ARMD that involves systemic immune activation detectable at both
the cellular (monocyte and T cell) and cytokine (MCP-1) levels.
Abnormal cellular and cytokine activation are correlated with
disease severity as assessed by drusen score. These data support a
disease model in which systemic immune activation contributes to
the evolution of ARMD regardless of "wet" versus "dry" clinical
diagnosis. FIG. 4C provides a schematic of this disease model.
[0165] Therefore, global therapeutic strategy should focus on the
immunological parameters as described herein, rather than the
clinical parameters (e.g., drusen accumulation, choroidal
neovascularization (CNV), and the like). Systemically activated
macrophages in ARMD provide targets for drugs to modulate these
immunologic aspects of ARMD pathogenesis. Efficacy of therapies
directed toward ARMD can be monitored through analysis of
macrophages and MCP-1 levels of blood specimens or other body
fluids (e.g., vitreal or aqueous fluid of the eye) or ocular
tissue. Examination of parameters of systemic immune response can
be performed in conjunction with retinal examination and other
conventional parameters of disease. Further, the immune parameters
discussed above define immunological differences between the wet
and dry forms of disease, and thus useful in disease monitoring and
development of effective novel therapies.
Example 2
Effect of Polyamine Analogs on Macrophage Proliferation in ARMD
Patients
[0166] Percoll gradient separation, which allows for denser cells
to be captured, was accomplished as follows. A two-step gradient
was prepared in 15 mL conical tubes: bottom layer of 1.087 density
Percoll, overlaid with 1.077 density. 1.5 mL whole heparinised
blood was mixed with an equal volume of isotonic saline. This
blood/saline was layered over the gradient and centrifuged. Cells
from the 1.077 and 1.087 interfaces were collected, combined, and
washed in 5 volumes of RPMI 1640.
[0167] 5.times.105 PBMCs from ARMD patients (both wet and dry) were
isolated through a 1.087 g/cc Percoll/saline gradient as described
above, and then were exposed to varied concentrations of polyamine
analog after baseline CD14/PCNA staining was performed. The cells
were cultured at 5.times.10.sup.5 cells in RPM1-1640/10% fetal calf
serum in a polypropylene tube (Falcon) at 37.degree. C. for five
days. After five days CD14/PCNA staining was performed on control
and agent-treated cultures. FACSCAN analysis of PCNA-positive cells
in control cultures was compared with agent-treated cultures and
the percentage of control PCNA/CD14 cells was calculated. Samples
from age-matched normal or Alzheimer's disease (AD) patients were
used as controls.
[0168] The results of such an experiment in which the effect of
polyamine analog SL-11047 on proliferating macrophages (as detected
by PCNA) from the blood of ARMD patients are shown in FIG. 5. This
polyamine analog significantly reduced the percentage of
proliferating macrophages by at lest 80% in about 60% of ARMD
patients. In contrast only 17% of unaffected age-matched subjects
had at least 80% killing of CD14/PCNA positive cells. CD14/PCNA
cells from age-matched AD patients were similarly killed at only
low levels (8% killing). The results of this example shows that
polyamine analog SL-11047 kills proliferating macrophages in ARMD.
Further, screening of polyamine analogs for activity against
proliferating macrophages to identify agents with the most
effective killing is warranted.
[0169] In culture in which the proliferating macrophages were
killed, cell supernatants were evaluated for MCP-1 production.
Cells were treated as described above, and MCP-1 collected over a 2
day period that followed a 4 day period of treatment with SL-11047.
Cells from an age-matched normal donor were as a control. FIG. 6
shows that ARMD PBMCs spontaneously produce high levels of MCP-1
("MCP-1 pre"). Exposure of these PBMCs to SL-11047 markedly
decreased MCP-1 production (FIG. 6, "MCP-1 post"). These data are
consistent with the data in FIG. 6. Thus, killing of CD14/PCNA
cells (proliferating macrophages), removes a source of MCP-1, a
factor implicated in the pathogenesis of ARMD as discussed
above.
Example 3
Polyamine Analog Treatment Improves the Monocyte Phagocytic Index
In Vivo
[0170] In order to assess the ability of polyamine analogs to
improve phagocytic function, whole blood was obtained from a
lymphoma patient pre- and post-administration of SL-11047 (25
mg/m.sup.2 by infusion per day for 5 days every 3 weeks (one
cycle), for a total four cycles) and the phagocytic index assessed
as described above. As shown in FIG. 7, abnormal baseline
phagocytic function improved significantly after administration of
four cycles of SL-11047 polyamine analog therapy. In addition,
during the course of therapy, CD16+ cells in the patient decreased
to a normal range of the total monocyte population.
[0171] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding it will be apparent to those skilled in the art
that certain changes and modifications will be practiced.
Therefore, the description and examples should not be construed as
limiting the scope of the invention, which is delineated by the
appended claims.
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