U.S. patent application number 09/855067 was filed with the patent office on 2002-04-18 for method of inhibiting membrane-type matrix metalloproteinase.
This patent application is currently assigned to The Research Foundation of State University of New York. Invention is credited to Golub, Lorne M., Lee, Hsi-Ming, Salo, Tuula, Sorsa, Timo, Teronen, Olli.
Application Number | 20020045603 09/855067 |
Document ID | / |
Family ID | 21976191 |
Filed Date | 2002-04-18 |
United States Patent
Application |
20020045603 |
Kind Code |
A1 |
Golub, Lorne M. ; et
al. |
April 18, 2002 |
Method of inhibiting membrane-type matrix metalloproteinase
Abstract
The invention is a method of inhibiting the activity of
membrane-type matrix metalloproteinase (MT-MMP) in biological
systems. Accordingly, the invention permits the treatment of
medical conditions in mammals that are characterized by MT-MMP
activity, and especially those conditions characterized by
excessive MT-MMP activity. The method employs a tetracycline
compound, preferably a non-antimicrobial tetracycline, and more
preferably 6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline
(CMT-3) or
6-.alpha.-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8)
to inhibit the MT-MMP activity.
Inventors: |
Golub, Lorne M.; (Smithtown,
NY) ; Lee, Hsi-Ming; (Setauket, NY) ; Sorsa,
Timo; (Helsinki, FI) ; Teronen, Olli;
(Helsinki, FI) ; Salo, Tuula; (Oulu, FI) |
Correspondence
Address: |
Ronald J. Baron, Esq.
HOFFMANN & BARON, LLP
6900 Jericho Turnpike
Syosset
NY
11791
US
|
Assignee: |
The Research Foundation of State
University of New York
|
Family ID: |
21976191 |
Appl. No.: |
09/855067 |
Filed: |
May 14, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09855067 |
May 14, 2001 |
|
|
|
09052222 |
Mar 31, 1998 |
|
|
|
Current U.S.
Class: |
514/152 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 31/65 20130101; A61P 35/00 20180101 |
Class at
Publication: |
514/152 |
International
Class: |
A61K 031/65 |
Goverment Interests
[0002] This invention was made with Government support under Grant
No. R37-DE03987 awarded by the National Institutes of Health
through the National Institute of Dental Research. The Government
has certain rights in the invention.
Claims
What is claimed is:
1. A method of inhibiting membrane-type matrix metalloproteinase
activity in a biological system, comprising administering to the
system in need thereof an MT-MMP-inhibitory amount of a
tetracycline compound.
2. A method according to claim 1, wherein the tetracycline compound
is administered in an amount that has substantially no
antimicrobial activity.
3. A method according to claim 1, wherein the biological system has
excessive MT-MMP activity, and the method comprises administering
to the system an amount of the tetracycline compound sufficient to
inhibit the excessive MT-MMP activity.
4. A method according to claim 1, wherein the biological system
comprises cultured mammalian cells.
5. A method according to claim 4, wherein the mammalian cells
comprise tumor cells, osteoclasts, or fibroblasts.
6. A method according to claim 1, wherein the biological system is
a mammal.
7. A method according to claim 5, wherein the mammal has a
condition characterized by excessive MT-MMP activity.
8. A method according to claim 5, wherein the mammal has a
cancer.
9. A method according to claim 8, wherein the cancer is a
carcinoma, fibrosarcoma, or osteosarcoma.
10. A method according to claim 5, wherein the mammal has a
condition characterized by excessive osteoclast activity.
11. A method of inhibiting the invasive potential of carcinoma
cells, fibrosarcoma cells, or osteosarcoma cells, comprising
contacting the cells with an invasion-inhibitory amount of a
tetracycline compound.
Description
[0001] This Application is a Continuation Application of Ser. No.
09/052,222, filed on Mar. 31, 1998. The entire disclosure of the
aforementioned application is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] The invention relates to methods of inhibiting the activity
of membrane-type matrix metalloproteinase (MT-MMP) in biological
systems. More specifically, the invention relates to the use of
tetracycline compounds for the inhibition of MT-MMP in mammals.
[0004] Matrix metalloproteinases (MMPs, matrixins) are a family of
related proteolytic enzymes expressed by a wide variety of cells
and tissue types in both normal and abnormal situations. These
zinc-binding metalloendopeptidase enzymes are unified as a family
by virtue of structural homologies, and by proteolytic activity
against many components of the extracellular matrix (ECM). As a
result, the MMPs play important roles in development and
morphogenesis as well as in tissue remodeling following injury. In
particular, matrix metalloproteinases are distinguished from other
metalloproteinases by virtue of their susceptibility to activation
of the zymogen by thiol-modifying reagents (e.g., 4-aminophenyl
mercuric acetate (APMA) and other mercurial compounds,
N-ethylmaleimide, oxidized glutathione), their inhibition by a
group of endogenous substances known collectively as "tissue
inhibitors of metalloproteinases" or "TIMPs," and the presence of a
consensus sequence in the propeptide forms. Nagase (1996).
[0005] However, the MMPs have widely divergent substrate
specificities and activities, leading to their classification into
a series of subtypes. The collagenases include interstitial
collagenase (MMP-1), neutrophil collagenase (MMP-8), and
collagenase-3 (MMP- 13), and are characterized by their ability to
specifically degrade triple helical regions of collagens I, II, and
III. The gelatinases include gelatinase A (MMP-2; 72-kDa
gelatinase) and gelatinase B (MMP-9; 92-kDa gelatinase), and are
characterized by their ability to specifically cleave gelatins, but
they are further characterized by their ability to hydrolyze
collagen IV, elastin and other substrates. The stromelysins include
stromelysin 1 (MMP-3), stromelysin 2 (MMP-10), and in certain
systems stromelysin 3 (MMP- 11). The stromelysins 1 and 2
characteristically degrade a variety of substrates including
aggrecan, fibronectin, laminin, numerous collagen types, and play a
role in activation of other enzymes.
[0006] The MMP family also includes several other MMPs that do not
neatly fit into the collagenase, gelatinase, and stromelysin
subfamilies. Matrilysin (MMP-7; PUMP-1) differs substantially in
structure, lacking a C-terminal domain common to the other MMPs.
Metalloelastase (MMP-12) is characterized by its ability to degrade
elastin.
[0007] A more recently discovered MMP that is substantially
distinct from the conventional MMP structural subfamilies is
membrane-type matrix metalloproteinase (MT-MMP). MT-MMP differs
from other MMPs in a number of ways. For example, most MMPs are
secreted by the cells in which they are made, but MT-MMP, as is
implicit in its name, is expressed as a membrane-bound protein on
the surface of the cell. It is believed that this feature is unique
among the MMPs and that such expression may be attributable to a
hydrophobic domain of up to 24 amino acids. See Nagase (1996).
[0008] The MT-MMP enzyme also has characteristic substrate
specificity, with particular capacity for converting pro-MMP-2 and
pro-MMP-9 into the active forms of these gelatinases (Murphy et al.
1992; Zucker et al. 1995). However, MT-MMP is also characterized by
direct degradation of gelatin. MT-MMP may further act as a surface
receptor for TIMP-2, and the resulting MT-MMP/TIMP-2 complex may
then act as a receptor for pro-gelatinase A, which can activate the
gelatinase (Strongin et al. 1995; Cao et al. 1996; Lichte et al.
1996). This plasma membrane-activated MMP-2 can in turn activate
pro-MMP-9 and this process is inhibited by TIMPs (Fridman et al.
1995).
[0009] Unlike other MMPs, MT-MMPs and MMP-11 (stromelysin-3) are
activated by an intracellular proteinase, furin, a Golgi-associated
serine proteinase (Pei and Weiss 1995). Similar to MMP-11, the
mechanism of activation of MT-MMP is under investigation and may
also involve an intracellular activation through a sequence which
can be recognized by furin-like enzymes (Sato et al. 1996).
Recently, Cao et al. (1996) demonstrated that the 63 kDa pro-MT-MMP
expressed on the cell surface has not been processed
(contransfection with furin did not change the molecular weight of
MT-MMP). These authors hypothesize that conformational effects
induced by the plasma membrane localization of MT-MMP may provide
functional activity to pro-MMP-2 without cleavage of the molecule.
Receptor-bound TIMP-2 may participate in this process.
[0010] Investigators have identified MT-MMP expression by normal
tissues, e.g., placenta, lung, and kidney (Takino et al. 1994). A
form of MT-MMP, identified as membrane-type 1 MMP (MT.sub.1-MMP)
has also been found to be expressed by osteoclasts, the principal
cell type involved in bone resorption (Sato et al. 1997). Other
investigators have identified three different forms of MT-MMP
expressed by rat smooth muscle cells, that appear to be involved in
matrix remodeling of blood vessels (Shofuda et al 1997).
[0011] Lee et al. (1997) have observed expression of a gelatinase
activator that appears to localize to the Golgi membranes. Lee et
al. hypothesize that this activator may be MT-MMP, and that a
change in localization of this activator to the plasma membrane may
occur in tumor cells. Other investigators have identified excessive
MT-MMP expression by lung carcinomas (Sato et al. 1994). Still
others have seen MT-MMP expression by fibroblasts in tumor stroma
of colon, breast, head and neck carcinomas (Okada et al. 1995).
[0012] MT-MMP expressed on cancer cell membranes also is an
extracellular matrix-degrading enzyme sharing the substrate
specificity with interstitial collagenases (i.e., digestion of type
I, II, and III collagens into characteristic 3/4 and 1/4 fragments)
(Ohuchi et al. 1997; Cao et al. 1998). As noted above, MT-MMP also
exhibits gelatinolytic activity, as measured by gelatin zymography
(Imai et al. 1996). Thus, MT-MMP may play a dual role in
pathophysiological digestion of extracellular matrix through direct
cleavage of the substrates and activation of pro-MMP-2, which is
produced constitutively in relatively high concentrations by many
cell types including tumor cells (Sato et al. 1994; Sato et al.
1997).
[0013] Tetracycline and a number of its chemical relatives form a
particularly successful class of antibiotics. Certain of the
tetracycline compounds, including tetracycline itself, as well as
sporocycline, etc., are broad spectrum antibiotics, having utility
against a wide variety of bacteria. The parent compound,
tetracycline, has the following general structure: 1
[0014] The numbering system for the multiple ring nucleus is as
follows: 2
[0015] Tetracycline, as well as the 5-OH (terramycin) and 7-Cl
(aureomycin) derivatives, exist in nature, and are all well known
antibiotics. Semisynthetic derivatives such as
7-dimethylamino-tetracycli- ne (minocycline) and
6.alpha.-deoxy-5-hydroxy-tetracycline (doxycycline) are also known
antibiotics. Natural tetracyclines may be modified without losing
their antibiotic properties, although certain elements of the
structure must be retained to do so. Recently, however, a class of
compounds has been defined that are structurally related to the
antibiotic tetracyclines, but which have had their antibiotic
activity substantially or completely expunged by chemical
modification. The modifications that may and may not be made to the
basic tetracycline structure have been reviewed by Mitscher (1978).
According to Mitscher, modification at positions 5-9 of the
tetracycline ring system can be made without causing the complete
loss of antibiotic properties.
[0016] However, changes to the basic structure of the ring system,
or replacement of substituents at positions 1-4 or 10-12, generally
lead to synthetic tetracyclines with substantially less, or
essentially no, antibacterial activity. For example,
4-de(dimethylamino)tetracycline is commonly considered to be a
non-antibacterial tetracycline. These compounds, known as
chemically-modified tetracyclines (CMTs) have been found to possess
a number of interesting properties, such as the inhibition of
excessive collagenolytic activity both in vitro and in vivo. See,
for example, Golub et al. (1991).
[0017] More recently, it has been established that tetracyclines,
which are rapidly absorbed and have a prolonged plasma half-life,
exert biological effects independent of their antimicrobial
activity (Golub et al. 1991, Golub et al. 1992, Uitto et al. 1994).
Such effects include inhibition of some but not all matrix
metalloproteinases. Specific activity has been shown with respect
to collagenases (MMP-1; MMP-8; MMP-13) and gelatinases (MMP-2;
MMP-9), as well as prevention of pathologic tissue destruction
(Golub et al. 1991). However, Applicants are not aware of any
evidence in the prior art disclosing or suggesting that
tetracycline compounds could be of any use in inhibiting MT-MMP
activity, either in normal systems or in systems characterized by
abnormal or excessive MT-MMP activity.
[0018] Recent studies have suggested that, in some systems, certain
tetracyclines and inhibitors of metalloproteinases can inhibit
tumor progression (DeClerck et al. 1994) or angiogenesis (WIPO
publication WO 92/12717; Maragoudakis et al. 1994). Zucker et al.
(1985) showed that minocycline can inhibit melanoma cell activity
in vitro. Some tetracyclines may exhibit cytostatic effects against
some tumors (Kroon et al. 1984; van den Bogert et al. 1986).
Pro-gelatinase A (MMP-2) has been reported to be associated with
tumor spread (Yu et al. 1997).
6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3) has been
shown to experimentally suppress prostate and melanoma tumor growth
and metastasis in vivo (Lokeshwar et al. 1998; Seftor et al.
1998).
[0019] While tetracycline antibiotics are generally effective for
treating infection, the use of these compounds can lead to
undesirable side effects. For example, the long term administration
of antibiotic tetracyclines can reduce or eliminate healthy
microbial flora, such as intestinal flora, and can lead to the
production of antibiotic resistant organisms or the overgrowth of
yeast and fungi. Accordingly, chemically-modified tetracyclines, in
which the antimicrobial activity is attenuated or deleted, can be
preferred for use in applications in which anti-collagenolytic
activity is indicated.
[0020] In view of the above considerations, it is clear that there
is a need to supplement existing methods of inhibiting
proteinase-mediated physiological and structural changes in
biological systems, especially as therapeutic interventions in
disease processes. It is also apparent that there is a need for
advanced and precise inhibitors of MT-MMP activity in diagnostic
and therapeutic applications, especially in respect of those
medical conditions that are characterized by excessive MT-MMP
activity. In particular, it is desirable to identify new and
selective MT-MMP inhibitors with relatively high activity, i.e.,
being active at doses that are substantially free of harmful side
effects.
[0021] Accordingly, it is one of the purposes of this invention to
overcome the above limitations in modulation or control of
proteinase activity, by providing a compound and method for
inhibiting membrane-type matrix metalloproteinase, and inhibiting
deleterious effects of excessive activity of the enzyme in
biological systems. In particular, it is a one of the purposes of
the invention to provide new and selective MT-MMP inhibitors that
are active at doses that are substantially free of harmful side
effects.
SUMMARY OF THE INVENTION
[0022] It has now been discovered that these and other objectives
can be achieved by the present invention, which provides a method
for inhibiting the activity of membrane-type matrix
metalloproteinase in a biological system by providing a chemically
modified tetracycline to the system in an amount that is effective
to achieve the specified result.
[0023] In one embodiment, the invention is a method of inhibiting
membrane-type matrix metalloproteinase activity in a biological
system, comprising delivering to the system in need thereof an
MT-MMP-inhibitory amount of a tetracycline compound.
[0024] The tetracycline compound is preferably administered in an
amount that has substantially no antimicrobial activity. Preferred
tetracycline compounds include, for example,
6-demethyl-6-deoxy-4-dedimethylaminotetra- cycline (CMT-3) or
6-.alpha.-deoxy-5-hydroxy-4-de(dimethylamino)tetracycli- ne
(CMT-8).
[0025] The biological system can have or be characterized by
excessive MT-MMP activity, wherein the method comprises
administering to the system an amount of the tetracycline compound
sufficient to inhibit the excessive MT-MMP activity.
[0026] The biological system can comprise cultured mammalian cells,
such as tumor cells, osteoclasts, or fibroblasts. The biological
system can be a mammal, such as a mammal that has a condition
characterized by excessive MT-MMP activity. Thus the method can be
used to treat a mammal that has a cancer, such as a carcinoma,
fibrosarcoma, or osteosarcoma. The method can also be used if the
mammal has a condition characterized by excessive osteoclast
activity.
[0027] In an alternative embodiment, the invention is a method of
inhibiting MT-MMP activity in a mammal, comprising administering to
the mammal an amount of a tetracycline compound sufficient to
inhibit MT-MMP activity.
[0028] The tetracycline compound is preferably administered in an
amount that has substantially no antimicrobial activity. Preferred
tetracycline compounds include, for example,
6-demethyl-6-deoxy-4-dedimethylaminotetra- cycline (CMT-3) or
6-.alpha.-deoxy-5-hydroxy-4-de(dimethylamino)tetracycli- ne
(CMT-8).
[0029] In this method, if the mammal has a condition characterized
by excessive MT-MMP activity, the method comprises administering to
the mammal an amount of the tetracycline compound sufficient to
inhibit excessive MT-MMP activity. The method is useful in cases in
which the mammal has a cancer, such as a carcinoma, fibrosarcoma,
or osteosarcoma, or a condition characterized by excessive
osteoclast activity.
[0030] In another embodiment, the invention is a method of
inhibiting the invasive potential of carcinoma cells, fibrosarcoma
cells, or osteosarcoma cells, comprising contacting the cells with
an invasion-inhibitory amount of a tetracycline compound. Preferred
tetracycline compounds include, for example,
6-demethyl-6-deoxy-4-de(dime- thylamino)tetracycline (CMT-3) or
6-.alpha.-deoxy-5-hydroxy-4-de(dimethyla- mino)tetracycline
(CMT-8). More preferably in this embodiment, the method comprises
contacting the cancer cells with an amount of CMT-3 or CMT-8 that
is sufficient to inhibit membrane-type matrix metalloproteinase
activity of the cells.
[0031] These and other advantages of the present invention will be
appreciated from the detailed description and examples set forth
hereinbelow. The detailed description and examples enhance the
understanding of the invention, but are not intended to limit the
scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Preferred embodiments of the invention have been chosen for
purposes of illustration and description, but are not intended in
any way to restrict the scope of the invention. The preferred
embodiments of certain aspects of the invention are shown in the
accompanying drawings, wherein:
[0033] FIG. 1 is a graphical illustration of an autoradiographic
analysis of CMT-3-mediated inhibition of gelatinolytic activity
exhibited by a membrane fraction of MT1-MMP-transfected COS-1
cells.
[0034] FIG. 2 is a graphical illustration of a zymographic analysis
CMT-3-mediated inhibition of gelatinase activation by recombinant
MT1-MMP.
[0035] FIG. 3A is a graphical illustration of a zymographic
analysis of CMT-3-mediated inhibition of caseinolytic activity of
recombinant MT1-MMP.
[0036] FIG. 3B is a graphical illustration of a zymographic
analysis CMT-3-mediated inhibition of pro-gelatinase A activation
by recombinant MT1-MMP.
[0037] FIG. 4A is a bar chart showing dose-dependent inhibition of
MMP-2 expression by cultured MG-63 osteosarcoma cells.
[0038] FIG. 4B is a bar chart showing dose-dependent inhibition of
MMP-2 activation in cultured MG-63 osteosarcoma cells.
[0039] FIG. 5A is a bar chart showing CMT-3- and CMT-8-mediated
inhibition of Matrigel invasion by HT1080 fibrosarcoma cells.
[0040] FIG. 5B is a bar chart showing CMT- and CMT-8-mediated
inhibition of Transwell migration by HT1080 fibrosarcoma cells.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0041] In one embodiment, the present invention is directed to a
method for inhibiting cancer growth, including processes of
cellular proliferation, invasiveness, and metastasis in biological
systems. The method includes the use of a tetracycline compound as
an inhibitor of cancer growth. Preferably, the method is employed
to inhibit or reduce cancer cell proliferation, invasiveness,
metastasis, or tumor incidence in living animals, such as mammals.
The method is also readily adaptable for use in assay systems,
e.g., assaying cancer cell growth and properties thereof.
[0042] The tetracycline compound used in the method of the
invention may be any tetracycline compound, from those that have
potent antibiotic activity to those lacking any substantial
antimicrobial activity. However, it is preferred that the
tetracycline compound be administered in an amount that has
substantially no antimicrobial activity, but that is effective for
inhibiting the activity of an MT-MMP, preferably inhibiting a
pathological or excessive activity of MT-MMP. The method can,
therefore, take advantage of tetracycline compounds that have been
chemically modified to reduce or eliminate their antimicrobial
properties.
[0043] Such compounds include, for example, those that lack the
dimethylamino group at position 4 of the tetracycline ring
structure, e.g.,
[0044] 4-de(dimethylamino)tetracycline (CMT-1),
[0045] 6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline
(CMT-3),
[0046] 7-chloro-4-de(dimethylamino)tetracycline (CMT-4),
[0047] 4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),
[0048] 4-de(dimethylamino)-12.alpha.-deoxytetracycline (CMT-7),
[0049] 6-deoxy-5.alpha.-hydroxy-4-de(dimethylamino)tetracycline
(CMT-8),
[0050] 4-dedimethylamino- 12.alpha.-deoxyanhydrotetracycline
(CMT-9),
[0051]
7-dimethylamino-6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline
(CMT- 10),
[0052] 4-de(dimethylamino)-5-oxytetracycline,
[0053]
5.alpha.,6-anhydro-4-hydroxy-4-de(dimethylamino)tetracycline,
[0054]
4-de(dimethylamino)-11-hydroxy-12.alpha.-deoxytetracycline,
[0055] 12.alpha.-deoxy-4-deoxy-4-de(dimethylamino)tetracycline,
and
[0056]
12.alpha.,4.alpha.-anhydro-4-de(dimethylamino)tetracycline.
[0057] Further examples of tetracyclines modified for reduced
antimicrobial activity include
6-.alpha.-benzylthiomethylenetetracycline, the mono-N-alkylated
amide of tetracycline, 6-fluoro-6-demethyltetracycli- ne, 11
.alpha.-chlorotetracycline, tetracyclinonitrile (CMT-2),
[0058] and tetracycline pyrazole (CMT-5).
[0059] The preferred tetracycline compounds include:
[0060] 4-de(dimethylamino)tetracycline (CMT-1),
[0061] tetracyclinonitrile (CMT-2)
[0062] 6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline
(CMT-3),
[0063] 4-de(dimethylamino)-7-chlorotetracycline (CMT-4),
[0064] tetracycline pyrazole (CMT-5),
[0065] 4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),
[0066] 4-de(dimethylamino)- 1 2-deoxytetracycline (CMT-7),
[0067] 6-deoxy-5.alpha.-hydroxy-4-de(dimethylamino)tetracycline
(CMT-8),
[0068] 4-dedimethylamino-12.alpha.-deoxyanhydrotetracycline
(CMT-9), and
[0069]
7-dimethylamino-6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline
(CMT- 10),
[0070] Particularly preferred modified tetracyclines include CMT-1,
CMT-3, CMT-7, and CMT-8. Highly preferred modified tetracycline
compounds include CMT-3 and CMT-8. Combinations of these compounds,
as well as combinations with other tetracyclines and/or
pharmaceutical compounds can be employed.
[0071] The use of such chemically-modified tetracyclines (CMTs) is
preferred in the present invention since they can be used at higher
levels than antimicrobial tetracyclines, while avoiding the
disadvantages associated with antimicrobial activity as described
elsewhere herein. However, sub-antimicrobial doses of typically
antibacterial tetracyclines (e.g., doxycycline) can also be given
according to the invention.
[0072] Preferred compounds having substantial antimicrobial
activity include, for example, tetracycline, doxycycline, and
minocycline and other well known tetracycline antibiotics,
especially those already approved for use in humans.
[0073] These compounds exhibit their MT-MMP-inhibitory properties
at concentrations that lead to relatively few, and in some cases
are substantially free of side effects. For example, the useful
concentrations of preferred chemically-modified tetracyclines do
not present any significant antimicrobial activity. Such
non-antimicrobial tetracycline compounds are useful for extended
treatment protocols, where other compounds would exhibit
undesirable side-effects.
[0074] The tetracycline compounds useful according to the invention
possess a desirable but unusual combination of physicochemical
properties, including activity, bioavailability, solubility, and
reduction of side effects. These properties render the compounds
particularly desirable for the inhibition of MT-MMP activity in
mammals. In addition, it is believed that the properties of
hydrophilicity and hydrophobicity are well balanced in these
compounds, enhancing their utility both in vitro and especially in
vivo, while other compounds lacking such balance are of
substantially less utility. Specifically, the compounds have an
appropriate degree of solubility in aqueous media to permit
absorption and bioavailability in the body, while also having a
degree of solubility in lipids to permit traversal of the cell
membrane to a putative site of action. The compounds are maximally
effective if they can be delivered to the site or region of the
MT-MMP activity, and additional advantage can be obtained if the
ability of the compound to localize to expressed MT-MMP is
maximized.
[0075] In the treatment of certain localized conditions, the degree
of hydrophilicity of the non-antimicrobial tetracycline compound
can be of lesser importance. Such compounds as tetracyclinonitrile
(CMT-2) and 4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),
which have low solubility in aqueous systems, can be used in direct
or topical treatment of skin conditions characterized by MT-MMP, or
by implantation into the brain to topically treat brain conditions.
Animal experiments, in which adult rats are orally gavaged with
these two CMTs, have shown no detectable blood levels of these
compounds, indicating a lack of systemic absorption and/or
extraordinarily rapid excretion.
[0076] This embodiment of the invention has been developed based on
the unexpected observation by Applicants that certain tetracycline
compounds, chemically modified to eliminate substantially all
antimicrobial activity, are effective to inhibit the activity of
MT-MMP and to reduce deleterious or injurious effects associated
with MT-MMP activity. Of these, one especially preferred CMT, i.e.,
6-demethyl-6-deoxy-4-de(dimeth- ylamino)tetracycline (also referred
to as "CMT-3"), appears to possess an excellent balance of
properties, in that it is shown to possess unusually strong
activity in inhibiting MT-MMP activity. Another advantage of CMT-3
is that it has an unexpectedly long serum half-life (approximately
28 hr). Therefore, CMT-3 may only require periodic administration,
e.g., once or twice per week.
[0077] In another embodiment, the method of the invention is
effective to inhibit the enzymatic activity of membrane-type matrix
metalloproteinase associated with medical conditions in mammals.
The MT-MMP activity capable of inhibition may derive from MT-MMP
expression by the diseased tissue or from normal tissue. In
particular, the MT-MMP activity may be derived from such normal
tissues as epithelial tissue or stromal tissue. More preferably,
the method can be used to inhibit excessive MT-MMP activity
associated with cancerous tumors. This inhibition of observed
gelatinolytic activity may be due to inhibition of MT-MMP activity,
down-regulation of MT-MMP expression, or some other interference
with the physiology associated with this enzyme, such as inhibition
of activation of a precursor form of the enzyme.
[0078] Applicants have discovered that the chemically modified
tetracyclines decrease the level of activity of MT-MMP expressed by
several types of human cancer cell lines, including carcinoma,
fibrosarcoma, and osteosarcoma cells. Applicants believe that this
observation carries significant therapeutic implications for cancer
treatment. Applicants also understand that these CMTs and other
chemically and functionally related compounds would be useful for
inhibiting the consequences of other diseases characterized by
excessive MT-MMP expression or activity.
[0079] The use of the method of the invention in the inhibition of
cancer cell growth or proliferation can occur with less
cytotoxicity to normal cells or tissues than is found with
conventional cytotoxic cancer therapeutics, preferably without
substantial cytotoxicity to normal cells or tissues. For example,
it has been unexpectedly observed that a tetracycline, e.g., CMT-3,
can inhibit MT-MMP activity in cancer cells while producing little
or substantially no cytotoxicity in normal cells.
[0080] The data presented in the examples below, reveal that cancer
cells treated with these compounds results in a decrease in
extracellular gelatinolytic activity, a corresponding
dose-dependent decrease in the cells' in vitro invasive potential,
and a decrease in the cells' metastatic ability in vivo. Moreover,
the compounds can induce killing of tumor cells, and can do so
while being substantially non-cytotoxic to normal tissues.
Accordingly, these chemically-modified tetracyclines can be used to
suppress the formation and magnitude of metastases associated with
certain cancers, used as an adjunct to other treatment regimens,
and lead to greater efficacy in the treatment of metastatic
cancers.
[0081] The method of the invention can be used in any biological
system, whether in vitro, ex vivo, or in vivo. In vitro biological
systems typically include cultured cells or tissues, but may
involve isolated or purified organelles or cellular components. For
example, MT-MMP is expressed as a membrane-bound protein, and a
membrane fraction containing the enzyme can be isolated and tested
in vitro. Diagnostic tests, such as to measure an index of MT-MMP
activity, are typically performed in vitro. Ex vivo biological
systems typically include organ systems removed from a living
animal. In vivo uses are limited to biological systems that are
living animals, and such uses typically include therapeutic or
pharmaceutical interventions. Thus, embodiments of the invention in
which a tetracycline compound is administered to a mammal are
representative of in vivo methods.
[0082] The conditions treatable by means of the present invention
occur in mammals. Mammals include, for example, humans, as well as
pet animals such as dogs and cats, laboratory animals such as rats
and mice, and farm animals such as horses and cows.
[0083] The method of the invention is useful to treat medical
conditions characterized by MT-MMP activity. In particular, the
invention is useful in the treatment (e.g., palliation,
amelioration) of medical conditions characterized by excessive or
pathological levels of MT-MMP activity. Such conditions include,
but are not limited to conditions characterized by increased levels
of osteoclast cell activity, e.g., different types of arthritis,
osteoporosis and other conditions characterized by bone resorption.
Various inflammatory conditions are also characterized by excessive
MT-MMP activity, and the method can be used to inhibit MT-MMP
activity in those in mammals subject to or susceptible to such
conditions.
[0084] Excessive MT-MMP activity is also observed in association
with cancers. Accordingly, other conditions susceptible to
treatment according to the invention include cancerous conditions,
e.g., tumors or neoplasms. Neoplastic conditions treatable by the
present invention include all solid tumors, i.e., carcinomas and
sarcomas. Carcinomas include those malignant neoplasms derived from
epithelial cells which tend to infiltrate (invade) the surrounding
tissues and give rise to metastases. Adenocarcinomas are carcinomas
derived from glandular tissue or in which the tumor cells form
recognizable glandular structures. Sarcomas broadly include tumors
whose cells are embedded in a fibrillar or homogeneous substance
like embryonic connective tissue. Sarcomas include, for example,
osteosarcomas and fibrosarcomas.
[0085] The invention is particularly illustrated herein in
reference to treatment of certain types of experimentally defined
cancers. In these illustrative treatments, standard
state-of-the-art in vitro and in vivo models have been used. These
methods can be used to identify agents that can be expected to be
efficacious in in vivo treatment regimens, without regard to
whether the condition to be treated is cancerous. Thus, it will be
understood that the method of the invention is not limited to the
treatment of these tumor types, but extends to any medical
condition affecting any organ system, such as inflammatory
diseases, provided that the condition characteristically is
associated with MT-MMP activity, especially excessive MT-MMP
activity.
[0086] The observed inhibition of MT-MMP activity effect occurs
over a wide range of concentrations, including at concentrations
that are extraordinarily low. The amount of the tetracycline
compound used according to the invention is an amount that is
effectively inhibitory of MT-MMP activity. An amount of a
tetracycline compound is effectively inhibitory to MT-MMP activity
if it significantly reduces MT-MMP activity in a biological
system.
[0087] Preferably, the tetracycline compound is provided in an
amount that has little or no antimicrobial activity. A tetracycline
compound is not effectively antimicrobial if it does not
significantly prevent the growth of microbes. Accordingly, the
method can beneficially employ a tetracycline compound that has
been modified chemically to reduce or eliminate its antimicrobial
properties. The use of such chemically-modified tetracyclines is
preferred in the present invention since they can be used at higher
levels than antimicrobial tetracyclines, while avoiding certain
disadvantages, such as the indiscriminate killing of beneficial
microbes, and the emergence of resistant microbes, which often
accompanies the use of antimicrobial or antibacterial amounts of
such compounds over prolonged periods of time.
[0088] The tetracycline compounds useful according to the method of
the invention appear to exhibit their beneficial effect in a
dose-dependent manner. Thus, within broad limits, administration of
larger quantities of a tetracycline compound has been observed to
inhibit MT-MMP activity to a greater degree than does
administration of a smaller amount. Moreover, efficacy has been
observed at dosages below the level at which toxicity is seen in
normal cells or at the organismal level. Accordingly, one of the
advantages of the invention is that the debilitating side effects
that may be attendant upon conventional therapeutic regimens are
reduced, and preferably avoided.
[0089] The maximal dosage for a subject is the highest dosage that
does not cause undesirable or intolerable side effects. For
example, the tetracycline compound(s) can be administered in an
amount of from about 0.1 mg/kg/day to about 30 mg/kg/day, and
preferably from about 1 mg/kg/day to about 18 mg/kg/day. For the
purpose of the present invention, side effects may include
clinically significant antimicrobial or antibacterial activity, as
well as toxic effects. For example, a dose in excess of about 50
mg/kg/day would likely produce side effects in most mammals,
including humans. In any event, the practitioner is guided by skill
and knowledge in the field, and the present invention includes,
without limitation, dosages that are effective to achieve the
described phenomena.
[0090] The invention can also be practiced by including with the
tetracycline compound one or more other therapeutic agents, such as
any conventional inhibitor of other metalloproteinases. The
combination of the tetracycline compound with such other agents can
potentiate the therapeutic protocol. Numerous therapeutic protocols
will present themselves in the mind of the skilled practitioner as
being capable of incorporation into the method of the invention.
Any chemotherapeutic agent can be used, including alkylating
agents, antimetabolites, hormones, radioisotopes, non-steroidal
anti-inflammatory drugs (NSAIDs), enzyme substrates or inhibitors,
receptor agonists and antagonists, as well as natural products.
[0091] The preferred pharmaceutical composition for use in the
method of the invention includes a combination of the tetracycline
compound in a suitable pharmaceutical carrier (vehicle) or
excipient as understood by practitioners in the art.
[0092] Enteral administration is a preferred route of delivery of
the tetracycline, and compositions including the tetracycline
compound with appropriate diluents, carriers, and the like are
readily formulated. Liquid or solid (e.g., tablets, gelatin
capsules) formulations can be employed. It is among the advantages
of the invention that, in many situations, the tetracycline
compound can be delivered orally, as opposed to parenteral delivery
(e.g., injection, infusion) which is typically required with
conventional chemotherapeutic agents.
[0093] Parenteral use (e.g., intravenous, intramuscular,
subcutaneous injection) is also contemplated, and formulations
using conventional diluents, carriers, etc., such as are known in
the art can be employed to deliver the compound.
[0094] Alternatively, delivery of the tetracycline compound can
include topical application. Compositions deemed to be suited for
such topical use include as gels, salves, lotions, ointments and
the like. In the case of tumors having foci inside the body, e.g.,
brain tumors, the tetracycline compound can be delivered via a
slow-release delivery vehicle, e.g., a polymeric material,
surgically implanted at or near the lesion situs.
[0095] The following examples are provided to assist in a further
understanding of the invention. The particular materials and
conditions employed are intended to be further illustrative of the
invention and are not limiting upon the reasonable scope
thereof.
EXAMPLE 1A
[0096] Preparation of MT1-MMP- or pro-MMP-2-Transfected Cells
[0097] MT.sub.1-MMP- and pro-gelatinase A-transfected COS-1 cells
were prepared in general accordance with the protocol of Cao et al.
(1996). Specifically, COS-1 cells were cultured in DMEM (Life
Technologies) containing 10% fetal bovine serum (Atlanta
Biologicals) and 2 mM glutamine under 5% CO.sub.2/95% air
atmosphere. On the day of transfection with either pro-gelatinase A
or MT.sub.1-MMP, the COS-1 cells were washed with
phosphate-buffered saline, pH 7.4 (PBS), followed by the addition
of DMEM containing 10% NUSERUM.RTM., 300 mg/mL DEAE-dextran, 100 mM
chloroquine, and 1.25 mg/mL DNA. Plasmids containing cDNAs of
MT.sub.1-MMP or pro-gelatinase A were used for transfection.
[0098] The cells were then incubated for 4 hr at 37.degree. C. in a
5% CO.sub.2 atmosphere. Then the cells were washed once with DMEM
and incubated for 2 min in 10% DMSO in Ca.sup.2+/Mg.sup.2+-free PBS
at room temperature, and washed twice with PBS. Finally, the cells
were incubated for 1 day in DMEM containing 10% fetal bovine
serum.
[0099] EXAMPLE 1B
[0100] Preparation of Cell Membrane Fractions of
MT1-MMP-Transfected Cells
[0101] Confluent MT.sub.1-MMP-transfected COS-1 cells
(2.times.10.sup.7 cells) were harvested following treatment with
trypsin-EDTA and washed thoroughly. Cells were suspended in 25 mM
sucrose, 5 mM MgCl.sub.2 in Tris base, pH 7.4, and then subjected
to nitrogen (N.sub.2) cavitation at 1000 p.s.i. for 30 min at
4.degree. C. Whole cells and nuclei were removed by centrifugation
at 770.times.g for 10 min, and the postnuclear supernatant was
centrifuged at 6,000.times.g for 15 min to remove heavy organelles.
The supernatant was centrifuged at 100,000.times.g for 60 min, and
the cell membrane fraction, comprising plasma membranes, Golgi, and
ribosomes, was recovered in the pellet. The 100,000.times.g
supernatant was designated as cytosol.
EXAMPLE 2
[0102] Direct Digestion of Gelatin by MT.sub.1-MMP is Inhibited by
CMT-3
[0103] Radiolabeled (.sup.3H) collagen type I was denatured, to
unravel the triple helix to produce [.sup.3H-methyl]-gelatin. When
separated by SDS-PAGE, and examined by autoradiography, the
denatured collagen yields three bands: .alpha.(.about.100 kDa),
.beta.(.about.200 kDa), and .gamma.(.about.300 kDa), corresponding
to the various components of the collagen in the sample. The
tritiated gelatin substrate was incubated with a membrane fraction
obtained by MT.sub.1-MMP-transfected COS-1 cells as described
above. The gelatinolytic activity was calculated as % lysis of the
.alpha., .beta., and .gamma. intact gelatin components assessed by
laser densitometer scanning of the fluorogram.
[0104] MT.sub.1-MMP exhibited degradative activity using gelatin as
substrate, and CMT-3 at 5, 10, and 20 .mu.M inhibited this
gelatinolytic activity by 16.3%, 35%, and 32% respectively as shown
in FIG. 1:
1 Lane 1 [.sup.3H-methyl] gelatin, .alpha., .beta., and .gamma.
chains were intact; Lane 2 [.sup.3H-methyl] gelatin and
MT.sub.1-MMP incubated for 18 hr at 37.degree. C.; and Lanes 3-5
CMT-3 was added to the incubation mixture of [.sup.3H-methyl]
gelatin and MT.sub.1-MMP at final concentrations of 5 .mu.M (Lane
3), 10 .mu.M (Lane 4), and 20 .mu.M (Lane 5).
[0105] The MT-MMP caused a characteristic loss of each of the 100
kDa, 200 kDa, and 300 kDa bands, with the formation of low
molecular weight (<100 kDa) bands, corresponding to gelatin
digestion products. However, when the incubation was performed in
the presence of CMT-3 (5 .mu.M to 20 .mu.M), a dose-dependent
decrease in the low molecular weight bands was seen with a
corresponding increase in the residual intact gelatin bands. These
data show that CMT-3 inhibits the characteristic ability of MT-MMP
to directly degrade gelatin.
EXAMPLE 3
[0106] Activation of Gelatinasae A by MT1-MMP is Inhibited by
CMT-3
[0107] Recombinant pro-gelatinase A (72 kDa type IV gelatinase) can
be activated to from 62 kDa active gelatinase by MT-MMP-containing
plasma membrane fractions. To examine the ability of a tetracycline
compound to inhibit this gelatinase activating property of MT-MMP,
membrane fractions obtained according to Example 1B were incubated
with recombinant human pro-gelatinase A for 18 h at 37.degree. C.
with or without CMT-3, and examined by zymography to evaluate
enzyme activation.
[0108] Specifically, pro-MMP-2 was obtained from the
culture-conditioned medium from MMP-2-transfected COS-1 cells. The
MT.sub.1 MMP membrane fraction preparation exhibited pro-MMP-2
activating activity by inducing the characteristic molecular weight
shift from 72 kDa (pro-MMP-2) to 62 kDa (active MMP-2) as measured
by gelatin zymography, generally in accordance with the following
conventional protocol:
[0109] Conditioned media were collected from the cultures that had
been treated with CMT-3 for 2 days. Media were then incubated with
SDS-gel electrophoresis sample buffer for 30 min at room
temperature, and then analyzed by gel electrophoresis on
SDS-polyacrylamide gel (10%) containing gelatin (1 mg/mL; Novex,
Inc.). Following electrophoresis, the gels were washed twice with
0.25% TRITON.RTM. X-100 for 30 min each (to renature the
gelatinase), and incubated for 18-24 hr at 37.degree. C. in a
Tris-HCl buffer, pH 7.4 (see, e.g., Lokeshwar 1993). After
incubation, the gels were briefly rinsed in distilled water and
stained with 0.25% Coomassie brilliant blue R250. The location of
gelatinase activity in the gels is visible as a colorless area on
an otherwise uniform blue background, indicative of digested
gelatin. Note that under zymography, both the 72 kDa and the 62 kDa
forms of gelatinase exhibit gelatinolytic activity, thus permitting
one to distinguish these forms on the basis of their differences in
molecular weight.
[0110] The results of this experiment are illustrated in the
zymogram shown as FIG. 2:
2 Lane 1 Pro-gelatinase A Lane 2 Pro-gelatinase A + membrane
fraction control (membrane fraction from COS-1 cells transfected
with vector absent the MT.sub.1-MMP gene); Lanes 3-4 Pro-gelatinase
A + MT.sub.1-MMP (duplicates); Lane 5 Pro-gelatinase A +
MT.sub.1-MMP + 50 .mu.M CMT-3; Lane 6 Pro-gelatinase A +
MT.sub.1-MMP + 20 .mu.M CMT-3; and Lane 7 Pro-gelatinase A +
MT.sub.1-MMP + 10 .mu.M CMT-3.
[0111] This experiment demonstrated that recombinant pro-gelatinase
A can be activated to form 62 kDa active gelatinase by plasma
membrane fractions of MT.sub.1-MMP-transfected cells. Moreover, as
shown in FIG. 2, CMT-3 at final concentrations of 10, 20, and 50
.mu.M inhibited the MT.sub.1-MMP-induced activation of
pro-gelatinase A assessed by gelatin zymography in a dose-dependent
manner. Thus, CMT-3 blocked MT.sub.1-MMP-mediated activation of
pro-gelatinase A.
EXAMPLE 4A
[0112] In FIG. 3A, the proteolytic activity of MT.sub.1-MMP and its
inhibition by a tetracycline compound was assessed using casein
zymography:
3 Lane 1 21 kDa intact .beta.-casein substrate (52 .mu.M); Lane 2
degraded .beta.-casein, by 50 ng of MT.sub.1-MMP, into a smaller
molecular weight fragments; Lanes 3-5 progressive inhibition of
caseinolytic activity by increasing (0.5, 1.5, and 3.0 .mu.M,
respectively) concentrations of CMT-3; and Lanes 6-8 MT.sub.1-MMP
incubated with .beta.-casein substrate with proteinase inhibitors:
0.5 mM PMSF, 2 mM EDTA, and TIMP-2 (1:1 molar ratio),
respectively.
[0113] Consistent with MT.sub.1-MMP activity against casein
reflecting metalloproteinase and not serine proteinase activity,
PMSF did not inhibit caseinolytic activity (Lane 6) whereas EDTA
(Lane 7) and TIMP-2 (Lane 8) did inhibit the caseinolytic
activity.
EXAMPLE 4B
[0114] The proteolytic activity of MT.sub.1-MMP was assessed using
gelatinase A zymography. Pure recombinant pro-MMP-2 (72 kDa
gelatinase or pro-gelatinase A) and MT.sub.1-MMP were co-incubated
in the presence or absence of CMT-3 (0.5-3.0 .mu.M). After
incubation, the enzyme mixture was examined by SDS-PAGE and the
protein (enzyme) bands were stained with Coomassie brilliant blue.
The results are illustrated in FIG. 3B:
4 Lane 1 Pro-gelatinase A is shown as a single band (72 kDa); Lane
2 the conversion of pro-gelatinase A (72 kDa) to a smaller
molecular weight active gelatinase A (62 kDa) mediated by
incubation with 50 ng recombinant MT.sub.1-MMP; Lanes 3-6 the
gelatinase A activated with MT.sub.1-MMP was incubated with 0.5,
1.0, 1.5 and 3.0 .mu.M CMT-3, respectively; and Lane 7 molecular
weight standard.
[0115] FIG. 3B clearly shows progressive inhibition of the
conversion of pro-gelatinase A to active gelatinase A
(approximately 10 kDa lower molecular weight than the precursor) in
a dose-response fashion. Note in particular that 3.0 .mu.M CMT-3
(Lane 6) essentially completely inhibits the formation of the
smaller molecular weight species of gelatinase A by
MT.sub.1-MMP.
EXAMPLE 5
[0116] MG-63 cells, an osteosarcoma cell line, were grown in
culture and treated with 40 .mu.g/mL concanavalin A to induce
MT.sub.1-MMP-dependent activation of pro-MMP-2. Culture-conditioned
medium was collected, as a source of pro-MMP-2, and was incubated
in the presence of the membrane fraction of COS-1 cells transfected
to express MT.sub.1-MMP. The incubated medium showed
gelatin-degrading activity by zymographic analysis, which was
inhibited by 0.5 .mu.M to 3.5 .mu.M CMT-3, in a dose-dependent
manner (FIG. 4A). Moreover, the percent of activated MMP-2 in the
total expressed MMP-2 was decreased in a dose-dependent fashion
(FIG. 4B). Note in FIG. 3B, that at CMT-3 concentrations of 3.0
.mu.M and 3.5 .mu.M, no active MMP-2 was detected. Thus, CMT-3 not
only inhibited expression of gelatinase expressed by the
osteosarcoma cells, it also inhibited activation of the expressed
gelatinase.
EXAMPLE 6A
[0117] Inhibition of Matrigel invasion of HT-1080fibrosarcoma cell
by CMTs.
[0118] HT-1080 cells were allowed to invade through Matrigel
(Collaborative Research, Bedford, Mass.) for 24 hr in medium
containing 10% serum. CMT-3 or CMT-8 were added to the medium to
assess their ability to inhibit invasivity of the cells. CMT-3 was
tested at final concentrations of 1 .mu.M or 10 .mu.M; CMT-8 was
tested twice at a final concentration of 50 .mu.M. The invading
cells were counted, and the relative numbers of cells were
expressed as means of triplicate measurements. The results are
presented in FIG. 5A.
EXAMPLE 6B
[0119] HT-1080 cells were allowed to migrate in the presence of
medium containing 10% serum for 18 hr in Transwell chambers
(Costar, Cambridge, Mass.). The concentrations of CMT-3 and CMT-8
are indicated, and noteworthy a clear inhibition of HT-1080
fibrosarcoma cell migration is observed already at 0.5 .mu.M CMT-3
and CMT-8 concentrations. The results of this study are summarized
in FIG. 5B, showing the relative number of cells having traversed
to the undersurface of Transwell chambers. The data are expressed
as means of triplicate measurements.
[0120] In Examples 6A and 6B, CMT-3 and CMT-8 were found to
suppress the invasion (FIG. 5A) and migration (FIG. 5B) of HT-1080
fibrosarcoma cells starting at 1-10 .mu.M and reaching maximal
inhibition at 50 .mu.M. CMT-3 was slightly more effective in
comparison to CMT-8 in prevention of invasion (FIG. 5A). Effective
inhibition of HT-1080 cell migration was seen at concentrations as
low as 0.5 .mu.M CMT-3 and CMT-8 (FIG. 5B). The concentrations
required to inhibit 50% (IC.sub.50) MT.sub.1-MMP activity and
MT.sub.1-MMP-dependent activation of proMMP-2 as well as the in
vitro invasion and migration of HT-1080 cells were 1-3 .mu.M. CMT-3
and CMT-8 did not block cell surface integrins, did not prevent
initial attachment and spreading of studied cell lines on
extracellular matrix or Matrigel substrate, and was not cytotoxic
to the cells under the conditions used (data not shown).
[0121] Therapeutically attainable CMT concentrations (<2 .mu.M)
directly inhibit the MT.sub.1-MMP activity, the activation of
proMMP-2 by MT.sub.1-MMP and prevent in vitro invasion and
migration of malignant human cells, such as HT-1080 fibrosarcoma
cell line which are capable of expressing both MT.sub.1-MMP and
MMP-2. Thus CMTs can be regarded as drugs with MMP-inhibitory and
anti-invasive properties, and they could be considered not only for
the prevention of bone destruction but also for inhibition of soft
tissue destruction in the treatment of human inflammatory and
malignant diseases.
[0122] Thus, while there have been described what are presently
believed to be the preferred embodiments of the present invention,
those skilled in the art will realize that other and further
embodiments can be made without departing from the spirit of the
invention, and it is intended to include all such further
modifications and changes as come within the true scope of the
claims set forth herein.
Bibliography
[0123] The following publications, having been mentioned in the
foregoing specification, are incorporated herein by reference for
all that they disclose:
[0124] Cao J, Rehemtulla A, Bahou W, and Zucker S, "Membrane type
matrix metalloproteinase 1 activates pro-gelatinase A without furin
cleavage of the N-terminal domain," J Biol Chem 271:30174-30180
(1996).
[0125] Cao J, Lee H M, Bahou W, and Zucker S, "The propeptide
domain of membrane type 1-matrix metalloproteinase (MT1-MMP) is
required for progelatinase A activation and binding of TIMP-2,"
AACR Annual Meeting, Mar. 28-Apr. 1, New Orleans, La. (1998).
[0126] DeClerck Y A, Shimada H, Taylor S M, and Langley K E,
"Matrix metalloproteinases and their inhibitors in tumor
progression," Annals NY Acad Sci 732:222-232 (1994).
[0127] Fridman R, Toth M, Pena D, and Mobashery S, "Activation of
progelatinase B (MMP-9) by gelatinase A (MMP-2)," Cancer Res
55:2548-2555 (1995).
[0128] Golub L M, Ramamurthy N S, McNamara T F, Greenwald R A, and
Rifkin B R, "Tetracyclines inhibit connective tissue breakdown: New
therapeutic implications for an old family of drugs," Crit Rev Oral
Biol Med 2(2):297-322 (1991).
[0129] Golub L M, Sorsa T, and Suomalainen K, Curr Opin Dent
2:80-90 (1992).
[0130] Imai K, Ohuchi E, Aoki T, Nomura H, Fujii Y, Sato H, Seiki
M, and Okada Y, Cancer Res 56:2702-2710 (1996).
[0131] Kroon A M, Dontje B H J, Holtrop M, and van den Bogert C,
"The mitochondrial genetic system as a target for chemotherapy:
tetracyclines as cytostatics," Cancer Letts 25(l):33-40 (1984).
[0132] Lee A Y, Akers K T, Collier M, Li T, Eisen A Z, and Seltzer
J L, "Intracellular activation of gelatinase A (72-kDa type IV
collagenase) by normal fibroblasts," Proc Natl Acad Sci USA
94(9):4424-4429 (1997).
[0133] Lichte A, Kolkenbrock H, and Tschesche H, "The recombinant
catalytic domain of membrane-type matrix metalloproteinase-1
(MT.sub.1-MMP) induces activation of progelatinase A and
progelatinase A complexed with TIMP-2,"FEBS Lett 397:277-287
(1996).
[0134] Lokeshwar B L, Selzer M G, Block N L, and Gunja-Smith Z,
"Secretion of matrix metalloproteinases and the inhibitors (TIMPs)
by human prostate in explant cultures: Reduced tissue inhibitor of
metalloproteinase secretion by malignant tissues," Cancer Res
53:4493-4498 (1993).
[0135] Lokeshwar B L, Selzer M G, Dudak S M, Block N L, and Golub L
M, "Inhibition of tumor growth and metastasis by oral
administration of a non-antimicrobial tetracycline analog (CMT-3)
and doxycycline in a metastatic prostate cancer model," Cancer Res
(submitted for publication).
[0136] Maragoudakis M E, Peristeris P, Missirlis E, Aletras A,
Andriopoulou P, and Haralabopoulos G, Annals NY Acad Sci
732:280-293 (1994).
[0137] Mitscher L A, The Chemistry of the Tetracycline Antibiotics,
Ch. 6, Marcel Dekker, New York (1978).
[0138] Murphy G, Willenbrock F, Ward R V, Cockett M I, Eaton D, and
Docherty A J, Biochem J 283:637-641 (1992).
[0139] Nagase H, "Matrix metalloproteinases," Chapter 7, pp.
153-204, in Zinc Metalloproteinases in Health and Disease, Hooper N
M, ed., Taylor and Francis, London (1996).
[0140] Ohuchi E, Imai K, Fujii Y, Sato H, Seiki M, and Okada Y,
"Membrane type 1 matrix metalloproteinase digests interstitial
collagens and other extracellular matrix macromolecules," J Biol
Chem 272(4):2446-2451 (1997).
[0141] Okada Y, Bellocq J-P, Rouyer N, Chenard M-P, Rio M-C,
Chambon P, and Basset P, "Membrane-type matrix metalloproteinase
(MT-MMP) gene is expressed in stromal cells of human colon, breast,
and head and neck carcinomas," Proc Natl Acad Sci USA 92:2730-2734
(1995).
[0142] Pei D and Weiss S J, Nature 371:244-247 (1995).
[0143] Sato H, Takino T, Okada Y, Cao J, Shinigawa A, Yamamoto E,
and Seiki M, "A matrix metalloproteinase expressed on the surface
of invasive tumour cells," Nature 370:61-65 (1994).
[0144] Sato H, Kinoshita T, Takino T, Nakayama K, and Seiki M,
"Activation of a recombinant membrane type 1-matrix
metalloproteinase (MT 1 -MMP) by furin and its interaction with
tissue inhibitor of metalloproteinases (TIMP)-2,"FEBS Lett
393:101-104 (1996).
[0145] Sato T, del Carmen Ovejero M, Hou P, Heegaard AM, Kumegawa
M, Foged N T, and Delaisse J M, "Identification of the
membrane-type matrix metalloproteinase MT.sub.1-MMP in
osteoclasts," J Cell Science 110:589-596 (1997).
[0146] Seftor R E B, Seftor E A, DeLarco J E, Kleiner D E, Leferson
J, Stetler-Stevenson W G, McNamara T F, Golub L M, and Hendrix M J
C, "Chemically-modified tetracyclines inhibit human melanoma cell
invasion and metastasis," Clin Exp Metastasis 16 (In Press).
[0147] Shofuda K, Yasumitsu H, Nishihashi A, Miki K, and Miyazaki
K, "Expression of three membrane-type matrix metalloproteinases
(MT-MMPs) in rat vascular smooth muscle cells and characterization
of MT3-MMPs with and without transmembrane domain," J Biol Chem
272(15)9749-9754 (1997).
[0148] Strongin A Y, Collier I, Bannikov G, Marmer B L, Grant G A,
and Goldberg G I, "Mechanism of cell surface activation of 72 kDa
Type IV collagenase: Isolation of the activated form of the
membrane metalloproteinase," J Biol Chem 270(10):5331-5338
(1995).
[0149] Takino T, Sato H, Yamamoto E, and Seiki M, "Cloning of a
human gene potentially encoding a novel matrix metalloproteinase
having a C-terminal transmembrane domain," Gene 155:239-298
(1994).
[0150] Uitto V J, Firth J D, Nip L, and Golub L M, Annals NY Acad
Sci 732:140-151 (1994).
[0151] van den Bogert C, Dontje B H J, Holtrop M, Melis T E, Romijn
J C, van Dongen J W, and Kroon A M, "Arrest of the proliferation of
renal and prostate carcinomas of human origin by inhibition of
mitochondrial protein synthesis," Cancer Res 46(7):3283-3289
(1986).
[0152] Yu A E, Hewitt R E, Connor E W, Stetler-Stevenson W G,
"Matrix metalloproteinases, Novel targets for directed cancer
therapy," Clin Pharmacol 11:229-244 (1997).
[0153] Zucker S, Lysick R M, Ramamurthy N S, Golub L M, Wieman J M,
and Wilkie D P, "Diversity of plasma membrane proteinases in mouse
melanoma cells: Inhibition of collagenolytic activity and cytolytic
activity by minocycline," J Natl Cancer Inst 75:517-525 (1985).
[0154] Zucker S, Conner C, DiMassimo B I, Ende B I, Drews M, Seiki
M, and Bahou W F, J Biol Chem 270:23730-23738 (1995).
* * * * *