Note: Descriptions are shown in the official language in which they were submitted.
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USE OF FURIN AND FURIN-LIKE PROTEASE INHIBITORS IN THE
TREATMENT OF INFLAMMATORY OR MATRIX REMODELLING DISEASES
Field of the Invention
The present invention relates to the treatment of
diseases related to inflammatory or matrix remodelling
conditions in mammals.
Background of the Invention
Rheumatoid arthritis (RA) comprises a group of
autoimmune diseases afflicting 1~ of the population and
leading to significant morbidity. There are no known cures
for RA or other inflammatory autoimmune arthropathies.
Arthritis is a multifactorial disease for which single
therapies have so far yielded disappointing results. In
order to address this complex problem, several strategies
are possible such as treating the disease with a
combinatorial approach or by finding a unique biological
target with a large activity spectrum. Rheumatoid
arthritis is characterized by chronic inflammation in
joints and concomitant destruction of cartilage and bones.
In the joint, three aspects are important, including
synovial cell hyperproliferation/activation, inflammation
and matrix destruction. Many of these events are mediated
by bioactive proteins; among them are a number of
cytokines, growth factors and matrix metalloproteinases.
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Several biologically active peptides and proteins
are synthesized initially as larger and inactive precursor
proteins that are endoproteolytically cleaved to generate
the regulatory protein in a mature and biologically active
form. These include hormones, neuropeptides, enzymes,
growth factors, cell-surface receptors and viral envelope
glycoproteins. In many of these precursors,
endoproteolysis takes place after a sequence of two or more
basic residues (K or R). Seven closely related mammalian
subtilisin/kexin-like serine proteinases with this cleaving
specificity have been discovered in recent years
(references 1 and 2). They have been grouped under the
generic name of proprotein convertases (PC) and named
furin, PC1/PC3, PC2, PC4, PACE4, PC5/PC6A, PC5/PC6B and
PC7/PC8. The convertase furin was the first to be
discovered and is considered the prototype of the PC
family.
Furin is localized mainly in trans-Golgi network
(TGN) (references 3 and 4) and can also translocate between
the cell surface and the TGN. Substrate specificity
studies have revealed that furin requires an R-X-X-R motif
for cleavage while the R-X-K/R-R sequence provides an
optimum processing site (reference 5). According to their
tissue distribution, the proprotein convertases can be
classified into distinct subgroups where furin and PC7/PC8
are ubiquitously distributed, PACE4, PC5/PC6A and PC5/PC6B
are expressed to varying degrees in many tissues and
whereas the other convertases PC1, PC2 and PC4 are
restricted to specific tissues such as neural and endocrine
ones (PC1, PC2) and testicular spermatogenic cells (PC4).
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A common structural feature of some polypeptide
growth involved in growth and inflammation is the presence
of basic residues at the proteolytic processing site within
the molecule. This is the case of PDGF A-chain and B-chain
(references 6 and 7) and most members of the extended
family of TGF ~ (reference 8). The PC-like site in most of
these precursors corresponds to the R-X-K/R-R consensus
cleavage of furin and in some case PACE4 and PC5/6 or PC7
(reference 9). Dubois et a1, have shown by in vitro and
in vivo studies that TGF ~1 is efficiently processed by
furin releasing the genuine mature growth factor (reference
10). PDGF is expressed by synoviocytes or macrophages in
rheumatoid arthritis (RA) and are major contributors to
synovial hyperplasia and pannus formation by stimulation of
DNA synthesis in synovial fibroblast (references 11 and
12). PDGF is also able to sustain cartilage degradation
presumably due to its ability to stimulate collagenase
synthesis and neovascularization (references 12-15).
Other factors, such as transforming growth factor
beta (TGF ~), may be stimulatory or inhibitory depending on
the cell type and local concentration. TGF ~ is present in
the synovial fluid and synovial cell cultures in RA. When
applied intraarticularly, this factor has been shown to
cause synovitis through its ability to induce neutrophil
recruitment, synovial hyperplasia and loss of cartilage
proteoglycans (references 16-20). In contrast, TGF B when
administered systemically inhibits acute and chronic
symptoms of streptococcal cell wall-induced arthritis in
rats through its immunosuppressive effects (reference 21).
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Beside these growth factors, metalloproteinases
are other contributors in the pathogenesis of RA. Among
the metalloproteinases, both the TNF a-converting enzymes
TACE (MMP-17), aggrecenase (ADAMTS11, ADAMTS4) MDC-9, and
members of the MT-MMP family were found to be first
synthesized as inactive precursor molecules (references
22-29). Their amino acid sequences at the activation
cleavage site correspond to the consensus furin recognition
sequence. The contribution of furin or furin MDC-9
activation has been recently documented (reference 23).
The removal of the TACE prodomain in a late Golgi
compartment has also been documented (reference 28).
Clarke et al. disclosed that human TACE was processed in
yeast at the RVKRjR site (reference 29). For gelatinase A,
the contribution of furin-activated MT1-MMP in its
activation is also documented (references 30 and 31).
TACE, through the activation of TNF a, plays a
major and well-documented contribution in the chronic
inflammatory aspect of RA. In animal models and clinical
trials, blockage of this cytokine using neutralizing
antibodies has shown impressive anti-inflammatory effect
with dramatic reduction of acute phase protein detection in
the majority of patients and an influence on the erosive
aspect of the pathology (references 32-34). Therefore,
TACE represents an important clinical target for
therapeutic intervention in a variety of inflammatory
diseases including RA.
Recent findings indicate that gelatinase A is
expressed in the normal synovial lining and in its
pathological extension, the pannocytes of the invasive
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pannus tissue (reference 35). This way, gelatinase A may
participate in the remodelling of the normal lining and its
pathological extension, a tissue deeply involved in matrix
destruction in arthritis.
Destruction of the cartilage matrix protein
aggrecan is one of the early hallmarks of arthritis.
Depletion of aggrecan from cartilage compromises the
weight-bearing properties of the tissue and lead to further
mechanical disruption of the cartilage. Recently, a new
metalloprotease responsible for aggrecan cleavage
(aggrecanase) was isolated from bovine cartilage. This
protease is a member of the ADAM (a disintegrin and
metalloproteinase) family that cleaves aggrecan at the
Glu3'3-Ala3'4 bond (reference 24) resulting in the production
of aggrecan fragments indistinguishable from those found in
arthropathies (reference 36). Since then, another ADAM
family member with aggrecanase activity has been cloned and
called aggrecanase 2 or ADAMTS-11 (reference 25).
Protein-based serine protease inhibitors have
been evaluated to block furin activity. The most specific
one is an engineered variant of the endogenous elastase
inhibitor, the serpin al-antitrypsin (al-AT). To engineer
this derivative, Anderson et al. have mutated the natural
reactive site (Ala-Ile-Pro-Met358) of the serpin for an
Arg-Ile-Pro-Air 358 sequence (reference 37). This mutant
named PDX now mimics the minimum consensus sequence
(R-X-X-R) required for furin recognition and has been shown
to be a potent furin inhibitor in vitro and in cells
(references 37 and 38). Recent studies using purified
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enzymes have demonstrated that PDX is a potent inhibitor of
furin that also inhibits PC6 to some extent (references 39
and 40). PDX has also been shown to block furin activity
in an in vitro measles virus model resulting in loss of
syncitia formation (reference 41). Thus, PDX offers an
interesting approach to address the role of furin in
pathological conditions.
United States patent 6,022,855 issued on
February 8, 2000, herein incorporated by reference, is
related to the inhibition of furin convertase with variants
of the serpin al-antitrypsin.
Summary of the Invention
The present invention provides methods, uses and
compositions for treating inflammatory or matrix
remodelling diseases in mammals by inhibiting furin or
furin-like protease activity.
Particularly, there are provided methods, uses
and compositions PDX or a construct, variant, analog,
peptide, peptidomimetic, salt, complex or derivative
thereof for the treatment of inflammatory or matrix
remodelling diseases in mammals.
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Brief Description of the Drawings
Figure 1 depicts the in vitro production of PDX in rat
synoviocytes.
Figures 2A and 2B depict the on-off regulation of PDX and
GFP expression.
Figures 3A and 3B show that PDX blocks the proteolytic
maturation of a furin-specific substrate TGF l31 in rat
synovial cells.
Figure 4A shows that PDX blocks the production of bioactive
TGF l~l.
Figure 4B shows that PDX blocks the production of bioactive
PDGF by rat synovial cells.
Figure 5A depicts the proteolytic conversion of TACE in
LoVo cells.
Figure 5B depicts the inhibition of furin-mediated
processing of endogenous TACE by PDX.
Figure 6 depicts the inhibition of furin-mediated
processing of endogenous gelatinase A (MMP-2) by PDX.
Figure 7 depicts the inhibition of rat synovial cell growth
by PDX.
Figure 8 depicts the anti-inflammatory effect of PDX virus
in a collagen-induced arthritis model.
Figure 9 depicts the transfer vector pAdTRSF-DC-GFP derived
from pAdBM5.
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Description of Preferred Embodiments
In particular, the present invention relates to
the use of PDX or a construct, variant, analog, peptide,
peptidomimetic, salt, complex or derivative thereof for the
preparation of pharmaceutical compositions or for gene
therapy in systemic and intrasynovial uses of PDX related
to the inhibition of furin or furin-like protease activity
in inflammatory and erosive diseases. Furin-like protease
activity includes the activity of proprotein convertases
such as PACE4, PC5/6 or PC7. Erosive diseases are included
under matrix remodelling diseases.
Preferably, the diseases are arthritis
(rheumatoid arthritis, arthrosis), glomerulonephritis,
pulmonary fibrosis, abnormal wound healing, degenerative
cartilage loss following traumatic joint injury,
inflammatory bowel disease, Cheliac diseases and of type 11
mellitus diabetis, atheriosclerosis, psoriasis and other
diseases characterized by furin or furin-like protease
activity. More preferably, the disease is rheumatoid
arthritis.
The present invention also includes the use of
PDX or a construct, variant, analog, peptide,
peptidomimetic, salt, complex or derivative thereof in the
treatment of diseases related to synoviocytes/,
chondrocytes/ and other cell types/hyperplesia including
but not restricted to the articular joint.
Further provided are compounds and compositions
containing furin-related/ or proprotein convertases-
related/cleavage sites inhibitory activities and the use of
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such compounds and compositions as anti-inflammatory, anti-
hyperplesia and metalloprotease inhibitors (including but
not restricted to TACE, gelatinase A, aggrecanase) in
mammals including humans. For example, by inhibiting the
ability of furin or furin-like proteases to act at the
furin-related or proprotein convertases-related cleavage
sites of metalloproteases, PDX or a construct, variant,
analog, peptide, peptidomimetic, salt, complex or
derivative thereof also inhibits the activity of the
metalloproteases. Inhibiting the action of
metalloproteases is therefore useful in the treatment of
diseases in which metalloproteases are implicated.
In one embodiment, pharmaceutical compositions
comprise constructs, variants, analogs, peptides,
peptidomimetics, salts, complexes or derivatives of PDX for
use in the treatment of inflammatory or matrix remodelling
disorders.
In one preferred embodiment, pharmaceutical
compositions are used in gene therapy methods for systemic
or intraarticular delivery.
There is provided, for use in gene therapy,
pharmaceutically acceptable compositions comprising a
therapeutically effective amount of a gene therapy delivery
system encoding PDX or a construct, variant, analog,
peptide, peptidomimetic, salt, complex or derivative
thereof.
Gene therapy involves the introduction of nucleic
acid material into target cells of a patient. The nucleic
acid material introduced generally codes for a therapeutic
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agent that is effective against a specific disease,
particularly a disease that affects the target cells and/or
surrounding tissues.
A number of techniques can be used to introduce
nucleic acid material into the cells. These include:
~ Transfection using vectors such as viruses, particularly
retroviruses, adenoviruses and adeno-associated viruses.
~ Biolisitics using high-speed metal particles coated with
the nucleic acid material to pierce cells thus
introducing the material.
~ Injecting liposomes into the target tissue where the
liposomes encapsulate the nucleic acid material.
~ Using cationic lipids that aggregate with nucleic acid
material and are subsequently taken up by the target
cells.
~ Using peptides that link to the nucleic acid material as
a vector rather than whole viruses.
The use of adenoviruses is particularly preferred method of
gene therapy.
Gene therapy provides an advantage over existing
pharmacological methods since gene therapy enables specific
targeting of a therapeutic agent to a tissue or tissues.
Vectors can be expressed in specific tissues and are thus
useful in facilitating enhanced expression in tissues as
well as in targeting expression with tissue specificity. A
vector encoding a therapeutic product can be introduced
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into a tissue so that the tissue will express the
therapeutic product.
While it is possible that PDX compounds may be
administered as constructs, peptides or raw chemical
entities, it is preferable to administer the active
ingredient as a pharmaceutical formulation.
It will be appreciated that the amount of the
compounds required for use in treatment will vary not only
with the particular compound selected but also with the
route of administration, the nature of the condition being
treated, and the age and condition of the patient, and
ultimately will be at the discretion of the attendant
physician. Optimal administration rates for a given
protocol of administration can be readily ascertained by
those skilled in the art, using conventional dosage
determination tests conducted with regard to the foregoing
guidelines. See as a general guideline, Remington's
Pharmaceutical Science, 16th Edition, Mack (Ed.), 1980.
According to the present invention, a
"therapeutically effective amount" of a pharmaceutical
composition is an amount which is sufficient to achieve the
desired pharmacological effect. Generally, the dosage
required to provide an effective amount of the composition,
and which can be adjusted by one of ordinary skill in the
art, will vary, depending upon the age, health, physical
condition, sex, weight and extent of disease, of the
recipient. Additionally, the dosage may be determined by
the frequency of treatment and the nature and scope of the
desired effect. The desired dose may conveniently be
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presented in a single dose or as divided doses administered
at appropriate intervals, for example as two, three, four
or more sub-doses per day.
The compositions described herein may be
administered as part of a sustained release formulation
(i.e., a formulation such as a capsule or sponge that
effects a slow release of modulating agent following
administration). Such formulations may generally be
prepared using well known technology and administered by,
for example, oral, rectal or subcutaneous implantation, or
by implantation at the desired target site. Sustained-
release formulations may contain a modulating agent
dispersed in a carrier matrix and/or contained within a
reservoir surrounded by a rate controlling membrane.
Carriers for use within such formulations are
bio-compatible, and may also be biodegradable; preferably,
the formulation provides a relatively constant level of
modulating agent release. The amount of modulating agent
contained within a sustained release formulation depends
upon the site of implantation, the rate and expected
duration of release and the nature of the condition to be
treated or prevented.
The pharmaceutical composition may be
administered orally (including sublingually), parenterally
(including intramuscularly, intraarticularly,
sub-cutaneously or intravenously), by inhalation (including
spray), rectally or topically. The formulations may, where
appropriate, be conveniently presented in discrete dosage
units and may be prepared by any of the methods well known
in the art of pharmacy. All methods include the step of
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bringing into association the active compound with liquid
carriers or finely divided solid carriers or both and then,
if necessary, shaping the product into the desired
formulation.
Compositions generally include conventional
excipients; i.e., pharmaceutically acceptable organic or
inorganic carrier substances that do not deleteriously
react with the active compounds. Suitable pharmaceutically
acceptable carriers include, but are not limited to, water,
salt solutions, alcohol, vegetable oils, polyethylene
glycols, gelatin, lactose, amylose, magnesium stearate,
talc, silicic acid, viscous paraffin, perfume oil, fatty
acid monoglycerides and diglycerides, petroethral fatty
acid esters, hydroxymethylcellulose, polyvinylpyrrolidone,
etc. The pharmaceutical preparations can be sterilized and
if desired, mixed with auxilliary agents, e.g., lubricants,
preservatives, stabilizers, wetting agents, emulsifiers,
salts for influencing osmotic pressure, buffers, colorings,
flavoring and/or aromatic substances and the like which do
not deleteriously react with the active compounds.
For parenteral application, particularly suitable
vehicles consist of solutions, preferably oily or aqueous
solutions, as well as suspensions, emulsions, or implants,
including suppositories. Ampoules are convenient unit
dosages.
For enteral application, particularly suitable
are tablets, dragees or capsules having talc and/or a
carbohydrate carrier binder or the like, the carrier
preferably being lactose and/or corn starch and/or potato
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starch. A syrup, elixir or the like can be used wherein a
sweetened vehicle is employed. Sustained release
compositions can be formulated including those wherein the
active component is protected with differentially
degradable coatings, e.g., by microencapsulation, multiple
coatings, etc, or slow-release polymers or other compounds
formulated with or without inherent complementary or
tissue-specific physical intervention capabilities.
Proper fluidity can be maintained, for example,
by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of
dispersion and by the use of surfactants. Non-aqueous
vehicles such as cottonseed oil, sesame oil, olive oil,
soybean oil, corn oil, sunflower oil, or peanut oil and
esters, such as isopropyl myristate, may also be used as
solvent systems for compound compositions. Additionally,
various additives which enhance the stability, availability
(e.g. binding to heparan-sulfate proteoglycans at the
myofiber extracellular matrix), sterility, and isotonicity
of the compositions, including antimicrobial preservatives,
antioxidants, chelating agents, and buffers, can be added.
Prevention of the action of microorganisms can be ensured
by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, and
the like. In many cases, it will be desirable to include
isotonic agents, for example, sugars, sodium chloride, and
the like. Prolonged absorption of the injectable
pharmaceutical form can be brought about by the use of
agents delaying absorption, for example, aluminum
monostearate and gelatin. Retention of bioavailability in
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a tissue may be influenced by co-injection or
co-administration with a stabilizing agent that would
localize the invention as treatment to the fiber sarcoplasm
or to the extracellular matrix as desired. Any vehicle,
diluent, or additive used would have to be compatible with
the compounds.
Sterile injectable solutions can be prepared by
incorporating the active compounds in the required amount
of the appropriate solvent with various of the other
ingredients, as desired.
A pharmacological formulation can be administered
to the patient in an injectable formulation containing any
compatible carrier, such as various vehicle, adjuvants,
additives, vectors, and diluents; or the compounds utilized
in the present invention can be administered parenterally
to the patient in the form of slow-release subcutaneous
implants or targeted delivery systems such as monoclonal
antibodies, vectored delivery, iontophoretic, polymer
matrices, liposomes, and microspheres. Examples of
delivery systems useful in the present invention include
those described in: U.S. Patent Nos. 5,225,182; 5,169,383;
5,167,616; 4,959,217; 4,925,678; 4,487,603 4,486,194;
4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many other
such implants, delivery systems, and modules are well known
to those skilled in the art.
A pharmacological formulation of the compounds
utilized can be administered orally to the patient.
Conventional methods such as administering the compounds in
tablets, suspensions, solutions, emulsions, capsules,
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powders, syrups and the like are usable. Known techniques
which deliver it orally or intravenously and retain the
biological activity are preferred.
In one embodiment, the compounds of the present
invention can be administered initially by intravenous
injection to bring blood levels to a suitable level. The
patient's levels are then maintained by an oral dosage
form, although other forms of administration, dependent
upon the patient's condition and as indicated above, can be
used.
When desired, the above-described formulations
adapted to give sustained release or pro-drugs of the
active ingredient may be employed.
PDX and related-compounds may also be used in
combination with other therapeutically active agents, for
example, cytotoxic, corticosteroid, non-corticosteroid,
immunosuppressive and antiinflammatory drugs and gene
therapy agents.
The combinations referred to above may
conveniently be presented for use in the form of a
pharmaceutical formulation and these pharmaceutical
formulations comprising a combination as defined above
together with a pharmaceutically acceptable carrier
comprise a further aspect of the invention. The individual
components of such combinations may be administered either
sequentially or simultaneously in separate or combined
pharmaceutical formulations.
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The following examples further illustrate the
practice of this invention, but are not intended to be
limiting thereof. It will be appreciated that the
selection of actual amounts of PDX and related-compounds to
be administered to any individual patient (human or animal)
will fall within the discretion of the attending physician
and will be prescribed in a manner commensurate with the
appropriate dosages depending on the stage of the disease
and like factors uniquely within the purview of the
attending physician.
EXAMPLE 1
A method to produce high levels of PDX in mammalian cells
Recombinant adenoviruses, were constructed using
replication-defective adenoviral vectors deleted of
sequences spanning ElA, E1B, and a portion of the E3
region, impairing the ability of the virus to replicate
(reference 42). The gene encoding full length PDX mRNA was
inserted into the multiple cloning site of the transfer
vector pAd-TRSF-DC-GFP (Figure 9) and placed under the
control of a modified CMV promoter containing a
tetracycline (tet)-regulated expression cassette (reference
43) and expressed together with the green fluorescent
protein (GFP) tracer. Recombinant adenoviruses were
constructed by in vitro homologous recombination in
293 cells as described (reference 44). Recombinant viruses
were amplified and purified from cell lysates by cesium
chloride gradient ultracentrifugation followed by desalting
on a Sephadex G-50 column.
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To test the ability of AdTRS-PDX vector to direct
the expression of PDX, rat synovial cells were either
non-infected or infected in hybridoma serum-free medium
(Gibco BRL) at various multiplicity of infection (MOI) with
AdtrSPDX and/or AdtrSGFP in the presence of AdCMVtTA
(encoding the transactivator tTA) used at a MOI of 50. For
each sample, the MOI of AdtrSGFP was adjusted for a final
MOI of 300. Following 48 h infection, cell lysates are
prepared with 1~ NP-40-containing lysis buffer supplemented
with Complete Protease Inhibitor Cocktail (Roche
Diagnostics GmbH, Mannheim, Germany). Fifteen ug per
sample are then resolved into reducing 10~ SDS-PAGE gels.
Separated proteins are then transferred onto nitrocellulose
membranes, blocked for 1 h, and probed overnight with
affinity-purified anti-human al Antitrypsine IgG (1:500)
(ICN BIOMEDICALS, Costa Mesa, CA). Immunoreactive bands
are revealed using the ECL detection system (Amhersham
Canada Limited, Oakville, ON). Upon infection with
adenovirus-encoding PDX, rat synoviocyte cells produce the
55 kDa immunoreactive PDX that increases with the
multiplicity of infection of the PDX adenoviral vector (see
Fig. 1, lanes 3-9). This band has the same migration
pattern as pure recombinant human al-antitrypsin (lane 10).
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EXAMPLE 2
Regulatable production of PDX
The transduction system used in Example 1 was
used to test the ability of AdTRS-PDX to mediate
regulatable transgene expression in 293 cells. The cells
were infected with AdTRS-PDX and/or AdCMVtTA in the
presence or absence of doxycycline (Dox) (1 ug/ml) and 24 h
after infection, cell cultures were assessed for both
A) PDX production by Western blotting as described in
Example 1 and B) GFP fluorescence. For fluorescence
analysis, cells were pelleted, resuspended in PBS, and
fixed with paraformaldehyde (2~ final) for 30 min at 4°C.
GFP emission was evaluated using a FACScan flow cytometer
(BECTON DICKINSON, San Jose, CA) equipped with an argon-ion
laser and configured for analysis of fluoresceine. Results
were analyzed using CellQuest'~' software (BECTON DICKINSON).
Results in Figure 2 indicated that the addition of 1 ug/ml
Dox to the cultures abolished PDX production induced by the
transactivator tTa (lane 1 compared to lane 2) without
affecting basal levels of PDX (lane 4 compared to lane 3).
Parallel results were observed when cells are analyzed for
fluorescence intensity (Figure 2B).
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EXAMPLE 3
Inhibition of furin-mediated processing of human
transforming growth factor (31 by PDX
Synovial cells were infected with AdTRS-PDX
(encoding PDX) and/or AdTRS-TGF(encoding human TGF f~l
precursor) and/or AdTRSFUR (encoding human furin) and/or
control AdTRSGFP. Forty-eight hours after infection,
concentrated cell supernatants were assessed by Western
blotting for A) the production of proteolytic fragments in
immunoblotting using TGF 131-specific anti-LAP antibodies
(R&D Systems, Minneapolis, MN) and B) the production of
bioactive TGF 131 using a TGF f31-specific ELISA assay (R&D
Systems, Minneapolis, MN). Co-infection of cells with
AdTRS-PDX abrogated pro-TGF t~l proteolytic processing
mediated by endogenous cellular enzymes) (Figure 3A, lanes
3-5) while co-expression with the control virus (AdTRSGFP)
did not affect basal level of TGF !3 proteolytic processing
(Figure 3A, lane 2). As a control, co-infection of
synovial cells with AdTRSFUR, encoding furin, resulted in
complete processing of TGF !31 precursor (Figure 3A, lane 6)
which is also inhibited by PDX co-infection (Figure 3A,
lanes 7-10). In parallel, the amounts of active TGF f~l
released in cell culture medium were diminished by the
expression of al-PDX (Figure 3B).
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EXAMPLE 4
PDX inhibits furin-mediated production of the endogenous
mature form of TGF !31
In parallel experiments, applicant tested the
ability of AdTRS-PDX to inhibit the endogenous production
of mature TGF 131 by synovial cells. The cells were
infected as described in Example 1 with the MOI of AdtrSPDX
compensated with the control AdtrSGFP for a final MOI of
300. After 48 h incubation, the supernatants from infected
cells were assessed for their release of TGF (31 as
described in Example 3. Infection of cells with 0 to 250
MOI of AdTRS-PDX reduces the amounts of active TGF !31
released in cell culture (Figure 4A) with 60~ inhibition
observed at a MOI of 250. The results demonstrated in
Examples 3 and 4 indicate that PDX is capable of inhibiting
endoprotease-mediated processing of TGF 131 precursor
in vivo and immediately suggested a method for treating
TGF 13-related pathological conditions by inhibiting the
furin convertase.
EXAMPLE 5
PDX inhibits furin-mediated production of the endogenous
mature form of PDGF
Applicant also tested the ability of AdTRS-PDX to
inhibit the endogenous production of mature PDGF by
synovial cells. Supernatant from infected cells were
assessed for their release of PDGF in supernatants using a
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PDGF-specific ELISA assay (R&D Systems, Minneapolis, MN).
Cells were infected as described in Example 1. Infection
of cells with 0 to 250 MOI of AdTRS-PDX reduces the amounts
of active TGF f31 released in cell culture (Figure 4B) with
100 inhibition observed at a MOI of 150 and 250. For each
sample, the MOI of AdtrSPDX was compensated with the
control AdtrSGFP for a final MOI of 300. The results
demonstrated that PDX is capable of inhibiting
endoprotease-mediated production of the mature form of PDGF
and suggested a novel method for treating PDGF-related
inflammatory/growth conditions by inhibiting the furin
convertase.
EXAMPLE 6
Inhibition of furin-mediated processing of human TACE by
PDX
To assess the involvement of furin in TACE
endoproteolytic processing, applicant first used a furin-
deficient cell line, the LoVo cells. These are human colon
carcinoma cells which have a point mutation in both alleles
of the fur gene leading to production of a defective enzyme
(reference 45). These furin knock-out cells have been
extensively used to study the contribution of furin in a
cellular context (references 10, 38 and 46). LoVo cells
were transfected with wild type furin and two stable
transfectants LoVoFURl and LoVoFUR2 and control
transfectant LoVoNEO have been generated accordingly to
their acquired gentamycin resistance. Cell lysates were
assessed by Western blotting for the production of TACE
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proteolytic fragments in immunoblotting using TACE-specific
antibodies (Chemicon International, CA). As shown in
Figure 5A, LoVoNEO cells expressed about 50~ of processed
TACE as seen by the relative intensity of the mature TACE
and the proTACE immunoreactive bands. Genetic
complementation with furin increases their endogenous
production of the mature form of TACE with complete
processing observed with the LoVo FUR2 clone indicating the
requirement of intact furin for optimal processing of the
TACE precursor molecule.
Applicant also tested the ability of AdTRS-PDX to
inhibit the endogenous production of mature TACE by
synovial cells. Synovial cells were infected with AdTR5PDX
and/or control AdTRSGFP and stimulated or not with mouse
TNF a (20ng/ml). Forty-eight hours after infection, cell
lysates were assessed by Western blotting for the
production of TACE proteolytic fragments in immunoblotting
using TACE-specific antibodies (Chemicon International,
CA). Control synovial cells produce only the mature form
of TACE (Figure 5B, lane 1). Forty-eight hours of
stimulation of synovial cells with TNF a resulted in an
increase in the immunoreactive band corresponding to the
mature form of TACE (lanes 2 to 6). Infection of cells
with AdTRS-PDX resulted in the production of an additional
immunoreactive TACE band corresponding to the TACE
precursor form (proTACE) indicating that PDX can reduce
TACE processing mediated by endogenous furin enzyme. As an
additional control, the addition of 1 ug/ml Dox to the
cultures abolished PDX-mediated block in TACE processing.
23
CA 02312109 2000-06-23
EXAMPLE 7
Inhibition of furin-mediated processing of endogenous
gelatinase A (MMP-2) by PDX
Gelatinolytic activity in the harvested culture
media was detected by gelatin zymography with 10~s (w/v)
acrylamide gel containing gelatin (0.6 mg/ml; Difco) as
described previously (reference 48). When rat synoviocytes
were treated with Con A (10 ug/ml), a spontaneously
produced 72 kDa pro-MMP-2 was converted into a 62 kDa
active MMP-2 with the appearance of a 64 kDa intermediate
form (Figure 6). Infection of cells with AdTRS-PDX
inhibited the augmentation of pro-MMP-2 activation in
Con A-treated rat synoviocytes in a MOI-dependent manner.
These results demonstrated that PDX is capable of
inhibiting endoprotease-mediated processing of gelatinase A
precursor in vivo and suggested a method to inhibit
gelatinase A-mediated conjunctive tissue degradation by
inhibiting the furin convertase.
EXAMPLE 8
Inhibition of synovial cell growth by PDX
Effect of AdTRS-PDX on cell proliferation was
evaluated by growth curve. Rat synovial cells (5 x 104
cells per well) seeded in culture plates were either
non-infected or infected with control virus AdtrSGFP or
AdtrSPDX virus. On days 0, 2, 4, 6, and 8, cells were
recovered by trypsin-EDTA treatment, and their number was
24
CA 02312109 2000-06-23
counted using an hematocytometer under the microscope. The
proliferation rate of AdTRS-PDX-infected cells was
significantly reduced compared with cells infected with
control AdTRSGFP (Figure 7). Therefore, inhibition of
endogenous furin proteases results in reduction of the
proliferation capacity of synovial cells. These results
demonstrated that PDX, through inhibition of endogenous
furin convertase, has novel growth inhibitory properties
and suggested a method for treating growth-related
pathological conditions in which the furin convertase is
involved.
EXAMPLE 9
Inhibition of collagen-induced arthritis (CIA) by PDX
Collagen-induced arthritis (CIA) is induced in
female Lewis rats by i.d. administration of heterologous
(bovine) type II collagen (250 ug) solubilized in O.1M
acetic acid and emulsified in Freund's Complete Adjuvant
(FCA) while control groups receive FCA only (day 0).
Synovitis typically develops 12-15 days postimmunization in
80-90~ of the animals. The severity of the arthritis was
evaluated using an established macroscopic scoring system.
A score of 1 is given for isolated ankle involvement; 2,
for the ankle and the proximal 1/2 of the tarsal joint; 3,
for the ankle and the entire tarsal joint down to the
metatarsal joints, and 4, for the involvement of the entire
paw, inclucing the digits. The sum of the scores for each
paws is calculated as the arthritic index. Therapeutic
CA 02312109 2000-06-23
effects of virus injection were examined by arthritis score
and paw tickness measured using a caliper.
For treatment, stocks of recombinant virus
purified on cesium chloride gradient are used for
intraarticular injection. The induction of CIA and
monitoring of the disease is performed essentially as
described above. Five days after CIA induction, animals
were randomly distributed into two groups and AdTRrSPDX
(3x108 pfu in 12 ul buffer) resuspended in Tris (pH 7.4),
1mM MgCl2 and 10~ (v/v) glycerol was infused into the right
ankle using sterile technique and a medial approach (N=8).
The left ankle was injected with (3x108 pfu) control
AdTRSGFP adenovirus (n=8). The data represented in
Figure 8 indicates that injection of AdtrSPDX results in a
significant (p L 0.05, N=8) reduction of ankle joint
inflammation especially during the acute phase (days 12-20)
of the disease with mean GFP-treated group measure of
7.74 +/- 0.14 mm compared to 6.87 +/- 0.18 mm for the group
injected with control adenovirus ((p L 0.05, N=40).
Parallel results were obtained with the direct measurement
of arthritic index for days 11-20) where applicant observed
a mean group index of 3.0 +/- 0.10 for control adenovirus
compared with 2.25 +/- 0.15 (pL0.05 N=88) for AdtrSPDX
injected ankles. Therefore, PDX, through inhibition of
endogenous furin convertase, has novel in vivo anti-
inflammatory and anti arthritic properties suggesting a
method for treating inflammatory-related pathological
conditions in which the furin convertase is involved.
26
CA 02312109 2000-06-23
EXAMPLE 10
Involvement of the convertase furin in aqqrecanase
naturation/activation
To define the role of furin in the activation
aggrecanase-1, aggrecanase-1 cDNAs are cloned in an
appropriate mammalian expression vector. The generated
recombinant proteins are C-terminal His or V5 tagged for
convenient detection of the precursor and C-terminal mature
forms in Western blotting. Parental and FUR-1 and FUR-2
LoVo cells as described in Example 6 are transfected with
the aggrecanase-1 expressing vector.
The influence of furin expression in
metalloproteinase fragmentation is monitored. LoVo cell
lysates are analyzed for production of aggrecanase-1-
related digestion products by Western blot using anti-His
or V5 antibodies. Using this system, a ~ 77 kDa
immunoreactive bands represents the unprocessed recombinant
aggrecanase whereas the immunoreactive processed forms are
revealed at ~64 kDa.
To ensure that furin conversion results in the
enzymatically active form of the metalloproteinases,
results from Western blots are corroborated with the
measure of aggrecanase enzymatic activity. For
aggrecanase-1 activity, concentrated medium is incubated in
the presence of purified aggrecan substrate and the
generated aggrecan fragments evaluated in Western blots
using the anti-NITEGE antisera (BC-3 antibody). This
antisera reveals a neoepitope generated by cleavage of
27
CA 02312109 2000-06-23
aggrecan between Glu3'3 and Ala3'4 sequence (references 48
and 49). In this way, BC-3 immunoreactive bands are
detected ranging in molecular mass from approximately 250
to 40 kDa. As a control, there is used the BC-14 antibody
that reacts with the neoepitope generated by cleavage
between the aggrecanase unrelated site Asn3ai and Phe3az.
28
___T__
CA 02312109 2000-06-23
References
1. Van de Ven, W.J., Roebroek, A.J., and Van
Duijnhoven, H.L. 1993. Structure and function of
eukaryotic proprotein processing enzymes of the subtilisin
family of serine proteases. J. Crit. Rev. Onc. 4:115-
136.
2. Seidah, N.G., Hamelin, J., Mamarbachi, M., Dong,
W., Tardos, H., Mbikay, M., Chretien, M., and Day, R.
1996. cDNA structure, tissue distribution, and
chromosomal localization of rat PC7, a novel mammalian
proprotein convertase closest to yeast kexin-like
proteinases. Proc. Natl. Acad. Sci. USA. 93:3388-3393.
3. Molloy, S.S., Thomas, L., VanSlyke, J.K.,
Stenberg, P.E., and Thomas, G. 1994. Intracellular
trafficking and activation of the furin proprotein
convertase: localization to the TGN and recycling from the
cell surface. EMBO J. 13:18-33.
4. Molloy, S.S., Anderson, E.D., Jean, F., and
Thomas, G. 1999. Bi-cycling the furin pathway: from TGN
localization to pathogen activation and embryogenesis.
Trends Cell. Biol. 9:28-35.
5. Takahashi, S., Hatsuzawa, K., Watanabe, T.,
Murakami, K., and Nakayama, K. 1994. Sequence requirements
for endoproteolytic processing of precursor proteins by
furin: transfection and in vitro experiments. J. Biochem.
(Tokyo) 116:47-52.
29
CA 02312109 2000-06-23
6. Betsholtz C, Johnsson A, Heldin CH, Westermark
B, Lind P, Urdea MS,Eddy R, Shows TB, Philpott K, Mellor AL
and et a1.1986. cDNA sequence and chromosomal localization
of human platelet-derived growth factor A-chain and its
expression in tumour cell lines Nature 320 (6064), 695-699.
7. Collins T, Ginsburg D, Boss JM, Orkin SH, Pober
JS 1985. Cultured human endothelial cells express platelet-
derived growth factor B chain: cDNA cloning and structural
analysis. Nature 316(6030):748-50.
8. Kingsle D.M. 1994.The TGF-beta superfamily: new
members, new receptors, and new genetic tests of function
in different organisms. Genes Dev.8(2):133-46.
9. Bergeron F, Leduc R, Day R. 2000. Subtilase-like
pro-protein convertases: from molecular specificity to
therapeutic applications. J Mol Endocrinol. 2000, 24(1):1-
22.
10. Dubois, C.M., Laprise, M.H., Blanchette, F.,
Gentry, L.E. and Leduc, R. 1995. Processing of
transforming growth factor al precursor by human furin
convertase. J. Biol. Chem. 270:10618-24.
11. Keyszer, G.M., Heer, A.H., and Gay, S. 1994.
Cytokines and oncogenes in cellular interactions of
rheumatoid arthritis. Stem Cells. 12:75-86.
12. Remmers, E.F., Sano, H., and Wilder, R.L. 1991.
Platelet-derived growth factors and heparin-binding
(fibroblast) growth factors in the synovial tissue
CA 02312109 2000-06-23
pathology of rheumatoid arthritis. Sem. Arthritis Rheum.
21:191-199.
13. Butler, D.M., Leizer, T., and Hamilton, J.A.
1989. Stimulation of human synovial fibroblast DNA
synthesis by platelet-derived growth factor and fibroblast
growth factor. Differences to the activation by IL-1. J.
Immunol. 142:3098-103.
14. Smith, R.J., Justen, J.M., Sam, LM., Rohloff,
N.A., Ruppel, P.L., Brunden, M.N., and Chin, J.E. 1991.
Platelet-derived growth factor potentiates cellular
responses of articular chondrocytes to interleukin-1.
Arthritis Rheum. 34:697-706.
15. Wilder, R.L., Case, J.P., Crofford, L.J.,
Kumkumian, G.K., Lafyatis, R., Remmers, E.F., Sano, H.,
Sternberg, EM., and Yocum, D.E. 1991. Endothelial cells
and the pathogenesis of rheumatoid arthritis in humans and
streptococcal cell wall arthritis in Lewis rats. J. Cell.
Biochem. 45:162-166.
16. Reibman ,J., Meixler, S., Lee, T.C., Gold, L.I.,
Cronstein, B.N., Haines, K.A., Kolasinski, S.L., and
Weissmann, G. 1991. Transforming growth factor al, a
potent chemoattractant for human neutrophils, bypasses
classic signal-transduction pathways. Proc. Natl. Acad.
Sci. USA 88:6805-6809.
17. Brandes, M.E., Mai, U.E., Ohura, K., and Wahl,
S.M. 1991. Type I transforming growth factor-R receptors
on neutrophils mediate chemotaxis to transforming growth
factor-beta. J. Immunol. 147:1600-1606.
31
CA 02312109 2000-06-23
18. Wahl, S.M., Hunt, D.A., Wakefield, L.M.,
McCartney-Francis, N., Wahl, L.M., Roberts, A.B. and Sporn,
M.B. 1987. Transforming growth factor type a induces
monocyte chemotaxis and growth factor production. Proc.
Natl. Acad. Sci. USA 84:5788-5792.
19. McCartney,-Francis, N., Mizel, D., Wong, H.,
Wahl, L., and Wahl, S. 1990. TGF-a regulates production
of growth factors and TGF-a by human peripheral blood
monocytes. Growth Factors. 4:27-35.
20. Wahl SM. Allen JB. Costa GL. Wong HL. Dasch
JR.1993. Reversal of acute and chronic synovial
inflammation by anti-transforming growth factor ~. J. Exp.
Med. 177:225-30.
21. Brandes, M.E., Allen, J.B., Ogawa, Y., and Wahl,
S.M. 1991. Transforming growth factor ~1 suppresses acute
and chronic arthritis in experimental animals. J. Clin.
Invest. 87:1108-1113.
22. Black, R.A. et al. 1997. A metalloproteinase
disintegrin that releases tumour-necrosis factor-a from
cells. Nature 385:729-731.
23. Roghani, M., Becherer, J.D., Moss, M.L.,
Atherton, R.E., Erdjument-Bromage, H., Arribas, J.,
Blackburn, R.K., Weskamp, G., Tempst, P., and Blobel, C.P.
1999. Metalloprotease-disintegrin MDC9 . intracellular
maturation and catalytic activity. J. Biol. Chem.
274:3531-3540.
32
CA 02312109 2000-06-23
24. Tortorella MD, Burn TC, Pratta MA, Abbaszade I,
Hollis JM, Liu R, Rosenfeld SA, Copeland RA, Decicco CP,
Wynn R, Rockwell A, Yang F, Duke JL,Solomon K, George H,
Bruckner R, Nagase H, Itoh Y, Ellis DM, Ross H, Wiswall BH,
Murphy K, Hillman MC Jr, Hollis GF, Arner EC, et al. 1999.
Protein, Nucleotide Purification and cloning of
aggrecanase-1: a member of the ADAMTS family of proteins.
Science. 284(5420):1664-1666.
25. Abbaszade I., Liu R.Q., Yang F., Rosenfeld S.A.,
Ross O.H., Link J.R., Ellis D.M., Tortorella M.D., Pratta
M.A., Hollis J.M., Wynn R., Duke J.L., George H.J., Hillman
M.C. Jr, Murphy K., Wiswall B.H., Copeland R.A., Decicco
C.P., Bruckner R., Nagase H., Itoh Y., Newton R.C., Magolda
R.L., Trzaskos J.M., Burn T.C., et al. Cloning and
characterization of ADAMTS11, an aggrecanase from the
ADAMTS family. J Biol Chem. 13;274(33):23443-23450
26. Massova I., Kotra L.P., Fridman R., Mobashery S.
1998. Matrix metalloproteinases: structures, evolution, and
diversification. FASEB J. 12(12):1075-1095.
27. Pei D. Identification and characterization of the
fifth membrane-type matrix metalloproteinase MT5-MMP. 1999.
J Biol Chem. 26;274(13):8925-8932.
28. Schlondorff J, Becherer JD, Blobel CP. 2000.
Intracellular maturation and localization of the tumour
necrosis factor alpha convertase (TACE). Biochem J. 2000
347:131-138.
29. Clarke HR, Wolfson MF, Rauch CT, Castner BJ,
Huang CP, Gerhart MJ, Johnson RS, Cerretti DP, Paxton RJ,
33
CA 02312109 2000-06-23
Price VL, Black RA. 1998. Expression and purification of
correctly processed, active human TACE catalytic domain in
Saccharomyces cerevisiae. Protein Expr Purif. 13(1):104-10.
30. Maquoi E, Noel A, Frankenne F, Angliker H,
Murphy G, Foidart JM. 1998. Inhibition of matrix
metalloproteinase 2 maturation and HT1080 invasiveness by a
synthetic furin inhibitor. FEBS Lett. 13;424(3):262-266.
31. Sato T, Kondo T, Fujisawa T, Seiki M, Ito A.
1999. Furin-independent pathway of membrane type 1-matrix
metalloproteinase activation in rabbit dermal fibroblasts.
J Biol Chem. 274(52):37280-37284.
32. Paleolog, E. 1997. Target effector role of
vascular endothelium in the inflammatory response:
insights from the clinical trial of anti-TNF alpha antibody
in rheumatoid arthritis. Mol. Pathol. 50:225-233.
33. Moreland, L.W., Baumgartner, S.W., Schiff, M.H.,
Tindall, E.A., Fleischmann, R.M., Weaver, A.L., Ettlinger,
R.E., Cohen, S., Koopman, W.J., Mohler, D., Widmer, M.B.,
and Blosch, C.M. 1997. Treatment of rheumatoid arthritis
with a recombinant human tumor necrosis factor receptor
(p75)-Fc fusion protein. N. Engl. J. Med. 337:141-147.
34. Ohshima, S. Saeki, Y. Mima, T., Sasai, M.,
Nishioka, K., Ishida, H., Shimizu, M., Suemura, M.,
McCloskey, R., and Kishimoto, T. 1999. Long-term follow-
up of the changes in circulating cytokines, soluble
cytokine receptors, and white blood cell subset counts in
patients with rheumatoid arthritis (RA) after monoclonal
34
CA 02312109 2000-06-23
anti-TNF a antibody therapy. J. Clin. Immunol. 19:305-
313.
35. Konttinen Y.T., Ceponis A., Takagi M., Ainola M.,
Sorsa T., Sutinen M., Salo T., Ma J., Santavirta S., Seiki
M. 1998. New collagenolytic enzymes/cascade identified at
the pannus-hard tissue junction in rheumatoid arthritis:
destruction from above. Matrix Biol. 17(8-9):585-601.
36. Little, C.B., Flannery, C.R., Hughes, C.E., Mort,
J.S., Roughley, P.J., Dent. C., and Caterson, B. 1999.
Aggrecanase versus matrix metalloproteinases in the
catabolism of the interglobular domain of aggrecan in
vitro. Biochem. J. 344:61-68.
37. Anderson E.D., Thomas L., Hayflick J.S., Thomas
G. 1993. Inhibition of HIV-1 gp160-dependent membrane
fusion by a furin-directed al-antitrypsin variant. J. Biol.
Chem. 268(33):24887-91.
38. Pei D. Weiss S.J. 1995. Furin-dependent
intracellular activation of the human stromelysin-3
zymogen. Nature. 375(6528):244-7.
39. Jean, F. , Stella, K. , Thomas, L. , Liu, G. , Xiang,
Y., Reason, A.J., and Thomas, G. 1998. al-Antitrypsin
Portland, a bioengineered serpin highly selective for
furin: application as an antipathogenic agent. Proc. Natl.
Acad. Sci. USA 95:7293-7298.
40. Cui, Y., Jean, F., Thomas, G., and Chrisian, J.L.
1998. BMP-4 is proteolytically activated by furin and/or
CA 02312109 2000-06-23
PC6 during vertebrate embryonic development. EMBO J.
17:4735-4743.
41. Watanabe M., Hirano A., Stenglein S., Nelson J.,
Thomas G., Wong T.C. 1995. Engineered serine protease
inhibitor prevents furin-catalyzed activation of the fusion
glycoprotein and production of infectious measles virus. J.
Virol. 69(5):3206-10.
42. Hurwitz, D.R., and Chinnadurai, G. 1985.
Evidence that a second tumor antigen coded by adenovirus
early gene region Ela is required for efficient cell
transformation. Proc. Natl. Acad. Sci. USA. 82:163-167.
43. Manfred, G., and Bujard, H. 1992. Tight control
of gene expression in mammalian cells by tetracycline-
responsive promoters. Proc. Natl. Acad. Sci. USA 89:5547-
5551.
44 . Petrof, B. J. , Acsadi, G. , Jani, A. , Massie, B. ,
Bourdon " J., Matusiewicz, N., Yang, L. Lochmuller, H., and
Karpati, G. 1995. Efficiency and functional consequences
of adenovirus-mediated in vivo gene transfer to normal and
dystrophic (mdx) mouse diaphragm. Am. J. Resp. Cell & Mol.
Biol. 13:508-517.
45. Takahashi, S., Nakagawa, T., Kasai, K., Banno,
T., Duguay, S.J., Van de Ven, W.J., Murakami, K., and
Nakayama, K. 1995. A second mutant allele of furin in the
processing-incompetent cell line, LoVo. Evidence for
involvement of the homo B domain in autocatalytic
activation. J. Biol. Chem. 270:26565-26569.
36
CA 02312109 2000-06-23
46. Hallenberger, S., Bosch, V., Angliker, H., Shaw,
E., Klenk, H.D., and Garten, W. 1992. Inhibition of furin-
mediated cleavage activation of HIV-1 glycoprotein gp160.
Nature. 360:358-361.
47. Sato T, Ito A, Ogata Y, Nagase H, Mori Y. 1996.
Tumor necrosis factor alpha (TNFalpha) induces pro-matrix
metalloproteinase 9 production in human uterine cervical
fibroblast but interleukin lalpha antagonizes the inductive
effect of TNFalpha. FEBS Letter, 26;392(2):175-178
48. Lark, M.W., et al. 1995. Quantification of a
matrix metalloproteinase-geneated aggrecan G1 fragment
using monospecific and anti-peptide serum. Biochem J.
307:245-252.
49. Lark, M.W. et al. 1995. Cell-mediated catabolism
of aggrecan. Evidence that cleavage at the aggrecanase
site (G1u373-A1a374) is a primary event in proteolysis of
the interglobular domain. J. Biol. Chem. 270:2550-2556.
The disclosures of all the above-mentioned references are
herein incorporated by reference.
37
