(1) slow down joint damage, which include Non-Steroidal

(1) Question or problem to be addressed –


”Cure Of Rheumatoid Arthritis”. There is no cure for
rheumatoid arthritis. Complicated prescription of various combinations of
medications that when together taken can reduce inflammation & pain &
slow down joint damage, which include Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), steroids
& standard Disease
Modifying Anti-Rheumatic Drugs(DMARDs) or biologic
DMARDs. In severe cases, surgical procedures like joint
replacement(arthroplasty), arthradisis(joint is removed & bones are fused
together with bone graft), synavectomy(synovial membrane the surrounding joint
is removed), & in some cases, replacement of arthritic  joint by artificial joint have to be done.
(68 words)

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(2) Background to the problem –


Bones provide support for body & aid in its
movement. Place where two or more bones meet is called a joint. Joints may be
immoveable(skull), slightly moveable(sternum & ribs) or freely
moveable(knee joint). Synovial membrane surrounds moveable joints. Inside
membrane, synovial fluid lubricates & nourishes joint tissue such as
cartilage. Articular cartilage is a tough slippery covering on ends of bones,
which allows smooth joint movement.

Features of joints: Joints give body flexibility, precision
of movement & help in supporting body’s weight.


Basically, ”arthritis” is any disorder that affects
joints. It can cause pain & inflammation. ”Rheumatoid arthritis” is
second most common type of arthritis- joints most commonly affected in this case
are in wrists, hands, knees, ankles & feet. It typically occurs at same
joints on both sides of body. It can also affect other organs in body such as
eyes, skin, heart, lungs, kidneys, nervous system & digestive tract.
Rheumatoid arthritis is an autoimmune disorder this means body attacks itself
by mistake. In rheumatoid arthritis, immune system attacks joint & organ


arthritis is a chronic, auto-immune disease which is an example of a type-III
hypersensitivity. Mediated by immune complex(antigen-antibody complex), in
which, mainly IgG is involved. This disease has a potential to cause
substantial joint damage & disability. Rheumatoid arthritis affects almost 13% of the world
population and is associated with rapid functional loss and reduced life


The major problem is that an ”unknown” synovial
joint antigen present in synovial joint membrane causes host immune system to
trigger immune response against the unknown antigen in synovial joint membrane
which leads to activation of white blood cells primarily macrophages, &
monocytes, which move into joint & release a pro-inflammatory chemical
called TNF-alpha(a cytokine) primarily, which mediates both inflammatory
synovitis & articular matrix degradation.


induces production of other pro-inflammatory cytokines, stimulates endothelial
cells to express adhesion molecules that attract leukocytes into affected
joints, increases rate of synthesis of metallo-proteinases by synovial
macrophages, fibroblasts, osteoclasts, & chondrocytes & inhibits
synthesis of proteoglycans in cartilage, & also attack cells of synovial
membrane. These chemicals cause synovial cells to release other destructive
substances like pro-matrix metallo-proteinase 1(pro-MMP-1) pro-MMP-2, &
pro-MMP-3, in which pro-MMP-1 & pro-MMP-3 are possible markers of cartilage
destruction that are cleaved to produce MMP-1(fibroblast or tissue collagenase)
& MMP-3(stromelysin1), the active MMPs. MMP-3 in turn, participates, in
activation of MMP-1. High serum levels of pro-MMP-3 are associated with greater
articular damage & presence of shared epitope in rheumatoid arthritis


They also cause synovial membrane to grow new blood
vessels & form a thickened area called a ”panus”. Overtime, as panus
grows it invades & destroys areas of cartilage & bone inside joint due
to which inflammation is caused, causing fluid build-up in joint, making joint
swell. Eventually, without treatment joint space narrows & ”ankleosis”
can occur. Ankleosis is fusion or growing together of bones in joint, resulting
in loss of ability to move joint, pain & inflammation.


Pannus formation, articular cartilage destruction, and massive
destruction of fibrous tissue are observed in the advanced stages of disease(RA).


 Endothelial cells (EC) are among
the principal physiological targets of pro-inflammatory cytokines like TNF,
which uses receptors and associated adapter proteins to trigger multiple
signaling pathways leading to activation of JNK and NF-KB as well as other
pathways. Activation of JNK is also required for TNF-induced gene expression of
pro-angiogenic molecules including VEGF, bFGF, IL-8, and MCP.


(3) Experiment to address the problem –


experiments & trials have been done, on blockage & treatment of
Rheumatoid Arthritis by many institutions under the criteria given by the
American College Of Rheumatology(ACR).


(a) The ARMADA
Trial – One of them was the Anti-Tumor Necrosis Factor Research Study Program
of the Monoclonal Antibody Adalimumab in Rheumatoid Arthritis (ARMADA) trial
was supported by, Abbott laboratories & Knoll pharmaceuticals.


This study was a 24-week,
randomized, double-blind, placebo-controlled trial of adalimumab with concomitant
MTX therapy and was performed at 35 sites throughout the US and Canada. ARMADA was a 24 week study conducted to
study efficiacy & safety of adalimumab administered subcutaneously every other week to patients with active RA(Rheumatoid
Arthritis) despite long-term therapy with MTX(Methotrexate).


”Adalimumab” is the first
fully human(100% human germline peptide sequences) anti-tumor necrosis factor
alpha, therapeutic monoclonal antibody for treatment of Rheumatoid Arthritis in
patients taking concomitant Methotrexate. Adalimumab was safe & well tolerated.
Adalimumab is genetically engineered through phage display technology, & is
indistinguishable in structure & function from natural human IgG1. Adalimumab
has high specificity and affinity for TNF(tumor necrosis factor) alpha and not
TNF beta (lymphotoxin). Adalimumab has a terminal half-life comparable to that
of human IgG1 (approx.2 weeks).


This trial was conducted to
test whether administration of adalimumab to patients who were not responding adequately
to MTX would achieve additional benefit and whether the adalimumab plus MTX
combination would be safe and well tolerated.


(b) TEMPO – TEMPO (Trial of
Etanercept and Methotrexate with Radiographic Patient Outcomes) under the given
definition of ACR(American College of Rheumatology),was a randomised,
double-blind, parallel-group study with injectable and oral drugs, consisted of
three treatment drugs: etanercept only (25 mg twice a week subcutaneously and
oral placebo once a week), methotrexate only (7·5 mg escalated to 20 mg oral
capsules once a week within 8 weeks if patients had any painful or swollen
joints), and placebo subcutaneous week.


One of the most challenging
results of the TEMPO trial was that the combination of etanercept and
methotrexate resulted in significant lowering of signs & symptoms of
Rheumatoid Arthritis.

Combination treatment(etanercept
plus methotrexate) was more efficacious than methotrexate or etanercept alone
for control of rheumatoid arthritis disease activity.


(c)  The pioneering work conducted at
SmithKline Beecham and elucidation of the roles of p38 with potent and
selective inhibitors such as SB-203580, many pharmaceutical companies have
embarked upon p38 synthetic programs, as indicated by the ever-increasing
number of patents in this domain.


(d)  At Aventis, a rapid parallel
synthesis project led to the identification of RPR-200765A, a potent and
selective p38 inhibitor of the lipopolysaccharide-induced release of TNF-alpha
in vitro in mononuclear phagocytes and in vivo in the rat. It also reduces
disease incidence and progression in the rat streptococcal cell wall arthritis
model when administered orally in a therapeutic dosing regimen.


(e) Endostatin Gene Transfer Experiment –
In this experiment, effect of potent anti-angiogenic factor endostatin on pro-angiogenic
TNF alpha induced inflammatory arthritis was evaluated. In this expt. an
endostatin expressing lentiviral vector(having low immunogenicity & high
transduction efficiency) was injected directly into the joints of human
TNF-transgenic mice before onset of disease(RA). Histological analysis of the injected
joints 8 weeks later revealed that endostatin reduced blood vessel density
within the synovial tissues and an overall mean arthritis index. In vitro and In vivo examination of the
potential mechanism by
which endostatin inhibited the arthritis revealed that endostatin blocks TNF-induced activation of JNK
and JNK-dependent pro-angiogenic gene expression, which demonstrates a  novel mechanism by which endostatin inhibits
angiogenesis, and demonstrates the
potential utility of anti-angiogenic gene therapy for treatment of
inflammatory arthritis.


Endostatin is a 22-kDa carboxy-terminal fragment of collagen XVIII that
is present in circulation of normal and tumor-bearing individuals. It is a specific
inhibitor of endothelial cell proliferation, migration, and angiogenesis. It
has been shown that endostatin can inhibit

systemic angiogenesis and tumor growth in mouse models by inhibiting TNF
signalling in arthritic joints.


In this expt. VEGF was found
to be a potent angiogenic factor in RA, and an imbalance in production between
VEGF and endostatin has been found in RA patients. Data of this expt. had shown
that endostatin inhibited VEGF expression in arthritic joints, suggesting that
endostatin inhibits joint angiogenesis and arthritis progression in part by
suppressing VEGF production.


In this expt. it has been
established in RA patients that TNF triggers inflammatory and angiogenic
cascades, & the angiogenic activity of TNF is likely mediated by its
multiple actions on endothelial cells(EC). JNK activation and JNK-dependent gene
expression have been implicated in progression of RA. In this expt., it has
been highlighted that TNF-induced JNK activation and JNK-dependent gene expression
of angiogenic molecules were specifically inhibited by endostatin, highlighting
the importance of JNK activation in RA progression.


Data of this expt. showed a
dominant role of EC derived JNK dependent pro-angiogenic molecules in arthritis
& that, effect of endostatin in inhibition of RA is in large part due to
its anti-angiogenesis activity.


This experiment demonstrated
that anti-angiogenesis therapy provides an efficient approach to treat RA.


(4) Expected
outcomes and alternative strategy if necessary –


Other treatment approaches
have been effective in RA patients who are partially responsive to MTX, including
combinations of MTX with traditional DMARDs (cyclosporine, sulfasalazine, hydroxychloroquine,
or leflunomide) or biologic DMARDs (infliximab or etanercept). Introduction of
adalimumab, a biologic DMARD and the first fully human anti-TNF alpha monoclonal
antibody, increases the range of possible therapeutic combinations.


This clinical trial demonstrated
that adalimumab (20 mg, 40 mg, or 80 mg subcutaneously every other week) with
concomitant MTX therapy is more effective than MTX plus placebo at reducing the
signs and symptoms of RA. All 3 regimens of adalimumab plus MTX were well


Despite all these results
& outcomes, the complete termination & treatment of Rheumatoid
arthritis has not yet been developed. Following are some alternative strategies
of termination of RA :-


(a)In my view, the
alternative genetic level strategy, for termination of Rheumatoid Arthritis
will be the inhibition of pannus(thickened area formed by excessive
angiogenesis of synovial membrane) formation, by inhibiting & blocking the
excessive angiogenesis(growth of new blood vessels) & angiogenesis of
synovial membrane by using ”anti-angiogenic gene therapy”. This strategy, can
inhibit the growth & angiogenesis of pannus & terminate the destruction
of areas of cartilage & bone inside the joint. As a result, no
inflammation, no fluid build-up, & no swelling will occur inside the joint.
This will definitely, prevent & inhibit the narrowing of space of joint
& therefore the fusion of bones or growing together of bones in joint will
not occur( prevention of anklyosis). Therefore, no inflammation, no pain &
no loss in ability of movement of joint will happen.


Research over the past
decade has demonstrated the involvement of angiogenesis in the pathogenesis of
RA, which is the most prevalent example of inflammatory angiogenic disease in



Endostatin is a 22-kDa carboxy-terminal fragment of collagen XVIII that
is present in circulation of normal and tumor-bearing individuals. It is a specific
inhibitor of endothelial cell proliferation, migration, angiogenesis & JNK
activation in endothelial cells of joint synovial membrane. Because, JNK
activation is important for TNF-induced expression of pro-angiogenic genes such
as VEGF, bFGF, IL-8, MCP-1, and MMPs in the endothelial cells of joint synovial


Because, angiogenesis
is a key component of inflammatory diseases such as rheumatoid arthritis (RA).Endostatin,
is a  Endostatin
expression inhibited joint angiogenesis and prevented joint destruction.
Endostatin inhibits TNF-induced JNK activation in endothelial cells(EC). Endostatin
inhibits expression of JNK-dependent pro-angiogenic genes in endothelial cells
by TNF-alpha, because proinflammatory cytokines
such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta
(IL-1beta) are implicated in the disease onset and in the progression of bone
and joint destruction that characterizes chronic RA.


Endostatin inhibits JNK activity and JNK-dependent expression of
pro-angiogenic genes in RA. Endostatin significantly increased mRNA expressions
of thrombospondin-1 (TSP-1, by twofold), a potent anti-angiogenic factor which induces
anti-angiogenesis by activating caspase 3 via a CD36 receptor and Src
kinase-dependent pathway.
Endostatin inhibits TNF siganlling in arthritic joints.Endostatin regulates the
balance of angiogenic and anti-angiogenic factors & because joint
neovascularization has an important role in the progression of arthritic
disease, anti-angiogenic gene therapy is an effective termination method of RA.


(b) p38 is an intracellular
mitogen/stress-activated protein kinase (MAPK/SAPK) that regulates both the
release and the actions of TNF-alpha and IL-1beta. Inhibition of p38 kinase is
thus an important potential target for anti-Rheumatoid Arthritis drugs.


(c) Another last but not
the least alternative strategy, will be construction of  fully human(100% human germline peptide
sequences) anti-pro-matrix
metallo-proteinase 1(anti-pro-MMP-1) antibody, anti-pro-MMP-2 antibody, &
anti-pro-MMP-3 antibody, & also against the active matrix
metallo-proteinases(MMPs), MMP-1(fibroblast or tissue collagenase) &
MMP-3(stromelysin1), which are the active MMPs i.e., anti-MMP-1 antibody &
anti-MMP-3 antibody.


pro-MMP-1 & pro-MMP-3 are possible markers of cartilage destruction that
are cleaved to produce MMP-1(fibroblast or tissue collagenase) &
MMP-3(stromelysin1), the active MMPs, which are involved in the breakdown of extracellular
matrix of bone & cartilage in joints in
active RA.


(5) References –


1. Michael E.
Weinblatt, et al. Adalimumab a Fully Human Anti-Tumor Necrosis Factor Alpha Monoclonal Antibody, for the
Treatment of Rheumatoid Arthritis in Patients Taking Concomitant Methotrexate: The
ARMADA Trial. Arthritis & Rheumatism 2003;48:35-45.


2. Peter E. Lipsky, et al.,
Infliximab & Methotrexate in the treatment of rheumatoid arthritis. N Engl
J Med 2000;343:1594-602.


3. Lars Klareskog, et al.,
Therapeutic effect of the combination of etanercept and methotrexate compared
with each treatment alone in patients with rheumatoid arthritis: double-blind
randomised controlled trial. Lancet 2004;363:675-81


4. Foster, M.L., et al., Drug News
Perspect 2000;13(8):488


5. Guoyong Yin, et al., Endostatin Gene Transfer Inhibits Joint Angiogenesis

and Pannus Formation in
Inflammatory Arthritis. Molecular therapy 2002;5:547-54.


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