Attenuation of Inflammatory Polyarthritis in TNF Transgenic Mice By Diacerein: Comparative Analysis w/ Dexamethasone, Methotrexate & Anti-TNF Protocols

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Attenuation of Inflammatory Polyarthritis in TNF Transgenic Mice By

Diacerein: Comparative Analysis With Dexamethasone, Methotrexate and

Anti-TNF Protocols

Eleni Douni; Petros P Sfikakis; Sylva Haralambous; Fernandes;

Kollias

Arthritis Res Ther 6(1):R65-R72, 2003. © 2003 BioMed Central, Ltd.

Posted 12/23/2003

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The impact of diacerein, an effective cartilage targeted therapy that is

used in patients with osteoarthritis, on the development and progression

of chronic inflammatory arthritis was evaluated in a tumor necrosis

factor (TNF) transgenic mouse model (Tg197). The response to diacerein

at 2, 20, or 60 mg/kg daily, as well as the comparative effects of other

antiarthritis drugs including dexamethasone (0.5 mg/kg daily),

methotrexate (1 mg/kg three times weekly) and an anti-TNF agent (5 mg/kg

weekly), were assessed in the Tg197 mice. Treatment was initiated before

the onset of arthritis and was continued for 5 weeks. A significant

improvement in clinical symptoms was found in all three diacerein

treated groups in comparison with untreated groups. Confirming these

data, semiquantitative histopathologic analysis of the hind paws

revealed a significant reduction not only in cartilage destruction but

also in the extent of synovitis and bone erosion in diacerein treated

groups in comparison with untreated groups. At the most effective dose

tested (2 mg/kg daily), diacerein inhibited the onset of arthritis in

28% and attenuated the progression of arthritis in 35% of the Tg197

mice. Comparative analyses showed diacerein to be more potent than

methotrexate but not as effective as dexamethasone or anti-TNF agents in

suppressing the progression of the TNF mediated arthritis in this model.

These results indicate that diacerein has a disease modifying effect on

the onset and progression of TNF driven chronic inflammatory arthritis,

suggesting that the prophylactic or therapeutic potential of diacerein

in patients with RA should be further examined.

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disease

characterized by progressive destruction of cartilage and bone, leading

to functional decline and disability. Tumor necrosis factor (TNF) is

recognized as a central pathogenic molecule in RA because blockade of

TNF in human RA patients has been shown to retard joint damage

significantly.[1,2] Experimental animals overexpressing human TNF

develop synovitis with accompanying destruction of cartilage and bone

structures.[2,3] This murine disease resembles the destructive

polyarthritis of human RA and can be prevented by administration of

anti-TNF agents[3] as well as by IL-1 receptor blockade.[4]

Diacerein, a drug with IL-1 inhibitory activity in vitro[5-8] and in

vivo,[9] has been shown to be effective in the treatment of

osteoarthritis (OA)[10-12] – a degenerative process of the joints that

is characterized by the progressive destruction and erosion of the

cartilage. Diacerein belongs to the anthraquinone class of compounds.

Use of diacerein in animal models of OA,[13-15] as well as in the

spontaneous polyarthritis model in male NZB/KN mice,[16] revealed that

it consistently moderates cartilage degradation. Oral administration of

diacerein to patients with hip OA was associated with symptomatic

improvement and a significant structure modifying effect, coupled with a

good safety profile.[11]

In the present study we investigated whether diacerein, in addition to

its action on OA, is effective in treating chronic inflammatory

arthritic diseases using the TNF driven transgenic mouse model of

arthritis.[3] A comparative analysis of the effects of diacerein and

those of known antiarthritic agents including methotrexate,[17,18]

dexamethasone[19,20] and an anti-TNF agent[21,22] in the progression of

the TNF driven inflammatory arthritis was performed in parallel.

Materials and Methods

Transgenic Animals

The heterozygous Tg197 transgenic mouse was generated and described

previously by our research group.[3] Briefly, Tg197 mice carry a human

TNF transgene with its 3'-untranslated region replaced by a sequence

from the 3'-untranslated region of the beta-globin gene, allowing

deregulated human TNF gene expression. By age 4 weeks, all human TNF

Tg197 mice spontaneously develop a severe bilateral, symmetric, erosive,

and disabling polyarthritis similar to RA. All animal procedures were

conducted in accordance with the principles of the Declaration of Helsinki.

Reagents

Diacerein (Verboril) was provided by Laboratoire Medidom S.A. (Geneva,

Switzerland), dexamethasone was purchased from Merck & Co., Inc. (West

Point, USA), methotrexate was purchased from Lederle Parenterals Inc.

(Puerto Rico, USA), and the anti-TNF antibody CB0006 was kindly provided

by Celltech Ltd (Slough, UK).

Treatment and Clinical Assessment

We conducted one large study in transgenic mice (n = 65) separated into

eight groups: group 1 was left untreated (n = 10); group 2 received an

injection of water daily (n = 10); group 3 received 2 mg/kg diacerein

daily (n = 9); group 4 received 20 mg/kg diacerein daily (n = 8); group

5 received 60 mg/kg diacerein daily (n = 10); group 6 received 1 mg/kg

methotrexate three times weekly (n = 6); group 7 received 0.5 mg/kg

dexamethasone daily (n = 6); and group 8 received the antihuman TNF

antibody CB0006 at 5 mg/kg weekly (n = 6). Each transgenic mouse

received either oral administration of an aqueous solution containing

diacerein or intraperitoneal administration of the rest of the tested

compounds at 2 weeks of age (i.e. before the onset of arthritis). Body

weight and arthritis scores were recorded weekly for each mouse.

Arthritis was evaluated in ankle joints in a blinded manner using a

semiquantitative arthritis score ranging from 0 to 3: 0 = no arthritis

(normal appearance and grip strength); 1 = mild arthritis (joint

swelling); 2 = moderate arthritis (severe joint swelling and digit

deformation, no grip strength); and 3 = severe arthritis (ankylosis

detected on flexion and severely impaired movement). At 7 weeks of age

all mice were killed and the hind ankle joints were removed for histology.

Histologic Processing and Scoring of Joints

Ankle joints were removed from the transgenic mice and were fixed in 10%

buffered formalin overnight, decalcified in 30% formic acid for 4 days,

and then embedded in paraffin. Sections were stained with hematoxylin

and eosin, and the histopathologic score was evaluated microscopically,

as described previously,[23] in a blinded manner using a modified

scoring system as follows: 0 = no detectable pathology; 1 = hyperplasia

of the synovial membrane and presence of polymorphonuclear infiltrates;

2 = pannus and fibrous tissue formation and focal subchondral bone

erosion; 3 = articular cartilage destruction and bone erosion; and 4 =

extensive articular cartilage destruction and bone erosion.

The extent of synovitis, cartilage destruction, or bone erosion was

based on arbitrary scores described extensively below, which we use

regularly in our laboratory when scoring this TNF model of arthritis.

Synovitis was evaluated using a semiquantitative scoring from 0 to 4: 0

= normal; 1 = mild synovial hypertrophy (< 5 cell layers) with few

inflammatory cells; 2 = moderate synovial hypertrophy (< 20 cell layers)

with accumulation of inflammatory cells into intrasynovial cysts; 3 =

pannus and fibrous tissue formation; and 4 = pannus and fibrous tissue

formation on both sides of the ankle joint. Bone erosions were scored

from 0 to 4 as follows: 0 = normal; 1 = mild (focal subchondral

erosion); 2 = moderate (multiple subchondral erosions); 3 = high (as

above + focal erosion of talus); and 4 = maximum (multiple erosions of

tarsal and metatarsal bones). Cartilage damage was evaluated on the two

ankle joint bones, tibia and talus, after staining of tissue sections

with safranin-O (BDH Laboratory Supplies, Poole, UK). Proteoglycan

depletion and matrix erosion are associated with cartilage degradation,

as can be revealed by loss of safranin-O staining. Cartilage damage was

scored semiquantitatively from 0 to 4: 0 = intact; 1 = minor (< 10%); 2

= moderate (10–50%); 3 = high (50–80%); and 4 = severe (80–100%).

Statistical Analysis

All values are expressed as means ± standard error. Arthritic scores and

histologic scores were analyzed using the Mann–Whitney U test for

nonparametric data. P < 0.05 was considered statistically significant.

Results

Clinical Effects of Diacerein

We tested the efficacy of diacerein in preventing the development of

pathology in the Tg197 transgenic murine model of RA, in which

inflammatory polyarthritis is clinically detectable at 4 weeks of age,

with swelling and deformation of the ankle joints. Daily administration

of diacerein at doses of 2, 20, or 60 mg/kg to Tg197 transgenic mice

from age 2 weeks (before the onset of arthritis)to age 7 weeks

significantly reduced clinical scores compared with those in untreated

mice (Fig. 1). Whereas the clinical arthritis score increased

progressively in the control groups, the score was less in all groups

treated with diacerein at the end of the study (i.e. at age 7 weeks),

indicating a marked suppression of disease progression (P < 0.05). The

majority of joints from mice treated with diacerein (69–89%) developed

low to mild arthritis (arthritis score </= 1), whereas in the untreated

mice only 25% of the joints exhibited similar arthritis scores, with the

remaining 75% having scores greater than 1 (Table 1). No significant

differences were observed in body weight gain in treated mice during the

study (data not shown), indicating that dosages of 2–60 mg/kg diacerein

daily are not associated with adverse toxicity.

Joint Histopathology in Diacerein Treated Mice

To assess joint damage, histopathologic analysis of the hind paws was

conducted in the diacerein treated mice. Similar to RA, in the Tg197

murine model the synovial lining becomes markedly thickened because of

synovial cell proliferation and infiltration of inflammatory cells. This

proliferative mass – the pannus – invades and progressively destroys

articular cartilage and bone, leading to irreversible destruction of

joint structure and function. Histologically, changes characteristic of

chronic inflammatory arthritis develop in the hind paws of Tg197 mice

from age 3 weeks. Interestingly, in the Tg197 mice that received the

three different dosages of diacerein for 5 weeks there was a significant

reduction (P < 0.05) in the mean histopathologic score when compared

with control Tg197 groups (Fig. 2). Ankle joints in the low-dose

diacerein treated mice (2 mg/kg daily) had lower histopathologic scores

than did mice treated with higher doses of diacerein (20 mg/kg daily or

60 mg/kg daily), but these differences were not statistically

significant. Our findings clearly show that administration of diacerein

at all three doses inhibited the onset of arthritis in 20–28% of joints

in Tg197 mice, which had nearly normal histologic appearance (i.e.

histopathologic score 0 or 1; Table 2). Interestingly, at the most

effective dose tested (2 mg/kg daily) we observed an additional

inhibition in progression of arthritis in 35% of the Tg197 mice, whereas

similar effects occurred in fewer mice receiving the other two doses

(13% in the 20 mg/kg group and 20% in the 60 mg/kg group).

To assess specific effects of diacerein in synovitis, cartilage

destruction and bone erosion, we conducted a semiquantitative scoring

analysis for each of these pathologic parameters. Interestingly,

administration of diacerein at 2–60 mg/kg daily in Tg197 mice resulted

in a significant reduction (P < 0.05) in all three analytical

histopathologic scores as compared with those of control Tg197 mice,

which all developed synovitis with severe articular cartilage

degradation and bone erosions (Fig. 3). These findings indicate that

diacerein exerts its disease modifying effects mainly by suppressing

inflammation and synovial hyperplasia, which are seemingly the

initiating pathogenic events leading to further tissue destruction. Fig.

4 shows the beneficial effect of diacerein on joint structure in

representative tissue sections from ankle joints exhibiting the lowest

histologic scores in diacerein treated and control groups. Preservation

of the ankle joint structure can only be observed in diacerein treated

and in anti-TNF treated Tg197 mice.

Comparison of Diacerein With Dexamethasone, Methotrexate and Anti-TNF

Protocols

The disease modifying effect of diacerein in Tg197 mice, as assessed by

clinical and histopathologic analysis, was compared with that of

anti-inflammatory agents (i.e. dexamethasone, methotrexate, and an

anti-TNF antibody) that are used in patients with chronic inflammatory

arthritides, including RA. Findings of semiquantitative clinical

assessment based on arthritis scores of the ankle joints of untreated or

treated Tg197 mice are shown in Table 1. Of the ankle joints of the

untreated mice, 75% had moderate to severe arthritis with obvious digit

deformation and less strength on flexion (arthritis score > 1). In

contrast, only 11% of the joints of the mice treated with low dose

diacerein and none of the joints of mice treated with dexamethasone or

anti-TNF had a similar score, whereas in mice treated with methotrexate

50% of the joints were affected by the same degree of arthritis. A

significant difference (P < 0.05) in arthritis score was observed

between all diacerein treated groups, as well as dexamethasone or

anti-TNF treated groups, and the control groups (Table 1).

Histologically, more than 80% of the joints of the untreated control

groups and of the methotrexate treated group were moderately

(histopathologic score 3) to severely (histopathologic score 4) damaged

by the expansion of synovial pannus and destruction of cartilage and

bone structures (Table 2). In contrast, fewer than 50% of the joints of

diacerein treated groups and none of the joints from mice treated with

dexamethasone or an anti-TNF antibody had a similar histologic

appearance (histopathologic score >/= 3). Ankle joints from mice treated

with diacerein, dexamethasone, or an anti-TNF antibody had significantly

lower histopathologic scores (P < 0.05) compared with those from

untreated or methotrexate treated Tg197 mice.

Discussion

RA is a chronic polyarthritis that leads to joint destruction and

serious disability. Despite the use of a variety of medications,

treatment of RA is not fully effective in most patients and side effects

frequently limit their long-term use. Classic nonsteroidal

anti-inflammatory drugs are used to control the symptoms of RA but they

are associated with significant gastrointestinal toxicity, including a

risk for potentially life threatening gastroduodenal perforations,

ulcers, and bleeds.[24] Therapy of RA with slow acting, disease

modifying antirheumatic drugs such as methotrexate, which is generally

accepted as the standard for long-term treatment, leads to a significant

amelioration of symptoms but does not stop joint destruction.[17,18]

Glucocorticoids, which are among the most potent and clinically

important immunosuppressants, are used to control acute and severe

flare-ups of joint inflammation, but they are not used for chronic

therapy in most patients because of their significant adverse effects.

Novel therapeutic agents such as monoclonal antibodies, cytokine

receptor–human immunoglobulin constructs, or recombinant human proteins

have been tested in RA and in other chronic arthritides such as

ankylosing spondylitis or psoriatic arthritis with convincing evidence

of success. In particular, clinical trials testing anti-TNF agents,

either alone or in combination with methotrexate, have proven the

feasibility and efficacy of these novel approaches.[21,22] However,

therapy that is directed against TNF and IL-1 is clinically effective in

only 40–70% of patients, and importantly TNF antagonist therapies have

been associated with side effects including tuberculosis,[25]

listeriosis,[26] lymphomas,[27] and life-threatening histoplasmosis.[28]

The numbers are not high, but clinical vigilance is necessary to

minimize the risk. Treatments that are directed against osteoclasts such

as osteoprotegerin have shown great promise for the prevention of bone

destruction in experimental models of RA[29] but that therapy does not

affect the inflammatory tissue or symptoms of the disease.

Diacerein is an effective and well tolerated agent for the treatment of

OA[10-12] and it is unique among current anti-OA products in that it is

able to influence both the anabolism and catabolism of chondrocytes. The

effect of diacerein in chronic inflammatory arthritis was investigated

in the present study using an established TNF-mediated murine model of

RA – the Tg197 mouse.[3] Invasive growth of a hypertrophic synovial

membrane and local accumulation of inflammatory infiltrates are typical

features of RA and arthritis in TNF transgenic mice, and are considered

to be prerequisites for cartilage destruction and bone erosion. The

Tg197 model has been extensively used by various research groups in the

past as a reliable tool to assess the efficacy of potent antiarthritic

compounds, as well as to investigate the mechanisms that are involved in

the pathogenesis of chronic inflammatory arthritis.[29-31]

The results of the present study clearly demonstrate that diacerein has

antiarthritic activity, preventing the onset and suppressing the

progression of joint pathology, as shown by clinical and histopathologic

assessment of mice treated with three different doses (2, 20, and 60

mg/kg diacerein daily). Additional studies evaluating the effects of

diacerein at 4 and 40 mg/kg daily showed a beneficial response that was

clearly reproduced in the present study (data not shown). Although

pharmacokinetic studies were not performed, the lack of a dose effect

suggests that the beneficial response can be achieved even with the low

dose of 2 mg/kg daily, which is analogous to the dose used in humans

with OA.[10-12] The lowest effective dose of diacerein is yet to be

assessed in the TNF transgenic model. Our findings revealed a beneficial

effect of diacerein not only for cartilage protection, which is usually

the target of diacerein in OA studies, but also for synovitis and bone

erosion. Preservation of the joint architecture after diacerein

administration appears to be mainly due to the significant suppression

of the highly proliferative pannus-like tissue, which consists of

synovial fibroblasts, synovial macrophages, and various infiltrating

inflammatory cells. This is the first report providing evidence for

anti-proliferative and anti-inflammatory effects of diacerein in an

inflammatory model of arthritis.

In OA studies it has been shown that diacerein exerts its protective

action by down-regulating the production of cartilage degrading

enzymes[8,32,33] through inhibiting the IL-1/IL-1 receptor system and

increasing the production of tissue inhibitor of metalloprotease-1,[34]

whereas it activates cartilage repair in OA by stimulating the

production of transforming growth factor-?.[35] In the Tg197 transgenic

model of RA blockade of IL-1 receptor signaling prevented disease onset,

indicating that in TNF transgenic mice the IL-1 receptor acts as a

potent downstream mediator in the pathogenesis of chronic arthritis.[4]

Therefore, a possible mechanism to account for the attenuation of TNF

mediated joint damage by diacerein could be its inhibition of IL-1

production and hence of the downstream events that lead to production of

reactive oxygen species, nitric oxide, and matrix metalloproteases.

Quantitative analysis of the local production of cytokines such as IL-1

or transforming growth factor-? in joints of diacerein treated

transgenic mice could probably confirm such a hypothesis.

The anti-inflammatory effect of diacerein is linked to mechanisms that

have not yet been completely clarified. Recently, Tamura and

coworkers[36] reported anti-inflammatory activity of diacerein in acute

inflammatory models such as carrageenin, zymosan, and dextran induced

paw edema, as well as in adjuvant induced arthritis in rats. In addition

to its anti-inflammatory effects, diacerein reverses the change in bone

metabolism that is seen in ovariectomized rats, and maintains bone

mineral density by improving the balance of bone formation and bone

absorption.[36] Our finding that diacerein significantly reduced

synovitis, cartilage destruction, and bone erosion points to a

beneficial effect of diacerein on multiple cell types that are involved

in the pathogenesis and progression of arthritis. The cell type which is

the best target for the antiarthritic effect of diacerein in TNF

mediated arthritis remains to be studied.

Comparative analysis showed that diacerein administration in the Tg197

model of RA for 5 weeks was more potent than the 'slow acting' disease

modifying agent methotrexate. Even high dose methotrexate treatment (1

mg/kg given three times weekly) could not modify the progression of

joint destruction in Tg197 mice (Table 1 and Table 2). One possible

explanation for the beneficial actions of methotrexate in RA is

diminution of both the size and reactivity of the T cell population.[37]

The unresponsiveness of the Tg197 model to methotrexate administration

could be due to the minimal role of adaptive immunity in the development

of arthritis in TNF over-expressing transgenic models.[38,39] However,

it has been shown that methotrexate, at doses analogous to those used in

human regimens, is not particularly effective in collagen induced

arthritis either,[40] indicating the potential incompatibility of animal

models and human RA. On the other hand, diacerein was not as effective

as dexamethasone. Dexamethasone administration (0.5 mg/kg daily)

resulted in a dramatic suppression of inflammatory synovial tissue and

in preservation of cartilage and bone structures (P < 0.05).

Histologically, 100% of the mice treated with dexamethasone had nearly

normal histologic appearance (Table 2). Previous studies have shown that

the AU-rich region of TNF mRNA is required for inhibition of TNF

translation by dexamethasone.[39] In the present study dexamethasone was

expected to block TNF production partially (e.g. in the Tg197

macrophages) as a result of the absence of the AU-rich region from the

huTNF transgene.[3,39] It may therefore be postulated that the dramatic

effectiveness of dexamethasone seen in the Tg197 arthritic model works

through the inhibition of additional targets downstream of the TNF

production.

Our results clearly show that diacerein is effective in the TNF

transgenic model of RA if it is administered before the onset of

arthritis. It would certainly be even more informative if similar

studies could be performed in Tg197 mice after the establishment of

arthritis, thus providing a more accurate reflection of therapeutic

intervention in the human disease. Further studies are needed to

delineate the exact mechanisms of action of diacerein, as well as to

determine the efficacy of this treatment in mice with established

chronic disease. However, we suggest that the beneficial effects of

diacerein on progression of TNF mediated inflammatory arthritis render

this agent worthy of consideration for the prophylaxis of bone damage in

human arthritic conditions.

Conclusion

Diacerein is able to prevent TNF mediated structural damage in a murine

model of chronic joint inflammation, as shown by significant attenuation

of clinical and histologic scores. This indicates that the prophylactic

or therapeutic potential of diacerein in patients with RA should be

examined further.

Competing Interests

This work was supported in part by a research grant from Laboratoire

Medidom S.A. Dr Fernandes who is a co-author in the present study

receives a salary from Laboratoire Medidom S.A. To our knowledge, there

are no benefits from commercial sources for the work reported on in this

report or financial interests of the authors that could create a

potential conflict of interest or the appearance of a conflict of

interest with regard to the work.

Correspondence Address

Dr Kollias, Institute of Immunology, Biomedical Sciences Research

Center ‘ Fleming´, 34 Fleming Str, Vari 16672 Greece. Tel. +30

210 965 6507; fax: +30 210 965 6563; e-mail: g.kollias@...

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Abbreviation Notes

IL, interleukin; OA, osteoarthritis; RA, rheumatoid arthritis; TNF,

tumor necrosis factor

1Institute of Immunology, Biomedical Sciences Research Center '

Fleming', Athens, Greece

2First Department of Propedeutic and Internal Medicine, Laikon Hospital,

Athens University Medical School, Athens, Greece

3Hellenic Pasteur Institute, Athens, Greece

4Trans Bussan Chemedica International, Geneva, Switzerland

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