Treatment with spironolactone after the onset of collagen induced arthritis reduces joint inflammation,

cartilage destruction and bone erosion

Inderjeet VermaDoctoral Research Fellow

Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, India &

Healing Touch City Clinic, Chandigarh

Disclosures…

This study was supported by University Grant

Commission (UGC), New Delhi, F.No. 10-15/2007

(SA-1)

No other relevant financial involvement with any other

organization or entity with a financial interest in or financial

conflict with the subject matter or materials discussed in the

presentation apart from those disclosed.

No assistance was utilized in the production of this presentation

AcknowledgementsDr. Javed N Agrewala

Chief Scientist, Department of Immunology,

IMTECH, Chandigarh, India.

Dr. Asif Baliyan

Junior Resident, Department of Pathology,

GMCH, Chandigarh, India.

Dr. B.N. Datta

Former Professor, Department of Pathology,

PGIMER, Chandigarh, India

Study Background….. Synthetic DMARDs though effective but

do not produce relief in all Rheumatoid

Arthritis patients.

A number of patients have contraindications

or remain unrelieved from disease activity

& cardiovascular morbidity from these

drug. Biologic DMARDs is efficacious but has limitations.

Given the anti-inflammatory and TNF inhibiting potential of

spironolactone was first reported as 1961.1

Rev Can Biol 1961;20:82932

Study Objective….

To evaluate the therapeutic effect

of spironolactone (SPIR) in

collagen-induced arthritis model in

mice.

Materials and MethodsCollagen Induced Arthritis (CIA)

Male DBA/1 mice, 7–10 weeks of age

Immunized intradermal at the base of the tail with bovine collagen (CII) + CFA (4mg/ml)A booster inj. of CII + FIA was injected on day 21st. 2

IAEC No- 107/99/CPCSEA

Nat Protoc.2007; 2:1269–75.

Experimental Groups

Group I : Normal Control Group II : Arthritic Control/Group III : SPIR 20 (mg/kg/day)Group IV : SPIR 40 (mg/kg/day)Group V : SPIR 80 (mg/kg/day)Group VI : MTX (5 mg/kg/week)Group VII : CMC ControlGroup VIII : PBS Control

Forty-eight DBA/1 mice were randomly divided into eight groups each comprising six animals.

Outcome Parameters:

Materials and Methods

Arthritis score and paw thickness

Cytokine measurement (TNF-α & IL-6)

Oxidative markers (TBARS3 & NO4)

Histological analysis 

3Anal Biochem. 1979;95: 351-358.4 Analytical Biochemsitry . 2002; 306: 79-82

21 24 27 30 33 36 39 410

1

2

3

4

5

6

7

8

NC

PC

MTX

SPIR 20

SPIR 40

SPIR 80

Art

hriti

s sc

ore

Day after CII immunization

Results

Results were expressed in Means SEM.Error bar have been omitted for clarity of figure. NC; normal control, PC; positive/arthritis control, MTX; methotrexate control, SPIR; spironolactone (20/40/80 mg/kg/day).

Arthritis scores of CIA mice in different study groups

Values are given as mean ± SEM (n=6), **p ≤ 0.01 and ***p ≤ 0.001 vs. arthritis control). Statistical analysis: One way ANOVA followed by a followed by post hoc analysis. It represents the paw thickness of normal and CIA mice after treatment.

Paw thickness of normal and CIA mice after treatment

Effect of drugs on serum cytokines (TNF-α and IL-6)

All the values are the mean and SEM. *p <0.05, **p 6 0.01 compared with positive control group.Reduction of TNF-α and IL-6 level in CIA mice by SPIR and MTX treatment.

Effect of SPIR on the levels of oxidative markers

Values are given as mean ± SEM (n=6). *p < 0.05, **p ≤ 0.01.CIA mice were treated with SPIR 20, 40 and 80 mg/kg/day and MTX 5mg/kg/week for 3 weeks. SPIR 40 and 80 mg/kg/day produced significant effect on TBARS and NO level. MTX does not have shown any significant change in TBARS. But MTX significantly reduced NO level (p <0.05).

Histopathological images of the ankle joint of DBA1 mice

A; Normal control group: normal bone, cartilage and joint tissue. B; Arthritis control group: infiltration of inflammatory cells, erosive bone and cartilage destruction. C; Methotrexate treated group: no infiltration of inflammatory cells with minimal cartilage destruction and inflammation. D; Spironolactone 20 treated group: erosive bone and cartilage destruction showing marked infiltration of inflammatory cells in synovial tissue. E; Spironolactone 40 treated group: moderate erosive bone and cartilage destruction with moderate infiltration of inflammatory cells in synovial tissue. F; Spironolactone 80 treated group: mild infiltration of inflammatory cells in the synovial tissue. There is no bone and cartilage destruction.

The Present study concluded that SPIR (40 ,80 mg/kg) significantly reduced the paw swelling and arthritis severity indicated by improvement in arthritis score

Improvement in antioxidant status was evident by decreased level of TBARS and NO in mice treated with SPIR (40 ,80 mg/kg) when compared with arthritis control.

Significantly decreased inflammatory cytokines TNFα and IL-6 and histopathological analysis revealed that treatment with SPIR (40 ,80 mg/kg) led to restoration of joint architecture.

Results of current study highlights that SPIR possess anti-inflammatory and immunomodulatory effect that is extremely similar to MTX, suggesting that SPIR may be a potential alternative DMARD for the treatment of human arthritis.

Tidal wave of data: challenge and opportunity…..

THANK YOU