Tubastatin, a selective histone deacetylase 6 inhibitor shows anti-inflammatory and anti-rheumatic effects

International Immunopharmacology 16 (2013) 72–78

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Tubastatin, a selective histone deacetylase 6 inhibitor shows anti-inflammatory and anti-rheumatic effects Santosh Vishwakarma a,⁎, Lakshmi R. Iyer a, 1, Milind Muley a, 1, Pankaj Kumar Singh a, Arun Shastry a, Ambrish Saxena a, Jayanarayan Kulathingal a, G. Vijaykanth a, J. Raghul a, Navin Rajesh a, Suresh Rathinasamy b, Virendra Kachhadia b, Narasimhan Kilambi b, Sridharan Rajgopal b, Gopalan Balasubramanian b, Shridhar Narayanan c a b c

Department of Biology, Drug Discovery Research, Orchid Chemicals and Pharmaceuticals Ltd., Old Mahabalipuram Road, Sozhanganallur, Chennai – 600119, India Department of Medicinal Chemistry, Drug Discovery Research, Orchid Chemicals and Pharmaceuticals Ltd., Old Mahabalipuram Road, Sozhanganallur, Chennai – 600119, India Infection iScience, AstraZeneca India Pvt. Ltd. Hebbal, Bangalore, India

a r t i c l e

i n f o

Article history: Received 10 December 2012 Received in revised form 22 January 2013 Accepted 15 March 2013 Available online 27 March 2013 Keywords: Tubastatin HDAC6 inhibitor Rheumatoid arthritis Anti-inflammatory

a b s t r a c t Epigenetic modifications represent a promising new approach to modulate cell functions as observed in autoimmune diseases. Emerging evidence suggests the utility of HDAC inhibitors in the treatment of chronic immune and inflammatory disorders. However, class and isoform selective inhibition of HDAC is currently favored as it limits the toxicity that has been observed with pan-HDAC inhibitors. HDAC6, a member of the HDAC family, whose major substrate is α-tubulin, is being increasingly implicated in the pathogenesis of inflammatory disorders. The present study was carried out to study the potential anti-inflammatory and anti-rheumatic effects of HDAC6 selective inhibitor Tubastatin. Tubastatin, a potent human HDAC6 inhibitor with an IC50 of 11 nM showed significant inhibition of TNF-α and IL-6 in LPS stimulated human THP-1 macrophages with an IC50 of 272 nM and 712 nM respectively. Additionally, Tubastatin inhibited nitric oxide (NO) secretion in murine Raw 264.7 macrophages dose dependently with an IC50 of 4.2 μM and induced α-tubulin hyperacetylation corresponding to HDAC6 inhibition in THP-1 cells without affecting the cell viability. Tubastatin showed significant inhibition of paw volume at 30 mg/kg i.p. in a Freund's complete adjuvant (FCA) induced animal model of inflammation. The disease modifying activity of Tubastatin was also evident in collagen induced arthritis DBA1 mouse model at 30 mg/kg i.p. The significant attenuation of clinical scores (~70%) by Tubastatin was confirmed histopathologically and was found comparable to dexamethasone (~90% inhibition of clinical scores). Tubastatin showed significant inhibition of IL-6 in paw tissues of arthritic mice. The present work has demonstrated anti-inflammatory and antirheumatic effects of a selective HDAC6 inhibitor Tubastatin. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Histone Deacetylase inhibitors (HDACi) were initially studied for their ability to increase gene expression, however today there is increasing number of orally active, synthetic HDACi being primarily developed to treat cancer [1,2]. Recently HDACi have emerged as potent anti-inflammatory agents [3]. Histone hyperacetylation results in up-regulation of cell cycle inhibitors (p21Cip1, p27Kip1, and p16INK4, repression of inflammatory cytokines [interleukin (IL)-1, IL-8, tumor necrosis factor-α (TNF-α), down-regulation of immune stimulators (IL-6, IL-10, and CD154) [4]. HDACi like MS-275, Trichostatin A and Suberoylanilide hydroxamic acid (SAHA) have emerged to be potent

⁎ Corresponding author. Tel.: +91 98 4024 1027; fax: +91 44 2450 1396. E-mail addresses: [email protected], [email protected], [email protected] (S. Vishwakarma). 1 The second and third authors contributed equally to the manuscript and should be considered as joint second authors. 1567-5769/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2013.03.016

anti-inflammatory agents in murine and human monocytes [5,6]. Pan HDACi like Givinostat (ITF-2357); SAHA and MS-275 have been shown to ameliorate disease symptoms in animal models of Rheumatoid Arthritis (RA) [7,8]. Rheumatoid arthritis (RA) is characterized by persistent synovitis, systemic inflammation, and autoantibodies particularly to rheumatoid factor and citrullinated peptide. About 1% to 2% of the world’s population is affected by RA and women are three times more likely than men to develop RA between the ages of 35 and 50 years. RA is a common chronic autoimmune inflammatory disease [9,10]. The inflamed synovium is central to the pathogenesis of RA and formation of tumorlike synovial tissue, called ‘pannus’ is a characteristic feature of RA [11,12]. Traditionally available treatments of RA have included mainly disease modifying anti-rheumatic drugs (DMARD's). Approved treatments for RA include non-steroidal anti-inflammatory drugs, antimetabolites such as methotrexate and leflunomide, corticosteroids like dexamethasone, and various biologics. Unfortunately, therapies targeting the disease are still in their infancy and have various undesirable side effects [13].

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Most of the promising HDACi showing pro-inflammatory properties target multiple HDAC (Histone Deacetylase) isoforms. Pan-HDAC inhibition might accompany several undesirable side effects such as fatigue, diarrhea, nausea, neutropenia and thrombocytopenia. On the contrary HDACi highly selective for individual isoforms, may exhibit reduced side effects compared to the pan-HDACi, while still retaining capability for target modulation. Recently HDAC6, a member of the HDAC family, whose major substrate is α-tubulin, has been convincingly implicated in the pathogenesis of inflammatory disorders. The present work was carried out to study the potential anti-inflammatory and anti-rheumatic effects of Tubastatin. In the present study, Tubastatin; a selective HDAC6 inhibitor prevented the release of pro-inflammatory cytokines like TNF-α and IL-6 from human monocytes. Further, in animal models, Tubastatin treatment significantly improved paw edema in the Freund's complete adjuvant induced paw inflammation and anti-rheumatic activity in collagen induced arthritis mouse model with amelioration in the arthritic clinical scores that was corroborated histopathologically. All these findings strongly support the fact that HDAC6 selective inhibition has therapeutic potential for the treatment of RA.

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2.4. Cytotoxicity assay by sulforhodamine (SRB) method RAW 264.7 and THP-1 cells (10,000/well) were seeded in 96-well plate and treated with test compounds at various concentrations for 24 h and cell viability was assessed using SRB assay. The colorimetric readings were measured at 530 nm in a spectrophotometer (Tecan; Infinite M1000). % survival was calculated by comparing the treated cells with untreated cells [14]. 2.5. Cytokine secretion assay ELISA kits for human TNF-α and IL-6 was purchased from R&D systems (USA). THP1 cells (10,000/well) were seeded in 24-well plates along with 32nM phorbol 12-myristate 13-acetate and incubated for 24 h. Post incubation culture media was exchanged with fresh media and incubated for further 24 h. Next, cells were treated with Tubastatin, dexamethasone or SAHA at specified concentrations for 24 h; followed by LPS stimulation (10 ng/ml LPS for 4 h for TNF-α stimulation and 100 ng/ml LPS for 8 h for IL-6 stimulation). The conditioned media was collected and ELISA was performed with the same as per the manufacturer's instructions. The optical density was recorded using a multi-plate reader at 450 nm [15,16].

2. Materials and methods 2.6. NO secretion 2.1. Cell lines and cell culture Human acute monocytic leukemia cell line THP-1 (TIB-202) and murine macrophage cell line RAW 264.7 (TIB-71) was purchased from American Type Culture Collection (ATCC; VA, USA). THP-1 cells were cultured in RPMI-1640 medium (Invitrogen, CA, USA) supplemented with 10% (V/V) heat inactivated fetal bovine serum (FBS; Invitrogen, USA). RAW 264.7 cells were cultured in DMEM (Invitrogen, USA) supplemented with 10% Fetal Bovine Serum. Cell culture media was further supplemented with penicillin/streptomycin. All cell cultures were maintained at 37 oC in a humidified atmosphere with 5% CO2.

2.2. Animals DBA1 mice (Female, 6–8 weeks, 20–28 g) and Wistar rats (male, 6– 8 weeks, 200–225 g) were obtained from animal facility, Drug Discovery Research; Orchid Chemicals and Pharmaceuticals Ltd., (Chennai, India). Animals were maintained in controlled environment with temperature (22 ± 2 °C), humidity (44–56%) and 12 h light–dark cycle and were provided with standard diet (Nutrilab Rodent) and water ad libitum. The study was approved by Institutional Animal Ethics Committee. (DBA1 mice:-Protocol.No.18/IAEC-03/PPK/2010; Wistar rats:-Protocol No.13 /IAEC-01/PCP/2012)

2.3. Drugs and chemicals Mycobacterium tuberculosis H37Ra, bovine collagen Type II, Complete Freund's Adjuvant (CFA) and incomplete Freund's adjuvant (IFA) were obtained from Difco Laboratories (Detroit, MI). Dexamethasone and LPS was received from Sigma–Aldrich, St Louis, MO, USA. Tubastatin and SAHA were synthesized by the department of Medicinal Chemistry (Drug Discovery Research) at Orchid Chemicals and Pharmaceuticals Ltd., (Chennai, India). The test compound was handled and stored as per the specifications recommended for the test compound. Tubastatin was solubilized in 10% Dimethyl sulfoxide (DMSO) 10% Polyethylene glycol (PEG) 400 and 80% (40% of hydroxy propyl beta cyclodextrin) and dexamethasone was prepared as a suspension in 0.25% sodium carboxmethylcellulose. For in-vitro studies compounds were dissolved in DMSO only.

The secretion of NO was studied in RAW 264.7 cells (1 million). Briefly, cells were seeded in 24 well plates and incubated for 24 h. Cells were then treated with indicated compounds for 24 h followed by LPS stimulation (100 ng/ml) for 24 h. Nitric oxide content was estimated in conditioned media using Greiss reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid) the mixture was incubated at room temperature for 10 min. Upon development of purple/magenta color, absorbance was measured at 570 nm within 30 min. The quantity of nitrite was determined from a sodium nitrite standard curve and the values obtained were represented graphically [17]. 2.7. Freund's complete adjuvant (FCA) induced paw inflammation in Wistar rats Animals were randomized into 4 groups (n = 6) on the basis of their paw volume. Group I and II served as normal control and disease control. Group II received the vehicle, Group III received Tubastatin 30 mg/kg/day i.p. for 5 days, whereas Group IV received dexamethasone 1 mg/kg p.o. on day 5. All animals, except normal were injected with 100 μg of M. tuberculosis in 50 μl incomplete Freund’s adjuvant (IFA) in the sub-plantar region of the both hind paws on day 5. Treatment group received FCA 1 hour after dosing Tubastatin and dexamethasone. Paw volume was measured at 2, 6 and 24 hours after FCA Injection using LE 7500, Panlab, Spain instrument [18]. 2.8. Induction of Collagen induced Arthritis (CIA) in DBA1 mice and Experimental design Primary immunization of the animals with antigen was done on day 0, a booster injection was administered on day 21 and to accelerate the onset of arthritic clinical scores; LPS (50 μg/animal) was injected intraperitoneally on day 28. on day 0 commercially available collagen type II in 0.05 M acetic acid to a concentration of 2 mg/ml and M. tuberculosis H37 Ra 5 mg/mL of CFA was used for immunization. Each animal was injected 100 μl (consisting of 100 μg of collagen + 250 μg CFA intradermally at the base of the tail. On day 21 post primary immunization, mice were boosted with collagen Type II (2 mg/mL); emulsified with equal volume of IFA using homogenizer. The animals were injected 100 μl intradermally, proximal to the primary injection site. Lipopolysaccharide (LPS) 50 μg prepared in 100 μl of PBS was administered intra-

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peritoneally to each animal on day 28 to accelerate the onset of disease. This resulted in full-blown disease within 3 days of LPS injection [8]. The clinical assessment of scores was done daily once the treatment was initiated [19]. Animals were randomized to four groups of 6 animals each. The animals of the first group were not immunized (normal control); whereas the groups 2 to 4 were immunized with bovine type II collagen. Group 2 animals were treated with vehicle (disease control). Animals of the Group 3 were treated orally with dexamethasone (0.1 mg/kg, q.d.). Group 4 animals received intra-peritoneal injection of Tubastatin administered at 30 mg/kg, q.d. The treatment was given from day 21 to day 36. The body weight of animals was recorded once in 2 days after dosing regime was initiated. On day 37, animals were sacrificed; the right fore and hind paws were removed from each animal for histological analysis and left fore and hind paws were collected for tissue cytokines. The spleen and thymus weights were measured. The histological samples were paraffin-embedded, sectioned, stained with hematoxylin and eosin and scored as described [20]. Safranin O was used to identify the loss of proteoglycans in the articular [21].

RAW 264.7 Cells

125

THP-1 Cells 100

Cell Survival, %

74

75

50

25

0 100

1000

10000

100000

Tubastatin, nM Fig. 2. Cytotoxicity assay in RAW 264.7 and THP-1 macrophages. Values indicate mean ± SD, (n = 3).

3. Results 2.9. IL-6 estimation in paw tissue

3.1. Tubastatin inhibits TNF-α and IL-6 secretion in THP-1 cells

Estimation of IL-6 in the frozen tissues (whole joints including synovium, adjacent tissues and bones) were pulverized using a mortar and pestle filled with liquid nitrogen. The pulverized samples were transferred to eppendorf tubes filled with 200 μl of T-PER (Tissue Protein Extraction Reagent) and homogenized using a Polytron tissue homogenizer. Mouse joint homogenates were centrifuged for 10 min at 500 g at 4 °C. Supernatant was separated and used for protein estimation (BSA method) and IL-6 analysis was carried out using GE Healthcare ELISA kits according to the manufacturer's instructions. Data was expressed as IL-6 pg/ml of 1 mg of protein [22].

Tubastatin was able to inhibit TNF-α and IL-6 production from LPS-stimulated THP-1 cells in a dose dependent manner with an IC50 of 272.5 nM and 712.9 nM respectively. Dexamethasone was used as positive control for the study. Dexamethasone and pan HDAC inhibitor SAHA at 1 μM completely inhibited TNF-α and IL-6 secretion in these cells (Fig. 1). Further, the cell survival/proliferation was unaffected by the compound treatment (Fig. 2). These results clearly point towards the efficient inhibition of pro-inflammatory cytokines by Tubastatin in LPS-induced THP-1 cells. 3.2. Tubastatin attenuates NO production in RAW 264.7 cells

2.10. Statistical analysis All the results of in vitro data were expressed as mean ± standard deviation (S.D), while the in vivo results were expressed as mean ± standard error of means (S.E.M.). The data was analyzed by one way ANOVA followed by Dunnett’s multiple comparison tests using Graph pad prism version 4.0. In all the tests p b 0.05 was considered as statistically significant.

RAW 264.7 cells were used to study the effect of Tubastatin on LPS induced NO secretion. Tubastatin treatment showed a dose-dependent attenuation of NO secretion post LPS stimulation. Treatment with 10 μM of Tubastatin showed potent 66% inhibition of NO secretion where as 1 μM SAHA showed 89.5% inhibition while 10 μM dexamethasone was able to inhibit 36% of NO secretion (Fig. 3). 3.3. Anti-inflammatory activity of tubastatin

110 100

IL-6

Tubastatin

Tubastatin

SAHA

SAHA

Dexamethasone

Dexamethasone

Injection of FCA into the hind paws produced an increase in paw volume to 2.02 ± 0.05 ml, 2.55 ± 0.07 ml and 2.84 ± 0.1 ml in Disease 100

80

Tubastatin

90 70

SAHA

80

60

NO Inhibition, %

Cytokine Inhibition, %

90

TNF-

50 40 30 20

Dexamethasone

70 60 50 40 30 20

10

10 0

0 1

10

100

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HDAC Inhibitor, nM Fig. 1. Effect of tubastatin on TNF-α and IL-6 secretion in THP-1 cells. Values indicate mean ± SD, (n = 3).

10

100

1000

10000

100000

HDAC Inhibitor, nM Fig. 3. Effect of tubastatin on NO secretion in RAW 264.7 cells. Values indicate mean ± SD, (n = 3).

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75

##

3.0

Paw Volume (ml)

##

**

2.5

**

** ## 2.0

*

$

*

**

2hr

6hr

1.5

1.0 2hr

6hr

24hr

2hr

Normal

6hr

24hr

2hr

Disease control

6hr

4hr

Tubastatin 30 mg/kg i.p.

24hr

Dexamethasone 1 mg/kg p.o.

Fig. 4. Effect of tubastatin and dexamethasone on paw volume (ml) of FCA injected rats. The data represent Mean ± SEM of six rats. ##p b 0.01 as compared to normal control and *p b 0.05, **p b 0.01 as compared to disease control; $P b 0.01 as compared to Tubastatin at 6th hour (one way ANOVA followed by Dunnett's multiple comparison test).

control group at 2, 6 and 24 h respectively as compared to normal control (1.76 ± 0.03 ml). In-house experiments indicate administration of 10% DMSO, 10% PEG 400 and 80% (40% of hydroxy propyl beta cyclodextrin) and 0.25% CMC to disease control does not affect the progression of disease (Data not shown). Treatment with Tubastatin and Dexamethasone produced significant decrease in paw volume at 2 h 1.84 ± 0.05 ml and 1.85 ± 0.03 ml (71.90 and 65.36% inhibition), at 6 h 2.23 ± 0.04 ml and 1.84 ± 0.02 ml (40.34 and 90.45% inhibition) and at 24 h 2.43 ± 0.07 ml and 2.31 ± 0.06 ml (38.58 and 49.54% inhibition) as compared to disease control at respective time points (Fig. 4). 3.4. Anti-rheumatic effects of tubastatin and dexamethasone in DBA1 mouse semi-therapeutic collagen induced Arthritis model

disease control (Fig. 5). Tubastatin and dexamethasone showed 59% and 66% inhibition of IL-6 in paw tissues of arthritic mice (Fig. 6). The scored histopathological findings of cartilage erosion, synovial hyperplasia and inflammation for Tubastatin and dexamethasone was in agreement with the clinical scorings and showed similar significant inhibitions of 71% and 100% respectively. They also resulted in a mild cartilaginous change like proteoglycan loss (Fig. 7a, b). The animals treated with dexamethasone produced significant decrease in body weight; whereas Tubastatin treated animals showed insignificant changes in body weight. Unlike dexamethasone, Tubastatin did not show immunosuppressant effect on spleen and thymus weights (Table 1). 4. Discussion

The intra-dermal immunization of mice in the tail with CFA and collagen type II results in arthritis. The first signs of arthritis development are visible between days 25 and 29 after immunization [8,19]. The clinical scores of the disease control group increased gradually after LPS administration and it reached maximum score of 9.8 ± 2.2 by day 36. Chronic treatment with Tubastatin at (30 mg/kg/day, i.p.) and dexamethasone at (0.1 mg/kg/day, p.o.) showed significant attenuation of arthritic clinical scores. The average clinical scores of the mice treated with Tubastatin and dexamethasone were 2.6 ± 0.98 and 0.75 ± 0.48 respectively which were lower than the vehicle treated (disease control) group and resulted in 73 % and 92 % significant inhibition of clinical scores as compared to

14

Clinical score

12 10 8 6 4 2

*

*

*

*

*

**

*

**

33

34

35

36

Normal control

Tubastatin (30 mg/kg i.p.)

Disease control

Dexamethasone (0.1 mg/kg p.o.)

60

Tubastatin (30 mg/kg i.p.) Dexamethasone (0.1mg/kg p.o.)

Normal Disease control

## IL-6 (pg/ml/mg of protein)

16

In this study, we report anti-inflammatory and anti-rheumatic effects of Tubastatin. In concordance with previous reports our in-house synthesized Tubastatin demonstrated potent and selective inhibitory activity against HDAC6 with an enzymatic IC50 value of 11nM [23]. The exact role of histone acetylation in progression of RA remains to be completely understood still there is enough evidence to support a strong role of HDAC inhibition as an anti-inflammatory agent thereby

50

40

30

20

*

*

10

0 28

29

30

31

32

37

38

0

Days Fig. 5. Effect of Tubastatin and dexamethasone on Clinical score of Collageninduced arthritic mice. The data represent Mean ± SEM of six mice. * p b 0.05, as compared to Disease control, ** p b 0.01, as compared to Disease control.

Fig. 6. Effect of tubastatin and dexamethasone on IL-6 in paw tissues of collagen-induced arthritic mice. The data represent Mean ± SEM of six mice. ##p b 0.01 as compared to normal control and *p b 0.05, as compared to disease control (one way ANOVA followed by Dunnett's multiple comparison test).

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a

Fig A

Fig C

Fig B

Fig D

b

Fig A

Fig C

Fig B

Fig D

Fig. 7. a. Effect of Tubastatin and dexamethasone on histopathology of Collageninduced arthritic mice. Fig A from normal control with a normal quiescent synovial (s) membrane. Fig B treated with vehicle alone showed articular cartilage erosion, synovial hyperplasia(s), pannus formation (arrow) and inflammatory cell infiltration. Fig C treated with Tubastatin (30 mg/kg/bw) showed reduced cartilage destruction and inflammation. Fig D dexamethasone (0.1 mg/kg) treated group showed no noticeable reduction in cartilage damage or pannus formation and inflammation. Note: Depicted images were histological representation from each group. Original magnification ×200. [Images captured from Nikon eclipse E200]. b. Effect of tubastatin and dexamethasone on cartilage of Collageninduced arthritic mice. Fig A Intact columns of chondrocytes deeply stained for glycosamingolycans with smooth articular surface (red colour marked with arrow). Fig B treated with vehicle showed depleted, irregular columns of chondrocyte with eroded uneven, articular surface and lack of staining due to loss of glycosaminoglycans (arrow). Fig C and Fig D represents treatment of Tubastatin (30 mg/kg/bw) & dexamethasone (0.1 mg/kg) respectively, showed intact cartilage with less intense staining of Safranin attributed to loss of glycosaminoglycans Note: Depicted images were histological representation from each group. Original magnification ×400. [Images captured from Nikon eclipse E200].

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Table 1 Effect of tubastatin and dexamethasone on spleen, thymus weight and % change in body weight of collagen-induced arthritic mice. Groups & treatment

Spleen weight (mg)

Thymus weight (mg)

Change in body weight (%)

Normal Control Disease Control Tubastatin (30 mg/kg, i.p.) Dexamethasone (0.1 mg/kg, p.o.)

109.5 226.4 182.0 52.50

37.50 12.20 17.50 6.50

5.44 −25.03 −1.07 −13.9

± ± ± ±

1.50 11.03## 7.0 13.50⁎⁎

± ± ± ±

0.50 1.5## 2.5 0.50

± ± ± ±

0.32 5.22 0.23 4.92

The data was expressed as mean ± SEM. (n = 6) and statistical analysis was done by one way ANOVA followed by Dunnett's multiple comparison test. % change in body weight with reference to day 22 (appearance of inflammation) till the day of euthanization (day 36). ## p b 0.01, compared with normal control. ⁎⁎ p b 0.01 compared with disease control.

suggesting a role of HDAC inhibitors in treatment of RA [3,7,8]. Literature evidence supports the fact that synovial fibroblasts play an important role in RA pathogenesis and work actively to drive joint destruction [24–26]. HDAC6 inhibition induces hyperacetylation of α-tubulin which was also observed in our study with THP-1 cells (data not shown). Recently, it has also been reported that specific HDAC6 inhibition leads to impairment of synovial fibroblast motility thereby inhibiting cell motility and probably metastasis and hence might be beneficial in arrest of RA pathogenesis. [23,27]. The anti-rheumatic mechanism for the HDACi could also be mediated through inhibition of the NF-kB pathway. The transcriptional factor NF-kB is a pivotal regulator of inflammation in RA [28–30]. The secretion of inflammatory mediators by macrophages is involved in both the innate and adaptive immune responses of autoimmune diseases, such as rheumatoid arthritis (RA) [31]. LPS, is known to activate a number of cellular signals in macrophages [32]. Upon activation, macrophages produce large amounts of nitric oxide (NO) and pro-inflammatory cytokines (TNF-α, IL-1β and IL-6). A number of animal studies have demonstrated increases in production of NO in response to a variety of microbial products [33]. Through further exploration of innate inflammatory responses in RAW 264.7 and THP-1 cells, we have learned that NO production and cytokine secretion are modulated by HDACi in LPS-induced inflammatory responses in macrophages. We found that the degree of inhibition of cytokines (TNF-α, IL-6) and NO was comparable with the published reports for SAHA and dexamethasone [34,35]. Our experimental results showed that Tubastatin significantly inhibited the release of TNF-α and IL-6 from THP-1 cells post LPS stimulation in a dose dependent manner. This effect was corroborated with IL-6 inhibition in paw tissue samples of collagen induced arthritic mice which in turn supports the anti-inflammatory property of Tubastatin. To observe anti-inflammatory effects of Tubastatin at cellular level to in vivo condition, we carried out efficacy profile of Tubastatin in animal models of inflammation and rheumatoid arthritis. FCA induced paw inflammation animal model is one of the widely used rat model to study the effect of new chemical entities on inflammation. We showed that administration of selective class IIb HDAC6 inhibitor; Tubastatin significantly reduced the paw edema induced by intraplantar administration of FCA indicating its anti-inflammatory effect. Dexamethasone was used as positive control for the study. Anti-rheumatic activity of Tubastatin was assessed in collagen induced arthritis DBA1 mouse model. Tubastatin showed significant 73% inhibition of arthritic clinical scores, which is comparable to inhibition produced by dexamethasone (92%), positive control for the study. The efficacy was very well translated histopathologically and the percent inhibition produced by Tubastatin and dexamethasone was 71% and 100% respectively which in is in line with the inhibition of arthritic clinical scores. We observed significant inhibition of synovial hyperplasia; cartilage degradation and inflammation in histopathological haemotoxylin and eosin stained sections. These results further demonstrate the anti-rheumatic activity of Tubastatin. RA is a chronic, systemic inflammatory disease which requires a life-long therapy [24,36]. Clinically, loss of body mass is associated with RA [37–40] which is probably due to inflammatory cytokines, pain, loss of appetite, increased energy expenditure and enhanced protein catabolism [37,40–42]. Thus, the changes in body weight after the onset of

arthritis could be used as a nonspecific end point to evaluate the prophylactic efficacy of HDACi. Adverse effects of pan HDACi might be a more serious issue for application to RA; however class IIb selective HDAC6i Tubastatin did not show any significant changes in body weight. Further, the immunosuppressant activity observed with dexamethasone as decrease in spleen and thymus weights were lower with Tubastatin treatment. No significant changes observed in body weight, spleen and thymus weight indicate the advantage of Tubastatin over immunosuppressants and disease modifying anti-rheumatoid drugs. The toxicity of anti rheumatic drugs is one of the major reasons that prompt patients to switch between different therapies [9,36,43]. Thus the development of new generations of selective HDACi may reduce clinical toxicities and side effects observed with pan-HDACi. All together, the current study confirms that Tubastatin selective HDAC6i exerts clear anti-inflammatory and anti-rheumatic activities in animal models possibly by inhibiting the release of pro-inflammatory cytokines like IL-6 and TNF-alpha and chemokines like NO. These findings support the role of HDAC6 inhibition in the pathogenesis of RA. Further detailed mechanistic studies between HDAC6 inhibition and RA pathogenesis shall be valuable for the development of novel disease modifying agent for RA. Acknowledgement Authors acknowledge the generous support received from management of Orchid Chemicals and Pharmaceuticals limited, Chennai, Tamil Nadu, India for this research work. References [1] Johnstone RW. Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat Rev Drug Discov 2002;1:287–99. [2] Piekarz R, Bates S. A review of depsipeptide and other histone deacetylase inhibitors in clinical trials. Curr Pharm Des 2004;10:2289–98. [3] Blanchard F, Chipoy C. Histone deacetylase inhibitors: new drugs for the treatment of inflammatory diseases? Drug Discov Today 2005;10:197–204. [4] Chung YL, Lee MY, Wang AJ, Yao LF. A therapeutic strategy uses histone deacetylase inhibitors to modulate the expression of genes involved in the pathogenesis of rheumatoid arthritis. Mol Ther 2003;8(5). [5] Glauben R, Batra A, Fedke I, Zeitz M, Lehr HA, Leoni F, et al. Histone hyperacetylation is associated with amelioration of experimental colitis in mice. J Immunol 2006;176(8): 5015–22. [6] Leoni F, Zaliani A, Bertolini G, Porro G, Pagani P, Pozzi P, et al. The antitumor histone deacetylase inhibitor suberoylanilide hydroxamic acid exhibits antiinflammatory properties via suppression of cytokines. Proc Natl Acad Sci 2002;99:2995–3000. [7] Leo ABJ, Flavio L, Sajeda M, Paolo M. Inhibition of HDAC activity by ITF2357 ameliorates joint inflammation and prevents cartilage and bone destruction in experimental arthritis. Mol Med 2011;17(5–6):391–6. [8] Lin HS, et al. Anti-rheumatic activities of histone deacetylase (HDAC) inhibitors in vivo in collagen-induced arthritis in rodents. Br J Pharmacol 2007;150:862–72. [9] American College of Rheumatology (ACR) subcommittee on rheumatoid arthritis guidelines. Guidelines for the management of rheumatoid arthritis. Arthritis Rheum 2002;46:328–46. [10] Haringman JJ, Ludikhuize J, Tak PP. Chemokines in joint disease: the key to inflammation? Ann Rheum Dis 2004;63:1186–94. [11] Lee DM, Weinblatt ME. Rheumatoid arthritis. Lancet 2001;358:903–11. [12] Firestein GS. Evolving concepts of rheumatoid arthritis. Nature 2003;423:356–61. [13] Chan AC, Carter PJ. Therapeutic antibodies for autoimmunity and inflammation. Nat Rev Immunol 2010;10:301–16. [14] Vichai V, Kirtikara K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc 2006;1(3):1112–6.

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