International Immunopharmacology 16 (2013) 72–78

Contents lists available at SciVerse ScienceDirect

International Immunopharmacology

j ourna l homepage: www.e lsev ie r .com/ locate / in t imp

Tubastatin, a selective histone deacetylase 6 inhibitor showsanti-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 Department of Biology, Drug Discovery Research, Orchid Chemicals and Pharmaceuticals Ltd., Old Mahabalipuram Road, Sozhanganallur, Chennai – 600119, Indiab Department of Medicinal Chemistry, Drug Discovery Research, Orchid Chemicals and Pharmaceuticals Ltd., Old Mahabalipuram Road, Sozhanganallur, Chennai – 600119, Indiac Infection iScience, AstraZeneca India Pvt. Ltd. Hebbal, Bangalore, India

⁎ Corresponding author. Tel.: +91 98 4024 1027; faxE-mail addresses: santoshv@orchidpharma.com, san

ipm@orchidpharma.com (S. Vishwakarma).1 The second and third authors contributed equally to

considered as joint second authors.

1567-5769/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.intimp.2013.03.016

a b s t r a c t

a r t i c l e i n f o

Article history:Received 10 December 2012Received in revised form 22 January 2013Accepted 15 March 2013Available online 27 March 2013

Keywords:TubastatinHDAC6 inhibitorRheumatoid arthritisAnti-inflammatory

Epigenetic modifications represent a promising new approach to modulate cell functions as observed in autoim-mune diseases. Emerging evidence suggests the utility of HDAC inhibitors in the treatment of chronic immuneand inflammatory disorders. However, class and isoform selective inhibition of HDAC is currently favored as itlimits 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 disor-ders. The present study was carried out to study the potential anti-inflammatory and anti-rheumatic effects ofHDAC6 selective inhibitor Tubastatin. Tubastatin, a potent human HDAC6 inhibitor with an IC50 of 11 nM showedsignificant inhibition of TNF-α and IL-6 in LPS stimulated human THP-1macrophages with an IC50 of 272 nM and712 nM respectively. Additionally, Tubastatin inhibited nitric oxide (NO) secretion in murine Raw 264.7 macro-phages dose dependently with an IC50 of 4.2 μM and induced α-tubulin hyperacetylation corresponding toHDAC6 inhibition in THP-1 cells without affecting the cell viability. Tubastatin showed significant inhibition ofpaw volume at 30 mg/kg i.p. in a Freund's complete adjuvant (FCA) induced animal model of inflammation. Thedisease modifying activity of Tubastatin was also evident in collagen induced arthritis DBA1 mouse model at30 mg/kg i.p. The significant attenuation of clinical scores (~70%) by Tubastatinwas confirmed histopathologicallyand was found comparable to dexamethasone (~90% inhibition of clinical scores). Tubastatin showed significantinhibition of IL-6 in paw tissues of arthritic mice. The present work has demonstrated anti-inflammatory and an-tirheumatic effects of a selective HDAC6 inhibitor Tubastatin.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Histone Deacetylase inhibitors (HDACi) were initially studied fortheir ability to increase gene expression, however today there isincreasing number of orally active, synthetic HDACi being primarilydeveloped to treat cancer [1,2]. Recently HDACi have emerged as po-tent anti-inflammatory agents [3]. Histone hyperacetylation results inup-regulation of cell cycle inhibitors (p21Cip1, p27Kip1, and p16INK4,repression of inflammatory cytokines [interleukin (IL)-1, IL-8, tumornecrosis factor-α (TNF-α), down-regulation of immune stimulators(IL-6, IL-10, and CD154) [4]. HDACi like MS-275, Trichostatin A andSuberoylanilide hydroxamic acid (SAHA) have emerged to be potent

: +91 44 2450 1396.toshlmcp@gmail.com,

the manuscript and should be

rights reserved.

anti-inflammatory agents in murine and human monocytes [5,6]. PanHDACi like Givinostat (ITF-2357); SAHA and MS-275 have beenshown to ameliorate disease symptoms in animal models of Rheuma-toid Arthritis (RA) [7,8].

Rheumatoid arthritis (RA) is characterized by persistent synovitis,systemic inflammation, and autoantibodies particularly to rheumatoidfactor and citrullinated peptide. About 1% to 2% of the world’s popula-tion is affected by RA and women are three times more likely thanmen to develop RA between the ages of 35 and 50 years. RA is a com-mon chronic autoimmune inflammatory disease [9,10]. The inflamedsynovium is central to the pathogenesis of RA and formation of tumor-like synovial tissue, called ‘pannus’ is a characteristic feature of RA[11,12]. Traditionally available treatments of RAhave includedmainly dis-ease modifying anti-rheumatic drugs (DMARD's). Approved treatmentsfor RA include non-steroidal anti-inflammatory drugs, antimetabolitessuch as methotrexate and leflunomide, corticosteroids like dexametha-sone, and various biologics. Unfortunately, therapies targeting the diseaseare still in their infancy and have various undesirable side effects [13].

73S. Vishwakarma et al. / International Immunopharmacology 16 (2013) 72–78

Most of the promising HDACi showing pro-inflammatory propertiestarget multiple HDAC (Histone Deacetylase) isoforms. Pan-HDAC inhi-bition might accompany several undesirable side effects such asfatigue, diarrhea, nausea, neutropenia and thrombocytopenia. On thecontrary HDACi highly selective for individual isoforms, may exhibitreduced side effects compared to the pan-HDACi, while still retainingcapability for target modulation. Recently HDAC6, a member of theHDAC family, whose major substrate is α-tubulin, has been convinc-ingly implicated in the pathogenesis of inflammatory disorders. Thepresent work was carried out to study the potential anti-inflammatoryand anti-rheumatic effects of Tubastatin.

In the present study, Tubastatin; a selective HDAC6 inhibitorprevented the release of pro-inflammatory cytokines like TNF-α andIL-6 from human monocytes. Further, in animal models, Tubastatintreatment significantly improved paw edema in the Freund's com-plete adjuvant induced paw inflammation and anti-rheumatic activi-ty in collagen induced arthritis mouse model with amelioration in thearthritic clinical scores that was corroborated histopathologically. Allthese findings strongly support the fact that HDAC6 selective inhibi-tion has therapeutic potential for the treatment of RA.

2. Materials and methods

2.1. Cell lines and cell culture

Human acute monocytic leukemia cell line THP-1 (TIB-202) andmurine macrophage cell line RAW 264.7 (TIB-71) was purchased fromAmerican Type Culture Collection (ATCC; VA, USA). THP-1 cells werecultured in RPMI-1640 medium (Invitrogen, CA, USA) supplementedwith 10% (V/V) heat inactivated fetal bovine serum (FBS; Invitrogen,USA). RAW 264.7 cells were cultured in DMEM (Invitrogen, USA)supplementedwith 10% Fetal Bovine Serum. Cell culturemediawas fur-ther supplemented with penicillin/streptomycin. All cell cultures weremaintained at 37oC in a humidified atmosphere with 5% CO2.

2.2. Animals

DBA1mice (Female, 6–8 weeks, 20–28 g) andWistar rats (male, 6–8 weeks, 200–225 g) were obtained from animal facility, Drug Discov-ery Research; Orchid Chemicals and Pharmaceuticals Ltd., (Chennai,India). Animals were maintained in controlled environment with tem-perature (22 ± 2 °C), humidity (44–56%) and 12 h light–dark cycleand were provided with standard diet (Nutrilab Rodent) and waterad libitum. The study was approved by Institutional Animal EthicsCommittee.

(DBA1mice:-Protocol.No.18/IAEC-03/PPK/2010;Wistar rats:-ProtocolNo.13 /IAEC-01/PCP/2012)

2.3. Drugs and chemicals

Mycobacterium tuberculosis H37Ra, bovine collagen Type II, CompleteFreund's Adjuvant (CFA) and incomplete Freund's adjuvant (IFA) wereobtained from Difco Laboratories (Detroit, MI). Dexamethasone and LPSwas received from Sigma–Aldrich, St Louis, MO, USA. Tubastatin andSAHA were synthesized by the department of Medicinal Chemistry(Drug Discovery Research) at Orchid Chemicals and PharmaceuticalsLtd., (Chennai, India). The test compound was handled and stored asper the specifications recommended for the test compound. Tubastatinwas solubilized in 10% Dimethyl sulfoxide (DMSO) 10% Polyethyleneglycol (PEG) 400 and 80% (40% of hydroxy propyl beta cyclodextrin)and dexamethasone was prepared as a suspension in 0.25% sodiumcarboxmethylcellulose. For in-vitro studies compounds were dissolvedin DMSO only.

2.4. Cytotoxicity assay by sulforhodamine (SRB) method

RAW 264.7 and THP-1 cells (10,000/well) were seeded in 96-wellplate and treated with test compounds at various concentrations for24 h and cell viability was assessed using SRB assay. The colorimetricreadings were measured at 530 nm in a spectrophotometer (Tecan;Infinite M1000). % survival was calculated by comparing the treatedcells with untreated cells [14].

2.5. Cytokine secretion assay

ELISA kits for human TNF-α and IL-6 was purchased from R&Dsystems (USA). THP1 cells (10,000/well) were seeded in 24-wellplates along with 32nM phorbol 12-myristate 13-acetate and incu-bated for 24 h. Post incubation culture media was exchanged withfresh media and incubated for further 24 h. Next, cells were treatedwith Tubastatin, dexamethasone or SAHA at specified concentrationsfor 24 h; followed by LPS stimulation (10 ng/ml LPS for 4 h forTNF-α stimulation and 100 ng/ml LPS for 8 h for IL-6 stimulation).The conditioned media was collected and ELISA was performedwith the same as per the manufacturer's instructions. The opticaldensity was recorded using a multi-plate reader at 450 nm [15,16].

2.6. NO secretion

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 followedby LPS stimulation (100 ng/ml) for 24 h. Nitric oxide content was es-timated in conditioned media using Greiss reagent (1% sulfanilamideand 0.1% naphthylethylenediamine dihydrochloride in 2.5% phospho-ric acid) the mixture was incubated at room temperature for 10 min.Upon development of purple/magenta color, absorbance was mea-sured at 570 nm within 30 min. The quantity of nitrite was deter-mined from a sodium nitrite standard curve and the values obtainedwere represented graphically [17].

2.7. Freund's complete adjuvant (FCA) induced paw inflammation inWistar rats

Animals were randomized into 4 groups (n = 6) on the basis oftheir paw volume. Group I and II served as normal control and diseasecontrol. Group II received the vehicle, Group III received Tubastatin30 mg/kg/day i.p. for 5 days, whereas Group IV received dexametha-sone 1 mg/kg p.o. on day 5. All animals, except normal were injectedwith 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. Treat-ment group received FCA1 hour after dosing Tubastatin and dexameth-asone. Paw volume was measured at 2, 6 and 24 hours after FCAInjection using LE 7500, Panlab, Spain instrument [18].

2.8. Induction of Collagen induced Arthritis (CIA) in DBA1 mice andExperimental design

Primary immunization of the animalswith antigenwas done onday 0,a booster injection was administered on day 21 and to accelerate theonset of arthritic clinical scores; LPS (50 μg/animal) was injected intra-peritoneally on day 28. on day 0 commercially available collagen type IIin 0.05 M acetic acid to a concentration of 2 mg/ml and M. tuberculosisH37 Ra 5 mg/mL of CFA was used for immunization. Each animal wasinjected 100 μl (consisting of 100 μg of collagen + 250 μg CFA intra-dermally at the base of the tail. On day 21 post primary immunization,mice were boosted with collagen Type II (2 mg/mL); emulsified withequal volume of IFA using homogenizer. The animals were injected100 μl intradermally, proximal to the primary injection site. Lipopolysac-charide (LPS) 50 μg prepared in 100 μl of PBS was administered intra-

100 1000 10000 1000000

25

50

75

100

125

THP-1 Cells

RAW 264.7 Cells

Tubastatin, nM

Cel

l Su

rviv

al, %

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

74 S. Vishwakarma et al. / International Immunopharmacology 16 (2013) 72–78

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 treatmentwas initiated [19].

Animals were randomized to four groups of 6 animals each. The ani-mals of the first group were not immunized (normal control); whereasthe groups 2 to 4 were immunized with bovine type II collagen. Group2 animals were treated with vehicle (disease control). Animals of theGroup 3 were treated orally with dexamethasone (0.1 mg/kg, q.d.).Group 4 animals received intra-peritoneal injection of Tubastatin admin-istered 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 re-gimewas initiated. On day 37, animals were sacrificed; the right fore andhind paws were removed from each animal for histological analysis andleft fore and hind paws were collected for tissue cytokines. The spleenand thymus weights were measured. The histological samples wereparaffin-embedded, sectioned, stained with hematoxylin and eosin andscored as described [20]. Safranin O was used to identify the loss of pro-teoglycans in the articular [21].

2.9. IL-6 estimation in paw tissue

Estimation of IL-6 in the frozen tissues (whole joints includingsynovium, adjacent tissues and bones) were pulverized using a mor-tar and pestle filled with liquid nitrogen. The pulverized samplesweretransferred to eppendorf tubes filled with 200 μl of T-PER (TissueProtein Extraction Reagent) and homogenized using a Polytron tissuehomogenizer. Mouse joint homogenates were centrifuged for 10 minat 500 g at 4 °C. Supernatant was separated and used for protein esti-mation (BSA method) and IL-6 analysis was carried out using GEHealthcare ELISA kits according to the manufacturer's instructions.Data was expressed as IL-6 pg/ml of 1 mg of protein [22].

2.10. Statistical analysis

All the results of in vitro data were expressed as mean ± standarddeviation (S.D), while the in vivo resultswere expressed asmean ± stan-dard error of means (S.E.M.). The data was analyzed by one way ANOVAfollowed by Dunnett’s multiple comparison tests using Graph pad prismversion 4.0. In all the tests p b 0.05 was considered as statisticallysignificant.

1 10 100 1000 100000

10

20

30

40

50

60

70

80

90

100

110

Tubastatin

SAHA

Dexamethasone

Tubastatin

SAHA

Dexamethasone

TNF- IL-6

HDAC Inhibitor, nM

Cyt

oki

ne

Inh

ibit

ion

, %

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

3. Results

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

Tubastatin was able to inhibit TNF-α and IL-6 production fromLPS-stimulated THP-1 cells in a dose dependent manner with an IC50 of272.5 nM and 712.9 nM respectively. Dexamethasone was used as posi-tive control for the study. Dexamethasone and pan HDAC inhibitorSAHA at 1 μM completely inhibited TNF-α and IL-6 secretion in thesecells (Fig. 1). Further, the cell survival/proliferation was unaffected bythe compound treatment (Fig. 2). These results clearly point towardsthe efficient inhibition of pro-inflammatory cytokines by Tubastatin inLPS-induced THP-1 cells.

3.2. Tubastatin attenuates NO production in RAW 264.7 cells

RAW 264.7 cells were used to study the effect of Tubastatin on LPSinduced NO secretion. Tubastatin treatment showed a dose-dependentattenuation of NO secretion post LPS stimulation. Treatment with 10 μMof Tubastatin showed potent 66% inhibition of NO secretion where as1 μM SAHA showed 89.5% inhibition while 10 μM dexamethasone wasable to inhibit 36% of NO secretion (Fig. 3).

3.3. Anti-inflammatory activity of tubastatin

Injection of FCA into the hindpaws produced an increase in pawvol-ume to 2.02 ± 0.05 ml, 2.55 ± 0.07 ml and 2.84 ± 0.1 ml in Disease

10 100 1000 10000 1000000

10

20

30

40

50

60

70

80

90

100Tubastatin

SAHA

Dexamethasone

HDAC Inhibitor, nM

NO

Inh

ibit

ion

, %

Fig. 3. Effect of tubastatin onNO secretion inRAW264.7 cells. Values indicatemean ± SD,(n = 3).

1.0

1.5

2.0

2.5

3.0

2hr 6hr rh2rh42 6hr 4hr 2hr 6hr 24hr2hr 6hr 24hr

Normal Diseasecontrol

Tubastatin30 mg/kg i.p.

Dexamethasone1 mg/kg p.o.

##

##

##

*

**

**

* **

**

$

Paw

Vo

lum

e (m

l)

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).

Normal control Tubastatin (30 mg/kg i.p.)

75S. Vishwakarma et al. / International Immunopharmacology 16 (2013) 72–78

control group at 2, 6 and 24 h respectively as compared to normal con-trol (1.76 ± 0.03 ml). In-house experiments indicate administration of10%DMSO, 10% PEG 400 and 80% (40% of hydroxy propyl beta cyclodex-trin) and 0.25% CMC to disease control does not affect the progression ofdisease (Data not shown). Treatment with Tubastatin and Dexametha-sone 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 h2.23 ± 0.04 ml and 1.84 ± 0.02 ml (40.34 and 90.45% inhibition) andat 24 h 2.43 ± 0.07 ml and 2.31 ± 0.06 ml (38.58 and 49.54% inhibi-tion) as compared to disease control at respective time points (Fig. 4).

3.4. Anti-rheumatic effects of tubastatin and dexamethasone in DBA1mouse semi-therapeutic collagen induced Arthritis model

The intra-dermal immunization ofmice in the tail with CFA and colla-gen type II results in arthritis. The first signs of arthritis development arevisible between days 25 and 29 after immunization [8,19]. The clinicalscores of the disease control group increased gradually after LPS adminis-tration and it reached maximum score of 9.8 ± 2.2 by day 36. Chronictreatment with Tubastatin at (30 mg/kg/day, i.p.) and dexamethasoneat (0.1 mg/kg/day, p.o.) showed significant attenuationof arthritic clinicalscores. The average clinical scores of themice treatedwith Tubastatin anddexamethasone were 2.6 ± 0.98 and 0.75 ± 0.48 respectively whichwere lower than the vehicle treated (disease control) group and resultedin 73 % and 92 % significant inhibition of clinical scores as compared to

28 29 30 31 32 33 34 35 36 37 380

2

4

6

8

10

12

14

16 NormalDisease control

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

* * **

* *

**

*

*

Days

Clin

ical

sco

re

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

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, synovialhyperplasia and inflammation for Tubastatin and dexamethasonewas in agreement with the clinical scorings and showed similar sig-nificant inhibitions of 71% and 100% respectively. They also resultedin a mild cartilaginous change like proteoglycan loss (Fig. 7a, b). Theanimals treated with dexamethasone produced significant decreasein body weight; whereas Tubastatin treated animals showed insig-nificant changes in body weight. Unlike dexamethasone, Tubastatindid not show immunosuppressant effect on spleen and thymusweights (Table 1).

4. Discussion

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

0

10

20

30

40

50

60

Disease control Dexamethasone (0.1 mg/kg p.o.)

* *

##

IL-6

(p

g/m

l/mg

of

pro

tein

)

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

a

b

Fig A

Fig C Fig D

Fig B

Fig A

Fig C Fig D

Fig B

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 treatedwith vehicle alone showedarticular cartilage erosion, synovial hyperplasia(s), pannus formation (arrow)and inflammatory cell infiltration. Fig C treatedwith Tubastatin (30 mg/kg/bw) showedreduced 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 imageswere histological representation fromeach group. Originalmagnification×200. [Images captured fromNikon eclipse E200]. b. Effect of tubastatin anddexamethasone oncartilage of Collageninduced arthritic mice. Fig A Intact columns of chondrocytes deeply stained for glycosamingolycans with smooth articular surface (red colour marked with arrow). Fig Btreated 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 andFig 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 glycos-aminoglycans Note: Depicted images were histological representation from each group. Original magnification ×400. [Images captured from Nikon eclipse E200].

76 S. Vishwakarma et al. / International Immunopharmacology 16 (2013) 72–78

Table 1Effect 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 109.5 ± 1.50 37.50 ± 0.50 5.44 ± 0.32Disease Control 226.4 ± 11.03## 12.20 ± 1.5## −25.03 ± 5.22Tubastatin (30 mg/kg, i.p.) 182.0 ± 7.0 17.50 ± 2.5 −1.07 ± 0.23Dexamethasone (0.1 mg/kg, p.o.) 52.50 ± 13.50⁎⁎ 6.50 ± 0.50 −13.9 ± 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.

77S. Vishwakarma et al. / International Immunopharmacology 16 (2013) 72–78

suggesting a role of HDAC inhibitors in treatment of RA [3,7,8]. Litera-ture evidence supports the fact that synovialfibroblasts play an importantrole in RA pathogenesis and work actively to drive joint destruction[24–26]. HDAC6 inhibition induces hyperacetylation of α-tubulin whichwas also observed in our study with THP-1 cells (data not shown). Re-cently, it has also been reported that specific HDAC6 inhibition leads toimpairment of synovial fibroblast motility thereby inhibiting cell motilityand probably metastasis and hence might be beneficial in arrest of RApathogenesis. [23,27].

The anti-rheumatic mechanism for the HDACi could also bemediatedthrough inhibition of theNF-kB pathway. The transcriptional factor NF-kBis a pivotal regulator of inflammation in RA [28–30]. The secretion of in-flammatory mediators by macrophages is involved in both the innateand adaptive immune responses of autoimmune diseases, such as rheu-matoid arthritis (RA) [31]. LPS, is known to activate a number of cellularsignals in macrophages [32]. Upon activation, macrophages producelarge amounts of nitric oxide (NO) and pro-inflammatory cytokines(TNF-α, IL-1β and IL-6). A number of animal studies have demonstratedincreases in production of NO in response to a variety of microbial prod-ucts [33]. Through further exploration of innate inflammatory responsesin RAW 264.7 and THP-1 cells, we have learned that NO production andcytokine secretion aremodulated byHDACi in LPS-induced inflammatoryresponses in macrophages. We found that the degree of inhibition ofcytokines (TNF-α, IL-6) and NO was comparable with the published re-ports for SAHA and dexamethasone [34,35]. Our experimental resultsshowed that Tubastatin significantly inhibited the release of TNF-α andIL-6 from THP-1 cells post LPS stimulation in a dose dependent manner.This effect was corroborated with IL-6 inhibition in paw tissue sam-ples of collagen induced arthritic mice which in turn supports theanti-inflammatory property of Tubastatin.

To observe anti-inflammatory effects of Tubastatin at cellular level toin vivo condition, we carried out efficacy profile of Tubastatin in animalmodels of inflammation and rheumatoid arthritis. FCA induced pawinflammation animal model is one of the widely used rat model tostudy the effect of new chemical entities on inflammation. We showedthat administration of selective class IIb HDAC6 inhibitor; Tubastatinsignificantly reduced the paw edema induced by intraplantar administra-tion of FCA indicating its anti-inflammatory effect. Dexamethasonewas used as positive control for the study. Anti-rheumatic activity ofTubastatin was assessed in collagen induced arthritis DBA1 mousemodel. Tubastatin showed significant 73% inhibition of arthritic clinicalscores, which is comparable to inhibition produced by dexamethasone(92%), positive control for the study. The efficacywas verywell translatedhistopathologically and the percent inhibition produced by Tubastatinand dexamethasone was 71% and 100% respectively which in is in linewith the inhibition of arthritic clinical scores. We observed significantinhibition of synovial hyperplasia; cartilage degradation and inflamma-tion in histopathological haemotoxylin and eosin stained sections. Theseresults further demonstrate the anti-rheumatic activity of Tubastatin.

RA is a chronic, systemic inflammatory disease which requires alife-long therapy [24,36]. Clinically, loss of body mass is associated withRA [37–40] which is probably due to inflammatory cytokines, pain, lossof appetite, increased energy expenditure and enhanced protein catabo-lism [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 prophy-lactic efficacy of HDACi. Adverse effects of pan HDACi might be a moreserious issue for application to RA; however class IIb selective HDAC6iTubastatin did not show any significant changes in body weight. Further,the immunosuppressant activity observed with dexamethasone as de-crease in spleen and thymus weights were lower with Tubastatin treat-ment. No significant changes observed in body weight, spleen andthymus weight indicate the advantage of Tubastatin over immunosup-pressants and disease modifying anti-rheumatoid drugs.

The toxicity of anti rheumatic drugs is one of the major reasons thatprompt patients to switch between different therapies [9,36,43]. Thusthe development of new generations of selective HDACi may reduceclinical toxicities and side effects observed with pan-HDACi. All togeth-er, the current study confirms that Tubastatin selective HDAC6i exertsclear anti-inflammatory and anti-rheumatic activities in animal modelspossibly by inhibiting the release of pro-inflammatory cytokines likeIL-6 and TNF-alpha and chemokines like NO. These findings supportthe role of HDAC6 inhibition in the pathogenesis of RA. Further detailedmechanistic studies between HDAC6 inhibition and RA pathogenesisshall be valuable for the development of novel disease modifyingagent for RA.

Acknowledgement

Authors acknowledge the generous support received from manage-ment of Orchid Chemicals and Pharmaceuticals limited, Chennai, TamilNadu, India for this research work.

References

[1] Johnstone RW. Histone-deacetylase inhibitors: novel drugs for the treatment ofcancer. Nat Rev Drug Discov 2002;1:287–99.

[2] Piekarz R, Bates S. A review of depsipeptide and other histone deacetylase inhibitorsin clinical trials. Curr Pharm Des 2004;10:2289–98.

[3] Blanchard F, ChipoyC.Histonedeacetylase inhibitors: newdrugs for the treatment ofinflammatory diseases? Drug Discov Today 2005;10:197–204.

[4] Chung YL, Lee MY, Wang AJ, Yao LF. A therapeutic strategy uses histone deacetylaseinhibitors tomodulate the expression of genes involved in the pathogenesis of rheuma-toid arthritis. Mol Ther 2003;8(5).

[5] Glauben R, Batra A, Fedke I, Zeitz M, Lehr HA, Leoni F, et al. Histone hyperacetylation isassociated 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 histonedeacetylase inhibitor suberoylanilide hydroxamic acid exhibits antiinflammatoryproperties via suppression of cytokines. Proc Natl Acad Sci 2002;99:2995–3000.

[7] Leo ABJ, Flavio L, SajedaM, PaoloM. Inhibition of HDAC activity by ITF2357 amelioratesjoint inflammation and prevents cartilage and bone destruction in experimental arthri-tis. Mol Med 2011;17(5–6):391–6.

[8] Lin HS, et al. Anti-rheumatic activities of histone deacetylase (HDAC) inhibitors invivo in collagen-induced arthritis in rodents. Br J Pharmacol 2007;150:862–72.

[9] American College of Rheumatology (ACR) subcommittee on rheumatoid arthritisguidelines. Guidelines for the management of rheumatoid arthritis. Arthritis Rheum2002;46:328–46.

[10] Haringman JJ, Ludikhuize J, Tak PP. Chemokines in joint disease: the key to inflamma-tion? 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.

78 S. Vishwakarma et al. / International Immunopharmacology 16 (2013) 72–78

[15] Laurence H, Karima B, Philippe R, Ravi M, Palaniyandi R, Frank T, et al. Signaling path-ways involved in LPS induced TNFalpha production in human adipocytes. J Inflamm2010;7:1–12.

[16] Uma S, James T, Senthil KV, Sridevi D, Ishwarlal J. Development of an in vitroscreening assay to test the anti-inflammatory properties of dietary supplementsand pharmacologic agents. Clin Chem 2005;51(12):2252–6.

[17] Weon JY, Young MH, Sang SK, Byoung SY, Ji YM, Jong SB, et al. Suppression ofpro-inflammatory cytokines, iNOS, and COX-2 expression by brown algae Sargassummicracanthum in RAW 264.7 macrophages. EurAsian. J Biosci 2009;3:130–43.

[18] SeedMP, Gardner CR. The modulation of intra-articular inflammation, cartilage ma-trix and bone loss in mono-articular arthritis induced by heat-killed Mycobacteriumtuberculosis. Inflammopharmacology 2005;12(5–6):551–67.

[19] Nishida K, Komiyama T, Miyazawa S, Shen ZN, Furumatsu T, Doi H, et al. Histonedeacetylase inhibitor suppression of autoantibody-mediated arthritis in micevia regulation of p16INK4a and p21(WAF1/Cip1) expression. Arthritis Rheum2004;10:3365–76.

[20] Bancroft, Gamble Marilyn. Theory and Practice of Histological Techniques. 6th ed.Churchill Livingstone Elsevier Health Sciences; 2008. p. 333.

[21] Mohan, et al. Application of in vivo micro-computed tomography in the temporalcharacterisation of subchondral bone architecture in a rat model of low-dosemonosodium iodoacetate-induced osteoarthritis. Arthritis Res Ther 2011;13:R210.

[22] Rioja I, Bush KA, Buckton JA, DicksonMC, Life PF. Joint cytokine quantification in tworodent arthritis models: kinetics of expression, correlation of mRNA and proteinlevels and response to prednisolone treatment. Clin Exp Immunol 2004;137:65–73.

[23] Butler KV, Kalin J, Brochier C, Vistoli G, Langley B, Kozikowski AP. Rational design andsimple chemistry yield a superior, neuroprotective HDAC6 Inhibitor, tubastatin A.J Am Chem Soc 2010;132(31):10842–6.

[24] Lee DM, Weinblatt ME. Rheumatoid arthritis. Lancet 2001;358:903–11.[25] Huber LC, Distler O, Tarner I, Gay RE, Gay S, Pap T. Synovial fibroblasts: key players

in rheumatoid arthritis. Rheumatology 2006;45:669–75.[26] Firestein GS. Evolving concepts of rheumatoid arthritis. Nature 2003;423:356–61.[27] Agustin RG, Tara L, Alan KI, Tiffany SW, Cecilia F, Sakamoto KM. Role of the

aggresome pathway in cancer: targeting histone deacetylase 6–dependent proteindegradation. Cancer Res 2012;2008(68):2557–60.

[28] Feldmann M, Brennan FM, Maini RN. Role of cytokines in rheumatoid arthritis.Annu Rev Immunol 1996;14:397–440.

[29] Makarov SS. NF-kappa B, in rheumatoid arthritis: a pivotal regulator of inflamma-tion, hyperplasia, and tissue destruction. Arthritis Res 2001;3:200–6.

[30] Firestein GS. NF-kappa B: holy grail for rheumatoid arthritis? Arthritis Rheum2004;50:2381–6.

[31] Stoy N.Macrophage biology and pathobiology in the evolution of immune responses: afunctional analysis. Pathobiology 2001;69:179–211.

[32] Guha M, Mackman N. LPS induction of gene expression in human monocytes. CellSignal 2001;13:85–94.

[33] Chen LC, Pace JL, Russell SW, Morrison DC. Altered regulation of inducible nitricoxide synthase expression in macrophages from senescent mice. Infect Immun1996;64(10):4288–98.

[34] Choo QY, Ho PC, Tanaka Y, Lin HS. Histone deacetylase inhibitors MS-275 and SAHAinduced growth arrest and suppressed lipopolysaccharide-stimulated NF-kappaBp65 nuclear accumulation in human rheumatoid arthritis synovial fibroblastic E11cells. Rheumatology 2010;49(8):1447–60.

[35] Yoon WJ, Lee NH, Hyun CG. Limonene suppresses lipopolysaccharide-inducedproduction of nitric oxide, prostaglandin E2, and pro-inflammatory cytokines inRAW 264.7 macrophages. J Oleo Sci 2010;59(8):415–21.

[36] Bansback NJ, Regier DA, Ara R, Brennan A, Shojania K, Esdaile JM, et al. An overview ofeconomic evaluations for drugs used in rheumatoid arthritis: focus on tumour necrosisfactor-alpha antagonists. Drugs 2005;65:473–96.

[37] Rall LC, Roubenoff R. Rheumatoid cachexia: metabolic abnormalities, mechanismsand interventions. Rheumatology 2004;10:1219–23.

[38] Sims NA, Green JR, GlattM, Schlict S, Martin TJ, GillespieMT, et al. Targeting osteoclastswith zoledronic acid prevents bone destruction in collagen-induced arthritis. ArthritisRheum 2004;50:2338–46.

[39] Jou IM, Shiau AL, Chen SY,Wang CR, Shieh DB, Tsai CS, et al. Thrombospondin 1 as aneffective gene therapeutic strategy in collagen-induced arthritis. Arthritis Rheum2005;52:39–44.

[40] Shelton DL, Zeller J, Ho WH, Pons J, Rosenthal A. Nerve growth factor mediateshyperalgesia and cachexia in autoimmune arthritis. Pain 2005;116:8–16.

[41] Argiles JM, Lopez-Soriano FJ. Catabolic proinflammatory cytokines. Curr Opin ClinNutr Metab Care 2002;1:245–51.

[42] Walsmith J, Abad L, Kehayias J, Roubenoff R. Tumor necrosis factor-alpha production isassociated with less body cell mass in women with rheumatoid arthritis. J Rheumatol2004;31:23–9.

[43] Klinkhoff A. Biological agents for rheumatoid arthritis. Drugs 2004;64:1267–83.