The extraction and preparation of S. indicus was performed as described previously [12] . Various extracts were evaluated for their anti-TNF activity. The methanolic extract was found to be the most potent (data not shown). This extract was further evaluated for its anti-inflammatory potential, and will henceforth be referred to as Sphira (NPS31807).

The anti-inflammatory potential of Sphira was evaluated using various in vitro screening models of inflammation. Blood was collected from healthy donors after obtaining Independent Ethics Committee approval and written informed consent. Briefly, peripheral blood was collected in potassium EDTA vacutainer tubes (BD Biosciences). hPBMC were isolated by density gradient centrifugation using Histopaque-1077 solution (Sigma Aldrich; St. Louis, MO). Isolated hPBMC were re-suspended in Rosewell Park Memorial Institute (RPMI) 1640 culture medium (Sigma Aldrich) containing 10% heat inactivated fetal bovine serum (FBS; JRH), 100 U/ml penicillin (Sigma Chemical Co.; St. Louis, MO) and 100 µg/ml streptomycin (Sigma Chemical Co.). The hPBMCs were uniformly plated in 96-well tissue culture plates at a seeding density of 1 × 10⁶ cells/ml. Cells were then treated to different concentrations of Sphira (0.03 - 100 µg/ml) dissolved in DMSO (the final concentration of DMSO maintained at 0.5%), or 0.5% DMSO (control), and incubated for 30 minutes. Incubation in all the in vitro screening assays was in a humidified atmosphere at 37˚C, 5% CO₂.

Cytokine release assay: This assay measures LPS-induced cytokine release from hPBMCs, specifically to evaluate TNF-α inhibition. The procedure followed was similar to that described previously [13] . After being exposed to the test compounds, 1 µg/ml LPS (Escherichia coli 0127:B8, Sigma Chemical Co., St. Louis, MO) was added per well to stimulate cytokine production. The cells were further incubated for 5 h, following which supernatants were collected, stored at −70˚C and assayed later for TNF-α, IL-1β, IL-6 and IL-8 by ELISA (OptiEIA ELISA sets, BD Biosciences, Pharmingen). In all experiments, a parallel plate was run to ascertain the toxicity of test extracts. The toxicity was determined using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-car- boxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium) reagent.

IL-17 release assay: After treatment with test compounds, the cells were stimulated with 25 ng/ml of PMA (phorbol 12-myristate 13-acetate; SIGMA) and 1 µM ionomycin. The plates were further incubated for 48 h, after which the supernatant from the cells were collected and ELISA was performed to detect the levels of IL-17.

The ability of Sphira to inhibit the spontaneous release of cytokines from freshly isolated human synovial tissue cells was determined in a manner similar to that described by Brennan et al. [14] . After informed consent and Independent Ethics Committee approval, synovial tissue was obtained from RA patients undergoing knee replacement surgery. The tissue was minced and digested in RPMI medium containing 100 U/ml penicillin-G, 100 µg/ml streptomycin, 50 ng/ml amphotericin B (GIBCO), 1.33 mg/ml collagenase Type I (Worthington Biochemical Corporation, New Jersey), 0.5 µg/ml DNase Type I (Sigma Aldrich) and 8.33 U/ml heparin (Biological E. Limited, India) for 3 h at 37˚C, 5% CO₂. Isolated cells were uniformly plated in 96-well tissue culture plates at a seeding density of 1 × 10⁶ cells/ml and exposed to various concentrations of Sphira (0.03 - 100 µg/ml) dissolved in DMSO. The plates were incubated for 16 h. Subsequently, the supernatants were harvested and stored at −70˚C. The amounts of TNF-α, IL-1β, IL-6 and IL-8 in the supernatants were assayed using OptiEIA ELISA sets (BD BioSciences Pharmingen). The protocol followed was as per manufacturer’s instructions.

The anti-psoriatic potential of Sphira was evaluated in an in vitro screening model targeting p40, a subunit shared by human interleukin-12 and human Interleukin-23 (IL-12/IL-23 p40). Differentiated THP-1 cells were treated with varying concentrations of Sphir aranging from 0.1 to 100 µg/ml, and later on with a combination of TNF-α and IFN-γ. The levels of IL-12/IL-23p40, IL-23p19 and IL-12p70 were estimated by ELISA [15] .

CEM-κB (a kind gift from Dr. Shigeki Miyamoto, University of Wisconsin, Madison, WI) is a CEM cell line transfected with κB binding element linked to green florescence protein (GFP) promoter. CEM-κB was maintained in RPMI-1640 supplemented with 10% FBS and 0.1% G418 (Sigma Aldrich). The experiment was done as described previously [16] . Briefly, the cells were seeded at a density of 50,000 cells/ml, in a 6-well plate. The cells were treated with 0.1, 0.5, 1, 3, 10 and 30 µg/ml of Sphira dissolved in DMSO, 0.5% DMSO (vehicle control) or BAY 11-7082, an IKKα inhibitor (positive control), and incubated for 30 minutes. The cells were then stimulated with 0.5 ng/ml TNF-α and incubated for 16 h. After 16 h, the shift in NFκB expression was measured using BD FACS Calibur. The GFP fluorescence measured is directly proportional to NFκB expression.

The surface expression of endothelial cell adhesion molecules was quantified using cell ELISA, as described previously [17] . Briefly, confluent HUVECs (Cascade biologics) in 96-well fibronectin-coated plates were pretreated with various concentrations of Sphira for 30 minutes, before being stimulated with 1 ng/mL TNF-α for the indicated time. The expressions of ICAM-1 and VCAM-1 were evaluated after TNF-α stimulation for 4 h, and expression of E-selectin was evaluated after 6 h of stimulation. The cells were fixed with 1% paraformaldehyde and blocked using bovine serum albumin (2% in DPBS). The cells were then washed and incubated with monoclonal mouse anti-human ICAM-1, VCAM-1, E-selectin or the isotype control mouse IgG1 for 2 - 4 h at 4˚C. Subsequently, cells were washed and incubated with a horseradish peroxidase-conjugated goat anti- mouse IgG for 90 minutes. Binding of the secondary antibody was determined by incubating with 3, 3, 5, 5’ tetramethylbenzidine (TMB) substrate and then terminating the reaction by 2N sulphuric acid. Surface expression of adhesion molecules was quantified by measuring absorbance at 490 nm in an automated microtitre plate reader.

Monocytic cells and endothelial cell adhesion assay: The human monocytic leukemia cell line, THP-1 was used as a model for monocytic cells. Leukocyte adhesion assays were performed under static conditions as previously described by Kim HK [18] , with minor modifications. Briefly, THP-1 cells were labeled with BCECF- AM in serum-free RPMI 1640 media for 30 minutes at RT in the dark, then washed with DPBS to remove surplus dye and suspended in M200 medium. Confluent HUVEC monolayers were pretreated with various concentrations of Sphira for 30 minutes, prior to 4 h exposure to 1 ng/mL TNF-α. Labeled THP-1 (6 × 10⁵ cells/ml) were seeded onto control or treated HUVECs, and incubated for 10 minutes at RT in the dark. The unbound cells were removed by gentle washing with medium and the images were obtained at 485 nm excitation and 538 nm emission using a NIKON Eclipse 80i fluorescence microscope. For adhesion quantification, THP-1 cells bound to HUVECs were lysed with 0.1% Triton-X-100 in 0.1 M Tris buffer, pH 8.8, and fluorescence intensity was measured by a spectrofluorometer with emission at 520 nm and excitation at 485 nm. Adherent cells were quantified using standard curves of BCECF and expressed as % adhesion of added cells. Furthermore, a duplicate set of flasks were kept aside and counterstained with hematoxylin to simultaneously evaluate the morphology of HUVECs and monocytic cells.

To evaluate the anti-inflammatory effect of Sphira, the “Inhibition of Lipopolysaccharide (LPS)-induced TNF-α release in BALB/c mice” model was used. All animal experiments were approved by Institutional Animal Ethics Committee of Piramal Life Sciences Limited. The procedure followed is as described in Moreira, AL [19] . Balb/c mice (procured from The Jackson Laboratory, U.S.A.) of either sex weighing between 18 - 22 g (n = 10) were orally administered Sphira at a dose of 30, 100 and 300 mg/kg. All suspensions were freshly prepared in 0.5% CMC. One hour later, LPS (1 and 2 mg/kg, i.p. for TNF-α and IL-1β release, respectively) (Escherchia coli, serotype 0127:B8, Sigma Chemical Co., St. Louis, MO) dissolved in sterile pyrogen-free saline was administered intra-peritoneally to the control group, standard treatment group (Rolipram, 30 mg/kg, p.o.) and test groups (Sphira), except the negative control group which received only normal saline. Post LPS-challenge (1.5 h for TNF-α and 4 h for IL-1β), blood samples were collected from anesthetized mice, with heparin as an anti- coagulant (25 IU per sample), centrifuged at 10,000 rpm for 10 min, and the resultant plasma samples were analyzed for levels of cytokines by ELISA, as described by the manufacturer (OptiEIA ELISA sets, BD BioSciences Pharmingen). Percent inhibition of TNF-α and IL-1β release was calculated by comparing the levels of the treatment groups with those of the control group.

The anti-arthritic potential of Sphira was evaluated using collagen-induced arthritis mouse model in a manner similar to that described previously [20] . Male DBA/1J mice (procured from The Jackson Laboratory, U.S.A.), aged 8 - 10 weeks were immunized with an emulsion equivalent to 200 µg of type II collagen in Freund’s Complete Adjuvant, injected intra-dermally at the base of the tail. A booster shot with the same emulsion was given 21 days later. Sphira was tested in the therapeutic regime, from day 23 following primary immunization. Mice were examined once daily for signs of RA, using the articular index and paw thickness as parameters. Articular index scoring was performed employing the following criteria:

Scoring for forelimbs: Scale 0-3, where, 0: No redness or swelling; 1: Redness, but no swelling; 2: Redness and swelling of the paw; 3: Redness and severe swelling of the paw. Scoring for hind limbs: Scale 0-4, where, 0: No redness or swelling; 1: Redness and mild swelling of paw; 2: Redness and moderate swelling of paw and/or swelling of at least one of the digits; 3: Redness and moderate/severe swelling of paw, swelling of ankle joint and/or swelling of one or more digits; 4: Redness and severe swelling of paw, digits and ankle joint, with joint stiffness and altered angle of digits.

Mice with a minimum hind paw score of 2 were inducted into the study. Mice were randomized into the various study groups and orally administered the vehicle (0.5% CMC, 1 ml/kg), test compound (Sphira, 600 mg/kg, twice daily) or standard compound (Enbrel, 3 mg/kg, s.c., once daily). Each group had a minimum of 8 - 10 mice. The dosing of the compounds was done for 13 days. Parameters such as body weight, articular index, paw thickness (in mm, using a tension-free caliper) and any significant observation regarding the condition of the animal were observed and recorded daily. On the last day, 1 h after the compound treatment, the animals were sacrificed, blood withdrawn, and plasma collected for drug level analysis. Also, the limbs of all the animals were preserved for histopathological evaluations.

Histological analysis: Mice were humanely euthanized and the hind paws were harvested from each animal, fixed in 10% neutral buffered formalin, decalcified in 10% EDTA and embedded in paraffin. Section were stained with either hematoxylin and eosin or safranin O and evaluated microscopically. Histopathological changes were scored as follows: mild (score = 1), moderate (score = 2) or severe (score = 3) for the parameters of cellular infiltration, bone erosions and cartilage damage, graded separately. Cartilage depletion was indicated visually by diminished safranin O staining of proteoglycan matrix. The mean total score was compared to that of vehicle treated group. In case of histological scoring, Kruskal-Wallis analysis was followed by Dunn’s multiple comparison tests to evaluate the statistical difference between two groups. Values of P < 0.05 were considered significant.

All statistical analysis were performed by Student’s t-test, where ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, were considered statistically significant.

The anti-inflammatory potential of methanolic extract of S. indicus was confirmed in vitro. Sphira was studied in 12 donors at eight different concentrations (0.3 - 100 µg/ml) in LPS-induced TNF-α, IL-1β, IL-6 and IL-8 release in hPBMCs. It was observed that it dose-dependently inhibits the release of LPS-induced TNF-α (IC₅₀ 4.31 ± 0.78 µg/ml), IL-1β (IC₅₀ 1.96 ± 0.56 µg/ml), IL-6 (IC₅₀ 10.17 ± 10.47 µg/ml) and IL-8 (IC₅₀ 28.33 ± 2.97 µg/ml) in hPBMCs. It was also found to be non-toxic at the doses tested (Figure 1(A)). Thus, our data clearly demonstrated that the extract could significantly modulate inflammatory cytokine secretion from monocyte-macro- phage lineage in the advent of LPS mediated stimulatory signals.

To study the effect of the extract on T-cell mediated cytokines such as IL-17, hPBMC were treated with different doses of Sphira, and the supernatant evaluated for secreted IL-17 levels in presence of PMA-ionmycin stimulus. The effect of the extract on the viability of these cells was also measured simultaneously. It is well demonstrated that Sphira blocked IL-17 (IC₅₀ 2.1 ± 0.67 µg/ml) and TNF-α (IC₅₀ 1.8 ± 0.9 µg/ml) production by PMA-ionomycin stimulated hPBMCs. The extract did not have any toxic manifestations on normal resting cells, but effectively blocked proliferation of stimulated cells (Figure 1(C)).

Sphira was evaluated at eight different concentrations in synovial cells obtained from RA patients undergoing knee replacement surgery [14] . This study was performed in 10 donors. The extract inhibited the spontaneous release of TNF-α (IC₅₀ 13.10 ± 3.19 µg/ml), IL-1 β (IC₅₀ 5.33 ± 1.76 µg/ml), and IL-6 (IC₅₀ 16.33 ± 2.91 µg/ml) in a dose-dependent manner, in synovial cells. It was found to be toxic at the highest concentration (100 µg/ml) (Figure 1(B)).

Anti-IL-12/23 antibodies have successfully been targeted to the common IL-12p40 subunit; both cytokines are implicated in inflammatory diseases. Ustekinumab and ABT-874, inhibitors of IL-12p40, have shown efficacy in psoriasis [21] [22] . Our studies show that Sphira potently inhibits the release of IL-12/IL-23 p40 (IC₅₀ 1.0 ±

0.0001 µg/ml) and IL-23 p19 (IC₅₀ 0.6 ± 0.0001 µg/ml), with no toxicity up to the highest concentration tested (Figure 1(D)).

NF-κB is an important transcription factor required for T-cell proliferation and other immunological functions. Sphira dose dependently inhibited NFκB activation (IC₅₀ 6.9 µg/ml) in TNF-α stimulated CEM-κB cells (Figure 1(E)).

As cell adhesion molecules play an important role during inflammation, we analyzed the effect of different concentrations of Sphira on TNF-α-induced cell surface expression of these molecules. In accordance with previous studies, ICAM-1 and E-selectin were expressed at low levels in unstimulated HUVECs, but their expression increased after TNF-α stimulation (data not shown). As shown in Figure 2(A), Sphira, at the highest concentration of 10 µg/mL, significantly inhibited the expression of TNF-α-induced ICAM-1 (61% ± 14%), VCAM-1 (69% ± 8%) and E-selectin (90% ± 7%) respectively. The IC₅₀ values of ICAM-1, VCAM-1 and E-selectin expression

were 7.6, 3.7 and 2.9 µg/ml, respectively. Taken together, these findings indicate that Sphira specifically inhibits the cytokine-induced expression of ECAMs in a dose-dependent manner.

The functional significance of inhibition of cell adhesion molecules was evaluated by analyzing the adhesion of monocytic cells to endothelial cells in the presence of Sphira (Figure 2(B)). Unstimulated confluent HUVEC monolayers exhibited minimal binding to THP-1 (Figure 2(B), Panel a). However, there was a marked increase in the THP-1 cell adherence to HUVECs treated with TNF-α (Figure 2(B), Panel b). Pretreatment of confluent HUVECs with Sphira (1, 3 and 10 µg/mL) drastically inhibited THP-1 adhesion to HUVECs in a dose-depen- dent manner (Figure 2(B), Panels c-e), with maximum inhibition at 10 µg/mL (P < 0.01; n = 5). By counterstaining with hematoxylin, the bright-field images (100×) show that the morphology of HUVECs was maintained, and monocytic cells were shown to be intact in each individual condition (Figure 2(B), lower panel). These results demonstrate that Sphira is effective in blocking adhesion of THP-1 to endothelial cells by inhibiting the TNF-α-induced expression of ICAM-1, VCAM-1 and E-selectin.

The anti-inflammatory effect of oral formulations of Sphira was evaluated in the in vivo LPS model of inflammation [23] . Percent inhibition of TNF-α and IL-1β release was calculated by comparing the cytokine levels in the Sphira-treated groups with those of the control group. Dose-dependent inhibition of cytokine release was noted (Figure 3(A)).

The extract was evaluated in a chronic model of arthritis, i.e. the well-established mouse collagen-induced arthritis (CIA) model. As reported earlier [24] , the CIA in DBA/1J mice was manifested with significant increases in paw thickness and articular index, and these clinical signs of arthritic disease were significantly reduced in mice receiving Sphira 600 mg/kg, orally, twice daily (Figure 3(B)). Histological analyses of paw tissues of diseased mice treated with placebo revealed severe destruction in the joints characterized by synovitis, pannus formation, articular cartilage erosion and pronounced infiltration of inflammatory cells invading bony cortex at multiple foci (Figure 3(C)). In contrast, the hind paws of treated mice (either with 600 mg/kg Sphira orally, or Enbrel) showed maintenance of joint architecture with diminished pannus formation and reduced infiltration of inflammatory cells (Figure 3(C)). The degree of macroscopic protection provided by Sphira 600 mg/kg, p.o., was statistically similar to the protection demonstrated by Enbrel treated with placebo control (Figure 3(C)).