Journal of Pharmacogenomics & Pharmacoproteomics

Rheumatoid Arthritis (RA) is an inflammatory condition with multi-organ involvement. It is a relatively common condition and about 0.5% to 1% of the population is effected [1]. Evidence suggests that it becomes more prevalent correlating with increasing latitude and when it is compared between rural to urban populations [2]. RA is destructive and it affects the small joints predominantly. Increased prevalence of atherosclerotic disease and reduced participation in society are observed in RA patients [3]. Reduced work capacity as a result of RA puts an indirect cost to society and has been estimated to be as high as € 41.6 billion in the United States and € 45.3 billion in Europe [4].

It was initially thought that RA was infection driven that led to the use of gold and sulpha drugs with limited success. As research progressed in the treatment of RA, use of steroids as therapeutic agents gained popularity among physicians [5]. As our understanding of the disease has improved, we have moved on to more targeted therapies. The onset of RA is thought to be dependent on both environmental and genetic factors. Of these, the major modifiable environmental risk factor identified is that of smoking [6]. Therapies currently employed to combat RA include traditional treatments such as glucocorticoids, non-steroidal anti-inflammatory drugs (NSAIDs), and diseasemodifying antirheumatic drugs (DMARDs), classified broadly as synthetic (encompassing the traditional DMARDs and newer therapies such as JAK-2 inhibitors) and biological drugs.

Marked symptomatic relief was seen with the introduction of NSAIDs. Though not a DMARD, its beneficial effects have been long documented with Willow and poplar bark, the substrates for salicylic acid, commonly used in the treatment of inflammatory arthopathies [7]. Aspirin was in use by the early twentieth century in the treatment of RA, making it the oldest of the medications currently in use [8]. We now have a vast selection of NSAIDs available, all working to inhibit the cyclo-oxygenase pathway. Prostaglandins are made by two different enzymes, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). The prostaglandins made by the two different enzymes have slightly different effects on the body. COX-2 inhibitors are NSAIDs that selectively block the COX-2 enzyme and not the COX-1 enzyme. Blocking this enzyme impedes the production of prostaglandins by the COX-2 which causes the pain, swelling and other painful conditions. Because they selectively block the COX-2 enzyme and not the COX-1 enzyme, these drugs are uniquely different from traditional NSAIDs which usually block both COX-1 and COX-2 enzymes. Although NSAIDs are effective analgesics, they do not have any demonstrable effect on disease course and further they have side effects that include gastric irritation and nephrotoxicity [9].

The term DMARD (Disease modifying anti-rheumatic drug) is applied to medications which can alter the course of disease and thus prevent joint erosion [10]. The mechanisms through which DMARDs act are varied but a collective outcome is to help stem the destructive process of intertwined inflammatory cascades resulting in the degradation of soft tissue, cartilage and bone.

The advent of proteomics technologies has enabled large-scale analysis of proteins to identify biomarkers that delineate disease subtypes of rheumatoid arthritis, and to gain insights into the mechanisms underlying these subtypes. The aim of this study is to identify proteins that are differentially expressed between control and arthritis sera and to further look at the protein profiles of the COX-2 inhibitor, celecoxib vs DMARD, methotrexate, and treated sera profiles by proteomics approach. The aim of this study is to figure out differences at the protein level for the above two drugs since celecoxib is known for its anti-inflammatory properties in arthritis and methotrexate is known to target the cytokines.

Unless otherwise noted, all chemicals were purchased from Sigma (Sigma Chemicals, St. Louis, MO) and were of analytical grade. Trypsin is purchased from Promega (sequencing grade modified trypsin, Catalog No. V5111, Mannheim, Germany).

Animals

Male Lewis rats (150-180 gm) were obtained from Harlan Sprague Dawley (Indianapolis, IN), housed in micro isolator cages (Lab Products, Maywood, NJ), and supplied with sterilized standard rodent chow diet and acidified water (pH 2.8-3.0). The animals were handled under clean conditions and were acclimated to the new environment for a week before use.

Preparation of type II collagen

Native type II collagen was extracted from bovine articular cartilage, after digestion with papain. The extraction and purification procedures for collagen have been described previously [11]. The purified preparation was stored in O.05M acetic acid at -20°C.

Induction of arthritis

The mean body weight and mean hind paw volume of rats in each group were very similar at the start of every experiment. Type II collagen arthritis was induced by an intradermal injection, at the base of the tail, of an emulsion of 100 pg bovine type II collagen in 0.05M acetic acid with an equivalent volume of incomplete Freund’s adjuvant (ICFA).

Intravenous treatment with the antigens

Type II collagen (stock solution in O.05M acetic acid) was adjusted to pH 7, diluted with phosphate buffered saline (PBS), and stirred vigorously at 0°C to avoid any possible clumping. The collagen solution (0.5 mg) was injected intravenously into the tail vein of each rat, without the use of any anesthetic and with precautions to minimize trauma. The denatured type II collagen solution was prepared by heating native type II collagen at 60°C for 30 minutes. Some animals received M tuberculosis (2 mg in PBS) intravenously in the same fashion. These treatments were given to rats either 3 days prior to or 7 or 10 days after the immunization for induction of arthritis.

Evaluation of inflammatory response

The development and severity of arthritis were mainly assessed by the quantitation of hind paw swelling of the rats. Both hind paws of each rat were dipped in a mercury bath and the volume of displacement of mercury was measured twice a week [12,13]. Along with the swelling of hind paws, visual disfigurement, restricted movement, and radiologic evaluation of the joints were taken into consideration to determine the degree of arthritis.

Administration of celecoxib and methotrexate to arthritic rats

Seven rats with collagen induced arthritis were given 10 mg/kg per day of celecoxib for two weeks from the onset of arthritis. Seven rats with collagen induced arthritis were given 10 mg/kg per day of methotrexate for two weeks from the onset of arthritis. Seven rats with collagen induced arthritis served as normal controls and are not given either of these drugs.

Serum protein solubilization and sample application

Serum collected from normal rats, arthritic rats, and arthritic rats treated with celecoxib and arthritic rats treated with methotrexate, were processed as follows. The serum sample is passed through albumin/globulin removal columns (commercially supplied by Amersham). The eluate from the column is collected and the protein amount is determined. The protein is suitably solubilized in the 2-d gel buffers. To each 4-7 IPG strip, 300 μg protein is added and the strips are allowed to rehydrate overnight along with the sample.

2-d Gel electrophoresis

The IPG strips are rehydrated with the sample overnight and electrophoresis is carried out in the first dimension the following day. The strips are then placed on a regular SDS-PAGE gel and the electrophoresis is carried out in the second dimension. The gels are then silver stained.

2-d Gel analysis

After 2-D gels are stained, the protein patterns are digitized and analyzed across multiple gels (Control rat sera, arthritis rat sera, celecoxib treated rat sera and methotrexate treated rat sera) by Computer-assisted image analysis which is an indispensable tool for the evaluation of complex 2-D gels. PDQuest™ 2-D analysis software from Biorad is used to pick the spots and compare the spots of interest across the gels.

Spot picking, protein digestion and MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight) analysis

Using the Ettan spot handling workstation (Amersham Biosciences) selected spots were automatically cut from the gels, destained and enzymatically digested with trypsin. The tryptic peptides were then spotted onto a MALDI target plate. The MALDI target plates were loaded in a Micromass M@ldi MALDI-TOF mass spectrometer (Waters, Milford, USA) for analysis of the peptide masses.

Database search

Peptide masses retrieved from MALDI-TOF analysis spectra were submitted to a database (Mascot) for protein identification.

The rats receiving an intradermal injection of native type II collagen began to exhibit swelling of the hind paws in approximately 15-17 days, with maximum swelling occurring at days 21-25. Thereafter, the inflammation subsided slightly and remained constant until day 30. Then the hind paws were measured. Each treatment reduced inflammation, but both celecoxib and methotrexate prevented bone loss adjacent to inflamed joints and significantly decreased bone resorption. In contrast, no treatment affected bone formation parameters.

Some important proteins of interest in arthritis are labelled on the 2-d gel picture (Figure 1). The analysis of serum protein profiles from 2-d gels of normal and arthritic rats clearly shows that several proteins are up-regulated and several proteins are down regulated in arthritic animals compared to normal animals (Figure 2, Tables 1 and 2). A pie chart (Figure 3) shows the protein pattern observed in this study and their distribution. We can see that some proteins are differentially expressed between control and arthritis and some proteins are common between these conditions. Both Celecoxib and Methotrexate are found to be effective in inhibiting some of the proteins that are responsible for arthritis and are over expressed in arthritic condition. The 2-d gel serum protein patterns of celecoxib treated and methotrexate treated rats are shown along with normal and arthritis serum profiles (Figure 4).

Figure 1: Proteins from rat serum that are of significance in arthritic condition are labeled on the 2-d gel.

Figure 2: 2-d gel electrophoresis protein profiles between A) Normal and B) Arthritis rat sera. Proteins separated on 4-7 IPG strips.

Figure 3: Image Analysis of some proteins analyzed after 2-d gel electrophoresis. Image shows proteins that are differentially expressed between normal and arthritic conditions and proteins that are common in normal and arthritic sera.

Figure 4: 2-d gel showing the effects of Celecoxib and Methotrexate on arthritic protein profiles.

Table 1: Protein up-regulated more than 2-fold in Arthritis condition.

Table 2: Proteins down-regulated more than 2-fold in Arthritis condition.

Cathepsin K is a cysteine protease that plays an essential role in osteoclast function and in the degradation of protein components of the bone matrix by cleaving proteins such as collagen type I, collagen type II and osteonectin. Cathepsin K therefore plays a role in bone remodelling and resorption in diseases such as osteoporosis, osteolytic bone metastasis and rheumatoid arthritis. We found increased levels of cathepsin K compared with a healthy control group and found a significant correlation with radiological destruction. Inhibition of different proteins responsible for arthritic condition by celecoxib and Methotrexate Figures 5 (A-F).

Figure 5A: Methotrexate was found to be a better inhibitor of cathepsin compared to celecoxib.

Figure 5B: This protein is potentially inhibited upon celecoxib and methotrexate treatments, where celecoxib was found to be more effective compared to methotrexate.

Figure 5C: In our study CRP is significantly up-regulated in arthritic condition and it is better inhibited by celecoxib when compared to methotrexate.

Figure 5D: The increased levels of IL-6 are effectively inhibited by D both celecoxib and methotrexate treatments, with methotrexate showing more inhibitory effect compared to celecoxib.

Figure 5E: Increased levels of IL-2 are found in arthritic sera and it is inhibited to a great extent by celecoxib and to a moderate extent by methotrexate.

Figure 5F: Celecoxib to be a better inhibitor of MMPs compared to methotrexate.

A valuable approach to monitor arthritis would be by measuring biological markers of cartilage degradation and repair to reflect variations in joint remodeling. One such potential biological marker of arthritis is cartilage oligomeric matrix protein (COMP). In various studies, COMP has shown promise as a diagnostic and prognostic indicator and as a marker of the disease severity and the effect of treatment. We found increased levels of COMP in arthritic sera.

C-reactive protein (CRP) is one of the biomarkers for the diagnosis and assessment of disease activity in rheumatoid arthritis (RA). CRP is not only the by-product of inflammatory response, but also plays proinflammatory and prothrombotic roles.

Interleukin 6 (IL-6) is a pleiotropic cytokine with a pivotal role in the pathophysiology of rheumatoid arthritis (RA). It is found in abundance in the synovial fluid and serum of patients with RA and the level correlates with the disease activity and joint destruction. IL-6 can promote synovitis and joint destruction by stimulating neutrophil migration, osteoclast maturation and vascular endothelial growth factor (VEGF)-stimulated pannus proliferation. IL-6 may also be mediating many of the systematic manifestations of RA including inducing the acute-phase reaction [including C-reactive protein (CRP)], anemia through hecipidin production, fatigue via the hypothalamic-pituitary-adrenal (HPA) axis) and osteoporosis from its effect on osteoclasts. In addition, IL-6 may contribute to the induction and maintenance of the autoimmune process through B-cell maturation and TH-17 differentiation. All of the above makes IL-6 blockade a desirable therapeutic option in the treatment of RA.

IL-2 is generally considered a pro-inflammatory cytokine that exacerbates Th1-mediated disease states, such as autoimmune arthritis.The collagenases, MMP-1 and MMP-13, have predominant roles in RA and because they are rate limiting in the process of collagen degradation. MMP-1 is produced primarily by the synovial cells that line the joints, and MMP-13 is a product of the chondrocytes that reside in the cartilage. In addition to collagen, MMP-13 also degrades the proteoglycan molecule, aggrecan, giving it a dual role in matrix destruction. Expression of other MMPs such as MMP-2, MMP-3 and MMP-9, is also elevated in arthritis and these enzymes degrade non-collagen matrix components of the joints. Significant effort has been expended in attempts to design effective inhibitors of MMP activity and/or synthesis with the goal of curbing connective tissues destruction within the joints. To date, however, no effective clinical inhibitors exist.

Increasing our knowledge of the crystal structures of these enzymes and of the signal transduction pathways and molecular mechanisms that control MMP gene expression may provide new opportunities for the development of therapeutics to prevent the joint destruction seen in arthritis. In our study we studied inhibition of total MMPs by celecoxib and methotrexate treatments.

In conclusion, this study provides a comparative profile of the effects of therapeutic doses of celecoxib and methotrexate on arthritic rats. The results indicate that the two antiarthritics have varying degrees of side effects on bone metabolism, and these findings may help physicians figure out whether appropriate measure will be needed to better prevent the occurrence of osteopenia in RA treatment.