Use of biofluorescence imaging to compare the distribution of certolizumab pegol, adalimumab, and infliximab in the inflamed paws of mice with collagen-induced arthritis

Abstract

Exposure to a drug at the site of inflammation may be an important consideration for the effective treatment of inflammatory disorders such as rheumatoid arthritis (RA). The purpose of this in vivo study was to identify a methodology to enable effective quantification of antibody-type reagents in normal and inflamed tissue by investigating the distribution of the tumor necrosis factor-alpha (TNF-α) inhibitors, certolizumab pegol, adalimumab, and infliximab, in healthy and inflamed murine tissue using a novel non-invasive biofluorescence method. Certolizumab pegol, adalimumab, and infliximab were labeled with the low molecular weight dye alexa680. The agents were administered intravenously at a dose of 2 mg/kg in naïve DBA/1 mice and in DBA/1 mice with ongoing collagen-induced arthritis. Concentrations of the TNF inhibitors in the hind paws were measured using a Xenogen IVIS200 biofluorescence imager at multiple time points up to 26 h post-administration. In 2 independent experiments, the distribution of certolizumab pegol was compared with that of adalimumab and infliximab. Certolizumab pegol, adalimumab, and infliximab all distributed more effectively into inflamed tissue than non-inflamed tissue in this animal model of arthritis. However, the ratio of penetration of certolizumab pegol into inflamed arthritic paws compared with normal tissue was greater than that observed with adalimumab and infliximab. Furthermore, the duration of exposure in the inflamed versus normal tissue was more prolonged for certolizumab pegol than for both adalimumab and infliximab, and the accumulation of certolizumab pegol in diseased tissue was more responsive to the severity of inflammation when compared with adalimumab and infliximab. It is probable that these features of certolizumab pegol are conferred on the molecule by PEGylation. It is important to assess exposure to drug at the site of inflammation, because distinct structural features of certain agents may affect efficacy, tolerability, rapidity and/or sustainability of effect. The novel non-invasive biofluorescence method used in this study is an effective tool for comparing tissue penetration of therapeutic agents.

Introduction

There is currently limited precedence to test the relative distribution of biologics for the treatment of autoimmune human disease in animal models in vivo. This is despite the fact that the degree of penetration of therapeutic agents to the site of inflammation may affect efficacy, tolerability, or rapidity of effect. Specific penetration of therapeutic agents to the site of disease is particularly relevant in rheumatoid arthritis (RA). RA is a common autoimmune disease that affects approximately 1% of the population. It is a systemic disease, characterized by a chronic inflammatory reaction in the synovium of joints with subsequent cartilage degeneration and juxta-articular bone erosion (Lee and Weinblatt, 2001). Recently there have been advances in the field of imaging that have allowed the non-invasive evaluation of cellular and molecular events (Wunder et al., 2005). Nevertheless, at the present time it is not realistic to compare the distribution of various reagents readily in human studies.

The tumor necrosis factor-alpha (TNF-α) inhibitors infliximab and adalimumab are currently licensed for use in RA and have shown similar efficacy in randomized, controlled clinical trials (Alonso-Ruiz et al., 2008). Adalimumab is a recombinant human monoclonal antibody and infliximab is a chimeric monoclonal antibody (Tracey et al., 2008). The structural differences between these TNF inhibitors contribute to their different pharmacokinetic and safety profiles (Keystone et al., 2004, Maini et al., 2004, Weinblatt et al., 2003).

Certolizumab pegol is the only PEGylated TNF inhibitor. In clinical trials it provided rapid and significant improvements in the signs and symptoms of disease as monotherapy and in combination with methotrexate in patients with RA (Fleischmann et al., 2009, Keystone et al., 2008, Smolen et al., 2009). Certolizumab pegol is indicated in patients with active RA who had an inadequate response to methotrexate. It is also indicated in the US for adults with moderate-to-severe active Crohn's disease who had an inadequate response to conventional therapy.

PEG (polyethylene glycol) is a polyether of repeating ethylene glycol subunits that can be of various molecular weights. PEG is commonly attached to proteins to increase their half-life in vivo. Two examples of such proteins are the PEGylated interferon α molecules, Pegasys® and PEG-Intron®. PEGylated molecules tend to diffuse relatively slowly out of the blood because of the hemodynamic properties of PEG (Chapman, 2002) and could therefore have a distribution pattern that may result in different exposure times in inflamed and non-inflamed tissues compared with an intact immunoglobulin (Ig) G. This is a well-described phenomenon for water-soluble polymers and has been investigated for the targeting of inflamed tissues (Wang et al., 2004).

The aim of this study was to develop a novel, non-invasive, biofluorescence method to investigate the distribution of antibody-like reagents using the TNF inhibitors, certolizumab pegol, adalimumab and infliximab in vivo in healthy and inflamed murine tissue.