[¹⁸F]DPA-714 as a biomarker for positron emission tomography imaging of rheumatoid arthritis in an animal model

Rheumatoid arthritis (RA) is a chronic inflammatory disorder that can affect many tissues and organs. This disease leads to progressive joint destruction principally due to synovial inflammation, but also to articular complications and functional disability. In addition to causing joint problems, RA can also affect the whole body with fevers and fatigue. RA is a systemic autoimmune disease that also leads to many disorders in organ systems and is associated with other diseases, including infections, malignancies and cardiovascular diseases. One percent of the world's population today is affected by RA with a repartition much more common in women than in men with ages ranging from 40 to 60 years [1]. Smoking is the main environmental risk factor for developing RA. Other potential risks, including alcohol abuse, vitamin D status, contraception use and coffee intake, have been reported too [2].

The detection of RA is important in clinical strategies that include early treatments which may slow down the disease progression and improve the long-term clinical outcome. Criteria proposed in 1987 by the American College of Rheumatology (ACR) 1987 are rather limited by the poor sensitivity and specificity for the classification of patients with early RA [3]. These criteria include morning stiffness, arthritis of three or more areas, arthritis of hand joints (>1 swollen joint), rheumatoid nodules, serum rheumatoid factor, radiographic changes (erosions). However, early events failed to be detected by those means. Until today, identification of bone changes in RA is traditionally the domain of plain radiography. In parallel to this, other techniques such as Magnetic Resonance Imaging and Ultrasound have demonstrated a superior sensitivity for the detection of erosive changes around the bone. Several teams have shown that subclinical arthritis can be detected by advanced imaging techniques such as sonography or power Doppler [4‐6]. The development of non-invasive, highly sensitive and 3D-imaging-based methods for detecting early arthritis could also offer an opportunity to initiate treatments at very early stages to the patient.

Molecular- and nuclear-based imaging techniques offer a large scale opportunity for detection of diseases. These techniques are based on the use of tracers labeled with radioactive isotopes and allow non-invasive in vivo detection of different physiologic and pathologic phenomena with high sensitivity. Positron Emission Tomography (PET) is one of those that are increasingly used to diagnose and characterize disease activity in the setting of inflammatory disorders such as RA [7‐10]. PET is even more sensitive than Single Photon Emission Computed Tomography (SPECT) and can also provide quantitative measurements. The low spatial resolution can make assigning the signal to specific anatomical structures difficult and can be partially compensated by combining PET with CT (X-ray Computed Tomography). [¹⁸F]Fluorodeoxyglucose ([¹⁸F]FDG), a radiofluorinated analogue of glucose and the most widely used radiopharmaceutical worldwide today, was proposed for imaging RA [11, 12]. Using this radiotracer, inflamed joints could be detected even though [¹⁸F]FDG is not a specific marker of inflammation. Macrophage infiltration has been identified in early stages of RA [13], and therefore a specific tracer of such a process would be more specific and possibly also enable an earlier detection of inflammation. Recently, expression of the folate receptor has been investigated in a rat model of RA using [¹⁸F]fluoro-PEG-folate [14] illustrating the interest for molecular imaging in this type of pathology.

The 18kDa translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is located in the outer mitochondrial membrane [15]. This protein is up-regulated in inflammatory processes, making radiolabeled TSPO ligands attractive imaging probes in a multitude of pathologies. Different TSPO ligands have already been used as imaging probes in different animal models of human diseases such as cancer [16], cerebral ischemia [17], lung [18] and liver diseases [19].

Today, [¹¹C]PK11195 is still considered as the reference radioligand for imaging in vivo TSPO expression mainly in central nervous system disorders and rarely in the case of peripheral inflammatory diseases. Numerous publications in the recent literature have, however, highlighted the limitations of this radiotracer for quantitative in vivo measurement of TSPO expression [20] and several novel TSPO radioligands have been developed today as alternatives to the use of [¹¹C]PK11195 [21‐23]. Among them, the pyrazolo [1, 5] pyrimidine acetamide DPA-714, labeled with fluorine-18 ([¹⁸F]DPA-714) is one of the most promising compounds. For the moment, [¹⁸F]DPA-714 is mainly used to image brain injury in various animal models [17, 24‐26] but also in the field of oncology with applications in breast cancer [27] and glioma [28]. Interest for the molecular imaging of peripheral inflammation has also been investigated recently in non-alcoholic fatty liver disease in rats [19] and lungs by the same team [18] using another radiolabelled TSPO ligand ([¹⁸F]FEDAC). Recently, [¹¹C]PK11195 has been shown to be of potential interest in imaging arthritis in humans [7, 10, 29]. The visualization of macrophages using [¹¹C]PK11195 PET seems to be useful for detecting early synovitis and for monitoring disease evolution during treatment.

The present study aims to investigate the potential of [¹⁸F]DPA-714 PET to image and quantify in vivo peripheral inflammation in an autoimmune adjuvant-induced RA rat model.

The present study used a readily accessible, small molecular weight, fluorine-18-labeled compound, coded DPA-714, as the radiotracer for the inflammatory processes. This TSPO radioligand has been largely used to monitor neuroinflammation. The results published recently in various articles illustrate the potential of [¹⁸F]DPA-714 to provide quantitative information about the expression of TSPO with correlation between the radioactive signal obtained by PET imaging and the molecular expression of the target, at a cellular level, obtained by Western blot analysis [16, 24]. In our inflammatory model, biochemical analysis could not be easily performed due to the presence of bones and cartilages. For this reason we chose to illustrate the increase of TSPO using IHC. This approach, done on fixed tissues, required a decalcification step to permit the slicing of the articular tissues. These analyses were done on control, mild and severe inflammatory grade permitting us to visualize an increasing number of CD68 and TSPO positive cells (data not shown). The combination of both [¹⁸F]DPA-714 PET imaging and IHC clearly illustrated the increase of TSPO expression in the ankles of treated rats in comparison to non-treated animals. IHC using CD68 showed the important infiltration of white blood cells, mainly macrophages, in the ankle. The CD68 positive cells represented around 25% of the cells counted on tissue samples from inflamed ankles. Co-labeling using a TSPO antibody showed that 95% of these CD68 marked cells were also TSPO positive. TSPO is expressed in mitochondria whereas CD68 is mainly localized in cytoplasmic granules. This non-overlap, adding to the counting error incertitude, does not permit us to affirm that some CD68 positives cells are really TSPO negatives. These types of cells (TSPO-; CD68+) represent less than one percent of total cells counted in the different fields of view for this quantitative approach. On the other hand, around 4% of total cells by field of view are TSPO + and CD68-. This cell population could represent other immune cells, such as lymphocytes or macrophages as suggested by the presence of Iba1+/CD68- or MRP8+/CD68- cells.

Conventional radiography is nowadays considered as the gold standard in RA imaging for the evaluation of structural damages. The main limitation with this technique is that its sensitivity is considered to be low and also that it is not able to assess the disease activity.

Recently, an international group of expert rheumatologists, radiologists and methodologists published some recommendations for the use of imaging of the joints in the clinical management of rheumatoid arthritis [34]. They compared the potential of different imaging techniques for diagnosis of RA, detecting inflammation and damage, predicting outcome and response to treatment, monitoring disease activity, progression and remission. These techniques, conventional radiography, MRI, CT, dual emission X-ray absorptiometry, digital X-ray radiogrammetry are anatomical images based mainly on density alteration of bones and cartilages. Two other techniques were included in this comparative study, SPECT and PET imaging. These two nuclear-based approaches mainly provide information on biological functions (molecular imaging) as opposed to anatomical imaging. In arthritis, anatomical alterations of bone, cartilage or at least severe stage tendon swelling, represents a late stage of the disease with clinical symptoms illustrative of a long molecular process of inflammation. Concerning the diagnosis of RA, precocity has been described as an important point to implement appropriate therapeutics and thus limit structural alterations and delayed invalidity associated with this disease. Molecular imaging, such as TSPO PET imaging, represents a possibility of following early events in the physiopathology of RA. It has, therefore, been proposed that [¹¹C]PK11195 PET imaging could be used for detecting early synovitis and for monitoring the evolution of the disease during treatment [7, 10]. The present work, performed in a rat RA model, demonstrates that [¹⁸F]DPA-714 can be used to image inflammation of joints non-invasively. The specificity of the uptake was assessed here by intravenous injection of a large amount of unlabeled DPA-714. This injection displaced specific binding of [¹⁸F]DPA-714 by saturation of TSPO binding site by a non-labelled compound. The phenomena took place in an inflamed area but also in every organ with a physiological high content of TSPO inducing plasma release of a large quantity of [¹⁸F]DPA-714. This effect is illustrated by the huge increase in an unspecific signal quantified in the muscle with a variation from 0.06 to 0.20% of ID/cc (Figure 5B). In the inflamed ankles, the displacement leads a decrease of the signal around 45%. After administration of unlabeled DPA-714, the ankle-to-muscle ratio in these animals was around 1:4 while this ratio was 7:8 before administration. This value illustrates that the signal quantified in the ankle after displacement is largely due to background corresponding to free [¹⁸F]DPA-714 (not bound to the TSPO 18kDa). In the inflamed areas it has been shown that the vascular density is also increased. This high vascular density can increase the volume of blood in the considered area and participate in the higher signal in comparison to normal ankles or muscle use as the reference area. Local inflammation also induces edema. Edematous areas are well known to present high oncotic pressure inducing extravasation of macromolecules. This phenomenon was not described for small ligands, such as DPA-714, with a molecular weight around 400 g/mol. This hypothesis is certainly not the explanation concerning the remaining radioactivity present in the ankle after displacement. The level of radiotracer accumulation in the swollen hind paws correlates to the thickness of the ankles, and is chosen as macroscopic criteria for the severity of the disease (Figure 6). In this graph, uptake results were expressed as density by number of the receptors by volume unit here extrapolated as Bequerel/cc corrected by the ID/cc. The fact that the increase of ankle volume was linked to an increase of the TSPO density (for example, [¹⁸F]DPA-714’s uptake) underlines the relation between the severity of the disease and the level of TSPO expression. This functional information represents an important point concerning the ability of imaging techniques to evaluate disease activity, a parameter simply not accessible using conventional anatomical imaging approaches. Another criterion that represents a large interest for rheumatologists is the potential of such non-invasive imaging tools and techniques to predict the evolution of the disease.

The results of the present work demonstrates that the use of the TSPO-targeting PET-ligand [¹⁸F]DPA-714 is well-adapted to the study of peripheral inflammation in an experimental rodent model of RA. [¹⁸F]DPA-714 PET imaging may provide new objective parameters to evaluate the early stages of the disease and to determine disease activity, also suggesting a strong potential of this technique for clinical investigation of peripheral inflammation.