In-vivo monitoring of anti-folate therapy in arthritic rats using [¹⁸F]fluoro-PEG-folate and positron emission tomography

Methotrexate (MTX) is the anchor drug in rheumatoid arthritis (RA) therapy, either as a single agent or in combination with disease-modifying anti-rheumatic drugs and biological agents [1‐4]. Membrane transport via carrier-mediated and receptor-mediated routes is the first regulatory step in the mechanism of action of MTX in immunological target cells [5‐7]. Notably, in RA (synovial) macrophages, the folate receptor β (FRβ) has been recognized as a major transport route for MTX, next to the reduced folate carrier [8, 9]. FRβ expression is confined to cells of the myeloid lineage [10, 11] as opposed to the α-isoform of FR (FRα), which is selectively expressed in specific types of cancer (ovary, breast) [12‐14]. Given the high binding affinity (low nanomolar Kd) of FR for folic acid, this receptor has been exploited for therapeutic targeting with folate-conjugated drugs [15, 16] as well as imaging of FRα-positive tumours and activated FRβ-positive macrophages in RA [17‐19]. FRs harbour several interesting properties for targeting with folate-based positron emission tomography (PET) tracers; for example, easy accessibility as an extracellular GPI-anchored membrane protein, high binding affinities for folates, and specific expression on activated macrophages in inflammatory diseases, allowing receptor targeting for imaging with folate-based PET tracers [9, 10].

In humans, macrophages have been identified as a sensitive biomarker for therapy monitoring, regardless of the choice of treatment [20]. Moreover, RA remission has been positively correlated with lower numbers of synovial macrophages [21]. These findings, however, were obtained by invasive histological studies. Clearly, non-invasive imaging of macrophages may be an attractive alternative approach to detect and monitor synovial activity in body tissues [22]. Animal models of arthritis can serve as a pre-clinical step to explore macrophage expression through novel imaging modalities. Beyond successful application of single-photon imaging agents—for example, EC20, a ^99mTc-labelled folate [23‐25]—to image arthritis, recently a folate-based PET tracer, [¹⁸F]fluoro-PEG-folate, has been synthesized [26]. Such a tracer could potentially employ the higher sensitivity of PET and its ability to quantify uptake, which is essential for intervention studies. The potential of [¹⁸F]fluoro-PEG-folate for imaging macrophages has been demonstrated in an arthritic animal model [26]. However, the potential of [¹⁸F]fluoro-PEG-folate to monitor the efficacy of therapeutic interventions, in particular using anti-folates, such as MTX, has not been explored.

In the present study, the potential of [¹⁸F]fluoro-PEG-folate as a macrophage-targeted PET agent for monitoring MTX therapy efficacy in arthritic rats was examined.

The present study, using [¹⁸F]fluoro-PEG-folate, investigated the feasibility of non-invasively monitoring efficacy of anti-folate therapeutic interventions in RA. Lower accumulation of [¹⁸F]fluoro-PEG-folate in arthritic knees corroborated with decreased numbers of active macrophages in MTX-treated rats compared with the untreated rats. This was illustrated for MTX, because this is the golden standard in clinically active RA treatment [1, 3, 4].

Folate receptor expression on activated macrophages has been exploited for imaging and therapeutic monitoring of arthritis with various folate PET tracers including 4-[¹⁸F]fluorophenylfolate and [⁶⁸Ga]-DOTA-folate [29]. These PET tracers showed a significantly improved specificity over a general inflammation tracer [¹⁸F]-FDG, which relates to increased glucose metabolism in, for example, activated macrophages. In the present study, we made use of a pegylated folate tracer, [¹⁸F]fluoro-PEG-folate, which harbours improved plasma pharmacokinetic properties over other folate tracers. In a side-by-side comparison in a rat model for RA [27], [¹⁸F]fluoro-PEG-folate demonstrated a 1.5× improved target to background ratio compared with the mitochondrial translocator protein targeted macrophage tracer (R)-[¹¹C]PK11195 [26]. Moreover, [¹⁸F]fluoro-PEG-folate also displayed promising PET imaging potential [26], which was taken a step further in the present study for monitoring therapeutic interventions, such as MTX therapy.

[¹⁸F]fluoro-PEG-folate PET combined with a CT has advantage over the previous reported [¹⁸F]fluoro-PEG-folate PET study [26], because the region of interest (ROI) around the synovium can be depicted more precisely. [¹⁸F]fluoro-PEG-folate showed a marked reduction in tracer uptake in arthritic knees of the rats following two different MTX treatment regimens. It is unlikely that reduced tracer uptake in the MTX-treated rats is due to direct competition of the radiolabelled tracer with MTX for FRβ for various reasons: PET scans were acquired in the last week after the last MTX dose and, based on MTX pharmacokinetics [30] at that time, residual plasma levels will be <10 nM; the binding affinity of FRβ for [¹⁸F]fluoro-PEG-folate outweighs the binding affinity for MTX by at least 100-fold; and also the binding affinity of the natural circulating plasma folate (i.e. 5-methyltetrahydrofolate) is 3-fold higher than the tracer [9, 26], and thus competitive effects are not anticipated. In addition, immunohistochemical analysis of the arthritic joints showed a significant reduction of macrophages in synovial tissue which was in line with reduced joint uptake of the folate tracer. Consistent with our PET results, Kelderhouse et al. [31] also demonstrated a markedly lower accumulation of the SPECT folate targeted imaging agent [^99mTc]-EC20 in a collagen-induced arthritis (CIA) model upon administration of anti-rheumatic drugs. In the same CIA model, OTL0038, a novel folate-conjugated near-infrared dye, also showed low accumulation following anti-rheumatic therapies [32]. Together, whereas SPECT and optical imaging each has proven value with folate-based imaging agents, PET folate harbours advantages over SPECT (low-resolution and low-sensitivity images) [26] and optical imaging (no deep tissue imaging) [32]. Although costs of PET are relatively high at this moment, it is anticipated that with the widespread application of PET technology worldwide, costs will come down in the near future as also happened in the past decennia for the other imaging techniques such as CT and MRI.

Previously, apart from prominent arthritis induction in the arthritic knee, signs of systemic inflammation were also observed [27], reflected by tracer uptake in macrophage-rich organs, especially the liver and spleen. Ex-vivo tissue distribution data indicated that MTX therapy also had systemic effects by reducing [¹⁸F]fluoro-PEG-folate uptake in these organs as well as in the contralateral knee. Independent of MTX therapy, the increased accumulation of [¹⁸F]fluoro-PEG-folate in kidneys and intestinal tissue could be attributed to tracer clearance and/or high expression of FRα on kidney proximal tubule cells [15, 17] and intestinal tissue [33] to which receptor the folate tracer also binds.

Immunohistochemical analyses indicated that markedly reduced numbers of macrophages in the synovium of MTX-treated arthritic rats accounted for reduced [¹⁸F]fluoro-PEG-folate tracer uptake. Interestingly, reduction in macrophages upon MTX treatment involved both ED1-positive and ED2-positive macrophages—although CD68 (and possibly its rat homologue ED1 used in the present study) is not an absolute marker for macrophages because its expression has also been demonstrated on fibroblasts. To extend on this point, an experienced pathologist examined the (arthritic) knee sections for morphological assessment of cellularity and immunohistochemical staining of ED1⁺ cells. ED1⁺ synovial fibroblasts were identified but morphologically discerned from macrophages and were not taken into account for macrophage scoring assessments. Moreover, from the perspective of macrophage polarization, we also examined numbers of ED2⁺ macrophages (the rat homologue of human CD163, so-called ‘M2’, ‘anti-inflammatory macrophages’) in synovial tissue before and after therapy. Notably, studies by Puig-Kroger et al. [34] showed that FRβ is differentially expressed on M2 macrophages upon ex-vivo skewing of monocytes with macrophage-colony stimulating factor. However, studies by Tsuneyoshi et al. [35] in human RA synovial tissue demonstrated mixed patterns of FRβ expression on ‘M1’ (‘pro-inflammatory’) and M2 macrophages. In this context it is also important to note that M2 macrophages in an RA environment with complex IgG autoantibodies and/or ACPA antibodies are triggered to produce pro-inflammatory cytokines [36, 37]. Thus, MTX targeting with respect to polarization of macrophages and the role of FRβ therein warrant further investigations. Given the fact that beyond MTX several other second-generation anti-folates have been developed which have demonstrated potential pre-clinical anti-arthritic activity [7, 9, 38‐40], [¹⁸F]fluoro-PEG-folate PET imaging may be useful for monitoring their efficacy.

The present study demonstrates the feasibility of in-vivo monitoring of MTX therapy in arthritic rats using [¹⁸F]fluoro-PEG-folate PET, paving the way for its future use in human clinical RA.