A New Dota-Folate Conjugate (CM09) For Imaging of Folate Receptor Expressing Macrophages in a Rat Model of Osteoarthritis

Purpose: Inflammation in OA animal models can be monitored using SPECT/CT with a DOTA-folate radioconjugate targeting the folate receptor that is specifically expressed on activated macrophages. However, the increase in folate receptor expression on macrophages in OA animal models is small and radioactivity accumulates mainly in the kidneys. A novel DOTA-folate conjugate (cm09) was recently developed with a low-molecular weight albumin-binding entity that enhances the blood circulation time of this conjugate, thereby improving the distribution of folate radioconjugates in the target tissue. In particular for its application in OA, where macrophages may be low abundant in the affected joint, this new radiotracer has potential applications. Our aim is to evaluate the in-vivo characteristics of the new 111In-Folate-conjugate (111In-cm09) in two different OA animal models compared to a control folate conjugate compound (111In-EC0800) without albumin binding entity.

Methods: In 16 weeks old wistar rats OA was induced by either an intra-articular injection of MIA (n=12) or groove surgery to induce local cartilage damage (n=10). Saline injections and sham surgery was performed in the contralateral leg as an internal control. Four weeks after MIA injections and 12 weeks after groove surgery, the folate conjugates were radiolabeled with 111InCl3 at a specific activity of 20MBq/nmol and administrated via the lateral tail vein (60MBq in 600 μL of saline) under sedation. In addition, the specificity of the technique was tested by blockade of the Folate Receptor-β (FRβ) by injection of an 500-fold excess of folic acid (100μg) immediately before radiofolate administration in 2 animals with MIA induced OA. SPECT/CT imaging with a high resolution rat multi-pinhole collimator (VECTor4CT, MILabs, Utrecht, The Netherlands) was performed 24 hours after radiolabeled folate administration. In-vivo whole-body and focused knee SPECT/CT images were obtained in all rats. Joint degeneration was assessed by histology using the OARSI histopathology score specific for the rat.

Results: Labeling of compound cm09 had a high efficiency and a radiochemical purity of more than 98%. An increase in joint degeneration was observed in the MIA injected knee joints compared to the saline injected control knee joints (10,2 ± 2,1 vs. 1,8 ± 1,1; p=0.011) together with a slightly increased synovial membrane inflammation (1.3 ± 0.8 vs 0.3 ± 0.7; p=0.07). Four weeks after MIA induction in-vivo imaging resulted in a higher uptake in the experimental knee joints compared to the contralateral knee joints (0,217 %ID/cm3 ± 0,049 vs. 0,208 %ID/cm3 ± 0,052; p=0.051, Figure 1). In parallel, in the groove operated knee joints, a mild joint degeneration was observed after 12 weeks, compared to the sham operated contralateral control knee joints (3,8 ± 1,1 vs. 1,56 ± 1,3; p=0.026) without synovial membrane inflammation (0.3 ± 0.5 vs 0.1 ± 0.3; p=0.32). In this mild non-inflammatory model no increase in macrophage activity was observed after 12 weeks compared to the sham operated knee joints (0,183 %ID/cm3 ± 0,029 vs. 0,182 %ID/cm3 ± 0,023; p=0.953). Looking more specifically to the effect of the new folate conjugate with albumin binding entity, no difference in macrophage activity in both models were detected compared to the control folate conjugate. However, the renal accumulation was significantly reduced in the new folate conjugate (see Figure 2) and the specificity of the signal is confirmed in the rats receiving an excessive amount of unlabeled folic acid. There the observed activity was 0,08 %ID/cm3 and equal in both the experimental MIA injected and contralateral saline injected control knee joints.

Conclusions: The present study showed that the new 111In-Folate-conjugate (111In-cm09) with albumin binding entity is suitable for SPECT/CT imaging of folate receptor positive cells, most likely activated macrophages, in small animal models of OA with specific binding to the folate receptor and non-inferior when compared to a control folate conjugate without the albumin binding entity (111In-EC0800). Moreover, a higher radiochemical yield is achieved in addition to a reduced renal accumulation of radioactivity. Besides, the new folate conjugate is safe to use and well tolerated in these animals. This makes the new folate conjugate a useful tool to study activated macrophages in the disease processes of OA.