Ethical approval for the study was obtained from our institution’s Human Research Ethics Committee.

Study Population and Design

This was a prospective cross sectional study which was conducted at the Department of Nuclear Medicine, University of the Free State/Universitas Academic Hospital in Bloemfontein, South Africa. Twenty two participants from the rheumatology clinic were recruited into this study. These participants were diagnosed with RA by an experienced rheumatologist (25 years experience), according to the ACR/EULAR classification criteria. They had disease involving either the wrist, small bones of the hands, and the knees. Recruitment period was between February and August 2022. Signed consent was obtained from all study participants.

Radiopharmaceutical Preparation

All commercial reagents and solvents were purchased from Sigma-Aldrich (Millipore Sigma, USA) or Merck Millipore (USA). All samples for 1H-NMR spectroscopy were prepared using CDCl3 or D2O and analyzed on a Bruker 300 MHz spectrometer (Massachusetts, USA). High-performance liquid chromatography (HPLC)-MS analysis was done using an Agilent Infinity 1200 Series system coupled to Agilent 6100 Series quadrupole MS system (Agilent, USA) with radiometric GABI Star gamma detector (Raytest GmbH, Straubenhardt, Germany).

Synthesis of ECDG

The ECDG ligand was synthesized according to the procedure described by Yang et al. [16]. Briefly, L-thiazolidine- 4-carboxylic acid (30.0 g) was dissolved in liquid ammonia (150 mL) followed by the slow addition of sodium metal (8.0 g, 1.5 eq) resulting in a deep blue-colored solution. The solution was stirred for 20 min at room temperature. The reaction was quenched with ammonium chloride (5.0 g), leading to the evaporation of ammonia solvent, and the resulting residue dissolved in water (200 mL). The pH was adjusted to 3.0, and the resultant precipitate was purified by recrystallization in ethanol to yield ethylenedicysteine 2HCl (EC) (38% yield).

EC (2.0 g) was dissolved in 2 M NaOH (30 mL) with ethanol (40 mL) and stirred vigorously for 20 min. Benzyl chloride (1.48 g, 2.0 eq) in dioxane (20 mL) was added dropwise to the solution and further stirred for 30 min, after which the organic solvents were removed. The pH of the resulting aqueous mixture was acidified to pH 3.0 resulting in the precipitation of the hydrochloride salt of S,S′-dibenzyl ethylene dicysteine (Bn-EC) (85% yield), which was used without further purification.

Benzyl chloroformate (4.90 g, 2.5 eq) in dioxane (150 mL) was added to a cooled (0 °C) solution of S,S′-dibenzyl ethylene dicysteine 2HCl (6.0 g) dissolved in 10% K2CO3 solution (150 mL), and the reaction was stirred for 2 h at 0 °C followed by stirring for 16 h at 25 °C. The solution was extracted with diethyl ether and the crude product precipitated by acidification of the aqueous phase. The product was redissolved and extracted using ethyl acetate to yield an amorphous solid product N,N′-dibenzyloxycarbonyl-S,S′-dibenzyl ethylene dicysteine (CBz-Bn-EC) (70% yield) upon drying.

CBz-Bn-EC (1.34 g) was activated by reaction with ethyl chloroformate (0.41 g, 2.0 eq) in chloroform (30 mL) with triethylamine (0.38 g, 2.0 eq) at − 15 °C for 15 min. To this reaction mixture, a solution of tetra-acetylglucosamine (1.58 g, 2.2 eq) and triethlyamine (0.42 g, 2.0 eq) in chloroform (30 mL) was added, and the combined reaction mixture was stirred for 1 h at 0 °C and then 12 h at 25 °C. Following an acid–base workup, the residue was purified by column chromatography (MeOH/EtOAc/hexane = 1:35:64 v/v ratio) to afford the fully protected ethylenedicysteine deoxyglucosamine (FP-ECDG) (70% yield).

FP-ECDG (1.0 g) underwent Birch reduction in liquid ammonia (80 mL) with sodium metal (0.5 g). The deep-blue-colored solution was stirred for 20 min at room temperature before quenching with ammonium phenylacetate (1.32 g, 12.0 eq). The formed solution was then dried under argon gas. The ammonium phenylacetate was extracted by stirring twice with isopropanol (50 mL and 25 mL) and separating using centrifugation (5 min, 4000 rpm). Residual isopropanol was removed by stirring and centrifuging with diethyl ether (2 × 50 mL), and the solid product was then dried under argon gas to afford ethylenedicysteine deoxyglucosamine (ECDG) (53% yield). The resultant product was confirmed MS and NMR analysis in accordance with the literature data (16). Calculated m/z 590.665 for C20H38O12N4S2 [M + H] = 591.1.

ECDG Kit Preparation

The ECDG kit preparation was done in a one vial procedure according to Zeevaart et al. [17]. All solutions (HCl (0.1 M), Na2HPO4 phosphate/citrate buffer solution and SnCl2 solution (1 mg/mL—ensuring the solution is clear and not milky), were freshly prepared with ultrapure, Milli-Q grade (> 18 MΩ/cm), and degassed water before production of the kits. Once prepared, all water and solutions were filtered through a sterile Millex-GP (polyethersulfone, 0.22 µm, 33 mm) syringe filter (Merck, Massachusetts, USA) into sterilized vials.

Citric acid (0.20 g) was added to a sterile vial containing Na2HPO4 (0.284 g) dissolved in water (pH 5.5) with the addition of SnCl2 solution (100 µL) and freeze-drying (Christ Alpha I-5 freeze-drier, Type 1050 (Medizinische Apparatebau, Harz, Germany) overnight. The ECDG (5 mg) was weighed into a vial under Ar (g) and dissolved in MeOH (0.75 mL). This solution was immediately transferred to the vial containing the Sn/buffer and flash frozen in liquid nitrogen followed by lyophilization overnight. The kits were sealed, capped, and placed in the − 80 °C freezer for storage.

Synthesis of 99mTc-ECDG

Radiosynthesis of 99mTc-ECDG was completed according to Zeevaart et al. [17] by adding Tc-99 m pertechnetate (99mTcO4 −) (50–60 mCi) to the prepared lyophilized ECDG kit vial (5 mg ECDG, citric acid/Na2HPO4, SnCl2). The solution was heated at 75 °C for 15 min.

Quality control was performed on the radiolabeled product by testing pH (pH 5.5) and radiochemical purity (RCP) (> 95%). RCP was determined using HPLC (Varian Prostar 325 UV/Vis (Varian Inc.) fitted with a radiometric GABI Star gamma detector (Raytest GmbH, Straubenhardt, Germany) and thin layer chromatography (TLC) (Raytest GmbH, Straubenhardt, Germany). HPLC analysis was performed using a C-18 reverse phase column (Agilent Luna-C18 column, 5 µm, 4.6 × 250 mm) with isocratic elution (4.5% MeCN in 2 mM ammonium formate (pH 3)) over 35 min. TLC analysis was completed using ITLC-SG (Agilent, USA) and Whatman (Millipore Sigma, USA) paper strips (10 cm) developed with saline and acetone as the mobile phase, respectively. Each strip was cut in half, and the activity was measured at the strip origin and front to determine the percentage of colloids (ITLC-SG) and percentage of labeled product (Whatman). The stability of the 99mTc-ECDG was determined up to 5 h after preparation using HPLC. The structure of 99mTc-ECDG is shown in Fig. 1.

Fig. 1

Structure of 99mTc-ethylenedicysteine-deoxyglucose (99mTc-glucosamine)

Radiopharmaceutical Administration

An aseptic dispensing process was followed for the preparation of 99mTc-ECDG patient dose. The sterility of the process was checked using air settle plates and finger dab plates which were cultured. The 99mTc-ECDG dose was prepared for injection by diluting the prepared radiolabeled solution with saline (approx. 3 mL) and filtering through Millex-GP, a sterile filter (polyethersulfone, 0.22 µm, 33 mm, Merck, Massachusetts, USA) into a sealed sterile vial. Additional saline (sterile, 1 mL) was added to the vial to ensure a final volume of around 5 mL. A patient dose of 20–25 mCi was withdrawn for intravenous administration. Fasting was not a requirement prior to the administration of the radiopharmaceutical. No adverse events were recorded after radiopharmaceutical administration.

Imaging Protocol

All 22 participants were scanned using a dual-head gamma camera (Siemens Symbia T16 True point SPECT-CT; Siemens medical solutions, USA). The SPECT/CT camera was equipped with a low-energy, high-resolution collimator (LEHR). Dynamic images of the clinically most symptomatic joints were acquired at the time of administering the radiopharmaceutical, with a frame rate of 1 fps for 60 s. This was followed by blood pool imaging of the hands, wrists, and knees. A delayed whole body image was acquired 2 h after radiotracer injection, followed by dedicated 5 min static images of the hands, wrists, and knees. SPECT images of the most clinically symptomatic joint (either the hands/wrist or knees) were also performed at 25 s/stop, with 3° steps, in a 128 × 128 matrix. This was followed by a low dose, non-contrast CT, with the patient in the same bed position.

Image Processing and Data Analysis

Images were processed using the Syngo workstation on the gamma camera. SPECT images were reconstructed using an iterative algorithm and SPECT/CT fusion images were obtained using the multimodality Syngo imaging software on the workstation.

The data of each patient were collected using an Excel 2019 spreadsheet (Microsoft, USA). Statistical analysis was performed using R, version 4.3.0 (R Foundation for Statistical Computing, Vienna, Austria).

Image Interpretation

Images were interpreted by a single nuclear medicine physician with 9 years experience. The dynamic flow images were assessed qualitatively for an increase, a decrease, or normal blood flow to the region imaged. The blood pool images were assessed for increased, decreased, or normal blood pool activity. Delayed images were interpreted for disease activity in the joints, using a slight modification of the scoring system used by Angelides et al. [14]:

Grade 0—Normal physiological joint uptake, defined as no/minimally increased radiotracer activity in joints (activity same as that of the neighboring muscle tissue).

Grade 1—Mild radiotracer uptake slightly more than that of the neighboring muscle tissue.

Grade 2—Moderate radiotracer uptake greater than that of grade 1.

Grade 3—Severe radiotracer uptake markedly greater than that of grade 1.

The whole body images were qualitatively assessed for the biodistribution of the radiotracer.