Synthesis of 99mTc-S-HYNIC CZP
All preparations were carried out under aseptic conditions working in a LAF IIa cabinet.
Derivatisation of CZP for injection with S-HYNIC
A 200-mg lyophilized CZP vial was reconstituted with water for injection (B. Braun, Melsungen AG, Germany); 100, 50 and 25 mg CZP was transferred to a Slide-A-Lyzer with cut-off of 10 kDa (Pierce Protein Research Products, Thermo scientific, Rockford, IL, USA) and dialyzed against 500 ml of a mixture of a Dulbecco’s phosphate-buffered saline (Lonza, Verviers, Belgium) and a self-prepared 0.9% m/v sodium chloride solution (Riedel-deHaën, Seelze, Germany) in 1:2 v/v ratio. Dialysis was maintained for 4 h at 2–8 °C, with the buffer refreshed after 1.5 h. Subsequently, 0.5 ml of a 8.4% sodium hydrogen carbonate (Merck, Darmstadt, Germany) solution was added to the solution followed by 10 5.0 μl portions of 1.7, 0.86 and 0.43% m/v solution of S-HYNIC (ABX GmbH, Radeberg, Germany) in dry DMSO (Merck, Darmstadt, Germany) at a pace of 1 portion/min . This yielded an average of 2.8 S-HYNIC groups per CZP. After 30 min incubation at room temperature in the dark, the reaction was quenched by adding 3.0 ml cooled 0.15 M acetate buffer pH 5.0 (Merck, Darmstadt, Germany). The unreacted S-HYNIC was removed by dialyzing the reaction mixture in a Slide-A-Lyzer (cutoff of 10 kDa) overnight at 2–8 °C against 500 ml acetate buffer, which was refreshed after 1, 2 and 3 h. The solution was diluted to 40.0 ml with 0.15 M acetate buffer pH 5.0 and membrane filtered (0.22 μm). Following dispensing into 1.0 ml portions, the glass vials were stored at −80 or 2–8 °C for 3 months. Three concentrations of CZP were obtained: 2.5, 1.25 and 0.625 mg of S-HYNIC-coupled CZP. Quality control was done by determination of the protein concentration (BCA protein reagent) and the p-NBA HYNIC assay to measure the number of S-HYNIC bifunctional chelator coupled to the protein.
Preparation of the co-ligand kit
A solution containing 4.66 mM tin(II) sulphate (Sigma Aldrich, Steinheim, Germany) and 55.81 mM tricine (Sigma Aldrich, Steinheim, Germany) dissolved in ultrapure sterile and pyrogen-free water was prepared.
Radiolabelling with 99mTc
Fifty-microliter co-ligand kit and 925 MBq (±10%) 99mTc pertechnetate were consecutively added to the S-HYNIC CZP vial (2.5, 1.25 and 0.625 mg). After 15-min incubation, physiological saline was added in order to obtain a volume of 3 ml. Quality control was carried out by instant thin layer chromatography (iTLC) with SilG as stationary phase and 0.9% NaCl solution as mobile phase. For the clinical study, the 1.25 mg S-HYNIC CZP vials stored at −80 °C were used and the radiochemical yield needed to exceed 90%.
The impact of aggregation on the chemical stability and radiochemical yield during storage of the formulation at three different concentrations (2.5, 1.25 and 0.625 mg) was studied over a 3-month period. Aggregate formation was assessed by size-exclusion HPLC (Agilent Zorbax Diol guard column), 4 × 12.5 mm, in series with a GF450, 9.4 × 250 mm and a GF250 size exclusion analytical column, 9.4 × 250 mm (Agilent Technologies, Diegem, Belgium). The mobile phase was composed of a mixture of a 200 mM phosphate buffer pH 7.0 and ethanol 90:10 v/v (1 ml/min, 30 min). Influence on the radiochemical incorporation of 99mTc was studied by iTLC as described earlier. Analyses were performed after preparation, at 2 weeks, 1 month and 3 months post production.
In vitro activity of 99mTc-S-HYNIC CZP against TNF-induced cytotoxicity
Murine fibrosarcoma TNF-sensitive L929s cells were cultured for 24 h by seeding 20,000 cells/well in 96-well plates, incubated at 37 °C with 5% CO2. The culture medium consisted of DMEM (GIBCO-BRL, 41965-062) supplemented with 10% fetal calf serum, 400 μM sodium-pyruvate (Sigma) and non-essential amino acids (Lonza). The following day, 55 μl human TNF (6.8 × 107 U/ml) produced by the Protein Service facility of VIB (Ghent, Belgium) was added at various concentrations (10 dilutions starting at 300 U/ml) to the test solutions at room temperature. The following solutions of antibodies directed against TNF were tested: CZP, S-HYNIC CZP, 99mTc-S-HYNIC CZP and infliximab (Remicade, Merck, Johnson & Johnson). Dilutions of 250, 50 and 10 ng/ml anti-TNF antibodies (55 μl) were preincubated with the human TNF for 45 min, as well as control samples without TNF blocking agents. Actinomycin D (Sigma, 1 mg/ml dissolved in absolute ethanol), at a final concentration of 1 μg/ml was also added. Twenty-four hours following the exposure to the test solutions, TNF cytotoxicity was measured using the MTT-test by adding 20 μl filter sterilized 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, 5 mg/ml). After 4 h, 80 μl stopping solution (10% SDS, 0.01 M HCL) was added and the read out was performed by a multichannel plate reader at a wavelength of 595 nm with a reference at 655 nm. The percentage of cell survival was estimated according to the formula below:
% cell survival = (absorbance treated cells − background) / (average absorbance non treated cells − background)*100.
The study was conducted in accordance with the Helsinki declaration and was approved by the ethical committee of the Ghent University Hospital. All patients signed an informed consent. Patient selection was limited to subjects aged 18–70 years and meeting the ‘American College of Rheumatology’ (ACR) criteria for RA  or the ‘Assessment of SpondyloArthritis international Society’ (ASAS) criteria for axial or peripheral SpA [7, 8]. Patients were not allowed if they had previously been treated with CZP or any other biological treatment. For more detailed information on the clinical eligibility criteria, we refer to the online detailed study protocol (EudraCT number: 2009-017998-37, http://clinicaltrials.gov/show/NCT01590966).
Scan procedure, blood and urine sampling
All patients were scanned on the same double-headed gamma camera (BrightView, Philips Healthcare, Best, the Netherlands). First, an attenuation map was obtained from a whole-body (WB) scan using a Cobalt-57 flood source. Subsequently, the patient was injected IV with 10.6 MBq/kg 99mTc-S-HYNIC CZP. The net IA was calculated following correction for rest activity in the syringe and exact time of administration (Veenstra ionization chamber VIK-202, Veenstra Instruments, the Netherlands). WB images (15 cm/min, 1024 × 512 matrix, pixel size 2.80 mm) were performed immediately following administration, at 1, 4–6 and 24 h post-injection (pi). A standard activity of approximately 5 MBq 99mTc in an unshielded syringe was always in the field of view for quantification purposes. Static images (5 min, 256 × 256 matrix, pixel size 2.33 mm) of hands and feet were acquired immediately following the first WB scan, at 4–6 and 24 h.
All visualized organs (heart, lungs, liver, spleen, kidneys), as well as regions of interest (ROIs) for the WB, background and standard activity were delineated manually on the four geometric mean images, generated from the WB scans (Nuclear Diagnostics, Stockholm, Sweden). For each organ, attenuation correction factors were calculated based on measured conversion factors on our camera from 57Co to 99mTc. These conversion factors were derived from sequential attenuation scans with a 99mTc flood source and a 57Co flood source in the first patient. The activity measured in the urine collections between the scans was used to correct the estimated activity in the WB compartment. Cumulated activities in the WB and various organs were estimated using mono-exponential fitting and converted to time integrated activity coefficients to be processed by OLINDA/EXM 1.1 software (Stabin M. Vanderbilt University, Nashville) to estimate the absorbed organ doses. The adult male and female mathematical reference phantoms were applied. A bladder voiding interval of 4 h was included in the model. In OLINDA/EXM 1.1, effective doses (ED60) were directly calculated using ICRP 60 tissue weighting factors . In order to account for the recent ICRP 103 recommendations, the effective dose was recalculated manually by combining the ICRP 103 tissue weighting factors and the organ doses from the OLINDA/EXM 1.1 output (ED103) . As some organs contributing to ED103 are not listed in the OLINDA/EXM 1.1, the sum of tissue-weighting factors for the available target organs were calculated (0.913 for men and 0.922 for women). In the ED103 calculation, these factors were accounted for by scaling .
Urinary tracer excretion was measured in sequential urine collections at 1, 4–6 and up to 24 h pi. Total volumes were recorded. Blood sampling was performed immediately following IV tracer injection from a vein in the contralateral arm and subsequently at 1, 4–6 and at 24 h pi. 99mTc activity was measured in 2 ml blood and urine samples in triplicate on a calibrated NaI(Tl) 3″ × 3″ gamma well counter (Cobra II, PerkinElmer, USA). The raw count data generated from the NaI(Tl) 3″ × 3″ detector were converted to kBq by a calibration curve. The standard samples—in the same geometry as the urine and blood samples—were prepared from a stock solution which was measured in the same calibrated dose calibrator (ionization chamber) used to measure the 99mTc-HYNIC CZP syringes. Results were decay corrected and expressed as kBq/ml and as %IA.
To assess the tracer accumulation in the peripheral joints, we scored each joint visually and semiquantitatively. The latter was done by manually drawing ROIs around each joint (Hermes, Nuclear Diagnostics, Sweden). For each individual scan, a background ROI was defined within the field of view, e.g. left supraclavicular region on WB, right forearm on static images of the wrists and hands, right distal tibia for the static images depicting the ankles and feet. These results were compared with the findings on clinical examination for each assessable joint. The nuclear medicine physician reading the immunoscans and the clinician performing the clinical examination were blinded to each other’s observations. Groups were compared by Mann-Whitney U test using IBM SPSSv21 software (NY, USA).