Development and characterisation of novel oxytocin analogues for PET imaging

Figures

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  Fig. 1. Chemical and radiochemical analogues studied. A Chemical structures of studied OT analogues and respective synthetic route. Aoa = aminooxy acetic acid; SFB = N-succinimidyl-4-fluorobenzoate; FDG = fluorodeoxyglucose (acyclic aldehyde representation); ESF = ethenesulfonylfloride; FBA = 4-Fluorobenzaldehyde. B Reaction scheme for the radioconjugation of dOTK8 using [18F]SFB. Aspects that were varied during optimisation are highlighted under the reaction arrow.

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  Fig. 2. Pharmacological characterisation of OT analogues. A OT analogues hydrophobicity test assessed by HPLC (HPLC Column: Vydac 218TP52, C18, 10 μm, 22 × 250 mm, 300 Å; gradient method: 0–40% B, 1%/min, 0.3 mL/min; solvent A: 0.05% TFA in H2O, solvent B: 0.043% TFA in 90% ACN). B Metabolic stability of OT analogues in rat and human serum. Each time point was analysed in three independent experiments (n = 3 technical triplicates, mean ± SEM). C Potencies (EC50) values for OT and analogues at the human OTR, V1aR, and V1bR, determined by a FLIPR calcium mobilisation assay (n = 3 technical triplicates, mean ± SEM). D Synoptic table of features tested for the analogues in this work and their similarity assessment with native OT. Desired criteria: anticipated optimal radioconjugation in < 1 h and activity yield > 15%; similar hydrophobicity with HPLC Rt ± 20% from OT; buffer t1/2 > 30 min at pH 7; metabolic t1/2 > 10 h in rat and human serum; EC50 at OTR < 50 nM; EC50 at V1aR and V1bR < 5×EC50(OTR) (n.a.: not assessed).

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  Fig. 3. Optimisation of radiolabelling of OT and dOTK8 precursors with [18F]SFB. A Radiochemical yields (RCY) for vessel radioconjugation of OT and dOTK8 with [18F]SFB evaluated by Radio-HPLC (n = 1). B Comparison of radiochemical yields for the radioconjugation of OT and dOTK8 with [18F]SFB, under different microfluidic conditions (n = 1). C Comparison of radiochemical yields for the radioconjugation of dOTK8 with [18F]SFB, under different flow rates at 50 °C (n = 1).

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  Fig. 4. Purification and analysis of the final product. A Radio-HPLC profiles for the conjugation reaction mixture injected in the semi-prep system (Chromolith RP semi-prep column (150 × 10 mm, 13 nm pores), using a 25/75 = ACN/H2O with 0.05% TFA (v/v) at a 2.5 mL/min flow rate), highlighting the fraction collected (F1-F4). B Expanded portion of radiochromatogram (method B, 4:00–6:30 min) analysing the 4 fractions collected in the semi-prep run (i-iv), compared with the unpurified mixture analysed in the same conditions (v); F2 (ii) revealed to be the desired product. C HPLC profiles for quality control of final [18F]dOTK8[SFB] product using gradient conditions (method B) to quantify radiochemical purity. D HPLC profiles for quality control of final [18F]dOTK8[SFB] product using isocratic conditions (method C) to quantify molar activity.

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  Fig. 5. Initial PET biodistribution assessment. A Radiotracer concentration (shown as %ID/g) in blood and plasma samples collected over 60 min (average of n = 5). B Biodistribution data at 60 min post injection (shown as % injected dose per gram of tissue weight, %ID/g) (n = 3, ± SEM, urine data excluded for clarity). C PET/CT 3D fusion render highlighting regions of specific uptake (green arrow: pituitary gland; blue arrows: mammary pads); in the lower region of the field of view it is possible to notice two more sets of mammary pads and substantial liver and intestines uptake.

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  Fig. 6. Pilot PET imaging study results. TACs for each of the pilot experiments (%ID/g vs min), compared with data from 20 min prior pre-blocking using non-radioactive isotopologue, in the heart (control, A), pituitary gland (B) and left (C) and right (D) mammary pads (it was not possible to locate the right mammary pad of T1 subject).

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  Fig. 7. Grouped PET imaging study and Patlak plot results. A-D TAC average curves (%ID/g vs min) for the three groups of animal experiments in four ROIs, compared with the data from the pilot blocking experiment (T4); positive error bar for baseline PET, negative error bar for blocking experiments, no error bar for heart content data (error bars: STD). E, F Patlak Kiref values of two tissues of interest (left and right mammary pads have been averaged) for the three groups (error bar: SEM); p values calculated with one-way ANOVA, with significant differences marked (*: p < 0.05); the parameter reported is the average of choosing the last 4, 5 and 6 time points of the Patlak plot.