Radiosynthesis of N-¹¹C-Methyl-Taurine–Conjugated Bile Acids and Biodistribution Studies in Pigs by PET/CT

Chemicals

CA, CDCA, DCA, UDCA, LCA, GCA sodium salt hydrate, TCA sodium salt hydrate, taurine, 1,2,2,6,6-pentamethylpiperidine (PMP), diethyl phosphoryl cyanide (DEPC), and anhydrous dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich and used as received. Nonradioactive MTCA, N-methyl-taurochenodeoxycholic acid (MTCDCA), N-methyl-taurodeoxycholic acid (MTDCA), MTUDCA, and MTLCA were prepared as described in the supplemental data (available at http://jnm.snmjournals.org). Nonradioactive MGCA, that is, cholylsarcosine (CSar), was prepared as described previously (14). The sodium and potassium salts of taurine were prepared by treating taurine with 1 equivalent of aqueous NaOH or KOH, respectively, followed by evaporation of the solvent and oven-drying. European Pharmacopoeia–grade acetonitrile and analytic-grade methanol, NaOH (solid), KOH (solid), and glacial acetic acid were obtained from VWR International Ltd. and used as received. Water (sterile), saline (sterile), ethanol (sterile), and 70 mM aqueous NaH₂PO₄ (sterile, pH 4.5) were obtained from the pharmacy at Aarhus University Hospital. Sep-Pak C8 Plus Short Cartridges (400 mg sorbent/cartridge, 37–55 μm particle size) were obtained from Waters and preconditioned before use with 10 mL of sterile ethanol followed by 10 mL of sterile water.

Radiosynthesis of N-¹¹C-Methyl-Taurine–Conjugated Bile Acids

The N-¹¹C-methyl-taurine–conjugated bile acids were prepared by a 2-step 1-pot radiosynthesis (Fig. 2): the radionuclide was produced by the ¹⁴N(p,α)¹¹C nuclear reaction using an IBA Cyclone 18/18 cyclotron. The applied target gas mixture (N₂ with 0.5% O₂) was bombarded at 30 μA for 15–30 min to yield ¹¹C-CO₂. The ¹¹C-CO₂ was delivered to the synthesis system (Tracerlab FXcPro; GE Healthcare) and converted to ¹¹C-CH₄ for further gas phase reaction with molecular iodine to ¹¹C-CH₃I. Fifteen minutes after the end of bombardment, the ¹¹C-CH₃I was released from the Porapak N at 190°C and transferred to the reactor of the synthesis system preloaded with a solution of the oven-dried precursor taurine sodium salt (1 mg) dissolved in anhydrous DMSO (300 μL). The ¹¹C-CH₃I was slowly bubbled through the precursor solution (helium flow, 15 mL/min) until maximum activity was trapped in the reactor. The ¹¹C-methylation of taurine was achieved by heating at 80°C for 3 min to give N-¹¹C-methyl-taurine. After the reactor was cooled to 65°C, freshly prepared solutions of the bile acid (12 mg) and PMP (5 μL) in anhydrous DMSO (200 μL), and DEPC (5 μL) in anhydrous DMSO (100 μL) were added successively through dry tubing directly into the reactor solution of N-¹¹C-methyl-taurine. The coupling reaction was allowed to proceed for 5 min at 65°C before quenching the reaction with sterile 20% aqueous ethanol (0.7 mL). The crude reaction mixture was purified by preparative reversed-phase high-pressure liquid chromatography using a Synergi Hydro-RP column (4 μm, 21.2 × 100 mm; Phenomenex) and with 30%–40% acetonitrile in sterile 70 mM aqueous NaH₂PO₄ buffer (pH 4.5) as the mobile phase (flow, 15 mL/min; λ = 220 nm). The fraction containing the N-¹¹C-methyl-taurine–conjugated bile acid (retention time, 5–10 min) was collected and diluted with 50 mL of sterile water and then extracted from this solution using a preconditioned C8-catridge. The purified product was eluted from the cartridge using sterile ethanol (1.0 mL) and finally formulated with sterile saline (9.0 mL). Total synthesis time, including ¹¹C-CH₃I production and tracer purification, was 40 min from the end of bombardment.

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FIGURE 2.

Radiosynthesis of N-¹¹C-methyl-taurine–conjugated bile acids (R¹, R² = H or α-OH; R³ = H, α-OH or β-OH).

The radiochemical conversion of the reactions (determined from samples of the crude reaction mixtures) and the identity and radiochemical purity of the isolated N-¹¹C-methyl-taurine bile acid conjugates were determined by liquid chromatography (LC) mass spectroscopy (MS) (LC: Dionex system; MS: Bruker Daltonics HCT Plus ion trap mass spectrometer running in negative ionization mode; the supplemental data provide information for further MS settings) connected in series to a Gabistar radiodetector (Nuclear Interface). The chromatographic column for analysis was a Synergi 4 μm MAX-RP 80A (2.0 × 100 mm; Phenomenex) with 30%–46% acetonitrile in aqueous ammonium acetate (1 mM; pH 3.0, adjusted using glacial acetic acid) as isocratic eluent with a flow of 0.9 mL/min (details are provided in the supplemental data). The chromatographic radio- and MS signals were analyzed using Dionex Chromeleon software (version 6.80) and Bruker Daltonics esquireControl (version 5.3), respectively. The identities of the ¹¹C-labeled bile acid conjugates were confirmed by comparison of retention time and mass spectrum with that of the corresponding ¹²C bile acid conjugates.

Determination of Lipophilicity by Reversed-Phase Thin-Layer Chromatography

The lipophilicity of MGCA (CSar), MTCA, MTCDCA, MTDCA, MTUDCA, MTLCA as well as GCA, TCA, and CA was determined by reversed-phase thin-layer chromatography (stationary phase, C18; mobile phase, methanol/aqueous ammonium acetate [pH 7.40]) and reported as values of R_Mw and S, where R_Mw reflects the lipophilicity of the compound—that is, the higher the value of R_Mw, the more lipophilic—and S the degree of responsiveness to changes in the mobile phase composition (details are provided in the supplemental data).

PET/CT Biodistribution and Dosimetry in Pigs

Biodistribution and dosimetry was determined in 2 pigs (3-mo-old female Danish Landrace and Yorkshire cross-breed) by PET/CT for ¹¹C-MTCA, ¹¹C-MTUDCA, and ¹¹C-MTLCA after an 18-h fasting with free access to water. The pigs were premedicated with midazolam and s-ketamine; anesthetized with a mixture of midazolam, s-ketamine, and propofol; and mechanically ventilated as previously described (19). A catheter (Cordis) was inserted into the femoral vein for administration of the tracers. Blood pressure; heart rate; body temperature; and arterial blood pO₂, pCO₂, pH, and glucose were monitored continuously and disturbances corrected for by appropriate procedures (19). After completion of the experiment, the animals were euthanized with an overdose of pentobarbital (100 mg/kg). The studies were performed according to the Danish Animal Experimentation Act and the European convention for the protection of vertebrate animals used for experimental and other purposes (European Treaty Series no. 123).

The PET/CT studies were performed with the pigs placed supine in a Siemens Biograph 64 Truepoint PET/CT camera with a 21.6-cm axial PET field of view. A CT scan (250 effective mAs with CARE Dose4D [Siemens], 120 kV, pitch of 1.0, and slice thickness of 2.0 mm) was obtained for definition of anatomic structures and attenuation correction of the PET recordings. Pig 1 (body weight, 41 kg) was administered ¹¹C-MTCA (511 MBq), ¹¹C-MTUDCA (508 MBq), and then ¹¹C-MTLCA (486 MBq), whereas pig 2 (body weight, 36 kg) was administered ¹¹C-MTLCA (536 MBq), ¹¹C-MTCA (571 MBq), and then ¹¹C-MTUDCA (553 MBq). The tracers were administered as intravenous bolus injections, and at least 7 physical half-lives of the ¹¹C radioisotope (half-life, 20.4 min) were allowed to pass between each administration. After administration of the tracer, 6 whole-body PET scans, each with 6 bed positions, were obtained with 3 min between scans and with a progressive increase in scan duration per bed position of 1, 1, 1.5, 2, 3, and 5 min. Thus, each whole-body scan was started 1, 10, 19, 30, 45, and 68 min after tracer injection. Whole-body images were reconstructed using 3-dimensional ordered-subset expectation maximization with 6 iterations and 21 subsets and a 2-mm Gauss filter. All tissues were visually inspected, and organs with accumulation of tracer above that of surrounding tissue were defined as source organs. These organs were liver tissue, gallbladder contents, stomach contents, and small intestine contents. No measurable radioactivity was observed in other organs including kidneys and urinary bladder; urine samples verified that no tracer was excreted in urine. Volumes of interest were manually drawn in each source organ to encompass all radioactivity of the respective organs. Intrahepatic bile ducts were included in the volume of interest of the liver, whereas the choledochus was included in the volume of interest of the small intestines.

For each source organ, the time course of the non–decay-corrected total radioactivity was extracted and recalculated into time courses of percentage injected dose using injected dose, organ masses, organ densities, and total weight for the reference adult man (20). Residence times were computed using the trapezoidal integration method to calculate the area under the curves assuming only physical decay after the last scan, without further biologic clearance. The remainder fraction was calculated by subtracting the individual source organ residence times from the total-body residence time (without voiding), which for ¹¹C is 0.49 h. Residence times for source organs and remainder were used in OLINDA/EXM 1.0 (21) to compute organ doses using the male reference phantom and to obtain an effective dose estimate according to International Commission on Radiological Protection publication 60 (22).