Advertisement

A high molar activity 18F-labeled TAK-875 derivative for PET imaging of pancreatic β-cells

  • Mark H. Dornan
  • Daniil Petrenyov
  • José-Mathieu Simard
  • Antonio Aliaga
  • Guoming Xiong
  • Julien Ghislain
  • Barry Bedell
  • Vincent Poitout
  • Jean N. DaSilvaEmail author
Open Access
Research article
  • 238 Downloads

Abstract

Background

The free-fatty acid receptor-1 (FFA-1) is expressed by β-cells and is a promising target for molecular imaging of functional β-cell mass. Recently, the ((3-[18F]fluoropropyl)sulfonyl)propoxy-derivative of the high-affinity FFA-1 agonist TAK-875 ([18F]7) was reported. Here we describe the preparation of this tracer in high molar activity using a purification method permitting separation of [18F]7 from a structurally-related by-product and evaluation of the tracer in rats as a potential FFA-1 PET imaging agent.

Results

The radiotracer was produced by nucleophilic radio-fluorination of the tosylate precursor and deprotection of the methyl ester. Semi-preparative HPLC with a C18 column revealed that [18F]7 co-eluted with a non-radioactive impurity. Mass spectrometry identified the impurity as the alkene-containing elimination by-product. A pentafluorophenyl-functionalized HPLC column was found to separate the two compounds and allowed for purification of [18F]7 in high molar activity. A strong anion-exchange resin was used to reformulate [18F]7 in high concentration. Starting from 96 to 311 GBq of [18F]fluoride, 3.8–15.4 GBq of pure [18F]7 (end of synthesis (EOS)) was prepared (RCY 8.3% ± 1.1% decay-corrected, n = 4) in high molar activity (166–767 GBq/μmol at EOS). This PET agent was evaluated in rats using dynamic microPET/CT imaging, ex vivo biodistribution, and radio-metabolite studies. MicroPET/CT exhibited high uptake of the tracer in the abdominal area. There was no measurable decrease of the PET signal in the pancreatic area in rats pre-treated with saturating doses (30 mg/kg) of TAK-875. Biodistribution studies corroborated the microPET/CT results. Radiometabolism analyses revealed high compound stability with only the parent molecule detected in the pancreas.

Conclusions

Analysis of the crude reaction mixture and identification of the elimination by-product allowed for the development of a fully automated process to prepare the TAK-875-derived PET agent [18F]7 in high purity and high molar activity. Even though the radiotracer exhibited high in vivo stability, microPET/CT and biodistribution results confirmed recent reports demonstrating that lipophilic analogs of TAK-875 display a high degree of non-specific binding, masking any specific binding to FFA-1 in pancreatic β-cells. Future development of TAK-875-derived PET tracers should focus on reducing non-specific binding in the pancreatic tissue.

Keywords

Pancreatic Beta-cell imaging Diabetes Free fatty acid receptor-1 Lipophilic analogs 

Abbreviations

AM

Accurate mass

CT

Computed tomography

EM

Expectation maximization

EOS

End of synthesis

ESI

Electrospray ionization

FFA

Free-fatty acid receptor

HPLC

High performance liquid chromatography

HR

High resolution

ID

Injected dose

NMR

Nuclear magnetic resonance

OSEM

Ordered subset expectation maximization

PET

Positron emission tomography

RCY

Radiochemical yield

ROI

Region of interest

SD

Standard deviation

SPECT

Single photon emission computed tomography

SUV

Standardized uptake value

T2DM

Type II diabetes mellitus

THF

Tetrahydrofuran

Background

Type II diabetes mellitus (T2DM) is the predominant form of diabetes. The disease is characterized by insulin resistance and, after an initial compensatory over-production of insulin by β-cells, a loss of β-cell functional mass (Kahn 2003). Current understanding of β-cell dysfunction in T2DM has largely come from post-mortem autopsy data. There have been considerable efforts made towards developing a non-invasive imaging method to monitor β-cell function for both research and clinical studies (Andralojc et al. 2012). Such research would allow better understanding of β-cell mass biology including the pathological mechanisms that lead to depletion during disease progression and could be used in longitudinal studies in the pre-clinical and clinical evaluation of new therapies.

Insulin-secreting β-cells are localized in small (20 to 600 μM in diameter) clusters of endocrine cells called the islets of Langerhans, which are dispersed throughout the pancreas and account for only 1 to 2% of the total pancreatic mass (Ionescu-Tirgoviste et al., 2015). The size of this anatomical structure and its placement deep within the body restricts potential imaging modalities to those with high sensitivity, spatial resolution, and penetration depth. Given this, nuclear molecular imaging using techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT) hold the most promise.

The free fatty acid receptor-1 (FFA-1) is a G-coupled protein receptor that is highly expressed in human and rodent β-cells (Itoh et al. 2003; Briscoe et al. 2002). FFA-1 has attracted significant attention as a potential therapeutic target to promote glucose-stimulated insulin secretion (Mancini and Poitout 2015). The FFA-1 partial agonist TAK-875 was developed as a potent (KD = 4.8 nM and 6.3 nM for human and rat FFA-1, respectively) and selective therapeutic agent that reached phase III clinical trials before development was halted due to liver toxicity (Negoro et al. 2010; Otieno et al. 2017). Structure-activity relationship and TAK-875/FFA-1 co-crystal structure studies suggest that the 3-(methylsulfonyl)propoxy-moiety of TAK-875 could be modified to allow for radionuclide incorporation without significantly impacting target binding (Negoro et al. 2012; Srivastava et al. 2014).

Bertrand and coworkers recently reported the synthesis of TAK-875-derived fluorescent probes for β-cell imaging (Bertrand et al. 2016a). These new probes were used to track specific labeling of cells overexpressing the FFA-1 receptor. Later in 2016, while we were working on the same tracer, Bertrand and coworkers published the radiosynthesis of the ((3-[18F]fluoropropyl)sulfonyl)propoxy-derivative of TAK-875 ([18F]7) in 16.7% ± 5.7% radiochemical yield (RCY) in low molar activity (≥0.6 GBq/μmol) (Bertrand et al. 2016b). We report here a fully automated process to produce [18F]7, including optimized HPLC purification and reformulation conditions allowing for removal of the elimination by-product to obtain a high purity, high molar activity, and concentrated formulation of [18F]7 for in vivo evaluation in rats.

Methods

General

All commercially available materials were used without further purification unless otherwise noted. The synthesis of 1 was completed as previously described (Negoro et al. 2012). Flash chromatography was carried out with 40–60 μm silica gel. 1H and 13C NMR spectra were acquired with a 300 MHz spectrometer (Bruker) at ambient temperature. Spectral data is reported in parts per million using residual solvent as a reference. High resolution and accurate mass (HR/AM) measurements were performed in positive and negative ion mode by flow injection analysis into the Thermo Scientific Q-Exactive Plus Orbitrap Mass Spectrometer (San Jose, CA, USA) interfaced with a heated electrospray ion source.

An automated synthesis platform (IBA Synthera®) containing two synthesis modules and one semi-preparative radio-HPLC module was used for automated radiotracer production. The production was performed with the Synthera Nucleophilic Integrated Fluidic Processor research and development cassettes (Huayi Isotopes). The cassettes were cleaned with water, ethanol, and dried with nitrogen gas before each usage. Sep-Pak C18 Plus Light cartridges (130 mg, Waters) were pre-conditioned with 10 mL of ethanol followed by 20 mL of deionized water. Sep-Pak QMA Plus Light cartridges (130 mg, Waters) were pre-conditioned by passing through 10 mL of sodium bicarbonate solution (8.4%), followed by 10 mL of deionized water, and drying with helium gas. Semi-preparative HPLC purification was carried out with a Phenomenex Luna PFP (2) column (250 × 10 mm, 5 μm, flow rate, 8 mL/minute, 50% CH3CN/H2O + 0.1% TFA). A Phenomenex Luna C18 (2) column (250 × 4.6 mm, 10 μm, flow rate, 2 mL/minute, 35% CH3CN/H2O + 0.1% TFA) was utilized for analytical HPLC with a Waters HPLC equipped with a Raytest Gabi Star radioactivity detector. Residual solvent analyses were conducted using an Agilent 7890B gas chromatograph (headspace sampler).

Chemistry

3-(3-(tert-Butyl-dimethyl-silanyloxy)-propylsulfanyl)-propan-1-ol (2). The product 2 was prepared using a previously published method (Wilke et al. 2014). 3,3′-Thiodipropanol (870 mg, 5.8 mmol) was added to acetonitrile (12 mL) and hexanes (48 mL). Triethylamine (0.97 mL, 6.95 mmol) and tert-butyldimethylsilyl chloride (873 mg, 5.8 mmol) were added and the biphasic mixture was stirred vigorously at room temperature for 8 h. The reaction was quenched with a solution of saturated NH4Cl and extracted with EtOAc. The combined organic fractions were washed with brine, dried over MgSO4, filtered, and concentrated. Compound 2 (903 mg, 3.41 mmol, 59%) was purified by silica column chromatography (10% to 30% EtOAc in hexanes). TLC Rf = 0.44 in 30% EtOAc/hexanes. 1H NMR (300 MHz, CDCl3) δ 3.73 (t, J = 6.1 Hz, 2H), 3.67 (t, J = 6.1 Hz, 2H), 2.69–2.52 (m, 4H), 1.97 (s, 1H), 1.89–1.71 (m, 4H), 0.87 (s, 9H), 0.04 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 61.9, 61.7, 32.7, 32.0, 28.9, 28.6, 26.0, 18.4, − 5.2. HRMS (ESI): Exact mass calcd for C12H29O2SSi [M + H]+: 265.1652. Found: 265.1652.

(S)-methyl-2-(6-((4′-(3-((3-((tert-butyldimethylsilyl)oxy)-propyl)thio)propoxy)-2′,6′-dimethyl-[1,1′-biphenyl]3-yl)methoxy)-2,3-dihydrobenzofuran-3-yl)acetate (3). Compound 1 (950 mg, 2.27 mmol, 1.0 equiv.), 2 (600 mg, 2.27 mmol, 1.0 equiv.), and P(n-Bu3) (0.906 mL, 3.63 mmol, 1.6 equiv.) were dissolved in toluene (45 mL). 1,1′-(Azodicarbonyl)dipiperidine (0.916 g, 3.63 mmol, 1.6 equiv.) was added portion wise. The reaction was stirred for 20 h under N2 atmosphere at room temperature. The reaction mixture was diluted with H2O and extracted with EtOAc. The desired compound 3 (1.05 g, 1.585 mmol, 70%) was purified by silica column chromatography (2% to 8% EtOAc in hexanes). TLC Rf = 0.35 in 15% EtOAc/hexanes. 1H NMR (300 MHz, CDCl3) δ 7.47–7.34 (m, 2H), 7.17 (s, 1H), 7.08 (d, J = 6.9 Hz, 1H), 7.02 (d, J = 8.0 Hz, 1H), 6.66 (s, 2H), 6.54–6.42 (m, 2H), 5.06 (s, 2H), 4.75 (t, J = 9.0 Hz, 1H), 4.26 (dd, J = 9.1, 6.1 Hz, 1H), 4.08 (t, J = 6.0 Hz, 2H), 3.87–3.75 (m, 1H), 3.75–3.64 (m, 5H), 2.81–2.49 (m, 6H), 2.13–1.95 (m, 8H), 1.88–1.75 (m, 2H), 0.91 (s, 9H), 0.07 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 172.7, 161.4, 160.3, 158.0, 141.5, 137.7, 137.4, 134.6, 129.5, 129.0, 128.9, 125.8, 124.6, 121.8, 113.5, 107.6, 97.8, 77.9, 70.6, 66.4, 61.9, 52.1, 39.8, 38.1, 33.0, 29.7, 29.0, 28.9, 26.3, 21.4, 18.6, − 5.0. Exact mass calcd for C38H53O6SSi [M + H]+: 665.3327. Found: 665.3326.

(S)-methyl-2-(6-((4′-(3-((3-hydroxypropyl)sulfonyl)propoxy)-2′,6′-dimethyl-[1,1′-biphenyl]3-yl)methoxy)-2,3-dihydrobenzofuran-3-yl)acetate (4). The compound 3 (275 mg, 0.413 mmol, 1.0 equiv.) was dissolved in CH3OH (5 mL) and THF (5 mL) and the mixture was cooled to 0 °C. Oxone (254 mg, 0.827 mmol, 2.0 equiv.) dissolved in H2O (5 mL) was added to the mixture dropwise. The reaction mixture was stirred from 0 °C to room temperature for 18 h. The reaction mixture was concentrated to remove the organic solvent and the aqueous residue was diluted with H2O. The mixture was extracted with EtOAc, washed with brine, dried over MgSO4, filtered, and concentrated. Product 4 (223 mg, 0.383 mmol, 93%) was purified by silica column chromatography (50% to 90% EtOAc in hexanes). TLC Rf = 0.43 in 90% EtOAc/hexanes. 1H NMR (300 MHz, CDCl3) δ 7.47–7.33 (m, 2H), 7.16 (s, 1H), 7.07 (d, J = 7.1 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.64 (s, 2H), 6.51–6.43 (m, 2H), 5.05 (s, 2H), 4.74 (t, J = 9.0 Hz, 1H), 4.26 (dd, J = 9.2, 6.1 Hz, 1H), 4.12 (t, J = 5.7 Hz, 2H), 3.88–3.74 (m, 3H), 3.71 (s, 3H), 3.32–3.12 (m, 4H), 2.74 (dd, J = 16.4, 5.5 Hz, 1H), 2.55 (dd, J = 16.4, 9.2 Hz, 1H), 2.42–2.28 (m, 2H), 2.21–2.07 (m, 2H), 1.99 (s, 6H), 1.72 (s, 1H). 13C NMR (75 MHz, CDCl3) δ 172.7, 161.4, 160.2, 157.4, 141.2, 137.9, 137.5, 135.1, 129.4, 129.0, 128.9, 125.9, 124.6, 121.8, 113.5, 107.6, 97.8, 77.9, 70.6, 65.7, 60.9, 52.1, 50.4, 50.3, 39.8, 38.1, 25.3, 22.7, 21.4. HRMS (ESI): Exact mass calcd for C32H39O8S [M + H]+: 583.2360. Found: 583.2366.

(S)-methyl-2-(6-((2′,6′-dimethyl-4′-(3-((3-(tosyloxy)propyl)sulfonyl)propoxy)-[1,1′-biphenyl] 3-yl)methoxy)-2,3-dihydrobenzofuran-3-yl)acetate (5). Product 5 was prepared as described previously (Bertrand et al. 2016b). p-Toluenesulfonyl chloride (196 mg, 1.03 mmol, 1.5 equiv.) dissolved in 1.9 mL of CH2Cl2 was added dropwise to a solution of 4 (400 mg, 0.686 mmol, 1.0 equiv.) dissolved in toluene (4.7 mL) and CH2Cl2 (4.7 mL), in the presence of triethylamine (0.143 mL, 1.03 mmol, 1.5 equiv.) and N,N,N′,N′-tetramethyl-1,6-hexanediamine (15 μL, 0.0686 mmol, 0.1 equiv.). The reaction was stirred for 18 h at room temperature. The mixture was quenched with H2O, extracted with EtOAc, washed with brine, dried over MgSO4, filtered and concentrated. The product 5 (463 mg, 0.628 mmol, 92%) was purified by silica column chromatography (30% to 50% EtOAc in hexanes). TLC Rf = 0.40 in 50% EtOAc/hexanes. 1H NMR (300 MHz, CDCl3) δ 7.79 (d, J = 8.3 Hz, 2H), 7.46–7.32 (m, 4H), 7.16 (s, 1H), 7.07 (dt, J = 7.1, 1.6 Hz, 1H), 7.02 (d, J = 8.0 Hz, 1H), 6.64 (s, 2H), 6.52–6.43 (m, 2H), 5.06 (s, 2H), 4.75 (t, J = 9.0 Hz, 1H), 4.26 (dd, J = 9.2, 6.1 Hz, 1H), 4.19 (t, J = 5.9 Hz, 2H), 4.11 (t, J = 5.7 Hz, 2H), 3.87–3.74 (m, 1H), 3.71 (s, 3H), 3.28–3.16 (m, 2H), 3.16–3.05 (m, 2H), 2.75 (dd, J = 16.4, 5.5 Hz, 1H), 2.55 (dd, J = 16.4, 9.2 Hz, 1H), 2.46 (s, 3H), 2.38–2.19 (m, 4H), 1.99 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 172.6, 161.4, 160.2, 157.4, 145.6, 141.2, 137.9, 137.5, 135.2, 132.9, 130.4, 129.4, 129.0, 128.9, 128.3, 125.9, 124.6, 121.8, 113.5, 107.6, 97.8, 77.9, 70.6, 68.3, 65.6, 52.1, 50.7, 49.4, 39.8, 38.1, 22.6, 22.2, 22.0, 21.4. Exact mass calcd for C39H45O10S2 [M + H]+: 737.2449. Found: 737.2442.

(S)-methyl-2-(6-((2′,6′-dimethyl-4′-(3-((3-fluoropropyl)sulfonyl)propoxy)-[1,1′-biphenyl] 3-yl)methoxy)-2,3-dihydrobenzofuran-3-yl)acetate (6). Product 6 was prepared as described previously (Bertrand et al. 2016b). 50% Deoxo-fluor (bis(2-methoxyethyl)aminosulfur trifluoride) in THF (37 mg, 0.166 mmol, 1.65 equiv.) was added to 4 (59 mg, 0.101 mmol, 1.0 equiv.) in CH2Cl2 at 0 °C. The reaction was allowed to react from 0 °C to room temperature for 18 h. The reaction mixture was diluted with H2O, extracted with EtOAc, dried over Na2SO4, filtered and concentrated. The desired product 6 (31 mg, 0.053 mmol, 53%) was purified by silica column chromatography (20% to 50% EtOAc in hexanes). TLC Rf = 0.51 in 50% EtOAc/hexanes. 1H NMR (300 MHz, CDCl3) δ 7.46–7.34 (m, 2H), 7.16 (s, 1H), 7.07 (d, J = 7.0 Hz, 1H), 7.02 (d, J = 8.0 Hz, 1H), 6.64 (s, 2H), 6.52–6.44 (m, 2H), 5.05 (s, 2H), 4.75 (t, J = 9.0 Hz, 1H), 4.68 (t, J = 5.6 Hz, 1H), 4.53 (t, J = 5.6 Hz, 1H), 4.26 (dd, J = 9.2, 6.1 Hz, 1H), 4.13 (t, J = 5.7 Hz, 2H), 3.86–3.74 (m, 1H), 3.72 (s, 3H), 3.29–3.14 (m, 4H), 2.75 (dd, J = 16.4, 5.5 Hz, 1H), 2.55 (dd, J = 16.4, 9.2 Hz, 1H), 2.42–2.19 (m, 4H), 1.99 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 172.67, 161.45, 160.25, 157.40, 141.25, 137.98, 137.49, 135.18, 129.45, 128.99, 128.89, 125.94, 124.61, 121.83, 113.52, 107.63, 97.79, 82.01 (d, JC,F = 168 Hz), 77.90, 70.61, 65.70, 52.13, 50.59, 49.55 (d, JC,F = 4 Hz), 39.81, 38.09, 23.57 (d, JC,F = 21 Hz), 22.67, 21.45. HRMS (ESI): Exact mass calcd for C32H39O8S [M + Na]+: 607.2136. Found: 607.2136.

(S)-2-(6-((4′-(3-((3-fluoropropyl)sulfonyl)propoxy)-2′,6′-dimethyl-[1,1′-biphenyl]3-yl)methoxy)-2,3-dihydrobenzofuran-3-yl)acetic acid (7). Product 7 was prepared as described previously (Bertrand et al. 2016b). NaOH(aq) (1.0 M, 0.17 mL) was added to a solution of 6 (20 mg, 0.034 mmol, 1.0 equiv.) in CH3OH (0.3 mL) and THF (0.6 mL), and the mixture was heated to 50 °C for 1 h. The reaction mixture was concentrated and 7 (17.3 mg, 0.030 mmol, 88%) was purified by silica column chromatography (80% EtOAc in hexanes + 0.5% CH3CO2H). TLC Rf = 0.40 in 80% EtOAc/hexanes + 0.5% CH3CO2H. 1H NMR (300 MHz, CDCl3) δ 7.47–7.33 (m, 2H), 7.16 (s, J = 10.1 Hz, 1H), 7.06 (t, J = 7.4 Hz, 2H), 6.64 (s, 2H), 6.53–6.43 (m, 2H), 5.05 (s, 2H), 4.75 (t, J = 9.0 Hz, 1H), 4.68 (t, J = 5.6 Hz, 1H), 4.53 (t, J = 5.6 Hz, 1H), 4.28 (dd, J = 9.2, 6.1 Hz, 1H), 4.12 (t, J = 5.7 Hz, 2H), 3.87–3.72 (m, 1H), 3.30–3.13 (m, 4H), 2.80 (dd, J = 16.7, 5.2 Hz, 1H), 2.60 (dd, J = 16.7, 9.3 Hz, 1H), 2.41–2.28 (m, 3H), 2.28–2.19 (m, 1H), 1.99 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 161.43, 160.31, 157.40, 141.26, 137.98, 137.45, 135.17, 129.48, 129.00, 128.89, 125.94, 124.63, 121.58, 113.53, 107.72, 97.84, 82.0 (d, JC,F = 168 Hz), 70.62, 65.70, 50.58, 49.55 (d, JC,F = 4 Hz), 37.91, 23.56 (d, JC,F = 21 Hz), 22.65, 21.45. Exact mass calcd for C31H35FNaO7S [M + Na]+: 593.1980. Found: 593.1984.

Production of [18F]7

Using an IBA Cyclone® 18/9 cyclotron, [18F]fluoride was produced by the 18O(p,n)18F nuclear reaction and was transferred to the IBA Synthera® radiochemistry platform which was configured with two synthesis units and an HPLC module. The [18F]fluoride was trapped on a pre-conditioned Waters Sep-Pak® QMA Plus Light anion-exchange cartridge. [18F]Fluoride was eluted from the cartridge into the reactor with 0.6 mL of a 1:1 mixture of acetonitrile/water containing 2.2.2-cryptand (22.6 mg) and potassium carbonate (4.2 mg). Acetonitrile was added, and the mixture was azeotropically dried under vacuum and heat. A solution of the tosylate precursor 5 (5 mg) in 1 mL of acetonitrile was added and the reactor was heated to 100 °C for 2.5 min. Following removal of the solvent (vacuum), the reactor was cooled and 1 mL of a 1:1 mixture of aqueous NaOH (2.5 M)/CH3OH was added. After 3 min, the reaction was quenched with 10 mL of H2O + 0.1% TFA and transferred through a pre-conditioned Waters Sep-Pak® C18 cartridge. Acetonitrile (1.2 mL) eluted the radiotracer from the C18 cartridge into a V-vial containing H2O + 0.1% TFA (1.8 mL) and injected onto semi-preparative HPLC equipped with a Phenomenex Luna PFP (2) column, which enabled separation of the radiotracer from the elimination by-product 8 (Fig. 1). The fraction containing [18F]7 was collected into a vessel containing aqueous NaOH (1.0 M, 30 mL) and transferred over a Waters Oasis® MAX anion-exchange resin cartridge using vacuum and N2 push in the second Synthera synthesis unit. Following deionized water wash (10 mL), [18F]7 was eluted with 1 mL EtOH + 0.5% HCl, then filtered (0.22 μm) into a sterile multidose vial containing 9 mL of isotonic bicarbonate-buffered saline. Analytical HPLC was performed to determine the chemical and radiochemical purity. Identity of [18F]7 was determined by HPLC co-elution with the non-radioactive standard 7. The molar activity was measured by comparing the UV (254 nm) peak of [18F]7 with an injection of the non-radioactive standard 7 with a comparable UV response.
Fig. 1

Semi-preparative HPLC chromatogram of a typical radiosynthesis of [18F]7. Resolution of [18F]7 (tR = 23 min) from the elimination by-product 8 (tR = 21 min) was achieved using a pentafluorophenyl functionalized HPLC column. Radioactivity (black) and UV (γ = 254 nm; blue)

Identification of the impurity 8

The radiosynthesis and HPLC purification of [18F]7 was conducted as described above using a semi-preparative Phenomenex Luna C18 column. The HPLC peak containing a co-elution of [18F]7 and a non-radioactive impurity was collected. The decayed fraction was diluted in methanol and analyzed by mass spectrometry. HRMS (ESI): Exact mass calcd for C31H35O7S [M + H]+: 551.2098. Found: 551.2101.

Animals

Male Sprague-Dawley rats (150–175 g, Charles River Laboratories, Montreal, Canada) were housed on a 12 h:12 h light/dark cycle and fed standard rat chow and water ad libitum. This study was carried out in accordance with Canadian Council on Animal Care guidelines. The protocol (protocol number IM16029JDSr) was approved by the institutional Animal Care Committees of the Centre de Recherche du Centre Hospitalier de l’Université de Montreal and the McGill University Health Centre.

Small animal PET imaging

General

Small animal PET/CT scans were performed with [18F]7 (7.4–14.8 MBq, 0.12–0.66 μg non-radioactive mass) in control animals (n = 3) on a Mediso nanoScan PET/CT (Mediso Medical Imaging Systems, Budapest, Hungary). To assess binding specificity to FFA-1 in the pancreas, additional rats (n = 3) were injected with TAK-875 (30 mg/kg, from a 20 mg/mL solution of TAK-875 in 10,5,1 saline 0.9%/H2O/sodium bicarbonate 8.4% + 10% DMSO) intravenously 5 min before administration of [18F]7 (12.8–13.5 MBq, 0.27–0.58 μg non-radioactive mass).

Image acquisition

Each PET/CT session consisted of a 60 min PET emission scan, followed by a 10 min CT transmission scan. PET/CT scans were conducted under anesthesia (isoflurane 2%, oxygen 0.6 L/min) delivered by a nose cone. Temperature and heart rate were monitored throughout the procedure using the Mediso system. After the animal was placed in the scanner, with the heart positioned at the center of the field of view, emission scans were initiated immediately after a bolus injection in the tail vein. List mode data was histogrammed into 29 sequential time frames of increasing duration (6 × 10 s, 4 × 30 s, 4 × 60 s, 4 × 120 s, 5 × 180 s, 6 × 300 s frames) over 60 min. Images were reconstructed using expectation maximization (EM), ordered subset expectation maximization (OSEM), normalized, and corrected for scatter, dead time, and decay.

Image analysis

Images were analyzed with Amide software (version 1.0.5 for Linux). Regions of interest (ROI) were defined on reconstructed images in the pancreas, kidney, liver and muscle to obtain time-activity curves. A 3-D sphere with a radius of 2.5 mm was drawn within the left ventricle using an average image of very early frames (10–60 s) to sample the blood input function. The sphere was centred at the point where the maximum activity was found inside this region. The ROIs for pancreas, kidney, liver and muscle were drawn using an average image of early frames (1–20 min post injection where highest tissue to background contract can be obtained). For kidney and liver, the ROIs are 3-D spheres located roughly in the middle of the tissue with a radius of 5 mm (kidney) and 6 mm (liver), respectively. For pancreas, the ROI was ellipsoid shaped and was created based on the anatomy of rats as both CT and PET images could not help with distinguishing this tissue from other organs.. The radii of the ROI were 1.5 mm (sagittal), 3 mm (transverse), and 3 mm (coronal), respectively. For muscle, the ROI was a 3-D sphere located on the muscle near left shoulder of the animals with a radius of 3 mm.

Statistical analysis

Results are presented as mean ± standard deviation. A two-tailed t-test was used to compare between two groups. P values < 0.05 were considered significant.

Biodistribution studies

Biodistribution studies were performed to evaluate tissue uptake and to measure specific and non-specific binding of the radiotracer to FFA-1. Briefly, rats were anesthetized (2–2.5% isofluorane) and injected via a lateral tail vein with 7.8–12.2 MBq tracer activity. Baseline group animals (n = 4) received tracer alone, whereas the blocking group animals (n = 4) received an intravenous dose of the FFA-1 agonist TAK-875 (30 mg/kg) 5 min prior to tracer administration. Rats were sacrificed by decapitation at 20 min post-injection. Trunk blood was collected in heparinized tubes. The following tissues were dissected and collected into pre-weighed tubes: pancreas, kidney, spleen, fat, bone, muscle, blood, plasma. Using a gamma counter (PerkinElmer Wizard2), all tubes were counted along with a 1:100 dilution of the injected tracer solution as a standard. The percent of the injected dose per gram of tissue (%ID/g) was calculated from the decay-corrected counts and expressed as a ratio to blood.

Radiolabeled metabolite analysis

General

Radiolabeled metabolite analysis studies were performed to evaluate the metabolic stability of [18F] in plasma and the pancreas. Rats were anesthetized (2–2.5% isofluorane) and injected via the lateral tail vein with 35.2–73.3 MBq of [18F]7. At 60-min post-injection, the animals were sacrificed by decapitation. Trunk blood was collected into heparinized tubes and the pancreas was dissected.

Sample preparation

Blood was centrifuged (4000 x g, 5 min, 4 °C) to obtain plasma. Urea (1 g) was dissolved in the plasma (1 mL) and the sample was filtered (0.22 μm) and injected onto the column-switching HPLC. Pancreas samples were suspended in 80:20 EtOH/H2O, homogenized using a polytron and centrifuged (14.8 rpm, 20 min, 4 °C). The supernatant was collected and dried by rotary evaporation. The residue was reconstituted in 1 mL of 1% CH3CN/H2O containing 0.4 g of urea to reduce protein binding, filtered (0.22 μm), and injected onto the column-switching HPLC. Most of the radioactivity from the pancreas (and plasma) was present in the injected sample.

Column-switching HPLC

A modification of the column-switching HPLC procedure was used (Kenk et al. 2008) to analyse the radioactive metabolites from plasma and pancreas samples. Eluted solvents were analyzed via two detectors in series: a UV absorbance detector (Waters 2489) and a radiation detector (Raytest Gabi Star), which were connected to a chromatography data integration system (PeakSimple, using PeakSimple data analysis software, version 4.44). Using mobile solvent A (1% CH3CN/H2O + 0.1% TFA, 1 or 2 mL/minute), samples were loaded onto an in-line refillable capture column (hand-packed with 20 mg of Oasis HLB polymeric reverse phase sorbent) fitted with 2.5 μm frits. The elution of biological macromolecules was monitored by UV absorbance (254 nm) and radioactivity. When the UV signal returned to baseline, the solvent flow was switched to elute the capture column loaded sample onto the analytical column (Phenomenex Luna C18, 250 × 4.6 mm, 10 μm) using mobile Solvent B (65% CH3CN/H2O + 0.1% TFA, 2 mL/minute flow rate). Retention times were calculated from the time of switch. Radioactivity data are expressed as the percentage of the total radioactivity signal. For standards, samples of blood and pancreas from control rats were spiked with [18F]7 and processed as described above for each formulation tested. Use of such controls allowed for measurement of the proportion of radiotracer retained and not-retained by the capture column during loading. If radioactivity was eluted during loading of the capture column, this activity will be identified as authentic [18F]7 and not as a hydrophilic labeled metabolite.

Results

Chemistry and radiochemistry

The monoprotected diol 2 was synthesized following a literature procedure (Wilke et al. 2014). A Mitsunobu coupling reaction was used to synthesize 3 in 70% yield. Treatment of 3 with Oxone oxidized the thioether to a sulfone and deprotected the terminal alcohol protecting group to afford 4 in 93% yield (Scheme 1). Tosylation of 4 provided the radiochemical precursor 5 in 92% yield. To produce the non-radioactive standard 7, the compound 4 was treated with Deoxo-Fluor (bis(2-methoxyethyl)aminosulfur trifluoride) followed by base-mediated ester hydrolysis, as described before (Bertrand et al. 2016b).
Scheme 1

Synthesis of the radiochemical precursor 5 and the non-radioactive standard 7

The radiosynthesis of [18F]7 was performed with an automated one-pot, two-step procedure. [18F]fluoride nucleophilic displacement of the tosylate leaving group was followed by base-mediated hydrolysis of the methyl ester (Scheme 2). The quenched reaction mixture was purified by C18 SPE cartridge followed by semi-preparative HPLC (Fig. 1). The peak corresponding to [18F]7 was collected into a vessel containing aqueous NaOH, loaded onto a weak anionic exchange resin, then eluted with acidified ethanol into isotonic bicarbonate-buffered saline. This procedure allowed for rapid reformulation in high concentration. Starting from 96 to 311 GBq of [18F]fluoride, 3.8–15.4 GBq of [18F]7 (at EOS) was prepared (decay-corrected RCY 8.3% ± 1.1%, n = 4) in 75–89 min. The molar activity ranged from 166 to 767 GBq/μmol at EOS.
Scheme 2

Radiosynthesis of [18F]7

MicroPET imaging

Preliminary microPET/CT scans performed with [18F]7 exhibited the highest radiotracer concentration in the liver (Fig. 2). At 10 min post-injection, control group tissue-to-blood ratios for the liver, kidney and pancreas were 12.34 ± 3.34, 0.62 ± 0.07, and 0.49 ± 0.05, respectively (n = 3). In the TAK-875 pre-treated group, tissue-to-blood ratios in the liver, kidney and pancreas were 6.50 ± 0.96, 0.66 ± 0.04, and 0.57 ± 0.09, respectively (n = 3).
Fig. 2

Representative microPET images (SUV, sum of all frames; pancreas ROI shown in yellow) with tissue time-activity curves in rats following injection of (a) [18F]7 and (b) [18F]7 with a pre-injection of TAK-875 (30 mg/kg). Time-activity curve data are presented as average SUV ± standard deviation (n = 3)

Ex vivo biodistribution

The average % ID/g tissue-to-blood ratios for the control and blocked groups are presented in Fig. 3. In the control group, the highest average activity concentrations were found in the plasma, kidney, and pancreas (average tissue-to-blood ratios 1.60 ± 0.03, 0.98 ± 0.02, and 0.54 ± 0.04, respectively; n = 3). Lower activity concentrations were detected in the muscle, spleen, bone, and fat tissue samples (average tissue-to-blood ratios 0.30 ± 0.1, 0.27 ± 0.02, 0.19 ± 0.01, and 0.16 ± 0.01, respectively; n = 3). Blocking the FFA-1 receptor did not result in any significant difference in the tissue average activity concentrations except for the kidney, which was reduced by 20% (P < 0.05).
Fig. 3

[18F]7 relative tissue uptake assessed by ex vivo biodistribution in controls (n = 4; blue), and in rats pre-injected with 30 mg/kg TAK-875 (n = 4; red). Ratios are expressed as %ID/g ratio to blood ± SD. * p < 0.05 vs. control

Radiolabeled metabolites analysis

Column-switching HPLC analysis of the control plasma and pancreas samples spiked with authentic [18F]7 revealed only one peak, corresponding to unchanged tracer (Rt = 5.5 min) (Fig. 4a-b). This demonstrated that the tissue processing method did not degrade or affect the radiotracer. No other peaks were detected in the control samples.
Fig. 4

Representative column-switching HPLC radio-metabolite analysis of controls and experimental samples. Column-switching from capture-column loading to elution of the capture column onto the analytical column starts at t = 0 min. Radioactivity (blue) and UV (254 nm, red) spectra are shown overlaid. (a) Control sample plasma spiked with [18F]7; (b) control sample pancreas spiked with [18F]7; (c) 60-min post-injection in plasma; (d) 60-min post-injection in the pancreas

Sixty minutes following intravenous administration of [18F]7 in rats (n = 4), one labeled hydrophilic metabolite was observed in rat plasma after the switch (Rt = 3.1 min) on analytical HPLC, while 88% ± 4% of the total radioactivity was attributed to unchanged radiotracer (Fig. 4c). In the pancreas homogenate, only the unchanged radiotracer was detected (n = 4) (Fig. 4d).

Discussion

A novel automated method was developed to produce the ((3-[18F]fluoropropyl)sulfonyl)propoxy-derivative of TAK-875 ([18F]7) in high molar activity and purity, allowing for in vivo evaluation in rats as a potential FFA-1 probe for β-cell PET imaging. The tosylate precursor 5 and the non-radioactive standard 7 were synthesized starting from the aromatic alcohol 1. The thioether intermediate 3 was produced by coupling the mono-protected diol 2 with the aromatic alcohol 1 using Mitsunobu-coupling conditions. Removal of the silyl protecting group and oxidation of the thioether was achieved in a single step by treatment with Oxone to give 4. The radiochemical precursor 5 was generated by tosylation of 4 as described previously (Bertrand et al. 2016b). The non-radioactive standard was synthesized by fluorination of 4 using Deoxo-Fluor, followed by base-mediated ester hydrolysis (Bertrand et al. 2016b).

The radiosynthesis of [18F]7 was completed with a nucleophilic radio-fluorination reaction followed by base-mediated ester hydrolysis in a one-pot, two-step process. Semi-preparative HPLC purification of [18F]7 was initially explored using a C18 column. Co-elution of the radiotracer with a non-radioactive by-product was observed. Despite efforts, chromatographic resolution of the tracer from this by-product was not achieved by adjusting the mobile phase composition. High resolution mass spectrometry indicated that the identity of the impurity was the alkene-containing elimination by-product 8 (Fig. 1). Resolution of [18F]7 from 8 was achieved employing a pentafluorophenyl-functionalized reverse phase column which enabled preparation of [18F]7 in high purity. Inspection of previously described structure-activity relationships and the TAK-875/FFA-1 co-crystal structure predicted that 8 would potentially compete with [18F]7 for binding to FFA-1 (Negoro et al. 2012; Srivastava et al. 2014). Thus, removal of 8 will increase the apparent molar activity of the final product and improve the potential for quantitative PET imaging of pancreatic FFA-1 and β-cell mass.

Reformulation of [18F]7 was performed using an anion exchange resin. The purified [18F]7 HPLC peak was collected into a vessel containing aqueous base to ensure ionization of the carboxylic acid functional group. From this solution, the carboxylate form of [18F]7 was efficiently captured by the resin. The residual HPLC solvent was removed by washing with water. The radiotracer was then eluted from the resin with acidified ethanol, which was filtered and diluted into an isotonic bicarbonate and saline solution. This efficiently buffered the pH of the final injectable solution and provided [18F]7 as a concentrated, injectable solution for animal PET studies.

Dynamic microPET/CT imaging (with or without a saturating dose of TAK-875) was conducted in rats to evaluate tracer kinetics and the potential of [18F]7 as an FFA-1-targeting agent. The images displayed high uptake of the tracer in abdominal organs. While tracer uptake could be detected in the pancreas ROI, there was no reduction in tracer retention in the TAK-875 pre-treatment group. This suggests that the signal from the pancreas is confounded by non-specific binding in the pancreatic tissue. To further understand the in vivo value of this tracer and confirm these results, the focus of investigation was shifted to the analysis of tissue samples ex vivo.

Ex vivo biodistribution studies have the added benefit of eliminating error introduced when drawing the microPET/CT pancreatic ROI, which is indistinguishable from surrounding organs and tissues in rodents by CT and could only be estimated based on known anatomy (Yin et al. 2015). Theses studies indicated very little uptake of the radiotracer in the fat, bone, and muscle tissues. [18F]7 was detected in the pancreas (0.54 ± 0.04 tissue-to-blood ratio). However, saturating FFA-1 by pre-treatment with TAK-875 did not lead to a decrease in tracer accumulation in the pancreas, which corroborated the microPET results. This led to an examination of the formation and presence of potential radiolabeled metabolites to characterize the radioactive signal that accumulated in the pancreas. Interestingly, HPLC radio-metabolite analysis exhibited only the presence of unchanged [18F]7 in the pancreas at 60-min post-injection. The formation of one minor metabolite was also detected in the plasma. Together with the absence of radioactivity accumulation in the bone in the biodistribution studies, this result confirmed the relatively high metabolic stability of [18F]7 in rats.

During the course of this work, a study found that [3H]TAK-875 exhibits a high level of off-target binding, likely related to the high degree of lipophilicity of the molecule (cLogP = 4.2) (Hellström-Lindahl et al. 2017). Given the similarity of [18F]7 with TAK-875 in terms of chemical structure and lipophilicity (cLogP = 4.58), and with the results described here, it can be concluded that non-specific binding of [18F]7 in the pancreatic tissue invalidates this tracer as an in vivo or ex vivo quantitative FFA-1 imaging agent. Future development of TAK-875-derived FFA-1 targeting radiotracers should be approached with a focus on decreasing off-target labeling in the pancreas and surrounding tissue by increasing the hydrophilicity.

Conclusions

A one-pot, two-step fully automated process for the preparation of [18F]7 was developed. The use of a pentafluorophenyl-functionalized HPLC column in this process allowed for the removal of the potential FFA-1 competitive impurity 8 and improved the purity and apparent molar activity of the final product. A strong anion-exchange resin was used to conduct rapid and efficient reformulation of the radiotracer in high concentration, enabling in vivo evaluation of [18F]7 as a quantitative PET agent. MicroPET/CT imaging and biodistribution results exhibited activity accumulation in the pancreas although no specific binding could be detected following saturation studies of FFA-1 receptor. Radio-metabolite analysis indicated that [18F]7 is sufficiently stable, ruling out the possibility that degradation was a contributing factor to the high degree of non-specific binding observed. While these results allow for the conclusion that non-specific binding of [18F]7 in the pancreas impedes the ability to measure specific binding, they are instructive and provide direction for the development of future PET tracers for FFA-1-based β-cell PET imaging.

Notes

Acknowledgements

The authors thank the staff from the Radiochemistry and Cyclotron platform at the CRCHUM and Fleur Gaudette of the CRCHUM Pharmacokinetics core facility for high-resolution and accurate mass measurements.

Funding

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Authors’ contributions

MHD, JG, BB, VP, JND designed the study; MHD, DP, JMS, and AA performed the experiments. MHD, DP, JMS, AA, GX, and JND analyzed the data; MHD and JND wrote the manuscript. All authors read and approved the final manuscript.

Ethics approval

This study was carried out in accordance with Canadian Council on Animal Care guidelines. The protocol (protocol number IM16029JDSr) was approved by the institutional Animal Care Committees of the Centre de Recherche du Centre Hospitalier de l’Université de Montreal and the McGill University Health Centre.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. Andralojc K, Srinivas M, Brom M, Joosten L, de Vries IJM, Eizirik DL, et al. Obstacles on the way to the clinical visualisation of beta cells: looking for the Aeneas of molecular imaging to navigate between Scylla and Charybdis. Diabetologia. 2012;55(5):1247–57.CrossRefGoogle Scholar
  2. Bertrand R, Hamp I, Brönstrup M, Weck R, Lukacevic M, Polyak A, et al. Synthesis of GPR40 targeting 3H- and 18F-probes towards selective beta cell imaging. J Label Comp Radiopharm. 2016b;59(14):604–10.CrossRefGoogle Scholar
  3. Bertrand R, Wolf A, Ivashchenko Y, Lo M, Scha M. Synthesis and characterization of a promising novel FFAR1/GPR40 targeting fluorescent probe for β-cell imaging. ACS Chem Biol. 2016a;11(6):1745–54.CrossRefGoogle Scholar
  4. Briscoe CP, Tadayyon M, Andrews JL, Benson WG, Chambers JK, Eilert MM, et al. The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids. J Biol Chem. 2002;278(13):11303–11.CrossRefGoogle Scholar
  5. Hellström-Lindahl E, Åberg O, Ericsson C, O’Mahony G, Johnström P, Skrtic S, et al. Toward molecular imaging of the free fatty acid receptor 1. Acta Diabetol. 2017;54(7):663–8.CrossRefGoogle Scholar
  6. Ionescu-Tirgoviste C, Gagniuc PA, Gubceac E, Mardare L, Popescu I, Dima S, et al. A 3D map of the islet routes throughout the healthy human pancreas. Sci Rep. 2015;5:14634.CrossRefGoogle Scholar
  7. Itoh Y, Kawamata Y, Harada M, Kobayash M, Fuji R, Fukusumi S, et al. Free fatty acids regulate insulin secretion from pancreatic beta-cells through GPR40. Nature. 2003;422:173–6.CrossRefGoogle Scholar
  8. Kahn SE. The relative contributions of insulin resistance and beta-cell dysfunction to the pathophysiology of type 2 diabetes. Diabetologia. 2003;46(1):3–19.CrossRefGoogle Scholar
  9. Kenk M, Greene M, Lortie M, Robert A, Beanlands RS, Dasilva JN. Use of a column-switching high-performance liquid chromatography method to assess the presence of specific binding of (R)- and (S)-[11C]rolipram and their labeled metabolites to the phosphodiesterase-4 enzyme in rat plasma and tissues. Nucl Med Biol. 2008;35(4):515–21.Google Scholar
  10. Mancini AD, Poitout V. GPR40 agonists for the treatment of type 2 diabetes: life after “TAKing” a hit. Diabetes Obes Metab. 2015;17(7):622–9.CrossRefGoogle Scholar
  11. Negoro N, Sasaki S, Mikami S, Ito M, Suzuki M, Tsujihata Y, et al. Discovery of TAK-875: a potent, selective, and orally bioavailable GPR40 agonist. ACS Med Chem Lett. 2010;1(6):290–4.CrossRefGoogle Scholar
  12. Negoro N, Sasaki S, Mikami S, Ito M, Tsujihata Y, Ito R, et al. Optimization of (2,3-Dihydro-1-benzofuran-3-yl)acetic acids: discovery of a non-free fatty acid-like, highly bioavailable G protein-coupled receptor 40/free fatty acid receptor 1 agonist as a glucose-dependent Insulinotropic agent. J Med Chem. 2012;55(8):3960–74.CrossRefGoogle Scholar
  13. Otieno MA, Snoeys J, Lam W, Ghosh A, Player MR, Pocai A, et al. Fasiglifam (TAK-875): mechanistic investigation and retrospective identification of hazards for drug induced liver injury. Toxicol Sci. 2017;163(2):1–11.Google Scholar
  14. Srivastava A, Yano J, Hirozane Y, Kefala G, Gruswitz F, Snell G, et al. High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875. Nature. 2014;513(7516):124–7.CrossRefGoogle Scholar
  15. Wilke BI, Dornan MH, Yeung J, Boddy CN, Pinto A. Hexanes/acetonitrile : a binary solvent system for the efficient monosilylation of symmetric primary and secondary diols. Tetrahedron Lett. 2014;55(16):2600–2.CrossRefGoogle Scholar
  16. Yin T, Coudyzer W, Peeters R, Liu Y, Cona MM, Feng Y, et al. Three-dimensional contrasted visualization of pancreas in rats using clinical MRI and CT scanners. Contrast Media Mol Imaging. 2015;10(5):379–87.CrossRefGoogle Scholar

Copyright information

© The Author(s) 2018

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  • Mark H. Dornan
    • 1
    • 2
  • Daniil Petrenyov
    • 1
  • José-Mathieu Simard
    • 1
  • Antonio Aliaga
    • 3
  • Guoming Xiong
    • 3
  • Julien Ghislain
    • 1
  • Barry Bedell
    • 3
  • Vincent Poitout
    • 1
    • 4
  • Jean N. DaSilva
    • 1
    • 2
    Email author
  1. 1.Centre de Recherche du Centre Hospitalier de l’Université de MontréalQuébecCanada
  2. 2.Département de radiologie, radio-oncologie et médecine nucléaireUniversité de MontréalQuébecCanada
  3. 3.Centre for Translation Biology, Research Institute of the McGill University Health CentreQuébecCanada
  4. 4.Département de médecineUniversité de MontréalQuébecCanada

Personalised recommendations