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Human biodistribution, dosimetry, radiosynthesis and quality control of the bile acid PET tracer [N-methyl-11C]cholylsarcosine
Accepted Manuscript Human biodistribution, dosimetry, radiosynthesis and quality control of the bile acid PET tracer [N-methyl-11C]cholylsarcosine
Kim Frisch, Kristoffer Kjærgaard, Jacob Horsager, Anna Christina Schacht, Ole Lajord Munk PII: DOI: Reference:
S0969-8051(19)30080-0 https://doi.org/10.1016/j.nucmedbio.2019.07.006 NMB 8077
To appear in:
Nuclear Medicine and Biology
Received date: Revised date: Accepted date:
1 April 2019 12 June 2019 11 July 2019
Please cite this article as: K. Frisch, K. Kjærgaard, J. Horsager, et al., Human biodistribution, dosimetry, radiosynthesis and quality control of the bile acid PET tracer [N-methyl-11C]cholylsarcosine, Nuclear Medicine and Biology, https://doi.org/10.1016/ j.nucmedbio.2019.07.006
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Human biodistribution, dosimetry, radiosynthesis and quality control of the bile acid PET tracer 11C-cholylsarcosine[N-methyl-11C]cholylsarcosine
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Kim Frisch1, Kristoffer Kjærgaard1,2, Jacob Horsager1, Anna Christina Schacht1, Ole Lajord Munk1
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Department of Nuclear Medicine and PET Center, Aarhus University Hospital, Aarhus, Denmark.
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Department of Hepatology and Gastroenterology, Aarhus University Hospital, Aarhus, Denmark.
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Corresponding Author:
Kim Frisch, Department of Nuclear Medicine and PET Center, Aarhus University Hospital, DK-8000 Aarhus C,
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Denmark. Phone: +45 7846 3031; E-mail: [email protected]
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Abbreviated Title: 11C-cholylsarcosine[N-methyl-11C]cholylsarcosine dosimetry in humans
Keywords: cholestatic liver disease, 11C-cholylsarcosine, conjugated bile acids, gallbladder, hepatobiliary
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Conflict of Interest:
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function, PET
The authors declare that they have no conflicts of interest.
Grant Support: The Danish Council for Independent Research (Medical Sciences, 12-125512) and Ejnar and Aase Danielsen’s Foundation (10-001051, 10-000579).
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Abstract Introduction: 11C-Cholylsarcosine (11C-CSar) [N-methyl-11C]cholylsarcosine ([11C]CSar) is a tracer for imaging and quantitative assessment of intrahepatic cholestatic liver diseases and drug-induced cholestasis by positron emission tomography (PET). The purpose of this study is to determine whole-body biodistribution
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and dosimetry of 11C-CSar[11C]CSar in healthy humans. The results are compared with findings in a patient with primary sclerosing cholangitis (PSC) and in a patient with primary biliary cholangitis (PBC) as well as
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with preclinical findings in pigs. A Radiosynthesis and quality control for preparation of 11C-CSar[11C]CSar for
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clinical use isare also presented.
Methods: Radiosynthesis and quality control of 11C-CSar[11C]CSar were set up in compliance with
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Danish/European regulations. Both healthy participants (3 females, 3 males) and patients underwent
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whole-body PET/CT to determine the biodistribution of 11C-CSar[11C]CSar. The two patients were under treatment with ursodeoxycholic acid at the time of the study. Dosimetry was estimated from the PET data using the Olinda 2.0 software.
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Results: The radiosynthesis provided 11C-CSar[11C]CSar in a solution ready for injection. The biodistribution
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studies revealed that gallbladder wall, small intestine, and liver were critical organs in both healthy participants and patients with the gallbladder wall receiving the highest dose (up to 0.5 mGy/MBq). The
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gender-averaged (± SD) effective dose for the healthy participants was 6.2 ± 1.4 µSv/MBq. The effective dose for the PSC and the PBC patient was 5.2 and 7.0 µSv/MBq, respectively.
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Conclusion: A radiosynthesis for preparation of 11C-CSar[11C]CSar for clinical use was developed and approved by the Danish Medicines Agency. The most critical organ was the gallbladder wall although the amount of 11C-CSar[11C]CSar in the gallbladder was found to vary significantly between individuals. The estimated effective dose for humans was comparable to that estimated in anesthetized pigs. However, although the absorbed dose estimates to some organs, such as the stomach, was different from those in humans.
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Advances in knowledge and implications for patient care: 11C-CSar[11C]CSar PET/CT enables detailed quantitative assessment of patients with cholestatic liver disease by tracing the individual separate hepatobiliary transport steps of endogenous bile acids. The present work offers a radiosynthetic method
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and dosimetry data suitable for clinical implementation of 11C-CSar[11C]CSar.
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1. Introduction Bile acids are essential for intestinal absorption of dietary lipids, for regulation of intestinal microflora, and in metabolic signaling [1-4]. They are formed in hepatocytes as the end-products of cholesterol catabolism and secreted into bile as conjugates (N-acyl amidates) of glycine or taurine. Under normal conditions, bile
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acids undergo enterohepatic circulation between the liver and the small intestine [1, 5]. However, during cholestasis, where the flow of bile from the liver is impaired, bile acids accumulate in the liver and in the
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systemic circulation. At high hepatic concentrations, the inherently toxic bile acids cause extensive
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hepatocyte injury, which may progress to chronic liver disease fibrosis and eventually cirrhosis treatable
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only by liver transplantation [6-8].
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In vivo quantification of the hepatobiliary transport of bile acids is important for clinical assessment of patients with cholestatic liver disease as well as for scientific studies of the underlying mechanisms. Therefore, we have developed 11C-cholylsarcosine (11C-CSar) [N-methyl-11C]cholylsarcosine ([11C]CSar) as
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tracer for PET of conjugated bile acids [9-13]. Similar to endogenous bile acid conjugates, 11C-CSar[11C]CSar
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accumulates in liver tissue of patients with intrahepatic cholestatic liver disease to a degree that depends on the severity of the cholestasis [11, 12]. Furthermore, 11C-CSar[11C]CSar PET/CT enables quantification of
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the individual separate transport steps of conjugated bile acids from blood to hepatocytes and from hepatocytes to bile or back into blood; steps that have been shown to vary functionally between different
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cholestatic conditions [11, 12] as well as in drug-induced liver injury [12]. Other imaging agents for characterization of cholestasis and/or quantification of hepatobiliary transport in general have been reported for PET, scintigraphy, single photon emission computed tomography (SPECT), and magnetic resonance imaging. These imaging agents, however, are either not derived from bile acids, and therefore transported through hepatocytes by different mechanisms than bile acids [14-16], or there is little or no human data available [17-26]. In particular, the most widely used imaging agents of hepatobiliary transport are currently 99mTc-labelled iminodiacetic acids ([99mTc]Tc-IDA), such as [99mTc]Tc-mebrofenin, for planar
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scintigraphy or SPECT. Although clinically useful for assessment of patients with cholestatic liver disease, the [99mTc]Tc-IDA tracers are transported through the liver by a mechanism similar to that of bilirubin rather than of bile acids [16]. Thus, [11C]CSar PET may complement [99mTc]Tc-IDA scintigraphy by providing information of hepatobiliary transport of bile acids, which play a key role in the liver injuries associated
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with cholestatic liver diseases.
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During our initial approval of 11C-CSar[11C]CSar for clinical trials, we determined the biodistribution and
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dosimetry of the tracer in a 40-kg anesthetized pig with extrapolation of the dosimetry data to a reference 74-kg male phantom [9]. The pig is generally considered a suitable model for human anatomy, physiology,
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metabolism, and bioavailability [27-29]. Nonetheless, for clinical use of 11C-CSar[11C]CSar, the biodistribution and dosimetry of the tracer is best determined directly in humans. Here we report a PET/CT
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study of whole-body biodistribution and dosimetry of 11C-CSar[11C]CSar in healthy humans. The results are compared with two cases of patients with cholestatic liver disease as well as with preclinical findings in
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pigs. In addition, we present a radiosynthesis and quality control of 11C-CSar[11C]CSar setup in compliance
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with Danish/European regulations for preparation of the tracer for clinical use.
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2. Materials and methods 2.1 Chemicals Cholic acid (≥98%), cholylglycine (≥97%), 1,2,2,6,6-pentamethylpiperidine (97%), diethylphosphorylcyanide (90%), diethylphosphate (95%), triethylphosphate (≥99.8%) and anhydrous dimethylsulfoxide (DMSO;
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>99.9%) were obtained from Sigma-Aldrich and used as received. Cholic acid, originating from ox or sheep bile, was pre-processed by the supplier under alkaline conditions (pH 13–14) at elevated temperature (60–
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125 oC) to inactivate any pathogenic viral agents. The supplier also certified that the compound batch was
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not contaminated with neuronal tissue or other animal materials. Glycine methyl ester hydrochloride (99%), also obtained from Sigma-Aldrich, was dried in an oven at 80 oC overnight before use. Non-
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radioactive cholylsarcosine (≥95%) and cholylsarcosine methyl ester (≥95%) were prepared as previously
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described [9]. Acetonitrile (≥99.9%; Ph. Eur.) was obtained from VWR International. 2 N ± 0.02 N aqueous NaOH (Ph. Eur.; sterile; diluted to 1 N with sterile water) and aqueous citrate buffer (Ph. Eur.; sterile) was obtained from ABX. Water (Ph. Eur.; sterile), ethanol (99%; Ph. Eur.; sterile), and 70 mM aqueous NaH2PO4
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(Ph. Eur.; sterile, pH 4.5) were obtained from the pharmacy at Aarhus University Hospital. Sep-Pak® C8C8
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Plus Short Cartridges (400 mg sorbent/cartridge, 37–55 m particle size) were obtained from Waters® and preconditioned before use with 10 mL sterile ethanol followed by 10 mL sterile water. Sterile filtration was
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performed using a Millex®-GV sterile filter (0.22 m) from Millipore Corporation.
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2.2 Radiosynthesis and quality control of 11C-CSar[11C]CSar The carbon-11 radionuclide was produced by the 14N(p,)11C nuclear reaction using an IBA 18/18 cyclotron. The target gas (N2 with 0.5% O2) was bombarded at 30 A for 15–30 minutes to yield 11C-CO2[11C]CO2, which was delivered to the synthesis system (GE Tracerlab FXcPro) and converted to 11C-CH4[11C]CH4 for further gas phase reaction with molecular iodine to give 11C-CH3I[11C]CH3I. 15 minutes after end-ofbombardment (EOB), the 11C-CH3I[11C]CH3I was transferred to the reactor of the synthesis system preloaded with glycine methyl ester hydrochloride (1 mg; 8 mol) and 1,2,2,6,6-pentamethylpiperidine (5 L; 28
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mol) in anhydrous DMSO (300 L). The 11C-CH3I[11C]CH3I was bubbled through the precursor solution (helium flow: 15 mL/min) at room temperature until maximum activity was trapped in the reactor. The 11Cmethylation of glycine methyl ester was achieved by heating at 80 oC for 3 minutes (Fig. 1; step a). After cooling to 65 oC, freshly prepared solutions of cholic acid (12 mg; 29 mol) in anhydrous DMSO (200 l) and
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diethylphosphorylcyanide (5 L; 33 mol) in anhydrous DMSO (100 L) were added successively through dry tubing directly into the reactor solution. The coupling reaction was allowed to proceed for 5 minutes at
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65 oC before quenching with sterile 35% aqueous ethanol (0.7 mL) (Fig. 1; step b).
The crude reaction mixture containing 11C-CSar[11C]CSar methyl ester was purified by preparative reverse-
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phase high-pressure liquid chromatography (RP-HPLC) using a Waters®-XTerra® Prep RP18 OBDTM (5 m, 19x100 mm) column with 33% acetonitrile in sterile 70 mM aqueous NaH2PO4 (pH 4.5) as mobile phase
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(flow 20 mL/min; = 220 nm). The fraction containing 11C-CSar[11C]CSar methyl ester (retention time: 13 ± 1 min) was collected, diluted with sterile water (70 mL), and loaded onto a preconditioned C8C8-cartridge.
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The cartridge was washed with sterile water (10 mL) before eluting with sterile ethanol (1.0 mL). Finally,
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sterile aqueous 1 N NaOH (2.0 mL) was passed through the cartridge into the ethanolic solution of the ester. The alkaline mixture was allowed to stand for 1 min at room temperature, neutralized with sterile
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aqueous citrate buffer (5.0 mL) and sterile water (2.0 mL), and then passed through a sterile filter into a sterile product vial (Fig. 1; step c). Total synthesis time, including 11C-CH3I[11C]CH3I production and tracer
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purification, was 40 min from EOB.
The radioactivity amount of 11C-CSar[11C]CSar was measured in a dose calibrator (Capintec) and the radiochemical yield was calculated relative to the amount of 11C-CH3I[11C]CH3I initially trapped in the reactor. Quality control of 11C-CSar[11C]CSar was carried out as described in Supplementary Material. The shelf-life of the tracer formulation was determined by re-testing the radiochemical purity, pH and color/particles of three individual separate batches 1 hour after end-of-synthesis.
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2.3 Inclusion of participants Six healthy human participants (3 females, 3 males) with no history of liver disease and two patients diagnosed with cholestatic liver disease were included in this study (Table 1). The healthy participants (ID 01 – 06) were included after responding to an advertisement in a local newspaper or online, while the PSC
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patient (ID 07) and the PBC patient (ID 08) were included via our outpatient clinic. All participants were investigated using the same setup. At the time of the study, both patients were under treatment with
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ursodeoxycholic acid (250 mg UrsocholTM, 3 times daily, oral). All participants fasted for at least 8 hours
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before the PET/CT scan, but were allowed to drink water. Blood glucose and total plasma bile acid concentrations were measured on the day of the study. The clinical study (EudraCT no. 2016-004031-20)
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was approved by the Danish Medicines Agency and the Central Denmark Region Committees on Health Research Ethics, and was conducted in accordance with the Helsinki II Declaration, and monitored by the
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Good Clinical Practice Unit at Aarhus University Hospital. Written informed consent was obtained from all
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individual participants included in the study. No complications to the procedures were observed.
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2.4 PET/CT biodistribution and dosimetry
The participant was placed in supine position in a Siemens BiographTM 64 TruePointTM PET/CT camera. A low
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dose CT scan (50 effective mAs with CARE Dose4D [Siemens], 120 kV, pitch of 0.8 mm, and slice thickness of 5.0 mm) was performed before the first PET scan for definition of anatomical structures and attenuation
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correction of the PET recordings. 11C-CSar[11C]CSar was administered as an intravenous bolus injection with a median dose of 114 MBq (range, 60–143 MBq) immediately before start of the first PET scan. Accurate cross-calibration between PET scanner and dose calibrator used to measure injected radioactivity was verified daily. After administration of the tracer, 6 PET scans, each covering top of skull to mid-thigh (7 bed positions), were performed with 2 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, the scans were started 1, 10, 19, 32, 48, and 71 min after tracer administration. The PET images were reconstructed using 3-dimensional ordered-subset expectation
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maximization with 4 iterations and 21 subsets, 3-mm Gauss filter, and a 168x168 matrix with voxel size 4x4x3 mm3. The fused PET/CT images were analyzed using the PMOD 3.7 software (PMOD Technologies Ltd, Zürich, Switzerland). All tissues were visually inspected by two operators and organs with accumulation of 11C-CSar[11C]CSar above that of surrounding tissue were defined as source organs. These organs were
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liver, gallbladder contents and small intestine. No radioactivity was found in the urine bladder, and no radioactivity was excreted by the participant during the scans. Volumes-of-interest (VOIs) were manually
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drawn on fused PET/CT images to encompass all radioactivity in each source organ. Intrahepatic bile ducts
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were included in the VOI of the liver, whereas extrahepatic bile ducts were included in the VOI of the small intestine. For each source organ, the time course of the non–decay-corrected total radioactivity was
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normalized to the injected radioactivity and recalculated into time courses of percentage injected radioactivity. Time-integrated activity coefficients (TIACs) were computed using the trapezoidal integration
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method to calculate the area under the curves assuming only physical decay after the last scan, without further biological clearance. For the gallbladder, this approach does not account for the natural voiding of
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the gallbladder content that occurs within a few hours. Thus, the dose estimate to the gallbladder wall was
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conservative and could be overestimated. The remainder TIAC was calculated by subtracting the individual source organ TIACs from the total body TIAC (without voiding), which for 11C is 0.49 h. TIACs for source
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organs and remainder were used in OLINDA/EXM 2.0 (HERMES Medical Solution AB, Sweden) [30] to compute organ-absorbed doses (µGy/MBq) and the effective dose (µSv/MBq) using anthropomorphic
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human body phantoms with organ masses based on ICRP89 [31] and ICRP103 tissue weighting factors [32]. Organ doses and effective dose results are given for the reference gender-averaged adult according to ICRP103.
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3. Results 3.1 Radiosynthesis and quality control of 11C-CSar[11C]CSar The radiosynthesis of 11C-CSar[11C]CSar (Fig. 1) proceeded with an overall radiochemical yield of 18% ± 3% (decay corrected, mean ± SD), 6% ± 1% (not decay corrected, mean ± SD) to give 0.6–1.6 GBq 11C-
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CSar[11C]CSar with a radiochemical purity ≥98% (n = 20). The formation of 11C-cholylsarcosine[11C]CSar methyl ester (steps a and b in Fig. 1) proceeded with a radiochemical conversion of 24% ± 4%, while the
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hydrolysis to give 11C-CSar[11C]CSar (step c) was quantitative (n = 20). Unreacted 11C-cholylsarcosine[N-
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methyl-11C]sarcosine methyl ester and unidentifiable radiochemical impurities were efficiently removed during purification of 11C-cholylsarcosine[11C]CSar methyl ester by preparative RP-HPLC (Supplementary Fig.
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S1). Unlabeled cholylglycine, formed as a by-product of reaction step b (Fig. 1) and unreacted cholic acid were also efficiently removed during RP-HPLC purification and were detected at low concentrations, ≤0.3
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and ≤3.8 µg/mL, respectively, in the final formulation of 11C-CSar[11C]CSar (Supplementary Fig. S1 and Table S1). After purification, the concentrations of diethylphosphate, triethylphosphate (both decomposition
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products of diethylphosphorylcyanide formed when quenching the reaction with aqueous ethanol),
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1,2,2,6,6-pentamethylpiperidine, and the methyl esters of CSar and cholylglycine were below the limit of detection, <0.1 µg/mL, in the final formulation of 11C-CSar[11C]CSar (Supplementary Table S1). The amount
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of unlabeled CSar in the final formulation was determined to 0.1–1.2 µg/mL and the molar radioactivity of C-CSar[11C]CSar was calculated to 10 ± 2 GBq/µmol (mean ± SD; n = 20). The final formulation 11C-
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CSar[11C]CSar was a sterile, pyrogen- and particle-free, colorless solution with a pH of 6–7 and with acceptably low contents of ethanol (≤10%), acetroniltrile (<100 µg/mL), and DMSO (<0.1 µg/mL). The tracer (up to 1.6 GBq) was stable in formulation for at least one hour, i.e. the radiochemical purity did not drop below 98%, the pH remained 6–7, and the formulation remained colorless with no visible particles.
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3.2 PET/CT biodistribution and dosimetry For the healthy participants, mean (SD) blood glucose and total plasma bile acid concentrations were 5.1 0.6 mM (range, 4.5–6.0 mM, n = 6) and 1.8 0.8 µM (range, 0.9–3.2 µM, n = 6), respectively (Table 1). Blood glucose and total plasma bile acid concentrations for the two patients (ID 07 and ID 08) were within
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the same ranges (Table 1). In the healthy participants, 11C-CSar[11C]CSar was rapidly taken up by the liver and excreted into bile extrahepatic bile ducts with subsequent distribution between the gallbladder and
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small intestine (Fig. 2 and 3). The highest amount of 11C-CSar[11C]CSar in liver tissue (56% in average and a
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maximum of 68% measured in ID 01) relative to injected dose was observed 10–17 minutes after tracer administration (Fig. 3). After 71–106 minutes, approximately 95% of the administrated dose was located in
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liver tissue, small intestine and gallbladder for both female and male participants. The remaining 5% of the dose was distributed non-specifically, i.e. no accumulation of radioactivity was observed in other organs
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than liver tissue, small intestine and gallbladder in any of the participants at any time of the PET/CT scans. At 71–106 minutes, the average amount of 11C-CSar[11C]CSar in the gallbladder and the small intestine was
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39% and 50%, respectively. In one participant, however, the majority of the injected tracer was found in the
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small intestine (94% in ID 02), while in another the majority was found in the gallbladder (78% in ID 05). The biodistribution of 11C-CSar[11C]CSar in the two patients (ID 07 and ID 08) was comparable to that of the
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healthy participants with 11C-CSar[11C]CSar found only in liver tissue, gallbladder and small intestine (Supplementary Fig. S2). The amount of 11C-CSar[11C]CSar in liver tissue was however higher in the patients
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compared with the averaged healthy participants (Fig. 4).
Critical organs were gallbladder wall (range, 7–492 µGy/MBq), small intestine (range, 14–83 µGy/MBq), and liver (range, 28–46 µGy/MBq) (Table 2 and Supplementary Table S2). In general, the gallbladder wall received the highest radiation dose, up to 492 µGy/MBq (ID 05). In the case of healthy participant ID 02, however, where little 11C-CSar[11C]CSar entered the gallbladder, the gallbladder wall received a low radiation dose (7 µGy/MBq) while the small intestine received a high dose (83 µGy/MBq). The gender-
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averaged effective dose of 11C-CSar[11C]CSar in the healthy participants was 6.2 ± 1.4 µSv/MBq (mean ± SD; n = 6). For the PSC patient (ID 07) and the PBC patient (ID 08) the effective dose was 5.2 µSv/MBq and 7.0
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µSv/MBq, respectively (Table 2 and Supplementary Table S3).
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4. Discussion In this study, we optimized our previously reported radiosynthesis of 11C-CSar[11C]CSar [9] and set up a quality control in compliance with Danish/European regulations for preparation of tracers for clinical use. Specifically, by modifying the conditions for the individual radiosynthetic steps and the purification method
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we minimized the amounts of unlabeled impurities and solvent residues in the final formulation of 11CCSar[11C]CSar without compromising the radiochemical yield of the radiosynthesis or the radiochemical
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purity of the tracer. In fact, the presented radiosynthesis provides 11C-CSar[11C]CSar with slightly higher
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yield compared to that previously reported, 18% vs. 13% (decay-corrected). The overall production, which provides radiochemically pure 11C-CSar[11C]CSar in a solution ready for injection, has beenwas approved by
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the Danish Medicines Agency as suitable for preparation of the tracer for clinical use.
In both healthy participants and the patients diagnosed with intrahepatic cholestatic liver diseases, the target organs of 11C-CSar[11C]CSar are gallbladder wall, small intestine, and liver tissue with no
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accumulation of the tracer in other organs. In particular, no accumulation of 11C-CSar[11C]CSar was
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observed in stomach, kidneys, urinary bladder, large intestine, or brain; neither in the healthy participants nor in the patients. No differences were observed when estimating the effective dose using data from the
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healthy participants and data from the patients (Table 2). With the estimated gender-average effective dose of 6.2 µSv/MBq, the total radiation dose received by an adult will be around 0.4–0.6 mSv when 60–
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100 MBq 11C-CSar[11C]CSar is administrated intravenously. This dose range is sufficient to obtain highquality PET images of the hepatobiliary excretion of 11C-CSar[11C]CSar (Fig. 2 and reference [11]). Furthermore, the administered dose can probably be reduced by at least 50% on the newest digital PET systems that have fast time-of-flight timing resolution and a NEMA sensitivity that are more than twice that of the scanner used in this study.
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The distribution of 11C-CSar[11C]CSar between the gallbladder and the small intestine corresponds well to that of endogenous bile acids in the average fasting human. However, large variations were observed between individuals, which ultimately results in large variation in the estimated absorbed doses for these organs (Table 2). Therefore, absorbed dose estimates for 11C-CSar[11C]CSar (and presumably other tracers
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that similarly enters the intestine through the biliary tract) should not be based on data from a single individual. Rather, the estimates should be based on a group of individuals including cases with low and
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high gallbladder filling such as healthy participant ID 02 and ID 05.
The ursodeoxycholic acid-treated PBC and PSC patients were included in this study, because they represent
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common cases of patients with intrahepatic cholestatic liver diseases encountered in the clinic. PBC and PSC are characterized by inflammation and fibrosis of intrahepatic bile ducts, and for PSC also extrahepatic
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bile ducts, which causes impaired bile flow and accumulation of toxic bile acids in blood and liver tissue if not treated. At the time of the PET/CT study, both patients had normal plasma bile acid concentrations
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suggesting effective treatment with the choleretic ursodeoxycholic acid (Table 1). Similar to the healthy
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participants, 11C-CSar[11C]CSar was excreted from the liver tissue (blood vessels, liver cells and intrahepatic bile ducts) into extrahepatic bile ducts in the patients, albeit more slowly in the PSC patient (ID 07) than in
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the remaining participants (Fig. 4). Nevertheless, the amount of 11C-CSar[11C]CSar in liver tissue measured by PET of the patients was higher than in the healthy participants. Concordantly, with the exception of one
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participant (ID 01), the absorbed dose estimates for the liver was higher for the patients than for the healthy participants (Table 2). It is expected that this difference will be even more pronounced in untreated patients with severe cholestasis, where 11C-CSar[11C]CSar in some cases is hardly excreted into bile at all resulting in significant accumulation of the tracer in liver tissue as previously observed [11].
On the basis of data from previous studies in anesthetized pigs (40-kg, 3-month-old female Danish Landrace and Yorkshire cross-breed) [9, 19], we have estimated the effective dose for 11C-CSar[11C]CSar as well as for
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N-[11C]methyl-cholyltaurine (11C-MTCA[11C]MTCA) and N-[11C]methyl-ursodeoxycholyltaurine (11CMTUDCA[11C]MTUDCA) using the ICRP103 tissue weighting factors embedded in the Olinda 2.0 software. 11
C-MTCA and 11C-MTUDCA [11C]MTCA and [11C]MTUDCA are tracers derived from different bile acid
conjugates than 11C-CSar[11C]CSar, but with comparable biodistribution [9, 19]. The mean effective doses estimated from the pig data are 5.7, 7.2, and 7.5 µSv/MBq for 11C-CSar, 11C-MTCA, and 11C-
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MTUDCA[11C]CSar, [11C]MTCA, and [11C]MTUDCA, respectively (Supplementary Table S4 S2), which are all in
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range of the effective dose of 11C-CSar[11C]CSar estimated from the human data (Table 2). However, we
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have found that 11C-CSar[11C]CSar and the similar N-[11C]methyl-taurine conjugated bile acid tracers in some cases enter the stomach of the anesthetized pigs resulting in an increased radiation dose to this organ
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(Supplementary Table S4 S2). In contrast, we observe no 11C-CSar[11C]CSar in the stomach of humans. Because pigs and humans have a similar pyloric motor function [33], we believe that the occasional reflux
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of 11C-CSar[11C]CSar into the stomach of the pigs should be attributed anesthesia rather than physiology. Therefore, the anesthetized pig does not make a suitable model for estimating absorbed doses for 11C-
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CSar[11C]CSar for individual organs in (un-anesthetized) humans. Significant differences between species
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have also been reported for other PET tracers, where in several cases the estimated whole-body effective
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dose is similar but the absorbed doses to individual organs vary significantly [34-37].
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5. Conclusion We have presented a radiosynthesis and quality control of the conjugated bile acid PET tracer 11CCSar[11C]CSar set up in compliance with Danish/European regulations and suitable preparation of the tracer for clinical use. Target organs of 11C-CSar[11C]CSar are gallbladder wall, small intestine, and liver. The
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gender-averaged effective dose of 11C-CSar[11C]CSar is estimated to 6.2 1.4 µSv/MBq in healthy humans (mean SD, n = 6). The most critical organ is the gallbladder wall with an absorbed dose estimate of up to
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0.5 mGy/MBq. However, the distribution of 11C-CSar[11C]CSar between the gallbladder and the small
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intestine varies significantly among individuals resulting in some cases in low radiation doses to the gallbladder wall and a concurrently high radiation dose to the small intestine. Our results also show that
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while the effective dose estimated preclinical in anesthetized pigs is comparable to that estimated in
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humans, the absorbed dose estimates to critical organs can differ significantly.
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Acknowledgement
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This study was supported by The Danish Council for Independent Research (Medical Sciences, 12-125512)
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and Ejnar and Aase Danielsen’s Foundation (10-001051, 10-000579).
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Figure Legends Fig. 1. Radiosynthesis of 11C-Cholylsarcosine (11C-CSar) [N-methyl-11C]cholylsarcosine ([11C]CSar). Reaction conditions: a) 11C-CH3I[11C]CH3I, 1,2,2,6,6-pentamethylpiperidine, DMSO, 80 oC, 3 min; b) cholic acid, diethylphosphorylcyanide, DMSO, 65 oC, 5 min; c) purification, then 1 N aqueous sodium hydroxide, room
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temperature, 1 min, then neutralization (citrate buffer) and sterile filtration.
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Fig. 2. Example of PET/CT whole-body scans of a healthy participant (ID 06) recorded after intravenous
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injection of 11C-CSar ([11C]CSar) (98 MBq) over the time frames indicated. Top row shows PET emission only (average radioactivity concentration); bottom row shows fused PET/CT images. Post injection, the tracer is
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rapidly taken up by the liver (a), excreted into bile (b), and subsequently distributed between the
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gallbladder and the small intestine (c–f). Each image shows the average radioactivity concentration measured by PET over the time frame indicated.
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Fig. 3. Time course of the distribution of 11C-CSar[11C]CSar measured by whole-body PET/CT after
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intravenous injection of the tracer in healthy participants. The figure shows mean (± SD; n = 6) percent of injected dose in liver tissue including intrahepatic bile ducts (black), small intestine including extrahepatic
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bile ducts (light grey) and gallbladder (dark grey). The data is decay corrected to the time of injection.
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Fig. 4. Percent of injected dose of 11C-CSar[11C]CSar in liver tissue (including intrahepatic bile ducts) measured during whole-body PET/CT scans of healthy human participants (ID 01 – ID 06; gender-averaged), a PSC patient (ID 07), and a PBC patient (ID 08). Both patients were under treatment with ursodeoxycholic acid at the time of the study. The data is decay corrected to the time of injection. The relatively high SDvalues are due to one participant (ID 01) who had a higher amount of 11C-CSar[11C]CSar in liver tissue than the remaining group of healthy participants.
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Tables Table 1. Characteristics of participants ID
Gender/Age Healthy/disease Body weight
Height Total bile acidsa
Glucoseb
11
(yr)
(m)
(mmol/L blood)
CSar[11C]CSar
(kg)
(µmol/L plasma)
C-
73
1.68
<1
02
Female/63
Healthy
85
1.74
1.8
03
Male/70
Healthy
103
1.85
04
Male/73
Healthy
82
05
Male/70
Healthy
84
06
Female/57
Healthy
93
07
Male/34
PSC
82
08
Female/61
PBC
74
112
4.8
116
2.3
5.9
60
1.90
1.3
6.0
143
1.76
3.2
4.5
141
1.75
1.4
4.8
98
1.81
1.6
5.9
77
1.72
2.2
5.3
126
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a
4.8
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Healthy
(MBq)
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Female/59
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01
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dose
Bile acid reference <10 µmol/L plasma, b Fasting glucose reference <7 mmol/L blood
Abbreviations: 11C-CSar 11C-cholylsarcosine [11C]CSar [N-methyl-11C]cholylsarcosine, PBC primary biliary
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cholangitis, PSC primary sclerosing cholangitis
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Table 2. Absorbed dose estimates Gender-averaged organ doses (µGy/MBq) and effective dose (µSv/MBq) for 11C-CSar[11C]CSar in healthy human participants (ID 01 – ID 06), the PSC patient (ID 07), and the PBC patient (ID 08) Patientsa
Healthy participants
Target organ
PSC
PBC
02 (F)
03 (M)
04 (M)
05 (M)
06 (F)
07 (M)
08 (F)
Adrenals
8.1
5.7
7.2
7.1
8.4
6.8
8.2
8.4
Brain
0.8
1.0
1.1
1.0
0.9
1.0
0.8
0.9
Breasts
1.7
1.6
1.7
1.7
1.6
1.6
1.7
1.7
Esophagus
3.5
2.7
3.2
3.2
3.2
3.0
3.7
3.5
0.9
1.0
1.1
170.0
7.4
4.2
0.9
1.0
0.8
0.9
193.0
177.0
492.0
130.0
18.5
251.0
5.7
4.6
4.7
4.5
5.0
3.8
4.1
35.4
82.7
39.9
43.5
14.1
57.3
40.3
24.4
Stomach wall
3.4
3.5
3.4
3.4
3.2
3.4
3.5
3.4
Right colon
5.8
5.0
6.1
6.0
8.3
5.8
4.5
6.4
Rectum
1.9
3.1
2.3
2.3
1.6
2.5
2.0
1.7
Heart wall
3.1
2.7
3.0
3.0
2.9
2.9
3.3
3.2
Kidneys
5.2
4.2
5.0
5.0
6.1
4.8
4.9
5.5
b
Gallbladder wall Left colon Small intestineb
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Eyes
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1.1
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01 (F)
46.1
27.8
35.1
35.3
34.1
34.5
54.2
45.9
Lungs
2.9
2.4
2.7
2.7
2.6
2.6
3.1
2.9
2.6
4.1
3.0
3.1
2.4
3.3
2.6
2.4
6.9
6.9
6.6
6.7
6.8
6.9
6.9
6.8
1.6
2.3
1.9
1.9
1.5
2.0
1.6
1.5
1.0
1.2
1.2
1.2
1.0
1.1
1.0
1.0
2.0
2.1
2.1
2.1
2.0
2.0
2.0
2.0
Osteogenic cells
1.4
1.5
1.5
1.5
1.4
1.4
1.4
1.4
Spleen
2.3
2.6
2.5
2.5
2.3
2.5
2.3
2.3
Testes
0.9
1.1
1.1
1.1
0.9
1.0
0.8
0.9
Thymus
1.8
1.7
1.9
1.9
1.8
1.8
1.9
1.9
Thyroid
1.3
1.3
1.4
1.4
1.3
1.3
1.3
1.3
Urinary bladder wall
1.6
2.3
1.9
1.9
1.4
1.9
1.6
1.5
Uterus
2.9
4.8
3.3
3.4
2.3
3.8
2.9
2.5
Total body
3.1
3.2
3.1
3.1
3.1
3.1
3.1
3.1
Effective dose
6.3
4.4
6.1
6.0
8.7
5.6
5.2
7.0
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Liverb Ovaries Prostate Salivary glands
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Red marrow
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Pancreas
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a
Both patients were under treatment with ursodeoxycholic acid at the time of the study, b Critical organs.
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Abbreviations: F female, M male, PBC primary biliary cholangitis, PSC primary sclerosing cholangitis.
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Compliance with Ethical Standards
Conflict of Interest
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The authors declare that they have no conflict of interest.
Ethical approval
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This clinical study (EudraCT no. 2016-004031-20) was approved by the Danish Medicines Agency and the
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Central Denmark Region Committees on Health Research Ethics, and was conducted in accordance with the
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Helsinki II Declaration, and monitored by the Good Clinical Practice Unit at Aarhus University Hospital.
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Informed consent:
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Written informed consent was obtained from all individual participants included in the study.
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References [1] Frisch K and Hofmann AF. Biliary secretion. In: Keiding S, Sørensen M, editors. Functional molecular imaging in hepatology. Sharjah, UAE: Bentham Science Publishers; 2012, pp. 49–75.
PT
[2] Hofmann AF and Hagey LR. Key discoveries in bile acid chemistry and biology and their clinical
RI
applications: history of the last eight decades. J Lipid Res 2014;55:1553–95.
SC
[3] Li T and Chiang JYL. Bile Acid Signaling in Metabolic Disease and Drug Therapy. Pharmacol Rev
NU
2014;66:948–83.
MA
[4] Thomas C, Pellicciari R, Pruzanski M, Auwerx J, and Schoonjans K. Targeting bile-acid signalling for metabolic diseases. Nat Rev Drug Discov 2008;7:678–93.
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(Landmark Ed) 2009;14:2584–98.
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[5] Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and functions. Front Biosci
CE
[6] Wagner M and Trauner M. Recent advances in understanding and managing cholestasis. F1000Research
AC
2016;5(F1000 Faculty Rev):705.
[7] Williamson KD and Chapman RW. New Therapeutic Strategies for Primary Sclerosing Cholangitis. Semin Liver Dis 2016;36:5–14.
[8] Zollner G and Trauner M. Mechanisms of Cholestasis. Clin Liver Dis 2008;12:1–26.
22
ACCEPTED MANUSCRIPT
[9] Frisch K, Jakobsen S, Sørensen M, Munk OL, Alstrup AKO, Ott P, et al. [N-Methyl-11C]cholylsarcosine, a novel bile acid tracer for PET/CT of hepatic excretory function: radiosynthesis and proof-of-concept studies in pigs. J Nucl Med 2012;53:772–8.
[10] Sørensen M, Munk OL, Ørntoft NW, Frisch K, Andersen KJ, Mortensen FV, et al. Hepatobiliary secretion
RI
PT
kinetics of conjugated bile acids measured in pigs by 11C-cholylsarcosine PET. J Nucl Med 2016;57:961–6.
SC
[11] Ørntoft NW, Munk OL, Frisch K, Ott P, Keiding S, and Sørensen M. Hepatobiliary transport kinetics of the conjugated bile acid tracer 11C-CSar quantified in healthy humans and patients by positron emission
NU
tomography. J Hepatol 2017;67:321–7.
MA
[12] Ørntoft NW, Frisch K, Ott P, Keiding S, and Sørensen M. Functional assessment of hepatobiliary secretion by 11C-cholylsarcosine positron emission tomography. Biochim Biophys Acta Mol Basis Dis
PT E
D
2018;1864:1240–4.
CE
[13] Dickson I. Liver: PET for bile transport kinetics. Nat Rev Gastroenterol Hepatol 2017;14:260–1.
[14] Testa A, Zanda M, Elmore CS, and Sharma P. PET Tracers To Study Clinically Relevant Hepatic
AC
Transporters. Mol Pharmaceutics 2015;12:2203–16.
[15] Pastor CM, Müllhaupt B, and Stieger B. The Role of Organic Anion Transporters in Diagnosing Liver Diseases by Magnetic Resonance Imaging. Drug Metab Dispos 2014;42:675–84.
[16] Hoekstra LT, Graaf Wd, Nibourg GAA, Heger M, Bennink RJ, Stieger B, et al. Physiological and biochemical basis of clinical liver function tests. Ann Surg 2013;257:27–36. Geisel D, Lüdemann L, Hamm B,
23
ACCEPTED MANUSCRIPT
Denecke T. Imaging-Based Liver Function Tests – Past, Present and Future. Fortschr Röntgenstr 2015;187:863–71.
[17] Jazrawi RP, de Caestecker JS, Goggin PM, Britten AJ, Joseph AE, Maxwell JD, et al. Kinetics of hepatic
PT
bile acid handling in cholestatic liver disease: effect of ursodeoxycholic acid. Gastroenterology
RI
1994;106:134–42.
SC
[18] Felton J, Cheng K, Said A, Shang AC, Xu S, Vivian D, et al. Using multi-fluorinated bile acids and in vivo
NU
magnetic resonance imaging to measure bile acid transport. J Vis Exp 2016;117:e54597.
[19] Schacht AC, Sørensen M, Munk OL, and Frisch K. Radiosynthesis of N-11C-methyl-taurine–conjugated
MA
bile acids and biodistribution studies in pigs by PET/CT. J Nucl Med 2016;57:628–33.
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[20] Frisch K, Stimson DHR, Venkatachalam T, Pierens GK, Keiding S, Reutens D, et al. N-(4-
PT E
[18F]fluorobenzyl)cholylglycine, a novel tracer for PETof enterohepatic circulation of bile acids:
CE
radiosynthesis and proof-of-concept studies in rats. Nucl Med Biol 2018;2018:56–62.
[21] Jia L, Jiang D, Hu P, Li X, Shi H, Cheng D, et al. Synthesis and evaluation of 18F-labeled bile acid
AC
compound: a potential PET imaging agent for FXR-related diseases. Nucl Med Biol 2014;41:495–500.
[22] Chong H-S, Chen Y, Kang CS, Sun X, and Wu N. Novel 64Cu-radiolabeled bile acid conjugates for targeted PET imaging. Bioorg Med Chem Lett 2015;25:1082–5.
24
ACCEPTED MANUSCRIPT
[23] Testa A, Dall’Angelo S, Mingarelli M, Augello A, Schweiger L, Welch A, et al. Design, synthesis, in vitro characterization and preliminary imaging studies on fluorinated bile acid derivatives as PET tracers to study hepatic transporters. Bioorg Med Chem 2017;25:963–76.
[24] Lombaerde SD, Neyt S, Kersemans K, Verhoeven J, Devisscher L, Vlierberghe HV, et al. Synthesis, in
PT
vitro and in vivo evaluation of 3β-[18F]fluorocholic acid for the detection of drug-induced cholestasis in
SC
RI
mice. PLos One 2017;12:e0173529.
[25] Neyt S, Vliegen M, Verreet B, Lombaerde SD, Braeckman K, Vanhove C, et al. Synthesis, in vitro and in
NU
vivo small-animal SPECT evaluation of novel technetium labeled bile acid analogues to study (altered)
MA
hepatic transporter function. Nucl Med Biol 2016;43:642–9.
[26] Anelli PL, Lattuada L, Lorusso V, Lux G, Morisetti A, Morosini P, et al. Conjugates of gadolinium
PT E
Chem 2004;47:3629–41.
D
complexes to bile acids as hepatocyte-directed contrast agents for magnetic resonance imaging. J Med
AC
2017;15:21–6.
CE
[27] Nykonenko A, Vávra P, and Zonča P. Anatomic Peculiarities of Pig and Human Liver. Exp Clin Transplant
[28] Kararli TT. Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals. Biopharm Drug Dispos 1995;16:351–80.
[29] Gonzalez LM, Moeser AJ, and Blikslager AT. Porcine models of digestive disease: the future of large animal translational research. Transl Res 2015;166:12–27.
25
ACCEPTED MANUSCRIPT
[30] Stabin MG and Siegel JA. RADAR dose estimate report: a compendium of radiopharmaceutical dose estimates based on OLINDA/EXM version 2.0. . J Nucl Med 2018;59:154–60.
[31] Basic anatomical and physiological data for use in radiological protection. reference values. A report of
PT
age- and gender-related differences in the anatomical and physiological characteristics of reference
RI
individuals. ICRP Publication 89. Ann ICRP. 2002;32(3–4):5–265.
SC
[32] The 2007 Recommendations of the International Commission on Radiological Protection. ICRP
NU
publication 103. Ann ICRP. 2007;37(2–4):1–332.
[33] Treacy PJ, Jamieson GG, and Dent J. Pyloric motor function during emptying of a liquid meal from the
MA
stomach in the conscious pig. J Physiol 1990;422:523–38.
D
[34] Sakata M, Oda K, Toyohara J, Ishii K, Nariai T, and Ishiwata K. Direct comparison of radiation dosimetry
PT E
of six PET tracers using human whole-body imaging and murine biodistribution studies. Ann Nucl Med
CE
2013;27:285–96.
[35] Hino-Shishikura A, Suzuki A, Minamimoto R, Shizukuishi K, Oka T, Tateishi U, et al. Biodistribution and
AC
radiation dosimetry of [18F]-5-fluorouracil. Appl Radiat Isot 2013;75:11–7.
[36] Petrulli JR, Hansen SB, Abourbeh G, Yaqub M, Bahceb I, Holdenb D, et al. A multi species evaluation of the radiation dosimetry of [11C]erlotinib, the radiolabeled analog of a clinically utilized tyrosine kinase inhibitor. Nucl Med Biol 2017;47:56–61.
26
ACCEPTED MANUSCRIPT
[37] Kranz M, Sattler B, Tiepolt S, Wilke S, Deuther-Conrad W, Donat CK, et al. Radiation dosimetry of the α4β2 nicotinic receptor ligand (+)-[18F]flubatine, comparing preclinical PET/MRI and PET/CT to first-in-human
AC
CE
PT E
D
MA
NU
SC
RI
PT
PET/CT results. EJNMMI Physics 2016;3:25.
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Figure 1
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Kim Frisch, Kristoffer Kjærgaard, Jacob Horsager, Anna Christina Schacht, Ole Lajord Munk PII: DOI: Reference:
S0969-8051(19)30080-0 https://doi.org/10.1016/j.nucmedbio.2019.07.006 NMB 8077
To appear in:
Nuclear Medicine and Biology
Received date: Revised date: Accepted date:
1 April 2019 12 June 2019 11 July 2019
Please cite this article as: K. Frisch, K. Kjærgaard, J. Horsager, et al., Human biodistribution, dosimetry, radiosynthesis and quality control of the bile acid PET tracer [N-methyl-11C]cholylsarcosine, Nuclear Medicine and Biology, https://doi.org/10.1016/ j.nucmedbio.2019.07.006
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Human biodistribution, dosimetry, radiosynthesis and quality control of the bile acid PET tracer 11C-cholylsarcosine[N-methyl-11C]cholylsarcosine
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Kim Frisch1, Kristoffer Kjærgaard1,2, Jacob Horsager1, Anna Christina Schacht1, Ole Lajord Munk1
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Department of Nuclear Medicine and PET Center, Aarhus University Hospital, Aarhus, Denmark.
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Department of Hepatology and Gastroenterology, Aarhus University Hospital, Aarhus, Denmark.
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Corresponding Author:
Kim Frisch, Department of Nuclear Medicine and PET Center, Aarhus University Hospital, DK-8000 Aarhus C,
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Denmark. Phone: +45 7846 3031; E-mail: [email protected]
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Abbreviated Title: 11C-cholylsarcosine[N-methyl-11C]cholylsarcosine dosimetry in humans
Keywords: cholestatic liver disease, 11C-cholylsarcosine, conjugated bile acids, gallbladder, hepatobiliary
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Conflict of Interest:
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function, PET
The authors declare that they have no conflicts of interest.
Grant Support: The Danish Council for Independent Research (Medical Sciences, 12-125512) and Ejnar and Aase Danielsen’s Foundation (10-001051, 10-000579).
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Abstract Introduction: 11C-Cholylsarcosine (11C-CSar) [N-methyl-11C]cholylsarcosine ([11C]CSar) is a tracer for imaging and quantitative assessment of intrahepatic cholestatic liver diseases and drug-induced cholestasis by positron emission tomography (PET). The purpose of this study is to determine whole-body biodistribution
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and dosimetry of 11C-CSar[11C]CSar in healthy humans. The results are compared with findings in a patient with primary sclerosing cholangitis (PSC) and in a patient with primary biliary cholangitis (PBC) as well as
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with preclinical findings in pigs. A Radiosynthesis and quality control for preparation of 11C-CSar[11C]CSar for
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clinical use isare also presented.
Methods: Radiosynthesis and quality control of 11C-CSar[11C]CSar were set up in compliance with
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Danish/European regulations. Both healthy participants (3 females, 3 males) and patients underwent
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whole-body PET/CT to determine the biodistribution of 11C-CSar[11C]CSar. The two patients were under treatment with ursodeoxycholic acid at the time of the study. Dosimetry was estimated from the PET data using the Olinda 2.0 software.
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Results: The radiosynthesis provided 11C-CSar[11C]CSar in a solution ready for injection. The biodistribution
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studies revealed that gallbladder wall, small intestine, and liver were critical organs in both healthy participants and patients with the gallbladder wall receiving the highest dose (up to 0.5 mGy/MBq). The
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gender-averaged (± SD) effective dose for the healthy participants was 6.2 ± 1.4 µSv/MBq. The effective dose for the PSC and the PBC patient was 5.2 and 7.0 µSv/MBq, respectively.
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Conclusion: A radiosynthesis for preparation of 11C-CSar[11C]CSar for clinical use was developed and approved by the Danish Medicines Agency. The most critical organ was the gallbladder wall although the amount of 11C-CSar[11C]CSar in the gallbladder was found to vary significantly between individuals. The estimated effective dose for humans was comparable to that estimated in anesthetized pigs. However, although the absorbed dose estimates to some organs, such as the stomach, was different from those in humans.
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Advances in knowledge and implications for patient care: 11C-CSar[11C]CSar PET/CT enables detailed quantitative assessment of patients with cholestatic liver disease by tracing the individual separate hepatobiliary transport steps of endogenous bile acids. The present work offers a radiosynthetic method
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and dosimetry data suitable for clinical implementation of 11C-CSar[11C]CSar.
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1. Introduction Bile acids are essential for intestinal absorption of dietary lipids, for regulation of intestinal microflora, and in metabolic signaling [1-4]. They are formed in hepatocytes as the end-products of cholesterol catabolism and secreted into bile as conjugates (N-acyl amidates) of glycine or taurine. Under normal conditions, bile
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acids undergo enterohepatic circulation between the liver and the small intestine [1, 5]. However, during cholestasis, where the flow of bile from the liver is impaired, bile acids accumulate in the liver and in the
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systemic circulation. At high hepatic concentrations, the inherently toxic bile acids cause extensive
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hepatocyte injury, which may progress to chronic liver disease fibrosis and eventually cirrhosis treatable
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only by liver transplantation [6-8].
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In vivo quantification of the hepatobiliary transport of bile acids is important for clinical assessment of patients with cholestatic liver disease as well as for scientific studies of the underlying mechanisms. Therefore, we have developed 11C-cholylsarcosine (11C-CSar) [N-methyl-11C]cholylsarcosine ([11C]CSar) as
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tracer for PET of conjugated bile acids [9-13]. Similar to endogenous bile acid conjugates, 11C-CSar[11C]CSar
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accumulates in liver tissue of patients with intrahepatic cholestatic liver disease to a degree that depends on the severity of the cholestasis [11, 12]. Furthermore, 11C-CSar[11C]CSar PET/CT enables quantification of
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the individual separate transport steps of conjugated bile acids from blood to hepatocytes and from hepatocytes to bile or back into blood; steps that have been shown to vary functionally between different
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cholestatic conditions [11, 12] as well as in drug-induced liver injury [12]. Other imaging agents for characterization of cholestasis and/or quantification of hepatobiliary transport in general have been reported for PET, scintigraphy, single photon emission computed tomography (SPECT), and magnetic resonance imaging. These imaging agents, however, are either not derived from bile acids, and therefore transported through hepatocytes by different mechanisms than bile acids [14-16], or there is little or no human data available [17-26]. In particular, the most widely used imaging agents of hepatobiliary transport are currently 99mTc-labelled iminodiacetic acids ([99mTc]Tc-IDA), such as [99mTc]Tc-mebrofenin, for planar
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scintigraphy or SPECT. Although clinically useful for assessment of patients with cholestatic liver disease, the [99mTc]Tc-IDA tracers are transported through the liver by a mechanism similar to that of bilirubin rather than of bile acids [16]. Thus, [11C]CSar PET may complement [99mTc]Tc-IDA scintigraphy by providing information of hepatobiliary transport of bile acids, which play a key role in the liver injuries associated
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with cholestatic liver diseases.
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During our initial approval of 11C-CSar[11C]CSar for clinical trials, we determined the biodistribution and
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dosimetry of the tracer in a 40-kg anesthetized pig with extrapolation of the dosimetry data to a reference 74-kg male phantom [9]. The pig is generally considered a suitable model for human anatomy, physiology,
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metabolism, and bioavailability [27-29]. Nonetheless, for clinical use of 11C-CSar[11C]CSar, the biodistribution and dosimetry of the tracer is best determined directly in humans. Here we report a PET/CT
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study of whole-body biodistribution and dosimetry of 11C-CSar[11C]CSar in healthy humans. The results are compared with two cases of patients with cholestatic liver disease as well as with preclinical findings in
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pigs. In addition, we present a radiosynthesis and quality control of 11C-CSar[11C]CSar setup in compliance
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with Danish/European regulations for preparation of the tracer for clinical use.
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2. Materials and methods 2.1 Chemicals Cholic acid (≥98%), cholylglycine (≥97%), 1,2,2,6,6-pentamethylpiperidine (97%), diethylphosphorylcyanide (90%), diethylphosphate (95%), triethylphosphate (≥99.8%) and anhydrous dimethylsulfoxide (DMSO;
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>99.9%) were obtained from Sigma-Aldrich and used as received. Cholic acid, originating from ox or sheep bile, was pre-processed by the supplier under alkaline conditions (pH 13–14) at elevated temperature (60–
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125 oC) to inactivate any pathogenic viral agents. The supplier also certified that the compound batch was
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not contaminated with neuronal tissue or other animal materials. Glycine methyl ester hydrochloride (99%), also obtained from Sigma-Aldrich, was dried in an oven at 80 oC overnight before use. Non-
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radioactive cholylsarcosine (≥95%) and cholylsarcosine methyl ester (≥95%) were prepared as previously
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described [9]. Acetonitrile (≥99.9%; Ph. Eur.) was obtained from VWR International. 2 N ± 0.02 N aqueous NaOH (Ph. Eur.; sterile; diluted to 1 N with sterile water) and aqueous citrate buffer (Ph. Eur.; sterile) was obtained from ABX. Water (Ph. Eur.; sterile), ethanol (99%; Ph. Eur.; sterile), and 70 mM aqueous NaH2PO4
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(Ph. Eur.; sterile, pH 4.5) were obtained from the pharmacy at Aarhus University Hospital. Sep-Pak® C8C8
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Plus Short Cartridges (400 mg sorbent/cartridge, 37–55 m particle size) were obtained from Waters® and preconditioned before use with 10 mL sterile ethanol followed by 10 mL sterile water. Sterile filtration was
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performed using a Millex®-GV sterile filter (0.22 m) from Millipore Corporation.
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2.2 Radiosynthesis and quality control of 11C-CSar[11C]CSar The carbon-11 radionuclide was produced by the 14N(p,)11C nuclear reaction using an IBA 18/18 cyclotron. The target gas (N2 with 0.5% O2) was bombarded at 30 A for 15–30 minutes to yield 11C-CO2[11C]CO2, which was delivered to the synthesis system (GE Tracerlab FXcPro) and converted to 11C-CH4[11C]CH4 for further gas phase reaction with molecular iodine to give 11C-CH3I[11C]CH3I. 15 minutes after end-ofbombardment (EOB), the 11C-CH3I[11C]CH3I was transferred to the reactor of the synthesis system preloaded with glycine methyl ester hydrochloride (1 mg; 8 mol) and 1,2,2,6,6-pentamethylpiperidine (5 L; 28
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mol) in anhydrous DMSO (300 L). The 11C-CH3I[11C]CH3I was bubbled through the precursor solution (helium flow: 15 mL/min) at room temperature until maximum activity was trapped in the reactor. The 11Cmethylation of glycine methyl ester was achieved by heating at 80 oC for 3 minutes (Fig. 1; step a). After cooling to 65 oC, freshly prepared solutions of cholic acid (12 mg; 29 mol) in anhydrous DMSO (200 l) and
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diethylphosphorylcyanide (5 L; 33 mol) in anhydrous DMSO (100 L) were added successively through dry tubing directly into the reactor solution. The coupling reaction was allowed to proceed for 5 minutes at
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65 oC before quenching with sterile 35% aqueous ethanol (0.7 mL) (Fig. 1; step b).
The crude reaction mixture containing 11C-CSar[11C]CSar methyl ester was purified by preparative reverse-
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phase high-pressure liquid chromatography (RP-HPLC) using a Waters®-XTerra® Prep RP18 OBDTM (5 m, 19x100 mm) column with 33% acetonitrile in sterile 70 mM aqueous NaH2PO4 (pH 4.5) as mobile phase
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(flow 20 mL/min; = 220 nm). The fraction containing 11C-CSar[11C]CSar methyl ester (retention time: 13 ± 1 min) was collected, diluted with sterile water (70 mL), and loaded onto a preconditioned C8C8-cartridge.
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The cartridge was washed with sterile water (10 mL) before eluting with sterile ethanol (1.0 mL). Finally,
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sterile aqueous 1 N NaOH (2.0 mL) was passed through the cartridge into the ethanolic solution of the ester. The alkaline mixture was allowed to stand for 1 min at room temperature, neutralized with sterile
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aqueous citrate buffer (5.0 mL) and sterile water (2.0 mL), and then passed through a sterile filter into a sterile product vial (Fig. 1; step c). Total synthesis time, including 11C-CH3I[11C]CH3I production and tracer
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purification, was 40 min from EOB.
The radioactivity amount of 11C-CSar[11C]CSar was measured in a dose calibrator (Capintec) and the radiochemical yield was calculated relative to the amount of 11C-CH3I[11C]CH3I initially trapped in the reactor. Quality control of 11C-CSar[11C]CSar was carried out as described in Supplementary Material. The shelf-life of the tracer formulation was determined by re-testing the radiochemical purity, pH and color/particles of three individual separate batches 1 hour after end-of-synthesis.
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2.3 Inclusion of participants Six healthy human participants (3 females, 3 males) with no history of liver disease and two patients diagnosed with cholestatic liver disease were included in this study (Table 1). The healthy participants (ID 01 – 06) were included after responding to an advertisement in a local newspaper or online, while the PSC
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patient (ID 07) and the PBC patient (ID 08) were included via our outpatient clinic. All participants were investigated using the same setup. At the time of the study, both patients were under treatment with
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ursodeoxycholic acid (250 mg UrsocholTM, 3 times daily, oral). All participants fasted for at least 8 hours
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before the PET/CT scan, but were allowed to drink water. Blood glucose and total plasma bile acid concentrations were measured on the day of the study. The clinical study (EudraCT no. 2016-004031-20)
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was approved by the Danish Medicines Agency and the Central Denmark Region Committees on Health Research Ethics, and was conducted in accordance with the Helsinki II Declaration, and monitored by the
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Good Clinical Practice Unit at Aarhus University Hospital. Written informed consent was obtained from all
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individual participants included in the study. No complications to the procedures were observed.
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2.4 PET/CT biodistribution and dosimetry
The participant was placed in supine position in a Siemens BiographTM 64 TruePointTM PET/CT camera. A low
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dose CT scan (50 effective mAs with CARE Dose4D [Siemens], 120 kV, pitch of 0.8 mm, and slice thickness of 5.0 mm) was performed before the first PET scan for definition of anatomical structures and attenuation
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correction of the PET recordings. 11C-CSar[11C]CSar was administered as an intravenous bolus injection with a median dose of 114 MBq (range, 60–143 MBq) immediately before start of the first PET scan. Accurate cross-calibration between PET scanner and dose calibrator used to measure injected radioactivity was verified daily. After administration of the tracer, 6 PET scans, each covering top of skull to mid-thigh (7 bed positions), were performed with 2 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, the scans were started 1, 10, 19, 32, 48, and 71 min after tracer administration. The PET images were reconstructed using 3-dimensional ordered-subset expectation
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maximization with 4 iterations and 21 subsets, 3-mm Gauss filter, and a 168x168 matrix with voxel size 4x4x3 mm3. The fused PET/CT images were analyzed using the PMOD 3.7 software (PMOD Technologies Ltd, Zürich, Switzerland). All tissues were visually inspected by two operators and organs with accumulation of 11C-CSar[11C]CSar above that of surrounding tissue were defined as source organs. These organs were
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liver, gallbladder contents and small intestine. No radioactivity was found in the urine bladder, and no radioactivity was excreted by the participant during the scans. Volumes-of-interest (VOIs) were manually
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drawn on fused PET/CT images to encompass all radioactivity in each source organ. Intrahepatic bile ducts
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were included in the VOI of the liver, whereas extrahepatic bile ducts were included in the VOI of the small intestine. For each source organ, the time course of the non–decay-corrected total radioactivity was
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normalized to the injected radioactivity and recalculated into time courses of percentage injected radioactivity. Time-integrated activity coefficients (TIACs) were computed using the trapezoidal integration
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method to calculate the area under the curves assuming only physical decay after the last scan, without further biological clearance. For the gallbladder, this approach does not account for the natural voiding of
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the gallbladder content that occurs within a few hours. Thus, the dose estimate to the gallbladder wall was
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conservative and could be overestimated. The remainder TIAC was calculated by subtracting the individual source organ TIACs from the total body TIAC (without voiding), which for 11C is 0.49 h. TIACs for source
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organs and remainder were used in OLINDA/EXM 2.0 (HERMES Medical Solution AB, Sweden) [30] to compute organ-absorbed doses (µGy/MBq) and the effective dose (µSv/MBq) using anthropomorphic
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human body phantoms with organ masses based on ICRP89 [31] and ICRP103 tissue weighting factors [32]. Organ doses and effective dose results are given for the reference gender-averaged adult according to ICRP103.
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3. Results 3.1 Radiosynthesis and quality control of 11C-CSar[11C]CSar The radiosynthesis of 11C-CSar[11C]CSar (Fig. 1) proceeded with an overall radiochemical yield of 18% ± 3% (decay corrected, mean ± SD), 6% ± 1% (not decay corrected, mean ± SD) to give 0.6–1.6 GBq 11C-
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CSar[11C]CSar with a radiochemical purity ≥98% (n = 20). The formation of 11C-cholylsarcosine[11C]CSar methyl ester (steps a and b in Fig. 1) proceeded with a radiochemical conversion of 24% ± 4%, while the
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hydrolysis to give 11C-CSar[11C]CSar (step c) was quantitative (n = 20). Unreacted 11C-cholylsarcosine[N-
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methyl-11C]sarcosine methyl ester and unidentifiable radiochemical impurities were efficiently removed during purification of 11C-cholylsarcosine[11C]CSar methyl ester by preparative RP-HPLC (Supplementary Fig.
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S1). Unlabeled cholylglycine, formed as a by-product of reaction step b (Fig. 1) and unreacted cholic acid were also efficiently removed during RP-HPLC purification and were detected at low concentrations, ≤0.3
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and ≤3.8 µg/mL, respectively, in the final formulation of 11C-CSar[11C]CSar (Supplementary Fig. S1 and Table S1). After purification, the concentrations of diethylphosphate, triethylphosphate (both decomposition
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products of diethylphosphorylcyanide formed when quenching the reaction with aqueous ethanol),
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1,2,2,6,6-pentamethylpiperidine, and the methyl esters of CSar and cholylglycine were below the limit of detection, <0.1 µg/mL, in the final formulation of 11C-CSar[11C]CSar (Supplementary Table S1). The amount
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of unlabeled CSar in the final formulation was determined to 0.1–1.2 µg/mL and the molar radioactivity of C-CSar[11C]CSar was calculated to 10 ± 2 GBq/µmol (mean ± SD; n = 20). The final formulation 11C-
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CSar[11C]CSar was a sterile, pyrogen- and particle-free, colorless solution with a pH of 6–7 and with acceptably low contents of ethanol (≤10%), acetroniltrile (<100 µg/mL), and DMSO (<0.1 µg/mL). The tracer (up to 1.6 GBq) was stable in formulation for at least one hour, i.e. the radiochemical purity did not drop below 98%, the pH remained 6–7, and the formulation remained colorless with no visible particles.
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3.2 PET/CT biodistribution and dosimetry For the healthy participants, mean (SD) blood glucose and total plasma bile acid concentrations were 5.1 0.6 mM (range, 4.5–6.0 mM, n = 6) and 1.8 0.8 µM (range, 0.9–3.2 µM, n = 6), respectively (Table 1). Blood glucose and total plasma bile acid concentrations for the two patients (ID 07 and ID 08) were within
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the same ranges (Table 1). In the healthy participants, 11C-CSar[11C]CSar was rapidly taken up by the liver and excreted into bile extrahepatic bile ducts with subsequent distribution between the gallbladder and
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small intestine (Fig. 2 and 3). The highest amount of 11C-CSar[11C]CSar in liver tissue (56% in average and a
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maximum of 68% measured in ID 01) relative to injected dose was observed 10–17 minutes after tracer administration (Fig. 3). After 71–106 minutes, approximately 95% of the administrated dose was located in
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liver tissue, small intestine and gallbladder for both female and male participants. The remaining 5% of the dose was distributed non-specifically, i.e. no accumulation of radioactivity was observed in other organs
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than liver tissue, small intestine and gallbladder in any of the participants at any time of the PET/CT scans. At 71–106 minutes, the average amount of 11C-CSar[11C]CSar in the gallbladder and the small intestine was
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39% and 50%, respectively. In one participant, however, the majority of the injected tracer was found in the
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small intestine (94% in ID 02), while in another the majority was found in the gallbladder (78% in ID 05). The biodistribution of 11C-CSar[11C]CSar in the two patients (ID 07 and ID 08) was comparable to that of the
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healthy participants with 11C-CSar[11C]CSar found only in liver tissue, gallbladder and small intestine (Supplementary Fig. S2). The amount of 11C-CSar[11C]CSar in liver tissue was however higher in the patients
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compared with the averaged healthy participants (Fig. 4).
Critical organs were gallbladder wall (range, 7–492 µGy/MBq), small intestine (range, 14–83 µGy/MBq), and liver (range, 28–46 µGy/MBq) (Table 2 and Supplementary Table S2). In general, the gallbladder wall received the highest radiation dose, up to 492 µGy/MBq (ID 05). In the case of healthy participant ID 02, however, where little 11C-CSar[11C]CSar entered the gallbladder, the gallbladder wall received a low radiation dose (7 µGy/MBq) while the small intestine received a high dose (83 µGy/MBq). The gender-
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averaged effective dose of 11C-CSar[11C]CSar in the healthy participants was 6.2 ± 1.4 µSv/MBq (mean ± SD; n = 6). For the PSC patient (ID 07) and the PBC patient (ID 08) the effective dose was 5.2 µSv/MBq and 7.0
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µSv/MBq, respectively (Table 2 and Supplementary Table S3).
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4. Discussion In this study, we optimized our previously reported radiosynthesis of 11C-CSar[11C]CSar [9] and set up a quality control in compliance with Danish/European regulations for preparation of tracers for clinical use. Specifically, by modifying the conditions for the individual radiosynthetic steps and the purification method
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we minimized the amounts of unlabeled impurities and solvent residues in the final formulation of 11CCSar[11C]CSar without compromising the radiochemical yield of the radiosynthesis or the radiochemical
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purity of the tracer. In fact, the presented radiosynthesis provides 11C-CSar[11C]CSar with slightly higher
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yield compared to that previously reported, 18% vs. 13% (decay-corrected). The overall production, which provides radiochemically pure 11C-CSar[11C]CSar in a solution ready for injection, has beenwas approved by
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the Danish Medicines Agency as suitable for preparation of the tracer for clinical use.
In both healthy participants and the patients diagnosed with intrahepatic cholestatic liver diseases, the target organs of 11C-CSar[11C]CSar are gallbladder wall, small intestine, and liver tissue with no
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accumulation of the tracer in other organs. In particular, no accumulation of 11C-CSar[11C]CSar was
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observed in stomach, kidneys, urinary bladder, large intestine, or brain; neither in the healthy participants nor in the patients. No differences were observed when estimating the effective dose using data from the
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healthy participants and data from the patients (Table 2). With the estimated gender-average effective dose of 6.2 µSv/MBq, the total radiation dose received by an adult will be around 0.4–0.6 mSv when 60–
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100 MBq 11C-CSar[11C]CSar is administrated intravenously. This dose range is sufficient to obtain highquality PET images of the hepatobiliary excretion of 11C-CSar[11C]CSar (Fig. 2 and reference [11]). Furthermore, the administered dose can probably be reduced by at least 50% on the newest digital PET systems that have fast time-of-flight timing resolution and a NEMA sensitivity that are more than twice that of the scanner used in this study.
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The distribution of 11C-CSar[11C]CSar between the gallbladder and the small intestine corresponds well to that of endogenous bile acids in the average fasting human. However, large variations were observed between individuals, which ultimately results in large variation in the estimated absorbed doses for these organs (Table 2). Therefore, absorbed dose estimates for 11C-CSar[11C]CSar (and presumably other tracers
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that similarly enters the intestine through the biliary tract) should not be based on data from a single individual. Rather, the estimates should be based on a group of individuals including cases with low and
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high gallbladder filling such as healthy participant ID 02 and ID 05.
The ursodeoxycholic acid-treated PBC and PSC patients were included in this study, because they represent
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common cases of patients with intrahepatic cholestatic liver diseases encountered in the clinic. PBC and PSC are characterized by inflammation and fibrosis of intrahepatic bile ducts, and for PSC also extrahepatic
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bile ducts, which causes impaired bile flow and accumulation of toxic bile acids in blood and liver tissue if not treated. At the time of the PET/CT study, both patients had normal plasma bile acid concentrations
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suggesting effective treatment with the choleretic ursodeoxycholic acid (Table 1). Similar to the healthy
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participants, 11C-CSar[11C]CSar was excreted from the liver tissue (blood vessels, liver cells and intrahepatic bile ducts) into extrahepatic bile ducts in the patients, albeit more slowly in the PSC patient (ID 07) than in
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the remaining participants (Fig. 4). Nevertheless, the amount of 11C-CSar[11C]CSar in liver tissue measured by PET of the patients was higher than in the healthy participants. Concordantly, with the exception of one
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participant (ID 01), the absorbed dose estimates for the liver was higher for the patients than for the healthy participants (Table 2). It is expected that this difference will be even more pronounced in untreated patients with severe cholestasis, where 11C-CSar[11C]CSar in some cases is hardly excreted into bile at all resulting in significant accumulation of the tracer in liver tissue as previously observed [11].
On the basis of data from previous studies in anesthetized pigs (40-kg, 3-month-old female Danish Landrace and Yorkshire cross-breed) [9, 19], we have estimated the effective dose for 11C-CSar[11C]CSar as well as for
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N-[11C]methyl-cholyltaurine (11C-MTCA[11C]MTCA) and N-[11C]methyl-ursodeoxycholyltaurine (11CMTUDCA[11C]MTUDCA) using the ICRP103 tissue weighting factors embedded in the Olinda 2.0 software. 11
C-MTCA and 11C-MTUDCA [11C]MTCA and [11C]MTUDCA are tracers derived from different bile acid
conjugates than 11C-CSar[11C]CSar, but with comparable biodistribution [9, 19]. The mean effective doses estimated from the pig data are 5.7, 7.2, and 7.5 µSv/MBq for 11C-CSar, 11C-MTCA, and 11C-
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MTUDCA[11C]CSar, [11C]MTCA, and [11C]MTUDCA, respectively (Supplementary Table S4 S2), which are all in
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range of the effective dose of 11C-CSar[11C]CSar estimated from the human data (Table 2). However, we
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have found that 11C-CSar[11C]CSar and the similar N-[11C]methyl-taurine conjugated bile acid tracers in some cases enter the stomach of the anesthetized pigs resulting in an increased radiation dose to this organ
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(Supplementary Table S4 S2). In contrast, we observe no 11C-CSar[11C]CSar in the stomach of humans. Because pigs and humans have a similar pyloric motor function [33], we believe that the occasional reflux
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of 11C-CSar[11C]CSar into the stomach of the pigs should be attributed anesthesia rather than physiology. Therefore, the anesthetized pig does not make a suitable model for estimating absorbed doses for 11C-
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CSar[11C]CSar for individual organs in (un-anesthetized) humans. Significant differences between species
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have also been reported for other PET tracers, where in several cases the estimated whole-body effective
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dose is similar but the absorbed doses to individual organs vary significantly [34-37].
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5. Conclusion We have presented a radiosynthesis and quality control of the conjugated bile acid PET tracer 11CCSar[11C]CSar set up in compliance with Danish/European regulations and suitable preparation of the tracer for clinical use. Target organs of 11C-CSar[11C]CSar are gallbladder wall, small intestine, and liver. The
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gender-averaged effective dose of 11C-CSar[11C]CSar is estimated to 6.2 1.4 µSv/MBq in healthy humans (mean SD, n = 6). The most critical organ is the gallbladder wall with an absorbed dose estimate of up to
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0.5 mGy/MBq. However, the distribution of 11C-CSar[11C]CSar between the gallbladder and the small
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intestine varies significantly among individuals resulting in some cases in low radiation doses to the gallbladder wall and a concurrently high radiation dose to the small intestine. Our results also show that
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while the effective dose estimated preclinical in anesthetized pigs is comparable to that estimated in
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humans, the absorbed dose estimates to critical organs can differ significantly.
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Acknowledgement
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This study was supported by The Danish Council for Independent Research (Medical Sciences, 12-125512)
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and Ejnar and Aase Danielsen’s Foundation (10-001051, 10-000579).
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Figure Legends Fig. 1. Radiosynthesis of 11C-Cholylsarcosine (11C-CSar) [N-methyl-11C]cholylsarcosine ([11C]CSar). Reaction conditions: a) 11C-CH3I[11C]CH3I, 1,2,2,6,6-pentamethylpiperidine, DMSO, 80 oC, 3 min; b) cholic acid, diethylphosphorylcyanide, DMSO, 65 oC, 5 min; c) purification, then 1 N aqueous sodium hydroxide, room
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temperature, 1 min, then neutralization (citrate buffer) and sterile filtration.
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Fig. 2. Example of PET/CT whole-body scans of a healthy participant (ID 06) recorded after intravenous
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injection of 11C-CSar ([11C]CSar) (98 MBq) over the time frames indicated. Top row shows PET emission only (average radioactivity concentration); bottom row shows fused PET/CT images. Post injection, the tracer is
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rapidly taken up by the liver (a), excreted into bile (b), and subsequently distributed between the
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gallbladder and the small intestine (c–f). Each image shows the average radioactivity concentration measured by PET over the time frame indicated.
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Fig. 3. Time course of the distribution of 11C-CSar[11C]CSar measured by whole-body PET/CT after
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intravenous injection of the tracer in healthy participants. The figure shows mean (± SD; n = 6) percent of injected dose in liver tissue including intrahepatic bile ducts (black), small intestine including extrahepatic
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bile ducts (light grey) and gallbladder (dark grey). The data is decay corrected to the time of injection.
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Fig. 4. Percent of injected dose of 11C-CSar[11C]CSar in liver tissue (including intrahepatic bile ducts) measured during whole-body PET/CT scans of healthy human participants (ID 01 – ID 06; gender-averaged), a PSC patient (ID 07), and a PBC patient (ID 08). Both patients were under treatment with ursodeoxycholic acid at the time of the study. The data is decay corrected to the time of injection. The relatively high SDvalues are due to one participant (ID 01) who had a higher amount of 11C-CSar[11C]CSar in liver tissue than the remaining group of healthy participants.
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Tables Table 1. Characteristics of participants ID
Gender/Age Healthy/disease Body weight
Height Total bile acidsa
Glucoseb
11
(yr)
(m)
(mmol/L blood)
CSar[11C]CSar
(kg)
(µmol/L plasma)
C-
73
1.68
<1
02
Female/63
Healthy
85
1.74
1.8
03
Male/70
Healthy
103
1.85
04
Male/73
Healthy
82
05
Male/70
Healthy
84
06
Female/57
Healthy
93
07
Male/34
PSC
82
08
Female/61
PBC
74
112
4.8
116
2.3
5.9
60
1.90
1.3
6.0
143
1.76
3.2
4.5
141
1.75
1.4
4.8
98
1.81
1.6
5.9
77
1.72
2.2
5.3
126
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a
4.8
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Healthy
(MBq)
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Female/59
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dose
Bile acid reference <10 µmol/L plasma, b Fasting glucose reference <7 mmol/L blood
Abbreviations: 11C-CSar 11C-cholylsarcosine [11C]CSar [N-methyl-11C]cholylsarcosine, PBC primary biliary
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cholangitis, PSC primary sclerosing cholangitis
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Table 2. Absorbed dose estimates Gender-averaged organ doses (µGy/MBq) and effective dose (µSv/MBq) for 11C-CSar[11C]CSar in healthy human participants (ID 01 – ID 06), the PSC patient (ID 07), and the PBC patient (ID 08) Patientsa
Healthy participants
Target organ
PSC
PBC
02 (F)
03 (M)
04 (M)
05 (M)
06 (F)
07 (M)
08 (F)
Adrenals
8.1
5.7
7.2
7.1
8.4
6.8
8.2
8.4
Brain
0.8
1.0
1.1
1.0
0.9
1.0
0.8
0.9
Breasts
1.7
1.6
1.7
1.7
1.6
1.6
1.7
1.7
Esophagus
3.5
2.7
3.2
3.2
3.2
3.0
3.7
3.5
0.9
1.0
1.1
170.0
7.4
4.2
0.9
1.0
0.8
0.9
193.0
177.0
492.0
130.0
18.5
251.0
5.7
4.6
4.7
4.5
5.0
3.8
4.1
35.4
82.7
39.9
43.5
14.1
57.3
40.3
24.4
Stomach wall
3.4
3.5
3.4
3.4
3.2
3.4
3.5
3.4
Right colon
5.8
5.0
6.1
6.0
8.3
5.8
4.5
6.4
Rectum
1.9
3.1
2.3
2.3
1.6
2.5
2.0
1.7
Heart wall
3.1
2.7
3.0
3.0
2.9
2.9
3.3
3.2
Kidneys
5.2
4.2
5.0
5.0
6.1
4.8
4.9
5.5
b
Gallbladder wall Left colon Small intestineb
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Eyes
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1.1
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01 (F)
46.1
27.8
35.1
35.3
34.1
34.5
54.2
45.9
Lungs
2.9
2.4
2.7
2.7
2.6
2.6
3.1
2.9
2.6
4.1
3.0
3.1
2.4
3.3
2.6
2.4
6.9
6.9
6.6
6.7
6.8
6.9
6.9
6.8
1.6
2.3
1.9
1.9
1.5
2.0
1.6
1.5
1.0
1.2
1.2
1.2
1.0
1.1
1.0
1.0
2.0
2.1
2.1
2.1
2.0
2.0
2.0
2.0
Osteogenic cells
1.4
1.5
1.5
1.5
1.4
1.4
1.4
1.4
Spleen
2.3
2.6
2.5
2.5
2.3
2.5
2.3
2.3
Testes
0.9
1.1
1.1
1.1
0.9
1.0
0.8
0.9
Thymus
1.8
1.7
1.9
1.9
1.8
1.8
1.9
1.9
Thyroid
1.3
1.3
1.4
1.4
1.3
1.3
1.3
1.3
Urinary bladder wall
1.6
2.3
1.9
1.9
1.4
1.9
1.6
1.5
Uterus
2.9
4.8
3.3
3.4
2.3
3.8
2.9
2.5
Total body
3.1
3.2
3.1
3.1
3.1
3.1
3.1
3.1
Effective dose
6.3
4.4
6.1
6.0
8.7
5.6
5.2
7.0
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Liverb Ovaries Prostate Salivary glands
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Red marrow
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Pancreas
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a
Both patients were under treatment with ursodeoxycholic acid at the time of the study, b Critical organs.
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Abbreviations: F female, M male, PBC primary biliary cholangitis, PSC primary sclerosing cholangitis.
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Compliance with Ethical Standards
Conflict of Interest
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The authors declare that they have no conflict of interest.
Ethical approval
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This clinical study (EudraCT no. 2016-004031-20) was approved by the Danish Medicines Agency and the
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Central Denmark Region Committees on Health Research Ethics, and was conducted in accordance with the
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Helsinki II Declaration, and monitored by the Good Clinical Practice Unit at Aarhus University Hospital.
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Informed consent:
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Written informed consent was obtained from all individual participants included in the study.
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References [1] Frisch K and Hofmann AF. Biliary secretion. In: Keiding S, Sørensen M, editors. Functional molecular imaging in hepatology. Sharjah, UAE: Bentham Science Publishers; 2012, pp. 49–75.
PT
[2] Hofmann AF and Hagey LR. Key discoveries in bile acid chemistry and biology and their clinical
RI
applications: history of the last eight decades. J Lipid Res 2014;55:1553–95.
SC
[3] Li T and Chiang JYL. Bile Acid Signaling in Metabolic Disease and Drug Therapy. Pharmacol Rev
NU
2014;66:948–83.
MA
[4] Thomas C, Pellicciari R, Pruzanski M, Auwerx J, and Schoonjans K. Targeting bile-acid signalling for metabolic diseases. Nat Rev Drug Discov 2008;7:678–93.
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(Landmark Ed) 2009;14:2584–98.
D
[5] Hofmann AF. The enterohepatic circulation of bile acids in mammals: form and functions. Front Biosci
CE
[6] Wagner M and Trauner M. Recent advances in understanding and managing cholestasis. F1000Research
AC
2016;5(F1000 Faculty Rev):705.
[7] Williamson KD and Chapman RW. New Therapeutic Strategies for Primary Sclerosing Cholangitis. Semin Liver Dis 2016;36:5–14.
[8] Zollner G and Trauner M. Mechanisms of Cholestasis. Clin Liver Dis 2008;12:1–26.
22
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[9] Frisch K, Jakobsen S, Sørensen M, Munk OL, Alstrup AKO, Ott P, et al. [N-Methyl-11C]cholylsarcosine, a novel bile acid tracer for PET/CT of hepatic excretory function: radiosynthesis and proof-of-concept studies in pigs. J Nucl Med 2012;53:772–8.
[10] Sørensen M, Munk OL, Ørntoft NW, Frisch K, Andersen KJ, Mortensen FV, et al. Hepatobiliary secretion
RI
PT
kinetics of conjugated bile acids measured in pigs by 11C-cholylsarcosine PET. J Nucl Med 2016;57:961–6.
SC
[11] Ørntoft NW, Munk OL, Frisch K, Ott P, Keiding S, and Sørensen M. Hepatobiliary transport kinetics of the conjugated bile acid tracer 11C-CSar quantified in healthy humans and patients by positron emission
NU
tomography. J Hepatol 2017;67:321–7.
MA
[12] Ørntoft NW, Frisch K, Ott P, Keiding S, and Sørensen M. Functional assessment of hepatobiliary secretion by 11C-cholylsarcosine positron emission tomography. Biochim Biophys Acta Mol Basis Dis
PT E
D
2018;1864:1240–4.
CE
[13] Dickson I. Liver: PET for bile transport kinetics. Nat Rev Gastroenterol Hepatol 2017;14:260–1.
[14] Testa A, Zanda M, Elmore CS, and Sharma P. PET Tracers To Study Clinically Relevant Hepatic
AC
Transporters. Mol Pharmaceutics 2015;12:2203–16.
[15] Pastor CM, Müllhaupt B, and Stieger B. The Role of Organic Anion Transporters in Diagnosing Liver Diseases by Magnetic Resonance Imaging. Drug Metab Dispos 2014;42:675–84.
[16] Hoekstra LT, Graaf Wd, Nibourg GAA, Heger M, Bennink RJ, Stieger B, et al. Physiological and biochemical basis of clinical liver function tests. Ann Surg 2013;257:27–36. Geisel D, Lüdemann L, Hamm B,
23
ACCEPTED MANUSCRIPT
Denecke T. Imaging-Based Liver Function Tests – Past, Present and Future. Fortschr Röntgenstr 2015;187:863–71.
[17] Jazrawi RP, de Caestecker JS, Goggin PM, Britten AJ, Joseph AE, Maxwell JD, et al. Kinetics of hepatic
PT
bile acid handling in cholestatic liver disease: effect of ursodeoxycholic acid. Gastroenterology
RI
1994;106:134–42.
SC
[18] Felton J, Cheng K, Said A, Shang AC, Xu S, Vivian D, et al. Using multi-fluorinated bile acids and in vivo
NU
magnetic resonance imaging to measure bile acid transport. J Vis Exp 2016;117:e54597.
[19] Schacht AC, Sørensen M, Munk OL, and Frisch K. Radiosynthesis of N-11C-methyl-taurine–conjugated
MA
bile acids and biodistribution studies in pigs by PET/CT. J Nucl Med 2016;57:628–33.
D
[20] Frisch K, Stimson DHR, Venkatachalam T, Pierens GK, Keiding S, Reutens D, et al. N-(4-
PT E
[18F]fluorobenzyl)cholylglycine, a novel tracer for PETof enterohepatic circulation of bile acids:
CE
radiosynthesis and proof-of-concept studies in rats. Nucl Med Biol 2018;2018:56–62.
[21] Jia L, Jiang D, Hu P, Li X, Shi H, Cheng D, et al. Synthesis and evaluation of 18F-labeled bile acid
AC
compound: a potential PET imaging agent for FXR-related diseases. Nucl Med Biol 2014;41:495–500.
[22] Chong H-S, Chen Y, Kang CS, Sun X, and Wu N. Novel 64Cu-radiolabeled bile acid conjugates for targeted PET imaging. Bioorg Med Chem Lett 2015;25:1082–5.
24
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[23] Testa A, Dall’Angelo S, Mingarelli M, Augello A, Schweiger L, Welch A, et al. Design, synthesis, in vitro characterization and preliminary imaging studies on fluorinated bile acid derivatives as PET tracers to study hepatic transporters. Bioorg Med Chem 2017;25:963–76.
[24] Lombaerde SD, Neyt S, Kersemans K, Verhoeven J, Devisscher L, Vlierberghe HV, et al. Synthesis, in
PT
vitro and in vivo evaluation of 3β-[18F]fluorocholic acid for the detection of drug-induced cholestasis in
SC
RI
mice. PLos One 2017;12:e0173529.
[25] Neyt S, Vliegen M, Verreet B, Lombaerde SD, Braeckman K, Vanhove C, et al. Synthesis, in vitro and in
NU
vivo small-animal SPECT evaluation of novel technetium labeled bile acid analogues to study (altered)
MA
hepatic transporter function. Nucl Med Biol 2016;43:642–9.
[26] Anelli PL, Lattuada L, Lorusso V, Lux G, Morisetti A, Morosini P, et al. Conjugates of gadolinium
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Chem 2004;47:3629–41.
D
complexes to bile acids as hepatocyte-directed contrast agents for magnetic resonance imaging. J Med
AC
2017;15:21–6.
CE
[27] Nykonenko A, Vávra P, and Zonča P. Anatomic Peculiarities of Pig and Human Liver. Exp Clin Transplant
[28] Kararli TT. Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals. Biopharm Drug Dispos 1995;16:351–80.
[29] Gonzalez LM, Moeser AJ, and Blikslager AT. Porcine models of digestive disease: the future of large animal translational research. Transl Res 2015;166:12–27.
25
ACCEPTED MANUSCRIPT
[30] Stabin MG and Siegel JA. RADAR dose estimate report: a compendium of radiopharmaceutical dose estimates based on OLINDA/EXM version 2.0. . J Nucl Med 2018;59:154–60.
[31] Basic anatomical and physiological data for use in radiological protection. reference values. A report of
PT
age- and gender-related differences in the anatomical and physiological characteristics of reference
RI
individuals. ICRP Publication 89. Ann ICRP. 2002;32(3–4):5–265.
SC
[32] The 2007 Recommendations of the International Commission on Radiological Protection. ICRP
NU
publication 103. Ann ICRP. 2007;37(2–4):1–332.
[33] Treacy PJ, Jamieson GG, and Dent J. Pyloric motor function during emptying of a liquid meal from the
MA
stomach in the conscious pig. J Physiol 1990;422:523–38.
D
[34] Sakata M, Oda K, Toyohara J, Ishii K, Nariai T, and Ishiwata K. Direct comparison of radiation dosimetry
PT E
of six PET tracers using human whole-body imaging and murine biodistribution studies. Ann Nucl Med
CE
2013;27:285–96.
[35] Hino-Shishikura A, Suzuki A, Minamimoto R, Shizukuishi K, Oka T, Tateishi U, et al. Biodistribution and
AC
radiation dosimetry of [18F]-5-fluorouracil. Appl Radiat Isot 2013;75:11–7.
[36] Petrulli JR, Hansen SB, Abourbeh G, Yaqub M, Bahceb I, Holdenb D, et al. A multi species evaluation of the radiation dosimetry of [11C]erlotinib, the radiolabeled analog of a clinically utilized tyrosine kinase inhibitor. Nucl Med Biol 2017;47:56–61.
26
ACCEPTED MANUSCRIPT
[37] Kranz M, Sattler B, Tiepolt S, Wilke S, Deuther-Conrad W, Donat CK, et al. Radiation dosimetry of the α4β2 nicotinic receptor ligand (+)-[18F]flubatine, comparing preclinical PET/MRI and PET/CT to first-in-human
AC
CE
PT E
D
MA
NU
SC
RI
PT
PET/CT results. EJNMMI Physics 2016;3:25.
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