PET/CT Scans Using the Tracer 11C-Csar, a Bile Acid Analog, to Depict and Visualize Cholestatic Disorders in Patients with Genetic Liver Disorders and Healthy Individuals

Background

Cholestasis is defined as a decreased flow of bile from the liver to the duodenum, either due to reduced excretion from the liver, defects in the enterohepatic circulation or blockage of the biliary tree. Bile acids are essential for the absorption of lipophilic compounds from the intestine. \[1\] When bile acids accumulate in the liver, it causes damage to the tissue.

Patients with cholestasis typically present with jaundice and pruritus caused by the bile acids accumulating in the skin. Elevated blood levels of alkaline phosphatases (AP), gamma-glutamyl transferase, and sometimes bilirubin are observed \[2\]. The clinical workup includes ultrasound sonography (US) to exclude tumours/stones and specific blood tests, including autoantibodies, immunoglobulins, etc. A thorough history of familial diseases and medicine intake is also important for workup \[1\]. A liver biopsy can be necessary to evaluate hepatic histopathological changes in the liver. Treatment depends on the specific cause. The present project deals with medical causes of cholestasis as described below, i.e., not mechanical obstruction such as cancer or gallstones.

Monitoring medical cholestatic disorders relies on continuous blood measurements of AP, bilirubin, etc. Still, these tests do not necessarily convey information about the degree of cholestasis from a functional point of view.

The enterohepatic circulation Intact enterohepatic circulation is important since de novo synthesis of bile acids does not play a significant role in enterohepatic circulation.\[3\] Bile acids are absorbed in the terminal ilium by the ileal bile acid transporter (ASBT/SLC10A2) and secreted into the portal vein, which delivers the bile acids back to the liver. Here, the Na+-taurocholate co-transporting peptide (NTCP) and organic anion- transporting proteins (OATPs) mediate bile uptake from the sinusoids to hepatocytes.\[4\] The bile salt export pump (BSEP) facilitates the transport from hepatocytes into bile canaliculi. The multidrug resistance proteins 3 and 4 (MRP3/ABCC3, MRP4/ABCC4) and the heteromeric organic solute transporter (OSTa/b/SLC51A/SLC51B) mediate backflux from hepatocytes to blood. \[3\] All these transporters can be defective in different genetic diseases and lead to cholestasis, which is the case in patients with progressive familial intrahepatic cholestasis (PFIC), including protein-truncating mutations in tight-junction protein 2 gene (TJP2) and mutations in bile salt export pump (BSEP). Some of these patients will debut with drug-induced liver disease (DILI) or have recurrent cholestasis.

The functional consequences of genetic mutations and variations affecting hepatic transporter and tight- junction proteins as the cause of cholestatic disorders have been challenging to study in vivo. Some disorders progress rapidly into cirrhosis and potentially hepatocellular carcinomas, whereas others do not become apparent until the partially defective transporter protein is as it can be seen in drug-induced liver injury (DILI), e.g., for substrates excreted into bile via bile salt exporting protein, BSEP \[5\]-\[9\] Studying the hepatobiliary handling of bile acids has been challenging, primarily due to the livers concealed position between the portal venous and systemic blood systems and because of the inherent difficulty in retrieving sampling materials from bile.

In the present project, investigators wish to generate novel insights into the functional effects of cholestasis by visualizing the handling of bile acids. Visualization is achieved by using functional positron emission tomography/computer tomography (PET/CT).

Previous studies The Department of Hepatology \& Gastroenterology and the Department of Nuclear Medicine \& PET Centre, both Aarhus University Hospital, have, in collaboration, developed a PET/CT method to study the enterohepatic circulation of bile acids. This method utilizes the carbon-11 labelled conjugated bile acid tracer cholylsarcosine (11C-CSAR) \[3\], \[10\]-\[12\].

11C-CSAR is a synthetic conjugate of N-methyl glycine (sarcosine) and cholic acid, which was chosen due to its similarity to taurocholic acid, one of the essential bile salts in humans. Also, this bile acid is not metabolized in the body or by bacteria in the intestines.

Investigators have validated the 11C-CSAR PET/CT method in patients with various cholestatic disorders and healthy controls. \[3\], \[11\]-\[14\] Investigators plan to expand our data pool using newer and more advanced scanners and to expand the method to patients with genetic mutations/variations in proteins involved in biliary secretion to learn about these conditions and functional effects. Examination of this patient group has not been done before.

Hypothesis

* The hepatobiliary secretion of 11C-Csar will be reduced in patients compared to healthy individuals. * Cholestatic disorders affect different steps in hepatobiliary secretion of bile acids from blood depending on the specific disease/genetic disorder * The functional effect of cholestatic disorders varies among patients with some patients being almost unaffected and others being severely affected. * The measurements from functional11C-CSar PET/CT provide a better evaluation/description/characterization of patients with cholestatic disorders than standard biochemistry.

Study design The study is an investigator-initiated observational study. Healthy volunteers, patients who present with cholestasis and patients with known genetic disorders in the hepatobiliary system will be offered participation in a functional 11C-CSAR PET/CT.

Participants arrive at the department at 8.00 AM after an overnight fast. The participants will get a hepatic vein catheter, two venous catheters and an artflon. Indocyanine green (ICG) will be administered intravenously to measure hepatic flow (Ficks principle).

11C-CSAR is administered intravenously at the beginning of a functional PET/CT. The PET/CT scan takes approximately 45 minutes. Blood samples will be drawn from the artflon and the hepatic vein catheter during the scan to measure the hepatic blood flow and the concentrations of 11C-CSAR.

Investigators aim to include 14 patients with different cholestatic disorders, including genetic disorders, and eight healthy volunteers.

Methods 11C-CSAR PET/CT 11C-CSAR is injected intravenously in micro-doses and enters the enterohepatic circulation of endogenous bile acids \[3\], \[10\]-\[13\]. The tracer is injected at the beginning of a dynamic PET/CT scan of the hepato-biliary system. By external detection, this method provides real-time measurements of the time course of tissue concentrations of 11C-CSAR in liver tissue, bile ducts, etc. Combined with arterial blood measurements from an artflon and hepatic blood flow measurements using ICG (Ficks principle), specific kinetic parameters of the livers handling of conjugated bile acids are calculated by fitting a mathematical model to data. These parameters include uptake from sinusoidal blood, back flux to sinusoids, transport from hepatocytes into biliary canaliculi, and bile flow. Mean transit time in hepatocyte and relative tissue concentrations are also calculated \[3\], \[10\]-\[13\].

The 11C-CSAR PET/CT scans will be performed after an overnight (8-hour) fast, and the participants cannot take their regular medication before the scan. The participants will be placed in a supine position in the Siemens Biograph Vision Quadra with the liver within the 106 cm field of view. A low-dose CT scan is performed to visualize the anatomy. 50 MBq 11C-Csar mixed in 10 ml of saline water is administered intravenously over 25 seconds at the beginning of a 45-minute functional 11C-CSAR PET/CT scan. During the scan, blood samples from the hepatic vein catheter are collected to measure concentrations of 11C-CSAR.

Tracer The 11C-CSAR tracer will be prepared at the Department of Nuclear Medicine and PET Centre, Aarhus University Hospital, using a 3-step radio-synthesis method. \[16\]

Preparation Patients with cholestasis are followed with blood samples in the departments. Before the scan of the volunteers, blood samples are taken for biochemical analysis (haemoglobin, leukocytes, thrombocytes, INR, CRP, alanine transferase, bilirubin, alkaline phosphatase, creatinine, eGFR, albumin, sodium, potassium, and possibly other relevant health-related parameters), to rule out unknown health issues related to the liver. The amount of blood is 20 ml, which will be drawn at the time of inclusion at the Department of Clinical Biochemistry or by us. The results of the blood samples will appear in the participants regular medical journals. The blood samples are discarded immediately after analysis.

Women participating in the study will be asked to take a pregnancy test on the day of the scan. The test will be performed on urine. The urine will be discarded afterwards.

Catheters Venflons are placed percutaneously in both cubital veins by a trained technician for intravenous administrations of 11C-CSAR and ICG. For blood sampling, an artflon catheter will be placed percutaneously in a radial artery by a trained anesthesiologist, and a catheter will be placed in the hepatic vein via the right jugular vein by a trained hepatologist.

Data assessment The PET data involves 11C-CSAR concentrations in liver and bile ducts and blood concentrations of 11C-CSAR in arterial and hepatic vein blood and hepatic blood flow. Investigators will calculate the kinetics of hepatobiliary excretion of 11C-CSAR from the patients with cholestasis and compare them with the healthy volunteers.

Data management Source data for each participant is registered in the participants PET journal or Case Report Form (CRF). The CRF is established as a project in REDCap and contains information on contact information and demography (sex, weight, height). Source data on blood work will be in Labka, EPJ (Electronic patient journal).

The PET journal contains all data considering tracer production, quality control, administered dose, and scan data (including side effects related to the scan). The localization of source data is kept in the trial master file (TMF). The signed consent forms are kept in digital form in REDCap, and a physical form is kept in a locker with the TMF. Data is held in the eCRF (REDCap) and the PET journal.

Source data on each participant includes all data regarding the production of tracer and quality control, administered dose, data on scan results and possible deviations and complications, as well as the participants health records and laboratory results. This data will be registered in each participants PET journal and CRF.

All research participants will perform data management and assessment. Sample sizes and statistics The number of healthy subjects (n=8) is based on our experience from previous PET studies, including 11C-CSar studies.

The patient group is expected to be very heterogeneous, with individual results ranging from numbers close to healthy subjects to values significantly different from healthy subjects. As there are only so many patients with genetic disorders, Investigators aim to include 14 during the period to get a broad range of patients.

The statistical analysis is carried out in STATA and GraphPad Prism in pseudonymized form. Data is deleted or anonymized after ten years.

Data will be analyzed per protocol and participants who lack data considering the primary endpoints will be excluded from the analysis. It will be stated in the results if any deviations from the statistical analysis plan occur. Should a participant withdraw from the study before completion, a new participant will be recruited to ensure to have a complete dataset of participants.

Risk, side effects and precautions Monitoring during 11C-CSAR PET/CT During 11C-CSAR PET/CT, participants will be under constant medical supervision. The Department of Nuclear Medicine and PET Centre h