Synthesis, in vitro and in vivo small-animal SPECT evaluation of novel technetium labeled bile acid analogues to study (altered) hepatic transporter function

Nucl Med Biol. 2016 Oct;43(10):642-9. doi: 10.1016/j.nucmedbio.2016.07.001. Epub 2016 Jul 12.


Introduction: Hepatobiliary transport mechanisms are crucial for the excretion of substrate toxic compounds. Drugs can inhibit these transporters, which can lead to drug-drug interactions causing toxicity. Therefore, it is important to assess this early during the development of new drug candidates. The aim of the current study is the (radio)synthesis, in vitro and in vivo evaluation of a technetium labeled chenodeoxycholic and cholic acid analogue: [(99m)Tc]-DTPA-CDCA and [(99m)]Tc-DTPA-CA, respectively, as biomarker for disturbed transporter functionality.

Methods: [99mTc]-DTPA-CDCA([(99m)Tc]-3a) and [99mTc]-DTPA-CA ([(99m)Tc]-3b) were synthesized and evaluated in vitro and in vivo. Uptake of both tracers was investigated in NTCP, OCT1, OATP1B1, OATP1B3 transfected cell lines. Km and Vmax values were determined and compared to [(99m)Tc]-mebrofenin ([(99m)Tc]-MEB). Efflux was investigated by means of CTRL, MRP2 and BSEP transfected inside-out vesicles. Metabolite analysis was performed using pooled human liver S9. Wild type (n=3) and rifampicin treated (n=3) mice were intravenously injected with 37MBq of tracer. After dynamic small-animal SPECT and short CT acquisitions, time-activity curves of heart, liver, gallbladder and intestines were obtained.

Results: We demonstrated that OATP1B1 and OATP1B3 are the involved uptake transporters of both compounds. Both tracers show a higher affinity compared to [(99m)Tc]-MEB, but are in a similar range as endogenous bile acids for OATP1B1 and OATP1B3. [(99m)Tc]-3a shows higher affinities compared to [(99m)Tc]-3b. Vmax values were lower compared to [(99m)Tc]-MEB, but in the same range as endogenous bile acids. MRP2 was identified as efflux transporter. Less than 7% of both radiotracers was metabolized in the liver. In vitro results were confirmed by in vivo results. Uptake in the liver and efflux to gallbladder + intestines and urinary bladder of both tracers was observed. Transport was inhibited by rifampicin.

Conclusion: The involved transporters were identified; both tracers are taken up in the hepatocytes by OATP1B1 andOATP1B3 with Km and Vmax values in the same range as endogenous bile acids and are secreted into bile canaliculi via MRP2. Dynamic small-animal SPECT imaging can be a useful noninvasive method of visualizing and quantifying hepatobiliary transporter functionality and disturbances thereof in vivo, which could predict drug pharmacokinetics.

Keywords: Bile acid analogues; Drug induced cholestasis; Hepatobiliary imaging; Small-animal SPECT; Technetium.

MeSH terms

  • Animals
  • Biological Transport
  • Cell Line
  • Chemistry Techniques, Synthetic
  • Chenodeoxycholic Acid / chemical synthesis
  • Chenodeoxycholic Acid / chemistry*
  • Chenodeoxycholic Acid / metabolism
  • Cholic Acid / chemical synthesis
  • Cholic Acid / chemistry*
  • Cholic Acid / metabolism
  • Female
  • Humans
  • Isotope Labeling
  • Liver-Specific Organic Anion Transporter 1 / metabolism*
  • Mice
  • Organic Anion Transporters, Sodium-Independent / metabolism*
  • Radiochemistry
  • Solute Carrier Organic Anion Transporter Family Member 1B3
  • Technetium / chemistry*
  • Tomography, Emission-Computed, Single-Photon / methods*


  • Liver-Specific Organic Anion Transporter 1
  • Organic Anion Transporters, Sodium-Independent
  • SLCO1B1 protein, human
  • SLCO1B3 protein, human
  • Solute Carrier Organic Anion Transporter Family Member 1B3
  • Chenodeoxycholic Acid
  • Technetium
  • Cholic Acid