Validation of methods to study the distribution and protein binding of tacrolimus in human blood

J Pharmacol Toxicol Methods. 2001 Jul-Aug;46(1):27-35. doi: 10.1016/s1056-8719(02)00158-2.


Introduction: Tacrolimus is a macrolide immunosuppressant that has a narrow therapeutic index, displays considerable variability in response, and has the potential for serious drug interactions. Therapeutic drug monitoring and dose individualisation for tacrolimus is complicated but essential. Few studies have investigated the blood distribution and protein binding of tacrolimus and the results of these studies are conflicting. The aim of the present study is to establish and validate methods to investigate the distribution of tacrolimus in human blood. To conduct these studies at clinically relevant concentrations the use of 3H-dihydro-tacrolimus instead of tacrolimus was investigated.

Methods: The use of radiolabelled tacrolimus was validated by conducting studies with a mixture of both labelled and unlabelled drug where tacrolimus was analysed by LC-MS/MS. The in vitro distribution of tacrolimus and 3H-dihydro-tacrolimus was investigated in blood collected from healthy subjects using Ficoll-Paque reagent and density gradient ultracentrifugation, respectively. The unbound fraction of tacrolimus in plasma was studied using equilibrium dialysis conducted at 37 degrees C.

Results: In blood, tacrolimus was found to be mainly associated with erythrocytes (85.3+/-1.5%), followed by diluted plasma proteins (14.3+/-1.5%) and lymphocytes (0.46+/-0.10%). In plasma, tacrolimus was found to mainly be associated with the soluble protein fraction (61.2+/-2.5%), high-density lipoproteins (HDL, 28.1+/-5.4%), low-density lipoproteins (LDL, 7.8+/-1.6%), and very low-density lipoproteins (VLDL, 1.4+/-0.3%). The unbound fraction of tacrolimus was found to be only 1.2+/-0.12%. Statistical comparison indicated that there was no significant difference in the blood distribution and plasma protein binding of 3H-dihydro-tacrolimus when compared with tacrolimus.

Discussion: These results have important implications for therapeutic drug monitoring of tacrolimus and subsequent studies of tacrolimus distribution in transplant recipients.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Chromatography
  • Dialysis
  • Drug Monitoring / methods*
  • Humans
  • Immunosuppressive Agents / blood
  • Immunosuppressive Agents / pharmacokinetics*
  • Mass Spectrometry
  • Protein Binding
  • Tacrolimus / blood
  • Tacrolimus / pharmacokinetics*
  • Tissue Distribution
  • Tritium


  • Immunosuppressive Agents
  • Tritium
  • Tacrolimus