The New LC-MS/MS Method for Determination of Unbound Tacrolimus in Plasma
The New LC-MS/MS Method for Determination of Unbound Tacrolimus in Plasma
Tacrolimus (TAC) is characterized by a narrow therapeutic window, as well as high inter- and
 intra-individual variability in pharmacokinetics. Both under- and overexposure may lead to
 severe adverse effects. Therapeutic drug monitoring (TDM) is an essential element of
 post-transplant patient care. Most transplantation centers use C0 to adjust TAC dosage. Some
 controversies remain about relationship between C0 and clinical outcome.

 It is generally accepted that only protein-unbound drug molecules can cross cellular
 membranes, which imply that TDM of free tacrolimus fraction may be of paramount importance
 and improve clinical management of organ recipients.

 Whole blood TAC concentrations and dose requirements are strongly associated with CYP3A5
 polymorphism. Routine CYP3A5 genotyping on the waiting lists might be useful to guide
 tacrolimus dosing.

 This interdisciplinary project tackles the research problem from three angles - biochemistry,
 genetics and clinical observation. The primary goal of the study is to evaluate clinical
 usefulness of different TDM protocols in patients after kidney and liver transplantation.
As there are over 15.000 patients in Poland on continuous tacrolimus (TAC) therapy, the
 identification and validation of more sensitive and specific biomarkers is of utmost
 importance. The investigators propose a multiple step assessment of TAC therapeutic drug
 monitoring (TDM) in kidney and liver recipients. A role of genetic profiling on drug
 concentration and clinical effects will also be addressed. The project will significantly
 contribute to understanding tacrolimus pharmacokinetics and body response to drug exposure.
 Moreover, the proposed project is the first attempt to integrate both different TDM measure
 methods and patient genetics in a rigorous, prospective study with the assessment of the
 clinical over- and underexposure TAC effects. It is expected to provide an argument for
 implementation of even more personalized, predictable immunosuppressive therapy.

 The investigators hypothesize that:

 1. There is a correlation of free TAC level with drug toxicity on one hand, and graft
 rejection and underimmunosuppression despite target whole blood concentration on the
 other.

 2. CYP3A5 expressors and non-expressors will present different levels of TAC in both whole
 blood C0 and free TAC C0 as well as different effectiveness and toxicity profiles.

 3. The concentration of free TAC is related to changes in the concentration of blood
 components, thus it is possible to derive the equation for calculating free TAC fraction
 as a useful tool for the drug dosage adjustment.

 Study design:

 The group: 110 consecutive adult kidney or liver transplant on TAC-based immunosuppression,
 who declare to attend the local outpatient clinic for at least one year. Another exclusion
 criteria are HBV, HCV and HIV infection, neurological disordesr, intake of drugs affecting
 TAC metabolism.

 Objectives: A primary objective of this study is to look for a correlation between unbound
 TAC level in an ultrafiltrate with symptoms of drug toxicity, and graft rejection episodes.
 Also, a complex comparison of different methods of determination of TAC concentration in
 whole blood, plasma and ultrafiltrate is planned. Secondary aim of the study is to calculate
 free fraction of TAC from hematocrit level, albumin concentration and routine whole blood TAC
 C0 to predict dose adjustment more accurately. The generated equation will be plot against
 CYP3A polymorphisms. The benefit of genotyping before administration of TAC for dose
 prediction will be evaluated.

 Database and endpoints: a Case Report Form (CRF) was designed. Primary endpoints are:
 treatment failure, 12-month overall patient and graft survival. Secondary end points are side
 effects of tacrolimus toxicity: kidney graft function, liver graft function, new-onset
 diabetes mellitus after transplantation or worsening of former diabetes control, new arterial
 hypertension or worsening of former hypertension control, metabolic disorders (dyslipidemia,
 hypomagesemia, hypokalemia), neurotoxicity (especially post-transplant tremors), digestive
 disorders, cardiovascular events, infections and neoplasms' occurrence.

 TAC measure: free TAC in ultrafiltrate, plasma and whole blood C0 will be measured (day: 3,
 5, 7, 14, 360).

 Genotyping: CYP3A4 and CYP3A5 polymorphisms will be recorded. Study duration: The study is
 scheduled for 3 years: 1-1.5 years for collection of samples, 1 year of follow-up after
 transplantation, 0.5 year for analysis and publication of the results.

 Efficacy variables: Standard monitoring of blood and urine laboratory parameters in de novo
 renal or liver transplant recipients during first year after transplantation, neurological
 examination including The Essential Tremor Rating Assessment Scale (TETRAS), whole blood TAC
 trough level (C0), plasma TAC concentration, free TAC concentration in plasma ultrafiltrate,
 TAC daily dose.
Inclusion Criteria:

 -  adult (>18 yo), recipient of kidney or liver transplant from the Regional
 Qualification Center, tacrolimus-based immunosuppression

 Exclusion Criteria:

 -  the use of agents significantly influencing TAC metabolism, double organ recipients,
 HBV, HCV and HIV infection, neurological disorders.