The New LC-MS/MS Method for Determination of Unbound Tacrolimus in Plasma

Study Overview
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Brief Summary
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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.

Detailed Description
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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: 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. 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. 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 concentration as a useful tool for the drug dosage adjustment Study design Objectives: Phase 1) A primary objective of this study is to develop and validate a new method for unbound tacrolimus measurement. - Published: 12 March 2022 (https://doi.org/10.3390/pharmaceutics14030632) Phase 2) A primary objective 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 plotted against CYP3A polymorphisms. Phase 3) A primary objective is to look for a correlation between unbound TAC level in an ultrafiltrate with graft rejection episodes. Secondary endpoints: A complex comparison of different methods of determination of TAC concentration in whole blood, plasma and ultrafiltrate is planned. The benefit of genotyping before administration of TAC for dose prediction will be evaluated. The studied groups: 40 consecutive kidney or liver transplant recipients on TAC-based immunosuppression. 300 kidney transplant recipients attending the local outpatient clinic. 40 kidney transplant recipients experiencing acute rejection of the renal allograft. TAC measurements: Measurements of unbound tacrolimus concentrations in plasma ultrafiltrate and tacrolimus concentrations in plasma and whole blood will be performed using a Nexera LC System with LCMS-8050 MS triple quadrupole with ascomycin and deuterated tacrolimus as internal standards. Genotyping: DNA of patients will be purified and analyzed using RT-PCR for CYP3A4 and CYP3A5 polymorphisms Study duration: The study is scheduled for 3 years: 2.5 years for collection of samples, 0.5 year for analysis and publication of the results. Efficacy variables: Standard monitoring of blood and urine laboratory parameters, whole blood TAC trough level (C0), plasma TAC concentration, free TAC concentration in plasma ultrafiltrate, TAC daily doses.

Official Title
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Development and Validation of New LC-MS/MS Method for Determination of Unbound Tacrolimus in Plasma in CYP3A5 Expressors and Non-Expressors

Conditions
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Immunosuppression, Kidney Transplant Rejection, Kidney Transplant; Complications, Liver Transplant Rejection, Liver Transplant; Complications, Metabolism Medication Toxicity, Genetic Predisposition

Intervention / Treatment
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* Drug: Tacrolimus
* Diagnostic Test: Unbound tacrolimus measurement
* Diagnostic Test: CYP3A4 and CYP3A5 genotyping


Participation Criteria
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Eligibility Criteria
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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.

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| De novo renal/liver transplant recipients<br>The group of 40 consecutive adult (age > 18 years) male and female recipients of deceased kidney or liver transplant from the Regional Qualification Center (Warsaw, Poland). | Drug: Tacrolimus<br>* Prevention of rejection in kidney or liver transplant: a standard immunosuppressive therapy according to international protocols.<br>* Other names: Advagraf;Diagnostic Test: Unbound tacrolimus measurement<br>* Unbound tacrolimus measurement in plasma ultrafiltrate.<br>Diagnostic Test: CYP3A4 and CYP3A5 genotyping<br>* DNA purification and genotyping<br>|
| Random renal transplant recipients<br>The group of 300 random adult (age > 18 years) male and female recipients of deceased kidney attending the local outpatient clinic. | Drug: Tacrolimus<br>* Prevention of rejection in kidney or liver transplant: a standard immunosuppressive therapy according to international protocols.<br>* Other names: Advagraf;Diagnostic Test: Unbound tacrolimus measurement<br>* Unbound tacrolimus measurement in plasma ultrafiltrate.<br>Diagnostic Test: CYP3A4 and CYP3A5 genotyping<br>* DNA purification and genotyping<br>|
| Renal transplant recipients experiencing graft rejection<br>The group of 40 consecutive adult (age > 18 years) male and female recipients of deceased kidney experiencing acute rejection of the renal allograft. | Drug: Tacrolimus<br>* Prevention of rejection in kidney or liver transplant: a standard immunosuppressive therapy according to international protocols.<br>* Other names: Advagraf;Diagnostic Test: Unbound tacrolimus measurement<br>* Unbound tacrolimus measurement in plasma ultrafiltrate.<br>Diagnostic Test: CYP3A4 and CYP3A5 genotyping<br>* DNA purification and genotyping<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Development and validation of the new LC-MS/MS measurement method | Development and validation of the extremely sensitive method for unbound tacrolimus determination using the EMA and FDA guidelines | 1 year | 
| A comparison of different TAC TDM protocols depending on the matrix | concentration of unbound tacrolimus in plasma ultrafiltrate concentration of tacrolimus in plasma concentration of tacrolimus in whole blood The measures will be obtained with the use of LC-MS/MS method. | 1 year | 
| Equation to calculate unbound TAC concentration | Development of the equation (using the concentration of free TAC, plasma and whole blood, and blood components) by statistical methods. | 1 year | 
| A correlation between free TAC and symptoms of underimmunosuppresion | Biopsy proven acute rejection | 1 year | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| A correlation between free TAC and nephrotoxicity | Calcineurin inhibitor toxicity confirmed with the biopsy | 1 year | 
| CYP3A expression | SNP genotyping to address CYP3A genetic variations (CYP3A4 and CYP3A5) | 1 year | 
| Blood components | Hematocrit, plasma proteins, albumins, LDL, HDL, total cholesterol, triglycerid | 1 year | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Kidney transplant, liver transplant, CYP3A, unbound tacrolimus, therapeutic drug monitoring