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

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

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.

Study Overview

• Status: Recruiting
• Conditions: Kidney Transplant Rejection; Liver Transplant; Complications; Immunosuppression; Metabolism Medication Toxicity; Genetic Predisposition; Kidney Transplant; Complications; Liver Transplant Rejection
• Intervention / Treatment: Drug: Tacrolimus; Diagnostic test: Unbound tacrolimus measurement; Diagnostic test: CYP3A4 and CYP3A5 genotyping

Detailed Description

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 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:

1. 40 consecutive kidney or liver transplant recipients on TAC-based immunosuppression.
2. 300 kidney transplant recipients attending the local outpatient clinic.
3. 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.

• Study Type: Observational
• Enrollment (Anticipated): 380

Contacts and Locations

• Study Contact: Name: Karola Warzyszyńska, MD; Phone Number: +48225021783; Email: karola.warzyszynska@gmail.com

Study Locations

• Poland
  • Warsaw, Poland, 02-006
    • Recruiting
    • Department of General and Transplant Surgery, Medical University of Warsaw
    • Contact: Karola Warszyszynska, MD; Phone Number: +48 225021784; Email: karola.warzyszynska@gmail.com
    • Principal Investigator: Karola Warzyszynska, MD

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years and older (ADULT, OLDER_ADULT)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

• Sampling Method: Probability Sample

Study Population

1. 40 consecutive de novo adult kidney or liver transplant recipients on TAC-based immunosuppression.
2. 300 adult kidney transplant recipients attending the local outpatient clinic.
3. 40 adult kidney transplant recipients experiencing acute rejection of the renal allograft.

Description

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.

Study Plan

How is the study designed?

Number of groups / cohorts

3

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
De novo renal/liver transplant recipients; 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; Prevention of rejection in kidney or liver transplant: a standard immunosuppressive therapy according to international protocols.; Other Names: Prograf; Advagraf; Diagnostic test: Unbound tacrolimus measurement; Unbound tacrolimus measurement in plasma ultrafiltrate.; Diagnostic test: CYP3A4 and CYP3A5 genotyping; DNA purification and genotyping
Random renal transplant recipients; The group of 300 random adult (age > 18 years) male and female recipients of deceased kidney attending the local outpatient clinic.	Drug: Tacrolimus; Prevention of rejection in kidney or liver transplant: a standard immunosuppressive therapy according to international protocols.; Other Names: Prograf; Advagraf; Diagnostic test: Unbound tacrolimus measurement; Unbound tacrolimus measurement in plasma ultrafiltrate.; Diagnostic test: CYP3A4 and CYP3A5 genotyping; DNA purification and genotyping
Renal transplant recipients experiencing graft rejection; 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; Prevention of rejection in kidney or liver transplant: a standard immunosuppressive therapy according to international protocols.; Other Names: Prograf; Advagraf; Diagnostic test: Unbound tacrolimus measurement; Unbound tacrolimus measurement in plasma ultrafiltrate.; Diagnostic test: CYP3A4 and CYP3A5 genotyping; DNA purification and genotyping

What is the study measuring?

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

Collaborators and Investigators

• Sponsor: National Science Centre, Poland
• Collaborators: Medical University of Warsaw
• Investigators: Principal Investigator: Karola Warzyszyńska, MD, Department of General and Transplant Surgery, Medical University of Warsaw

Publications and helpful links

General Publications

• Kidney Disease: Improving Global Outcomes (KDIGO) Transplant Work Group. KDIGO clinical practice guideline for the care of kidney transplant recipients. Am J Transplant. 2009 Nov;9 Suppl 3:S1-155. doi: 10.1111/j.1600-6143.2009.02834.x.
• Ekberg H, Tedesco-Silva H, Demirbas A, Vitko S, Nashan B, Gurkan A, Margreiter R, Hugo C, Grinyo JM, Frei U, Vanrenterghem Y, Daloze P, Halloran PF; ELITE-Symphony Study. Reduced exposure to calcineurin inhibitors in renal transplantation. N Engl J Med. 2007 Dec 20;357(25):2562-75. doi: 10.1056/NEJMoa067411.
• European Association for the Study of the Liver. Electronic address: easloffice@easloffice.eu. EASL Clinical Practice Guidelines: Liver transplantation. J Hepatol. 2016 Feb;64(2):433-485. doi: 10.1016/j.jhep.2015.10.006. Epub 2015 Nov 17. No abstract available.
• Bittersohl H, Schniedewind B, Christians U, Luppa PB. A simple and highly sensitive on-line column extraction liquid chromatography-tandem mass spectrometry method for the determination of protein-unbound tacrolimus in human plasma samples. J Chromatogr A. 2018 Apr 27;1547:45-52. doi: 10.1016/j.chroma.2018.03.010. Epub 2018 Mar 7.
• Brunet M, van Gelder T, Asberg A, Haufroid V, Hesselink DA, Langman L, Lemaitre F, Marquet P, Seger C, Shipkova M, Vinks A, Wallemacq P, Wieland E, Woillard JB, Barten MJ, Budde K, Colom H, Dieterlen MT, Elens L, Johnson-Davis KL, Kunicki PK, MacPhee I, Masuda S, Mathew BS, Millan O, Mizuno T, Moes DAR, Monchaud C, Noceti O, Pawinski T, Picard N, van Schaik R, Sommerer C, Vethe NT, de Winter B, Christians U, Bergan S. Therapeutic Drug Monitoring of Tacrolimus-Personalized Therapy: Second Consensus Report. Ther Drug Monit. 2019 Jun;41(3):261-307. doi: 10.1097/FTD.0000000000000640.
• Pascual J, Berger SP, Witzke O, Tedesco H, Mulgaonkar S, Qazi Y, Chadban S, Oppenheimer F, Sommerer C, Oberbauer R, Watarai Y, Legendre C, Citterio F, Henry M, Srinivas TR, Luo WL, Marti A, Bernhardt P, Vincenti F; TRANSFORM Investigators. Everolimus with Reduced Calcineurin Inhibitor Exposure in Renal Transplantation. J Am Soc Nephrol. 2018 Jul;29(7):1979-1991. doi: 10.1681/ASN.2018010009. Epub 2018 May 11.
• Stienstra NA, Sikma MA, van Dapperen AL, de Lange DW, van Maarseveen EM. Development of a Simple and Rapid Method to Measure the Free Fraction of Tacrolimus in Plasma Using Ultrafiltration and LC-MS/MS. Ther Drug Monit. 2016 Dec;38(6):722-727. doi: 10.1097/FTD.0000000000000351.
• Bouamar R, Shuker N, Hesselink DA, Weimar W, Ekberg H, Kaplan B, Bernasconi C, van Gelder T. Tacrolimus predose concentrations do not predict the risk of acute rejection after renal transplantation: a pooled analysis from three randomized-controlled clinical trials(dagger). Am J Transplant. 2013 May;13(5):1253-61. doi: 10.1111/ajt.12191. Epub 2013 Mar 8.
• Israni AK, Riad SM, Leduc R, Oetting WS, Guan W, Schladt D, Matas AJ, Jacobson PA; DeKAF Genomics Investigators. Tacrolimus trough levels after month 3 as a predictor of acute rejection following kidney transplantation: a lesson learned from DeKAF Genomics. Transpl Int. 2013 Oct;26(10):982-9. doi: 10.1111/tri.12155. Epub 2013 Jul 24.
• Kershner RP, Fitzsimmons WE. Relationship of FK506 whole blood concentrations and efficacy and toxicity after liver and kidney transplantation. Transplantation. 1996 Oct 15;62(7):920-6. doi: 10.1097/00007890-199610150-00009.
• Zahir H, McCaughan G, Gleeson M, Nand RA, McLachlan AJ. Changes in tacrolimus distribution in blood and plasma protein binding following liver transplantation. Ther Drug Monit. 2004 Oct;26(5):506-15. doi: 10.1097/00007691-200410000-00008.
• Zahir H, Nand RA, Brown KF, Tattam BN, McLachlan AJ. 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.
• Hendijani F, Azarpira N, Kaviani M. Effect of CYP3A5*1 expression on tacrolimus required dose for transplant pediatrics: A systematic review and meta-analysis. Pediatr Transplant. 2018 Jun 19:e13248. doi: 10.1111/petr.13248. Online ahead of print.
• Rojas L, Neumann I, Herrero MJ, Boso V, Reig J, Poveda JL, Megias J, Bea S, Alino SF. Effect of CYP3A5*3 on kidney transplant recipients treated with tacrolimus: a systematic review and meta-analysis of observational studies. Pharmacogenomics J. 2015 Feb;15(1):38-48. doi: 10.1038/tpj.2014.38. Epub 2014 Sep 9.
• Haufroid V, Wallemacq P, VanKerckhove V, Elens L, De Meyer M, Eddour DC, Malaise J, Lison D, Mourad M. CYP3A5 and ABCB1 polymorphisms and tacrolimus pharmacokinetics in renal transplant candidates: guidelines from an experimental study. Am J Transplant. 2006 Nov;6(11):2706-13. doi: 10.1111/j.1600-6143.2006.01518.x.
• Venkataramanan R, Swaminathan A, Prasad T, Jain A, Zuckerman S, Warty V, McMichael J, Lever J, Burckart G, Starzl T. Clinical pharmacokinetics of tacrolimus. Clin Pharmacokinet. 1995 Dec;29(6):404-30. doi: 10.2165/00003088-199529060-00003.
• Kuypers DR, Claes K, Evenepoel P, Maes B, Vanrenterghem Y. Clinical efficacy and toxicity profile of tacrolimus and mycophenolic acid in relation to combined long-term pharmacokinetics in de novo renal allograft recipients. Clin Pharmacol Ther. 2004 May;75(5):434-47. doi: 10.1016/j.clpt.2003.12.009.
• Undre NA, van Hooff J, Christiaans M, Vanrenterghem Y, Donck J, Heeman U, Kohnle M, Zanker B, Land W, Morales JM, Andres A, Schafer A, Stevenson P. Low systemic exposure to tacrolimus correlates with acute rejection. Transplant Proc. 1999 Feb-Mar;31(1-2):296-8. doi: 10.1016/s0041-1345(98)01633-9. No abstract available.
• Zong YP, Wang ZJ, Zhou WL, Zhou WM, Ma TL, Huang ZK, Zhao CC, Xu Z, Tan RY, Gu M. Effects of CYP3A5 polymorphisms on tacrolimus pharmacokinetics in pediatric kidney transplantation: a systematic review and meta-analysis of observational studies. World J Pediatr. 2017 Oct;13(5):421-426. doi: 10.1007/s12519-017-0035-4. Epub 2017 May 24.
• Nankivell BJ, Alexander SI. Rejection of the kidney allograft. N Engl J Med. 2010 Oct 7;363(15):1451-62. doi: 10.1056/NEJMra0902927. No abstract available.
• Randomised trial comparing tacrolimus (FK506) and cyclosporin in prevention of liver allograft rejection. European FK506 Multicentre Liver Study Group. Lancet. 1994 Aug 13;344(8920):423-8.
• U.S. Multicenter FK506 Liver Study Group. A comparison of tacrolimus (FK 506) and cyclosporine for immunosuppression in liver transplantation. N Engl J Med. 1994 Oct 27;331(17):1110-5. doi: 10.1056/NEJM199410273311702.
• de Mattos AM, Olyaei AJ, Bennett WM. Nephrotoxicity of immunosuppressive drugs: long-term consequences and challenges for the future. Am J Kidney Dis. 2000 Feb;35(2):333-46. doi: 10.1016/s0272-6386(00)70348-9.
• Elble R, Comella C, Fahn S, Hallett M, Jankovic J, Juncos JL, Lewitt P, Lyons K, Ondo W, Pahwa R, Sethi K, Stover N, Tarsy D, Testa C, Tintner R, Watts R, Zesiewicz T. Reliability of a new scale for essential tremor. Mov Disord. 2012 Oct;27(12):1567-9. doi: 10.1002/mds.25162. Epub 2012 Oct 2.

Study record dates

Study Major Dates

• Study Start (ACTUAL): August 1, 2020
• Primary Completion (ANTICIPATED): December 1, 2022
• Study Completion (ANTICIPATED): May 1, 2023

Study Registration Dates

• First Submitted: November 24, 2020
• First Submitted That Met QC Criteria: December 1, 2020
• First Posted (ACTUAL): December 8, 2020

Study Record Updates

• Last Update Posted (ACTUAL): April 22, 2022
• Last Update Submitted That Met QC Criteria: April 14, 2022
• Last Verified: April 1, 2022

More Information

Terms related to this study

• Keywords: Kidney transplant; liver transplant; CYP3A; therapeutic drug monitoring; unbound tacrolimus
• Additional Relevant MeSH Terms: Pathologic Processes; Disease Attributes; Disease Susceptibility; Genetic Predisposition to Disease; Physiological Effects of Drugs; Molecular Mechanisms of Pharmacological Action; Enzyme Inhibitors; Immunosuppressive Agents; Immunologic Factors; Calcineurin Inhibitors; Tacrolimus
• Other Study ID Numbers: 2019/33/N/NZ7/01631

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: All IPD that underlie results in a publication will be available
• IPD Sharing Time Frame: After publication of the study results (about 2023).
• IPD Sharing Access Criteria: Non-comercial, after request
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL; SAP; ICF

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: Yes
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No