Study of the Cellular Diffusion of Tacrolimus Across the Membrane of Mononuclear Cells

Rennes University Hospital26 enrolled

Overview

Pharmacokinetics of tacrolimus are highly variable and may result in graft rejection (underdosing) or toxicity (overdosing). The risk of transplant rejection and the toxicity of tacrolimus can be reduced by pharmacological therapeutic monitoring of the molecule, based on the measurement of residual blood concentrations. Nevertheless, some patients are victims of rejections or toxic signs even though their blood concentrations are in the therapeutic target. The aim of the study is to describe the pharmacokinetics of tacrolimus diffusion in mononuclear cells as well as the kinetics of effect of the drug on its target protein

Factors responsible for pharmacokinetic variability of tacrolimus are multiple: compliance, diet, drug interactions and also genetic polymorphism of cytochrome P450 3A5 (CYP 3A5) and efflux protein ABCB1 (P-glycoprotein, P-gp). The mechanism of action of tacrolimus is based on inhibition of calcineurin in T cells. Therefore, tacrolimus intra-lymphocyte concentration may be a finer marker of the risk of transplant rejection or toxicity. The degree of inhibition of calcineurin in the T lymphocyte could also be a pharmacodynamic marker more relevant than the blood concentration. The hypothesis that the ABCB1 efflux pump is the main factor limiting the diffusion of tacrolimus into mononuclear cells is advanced. The diffusion of tacrolimus into mononuclear cells and the impact of the ABCB1 efflux pump on this diffusion have not been studied to date. The effect kinetics of the drug on calcineurin in mononuclear cells is also unknown. The aim of the study is to describe the pharmacokinetics of tacrolimus diffusion in mononuclear cells as well as the kinetics of effect of the drug on its target protein: calcineurin in the presence or absence of an efflux pump inhibitor ABCB1 at room temperature and at 4 ° C (in order to inhibit all transport proteins) from blood obtained from 18 patients undergoing bleeding as part of maintenance treatment for hemochromatosis.

Study Type

OBSERVATIONAL

Enrollment

Conditions

Hemochromatosis

Interventions

Cell pharmacokinetics of tacrolimusBIOLOGICAL

Three levels of tacrolimus will be tested. Each aliquot will be supplemented with an amount of tacrolimus to achieve one of these three levels of concentration. At 0, 5, 15, 30, 60, 120, 240mn, the samples will be separated into 2 aliquots : one dedicated to the determination of tacrolimus in mononuclear cells, the other dedicated to the determination of calcineurin activity. in mononuclear cells.

Eligibility

Sex: ALLMin age: 18 Years

Medical Language ↔ Plain English

Inclusion Criteria: * adult (age \> 18 years old); * phlebotomy as part of the maintenance treatment of hemochromatosis; * having received the information on the protocol and not having indicated his opposition to participate; * not receiving immunosuppressive therapy; * not receiving drug treatment that can induce or inhibit the protein ABCB1 (Rifampicin, Carbamazepine, Phenobarbital, Phenytoin, Efavirenz, Amiodarone, azole antifungals, calcium channel blockers). Exclusion Criteria: * participation in another protocol whose procedures are incompatible with the realization of the study; * adults who are subject to legal protection (protection of justice, guardianship) and persons deprived of their liberty; * pregnant women.

Outcomes

Primary Outcomes

Diffusion kinetics of tacrolimus in mononuclear cells

Determination of tacrolimus in mononuclear cells of subjects Tacrolimus will be assayed by mass spectrometry with a limit of quantification of 10 pg / million cells, sufficient to determine the concentrations in the volunteers' blood.

Time frame: At the time of inclusion

Secondary Outcomes

Determination of the activity of calcineurin in mononuclear cells

These determinations will be carried out by Dr. Benoit Blanchet according to a validated and published method (cf references Blanchet et al, 2003; Blanchet et al, 2006). The method is based on high pressure liquid chromatography (HPLC coupled) with spectrophotometric detection.