Tacrolimus (FK506) is a calcineurin inhibitor. It mainly exerts an immunosuppressive effect by inhibiting the transcription of cytokine genes related to T cell activation and the expression of their proteins. It has the advantages of strong medicinal effect, low dosage, high survival rate of grafts, and low incidence of acute rejection.
Tacrolimus treatment window is very narrow, the dose is too high or too low will increase the risk of postoperative infection, malignancy, rejection and nephrotoxicity, so it is necessary to routinely carry out therapeutic drug monitoring (TDM) for dose adjustment, and the current clinical practice is mainly achieved by monitoring trough concentrations.
However, because the trough concentration does not reflect the distribution of tacrolimus in T cells, and the drug sensitivity varies from person to person, relying solely on this data does not fully reflect the biological effect of tacrolimus. That is, when patients need to increase the immunosuppressive effect, increasing the blood concentration of tacrolimus does not necessarily achieve the desired immunosuppressive effect, but the rejection reaction occurs and increases the risk of late transplant failure, and there are differences in the immune system of transplant patients, even if the blood concentration of tacrolimus is consistent, different individuals will have different immunosuppressive effects.
Currently reported tacrolimus pharmacodynamic markers include detection of calcineurin activity in peripheral blood mononuclear cells (PBMCs), NFAT-regulated gene expression (NFAT-RGE), and cytokine expression. However, due to the lack of complete methodological validation of many marker tests and the lack of reports of relevance to clinical outcomes, only the detection of NFAT-RGE is currently included in the recommendations of the 2019 tacrolimus TDM (Therapeutic Drug Monitoring) guidelines.
NFAT-RGE
NFAT-RGE is the gene expression regulated by NFAT, which mainly detects the expression of IL-2, IFN-γ and GM-CSF (granulocyte-macrophage colony stimulator) as THE gene expression of NAFT regulation by real-time quantitative polymerase chain reaction (qPCR). The method, first published in 2004 by Giese et al., activates the T cell signaling pathway by stimulating whole blood in vitro using forvolution of forvolution and ionomycin, and then determines the expression of downstream genes by RNA extraction, reverse transcription, and qPCR. The results found that IL-2, IFN-γ, and GM-CSF were the most inhibited among the 18 downstream genes tested, so the average expression of the above three genes was selected as a substitute for NFAT-RGE. In 2016, Abdel-Kahaar et al. validated the method by examining the quantity-efficiency relationship, intra-batch/batch analysis error, quantitative limits and stability in 2 laboratories, and found that the method was reproducible across different laboratories and met the guidelines for qPCR methodological validation.
The expression of NFAT-RGE is (E1.5/E0) ×100%, E1.5 was chosen because the existing studies found that NFAT-RGE had the greatest degree of inhibition at 1.5 h after taking the drug, while the ratio E1.5/E0 was selected to correct the difference in cytokine gene expression levels between individuals and more accurately reflect the biological effects of the drug after administration. Claudi-a administered tacrolimus (6.0 mg/day) to 262 kidney transplant patients, of which tacrolimus inhibited 143 people's NFAT regulator (IL-2, IFN-γ, and GM-CSF) gene expression (NFAT-RGE) < 30%, and 119 NFAT-RGE ≥ 30%. After renal biopsy, it was found that only 1.3% of less than 30% of patients experienced rejection, while 25.2% of patients with NFAT-RGE ≥ 30% experienced rejection, suggesting that monitoring the expression of NFAT regulatory genes in kidney transplant patients may be an effective pharmacodynamic method to prevent rejection.
Monitoring with NFAT-RGE is also flawed. First, although Sommerer et al. reported that the method is more exclusive to CNIs, it is not sensitive to other immunosuppressive drugs after transplantation, such as hormones and azathioprine. But there are also studies showing that NFAT-RGE levels may be affected by other immunosuppressive drugs such as glucocorticoids. Therefore, follow-up multicenter, prospective studies are needed to further determine the exclusivity of the marker tacrolimus.
As of today, the study of tacrolimus pharmacodynamic markers is still very limited, pharmacodynamic monitoring has not yet entered routine monitoring, and trough concentrations are still used as the main reference index for dose adjustment in clinical practice. The use of NFAT-RGE as a supplement to routine trough concentration monitoring allows for greater individualized administration of tacrolimus and can have a beneficial effect on patient outcomes. Therefore, follow-up multicentre, prospective studies are needed to further determine the relevance of this marker to clinical outcomes.
bibliography:
1. A review of NFAT-regulated gene expression as a pharmacodynamic marker of tacrolimus after transplantation
2. Research progress of tacrolimus pharmacodynamics
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