Tacrolimus: A Review of Laboratory Detection Methods and Indications for Use

Abstract

Tacrolimus (Tac) is an immunosuppressive drug that is used in preventing organ and tissue rejection in patients after transplantation. Tac administration requires frequent and diligent monitoring by physicians to ensure proper dosage and to limit the potential for harmful adverse effects, which can include renal damage, neurotoxicity, and other serious adverse events. Tac is a calcineurin inhibitor, which suppresses the function of T-cells. Its success as an immunosuppressive agent has been well documented in preventing graft-vs-host disease in several types of organ transplants. This literature review will discuss Tac metabolism and its role in preventing tissue and organ transplant rejection. A variety of detection techniques used in the clinical laboratory, including dried-blood-spot analysis, liquid chromatography–tandem mass spectrometry, and immunoassay also will be discussed.

tacrolimus, tandem mass spectrometry, immunoassay, graft-vs-host disease, drug monitoring, organ transplantation

Therapeutic drug monitoring of tacrolimus (Tac) and other immunosuppressive agents, such as cyclosporine and sirolimus, are important to ensure adequate drug therapy and to increase favorable patient outcomes after allogenic organ and tissue transplantation. Tac has previously been noted as an effective drug in preventing organ and tissue rejection; however, its administration requires frequent monitoring and dose adjustments because of its metabolism and narrow therapeutic window. Even at low trough levels (4–6 ng/mL), Tac has been linked to nephrotoxicity.¹ Other toxic adverse effects of Tac administration include reduction in renal blood flow and creatinine clearance, microangiopathic hemolytic anemia, hypertension, central-nervous-system demyelination, and decrease in pancreatic insulin production.² Due to the severity of potential adverse effects, it is imperative for physicians to routinely monitor any patients on a Tac-based immunosuppressive regimen. Also, it is important for clinical laboratories to carefully evaluate their methods of detection, thereby recognizing the immediate effects that the results may have on patient outcomes.

Tacrolimus, also known as FK-506 or by its trade names Prograf and Advagraf (both manufactured by Astellas Pharma, Inc), is a macrolide immunosuppressant derived from Streptomyces tsukubaensis. This drug was discovered in 1984 by Fujisawa Pharmaceutical Co Ltd. Tac is formulated as a prolonged-release drug that inhibits calcineurin, a protein phosphatase that is required for T-lymphocyte activation.^3,4 Today, its usage has surpassed that of cyclosporine; Tac has become one of the more frequently prescribed immunosuppressive drugs for use after lung, kidney, and pancreas transplants in the United States.^5,6

Tac binds to FKBP-12, an immunophilin responsible for signal transduction, and forms a pentameric complex with Ca²⁺, calmodulin, and calcineurin. The resulting formation inhibits the action of the nuclear factor of activated T-cells (NFAT). The expression of NFAT is needed for interleukin-2 (IL-2) to begin signaling the activation of T-lymphocytes.² Compared with cyclosporine, Tac produces a higher in vitro inhibitory action against T-cells and is useful for the prophylaxis of graft-vs-host disease (GVHD) when used by itself or in conjunction with other immunosuppressive drugs.⁷

GVHD occurs in 3 phases. The first progression of GVHD, the afferent phase, results from tissue damage that originates from the conditioning regimen for a transplant. The sequential release of interleukins and cytokines from the site of tissue damage activates the antigen-presenting cells (APCs), namely, dendritic cells, Langerhans cells, and B-cells, of the host. The T-cells of the donor recognize the alloantigens presented by the APCs. Donor T-cell activation marks the second progression of GVHD, known as the efferent phase. During this phase, T-cells proliferate and produce additional cytokines and interleukins, most notably IL-2, which amplifies additional T-cell and natural killer cell (NKC) responses. In the effector phase, the final progression, an acute response of cells (neutrophils, NKCs, natural killer T-cells, and macrophages) escalate further tissue damage.⁸ This disruption of the immune system remains an obstacle to successful organ or tissue transplant.

Tac has been noted in previous literature reviews to be highly efficient in the prevention of GVHD. As described by Amann et al,⁹ the Rogosin Institute (an affiliate of New York Presbyterian–Weill Cornell Medical Center) has previously developed a personalized immunosuppressive drug therapy for patients undergoing kidney transplantation.

The Rogosin Institute immunosuppressive protocol for kidney allograft recipients begins with induction therapy followed by maintaining a trough blood concentration of tacrolimus at 8–10 ng/mL during the first 3 months post-transplant followed by a dose reduction to achieve target trough levels of 6–8 ng/mL for the remainder of the first year. Thereafter, the dose is further reduced to achieve trough levels of 4–6 ng/mL. The future goal is to reduce the tacrolimus whole blood concentration even further, to around 2–4 ng/mL, on a patient by patient basis to minimize nephrotoxic effects and still maintain adequate immunosuppression.⁹

In the absence of a medication-administration protocol, Thölking et al¹ suggest that a formulaic approach has the potential to assist health care professionals in personalizing the immunosuppressive regiment for each patient, to help each one avoid severe adverse effects. To identify patients that incur risk for impaired kidney function after transplant, Thölking et al describe a ratio (C/D) in which Tac blood concentration (C) is divided by the daily dose (D). A C/D ratio of less than 1.05 may be linked to inferior kidney function after transplantation and should be considered a high indicator for potential risk of a patient developing complications.

Due to the metabolism of Tac and its potential adverse effects, careful monitoring is required to yield the desired therapeutic effect. Drug monitoring for many laboratories ultimately is suitable for 2 platforms, namely, immunoassay or liquid chromatography–tandem mass spectrometry (LC-MS/MS). Next, I will provide a brief background of and discuss advances in those technologies.

Discussion

Frequent and careful determinations of Tac whole blood trough levels are necessary to prevent the risk of adverse reactions, which largely consist of nephrotoxic and neurotoxic effects, as well as new-onset diabetes mellitus, after transplantation. Tac is primarily administered through oral capsules. Intravenous injection of Tac is generally only reserved for patients who cannot tolerate oral administration. As with many pharmaceuticals, dosage adjustment is based on the ability of the patient to tolerate the medication, the type of organ transplant performed, and other clinical and diagnostic information observed by the physician.¹⁰ For this reason, many clinical laboratories do not have established reference ranges for immunosuppressant drugs because these ranges tend to vary dramatically among patients. Results should only be interpreted by a physician and/or pharmacist and should be used in conjunction with other diagnostic presentations.

In some instances, Tac can be coadministered along with other immunosuppressants, such as cyclosporine, sirolimus, and everolimus, to reduce the impact of harmful adverse effects. With the influence of additional pharmacokinetic and pharmacodynamic elements affecting drug therapy, the importance of regular monitoring is further recommended.¹¹ Because physicians tend to maintain patients at low Tac trough concentrations (3–7 ng/mL), laboratories should employ methodologies that display low limits of quantification, ideally, 1 ng/mL.¹² Methodologies that can be used in the detection of Tac include LC-MS/MS, enzyme-multiplied immunoassay (EMIT), radioimmunoassay, enzyme-linked immunoabsorbant assay (ELISA), antibody-conjugated magnetic immunoassay (ACMIA), electrochemiluminescence immunoassay (ECLIA), cloned enzyme donor immunoassay (CEDIA), turbidimetric quantitative microsphere systems (QMS), and fluorescence polarization immunoassay (FPIA).

In a survey conducted by the International Association for Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT), LC-MS/MS was identified as the primary detection methodology in 53% of surveyed laboratories—76 total laboratories in 14 different countries. LC-MS/MS is a preferred methodology in most laboratories because of its high specificity. This technique allows for optimal separation of different molecules based on molecular fragmentation. The primary disadvantage observed in many nonchromatographic systems is the potential for crossreactivity between the parent drug and its metabolites. Crossreactivity can lead to falsely elevated concentration values. Also, many of these systems are costly and may not offer the ideal throughput that is demanded of the laboratory.¹¹

Ultimately, clinical laboratories will use chromatography-based methods or immunoassay. It should be noted that liquid chromatography with ultraviolet detection (LC-UV) is not generally recommended as a means of detection because Tac has no chromophores and circulates in low concentrations. In most laboratories, LC-MS/MS is used as the primary chromatographic method of detection. After a protein precipitation procedure, specimens can be analyzed using this technique with direct injection after gradient elution, 2-dimensional chromatography, or online solid-phase extraction. Specimens for chromatography testing are often pretreated with an organic solvent and zinc sulphate.¹³

Mass spectrometry techniques also can be applied to the analysis of dried blood specimens (DBS). This collection technique offers the advantage of being collected by the patient at home using the finger stick method. The DBS can then be mailed to the laboratory for measurement, lowering transportation costs and saving time for the patient. However, high hematocrits have been shown to affect the permeability of the paper to which the blood specimen of the patient is applied. Specimens from patients with increased cell volumes have lower permeability through the paper, creating a smaller spot, which affects the ability of the laboratory to generate accurate results.^14,15 Further application of LC-MS/MS also can be extended to measuring Tac concentrations in liver biopsies and peripheral blood mononuclear cells (PBMCs). Although the hepatic tissue concentrations method offers strong correlations with severity of rejection, its clinical application is not practical. Also, measurement of Tac levels in PBMCs have shown poor correlation with whole blood concentration; the clinical utility of this method is still being researched.¹³

Detection of Tac by immunoassay is another largely preferred method for quantitation. Similar to LC-MS/MS, specimen measurement in Tac immunoassay is performed after a pretreatment step, such as in the ARCHITECT i2000SR system (Abbott Diagnostics), which uses methanol/zinc sulfate to precipitate protein and to extract Tac from a whole blood specimen preserved with ethylenediaminetetraacetic acid (EDTA). The chemiluminescent microparticle immunoassay of the ARCHITECT i200SR system uses anti-Tac coated paramagnetic microparticles and an acridinium-tacrolimus tracer. Similar systems include the COBAS MIRA (Roche Diagnostics) and the Dimension RxL (Siemens Healthcare Dianostics), which use enzyme-multiplied immunoassay and antibody- conjugated magnetic immunoassay technologies, respectively.¹⁶

Antibodies used in immunoassay procedures are well known to demonstrate crossreactivity with a variety of metabolites, which results in an overestimation of drug concentration.⁶ Advances in immunoassay measurement involve automated specimen pretreatment, enhanced reagent stabilities to lower potential matrix effect, and new anti-Tac antibodies that provide more sensitivity and affinity to the target drug.¹² Immunoassay continues to be used in many laboratories across the country because of its ease of use and lower costs associated with services. Many laboratories find this option appealing because it does not involve a high level of technical skill from staff; the equipment can be leased; and the manufacturer often provides training, support, and maintenance for these systems.¹¹ As cited in Gounden and Soldin,⁶ the College of American Pathologists (CAP) noted vast imprecision amongst immunoassays in a longitudinal replicate study of immunosuppressive drugs, accounting for a total of 77% to 90% of total variance. The authors attributed this finding to interlaboratory factors, namely, variance amongst different lot numbers.

LC/MS-MS has been noted in previous literature to be an optimal method for evaluating Tac concentrations because of its high specificity.¹¹ However, to perform this type of testing, a high degree of technical ability and extensive training is required from staff members. This method also requires high upfront costs and full validation for use, often independent of approval from the United States Food and Drug Administration (FDA): the laboratory director usually validates these methods within the laboratory. In a study conducted by Agrawal et al¹⁷ of 839 whole blood specimens, a 40% discordance rate between Tac concentrations measured by LC-MS/MS and immunoassay was observed. The researchers stated that the rate of discord falls at 1.7 Sigma, a factor below the minimum Sigma threshold, which indicates that the process is inefficient; they believe that laboratory and medical professionals should view such a rate as unacceptable. Consequently, the authors strongly suggested the need for assay standardization.

Conclusion

Tac detection can be accomplished through a variety of techniques, most commonly LC-MS/MS or immunoassay. Ensuring that these systems are fully validated, well maintained, and properly operated is important for accurate measurements of immunosuppressive drugs. The consequences of poorly monitored immunosuppressive therapy can yield serious adverse effects for patients. Nevertheless, Tac has been successfully indicated in the prevention of GVHD and continues to be a highly prescribed medication for use after allogenic organ and tissue transplantation.

Personal and Financial Conflicts of Interest

None reported.

Abbreviations

 
• Tac

  tacrolimus

 
• NFAT

  nuclear factor of activated T-cells

 
• IL-2

  interleukin-2

 
• GVHD

  graft-vs-host disease

 
• APCs

  antigen-presenting cells

 
• NKC

  natural killer cell

 
• LC-MS/MS

  liquid chromatography–tandem mass spectrometry

 
• EMIT

  enzyme-multiplied immunoassay

 
• ELISA

  enzyme-linked immunoabsorbant assay

 
• ACMIA

  antibody-conjugated magnetic immunoassay

 
• ECLIA

  electrochemiluminescence immunoassay

 
• CEDIA

  cloned enzyme donor immunoassay

 
• QMS

  quantitative microsphere systems

 
• FPIA

  fluorescence polarization immunoassay

 
• IATDMCT

  International Association for Therapeutic Drug Monitoring and Clinical Toxicology

 
• LC-UV

  liquid chromatography with ultraviolet detection

 
• DBS

  dried-blood specimens

 
• PBMCs

  peripheral-blood mononuclear cells

 
• EDTA

  ethylenediaminetetraacetic acid

 
• CAP

  College of American Pathologists

 
• FDA

  United States Food and Drug Administration