Calcineurin inhibitors

The chemical structures of CsA and tacrolimus, calcineurin inhibitors commonly used in organ transplantation, are shown in Fig. 1. The calcineurin inhibitors block the activation and proliferation of CD4+ and CD8+ T lymphocytes by inhibiting IL-2 production (21,22). Under normal circumstances, binding of major histo compatibility complex-peptide complexes to T-cell receptors results in the formation of an activated form of the calcium/calmodulin-dependent serine/threonine phosphatase calcineurin. This leads to de-phosphorylation of the nuclear factor of activated T cells (NF-AT) (among others) and nuclear translocation of NF-AT. Once in the nucleus, NF-AT binds genes encoding pro-inflammatory cytokines such as IL-2, resulting in up-regulated gene transcription (23). CsA and tacrolimus freely cross lymphocyte membranes and form complexes with specific cytoplasmic binding proteins called

Cyclosporins Tacrolimus

Fig. 1. Chemical structures of the calcineurin inhibitors, cyclosporine (CsA) and tacrolimus. This figure was published in Pharmacology & Therapeutics, Volume 112, Masuda S, Inui KI, an up-date review on individualized dosage adjustment of calcineurin inhibitors in organ transplant patients, page 186, Copyright Elsevier 2006.

Cyclosporins Tacrolimus

Fig. 1. Chemical structures of the calcineurin inhibitors, cyclosporine (CsA) and tacrolimus. This figure was published in Pharmacology & Therapeutics, Volume 112, Masuda S, Inui KI, an up-date review on individualized dosage adjustment of calcineurin inhibitors in organ transplant patients, page 186, Copyright Elsevier 2006.

immunophilins. CsA binds to the immunophilin cyclophilin and tacrolimus binds to the immunophilin FK506-binding protein-12 (24,25). The drug-immunophilin complexes inhibit calcineurin activity, which prevents nuclear translocation of NF-AT. The end result is down-regulated cytokine gene transcription (26-28).

3.1. Cyclosporine

CsA is a small cyclic polypeptide (molecular weight of 1204) that was originally isolated from fungal cultures of Tolypocladium inflatum Gams in 1970 (29). It is currently approved in the USA as an immunosuppressive drug to prolong organ and patient survival in kidney, liver, heart and bone marrow transplants. CsA is available for both oral and intravenous administration (Sandimmune). A microemulsion formulation of CsA, called Neoral, exhibiting more reproducible absorption characteristics is also available for oral administration (30). In addition, several generic microemulsion formulations are now available and are often referred to as CsA modified (31,32).

3.1.1. Pharmacokinetics

Oral absorption of Sandimmune is low (5-30%) and highly variable, ranging from 4 to 89% in renal and liver transplant patients (33,34). Absorption of the microemulsion formulation is more consistent, averaging approximately 40% (35). Peak blood concentrations typically occur between 1-3 and 2-6 h following oral administration of Neoral and Sandimmune, respectively (33,36,37). Absorption can be delayed for several hours in a subgroup of patients. Because CsA is lipophilic, it crosses most biologic membranes and has a wide tissue distribution (38). CsA is highly bound to plasma proteins (>90% to lipoproteins), with the majority of CsA localizing in erythrocytes. The distribution of CsA between plasma and erythrocytes is temperature-dependent and varies with changes in hematocrit (39). Because of the potential for artifactural redistribution of CsA during specimen processing because of ambient temperature fluctuations, ethylenediaminetetraacetic acid (EDTA)-anticoagulated whole blood should be used to measure CsA concentrations (40-42).

CsA is extensively metabolized by cytochrome P450 enzymes (CYP3A isoenzymes) located in the small intestine and liver (43). There is also a cellular transporter of immuno-suppressive drugs, called P-glycoprotein, that influences metabolism by regulating CsA bioavailability. P-glycoprotein pumps some of the CsA out of enterocytes back into the lumen of the gut (44,45). This efflux pump probably contributes to the poor absorption rates observed after oral administration of CsA. CYP3A isoenzymes and P-glycoprotein genetic polymorphisms can also influence the oral bioavailability of CsA and are probably involved in the delayed absorption that has been noted in a subset of patients (44). CsA is oxidized or N-demethylated to more than 30 metabolites (46,47). Most of the metabolites do not possess immunosuppressive activity and are not clinically significant (48). However, there is growing evidence to indicate that a few of the inactive metabolites may contribute to CsA toxicity (48). Two of the hydroxylated metabolites, AM1 and AM9, exhibit 10-20% of the immunosuppressive activity of the parent compound (49,50) and can account for as much as 33% of the whole blood CsA concentration (51). The major route of CsA elimination is biliary excretion into the feces. As expected, dosage adjustments are necessary in patients with hepatic dysfunction. Only a small fraction (6%) of CsA and metabolites appear in the urine (36), making dosage adjustments unnecessary in patients with renal insufficiency.

3.1.2. Adverse Effects

Serious side effects related to CsA treatment are concentration-dependent and include nephrotoxicity, neurotoxicity, hepatotoxicity, hirsutism, hypertrichosis, gingival hypertrophy, glucose intolerance, hypertension, hyperlipidemia, hypomag-nesemia, hyperuricemia, and hypokalemia. In general, over-suppression leads to an increased risk for viral infections and lymphoproliferative disease, especially in children (52).

3.1.3. Drug Interactions

Numerous drugs influence the absorption and metabolism of CsA. Any drug that inhibits the cytochrome P-450 system or the P-glycoprotein efflux pump increases blood CsA concentrations because of increased absorption and decreased metabolism. Drugs having the opposite effect (P-450 and/or P-glycoprotein inducers) produce decreased CsA concentrations. Drugs causing increased CsA blood concentrations include calcium channel blockers, several antifungal agents, and the antibiotic erythromycin. Several anticonvulsants and antibiotics, including antituberculosis agents, reduce blood CsA concentrations. In addition, there are many other drugs that synergize with CsA and potentiate nephrotoxicity. There are several excellent reviews that discuss specific drug interactions with CsA (53,54). Not all of the interactions are caused by pharmaceuticals as various foods and herbal remedies can influence CsA concentrations. For instance, grapefruit juice increases CsA blood concentrations by increasing absorption whereas St John's wort decreases CsA concentrations by increasing metabolism (55).

3.1.4. Preanalytic Variables

Whole blood anticoagulated with EDTA is the recommended sample type based on numerous consensus documents (40-42). CsA in EDTA whole blood is stable at least 11 days at room temperature or higher temperatures (37°C) (56). For long-term storage, whole blood samples should be placed at —20°C and are stable for at least 3 years (57). As previously mentioned, CsA should only be measured in whole blood samples. Plasma is considered generally not acceptable because partitioning of CsA between plasma and erythrocytes is a temperature- and time-dependent process that can be altered during in vitro specimen processing (41). In addition, plasma CsA concentrations are twofold lower than whole blood concentrations and results in poor analytical precision at low plasma CsA concentrations.

The timing of specimen collection has always been right before administration of the next dose (i.e., trough levels) (40,41). For standardization purposes, the timing should be within 1 h before the next dose (42). However, the introduction of Neoral in 1995, a microemulsion CsA formulation with more predictable absorption kinetics, has resulted in higher peak concentrations and increased drug exposure, based on area under the concentration time curves (58). The highest and most variable CsA concentrations typically occur within the first 4h after Neoral dosing (59). However, similar trough concentrations are observed for both the conventional and the microemulsion CsA formulations, demonstrating that trough concentrations are not predictive of total drug exposure (60)-(62). Increased exposure to CsA using Neoral results in decreased rejection rates with slightly higher serum creatinine concentrations compared with conventional CsA therapy (58,63,64). Thus, a better predictor of immunosuppressive efficacy was needed when administering Neoral. Pharmacokinetic and pharmacodynamic studies demonstrated that maximal inhibition of calcineurin and IL-2 production was correlated with the highest CsA concentrations 1-2h after dosing (59,65), indicating that drug levels shortly after dosing may be a better predictor of total drug exposure and clinical outcome (66). Because multiple time points after dosing are not practical in a clinical setting, different time points were examined and CsA concentrations 2 h after dosing (called C2 monitoring) was shown to correlate best with total drug exposure and result in better clinical outcomes (67-70). These findings have resulted in C2 monitoring of CsA becoming standard practice at many transplant centers. Unfortunately, this creates various nursing/ phlebotomy challenges because blood samples have to be drawn very close to the 2-h time point after dosing, ideally 10min on either side of the 2-h mark (71). At the author's institution, C2 testing is performed on 16% of all whole blood samples (annual volume ~ 14,000) received in the laboratory for CsA testing. To avoid confusion and prevent testing delays because of the need for sample dilution of C2 specimens, our laboratory has created a separate test for C2 monitoring and reports all CsA C2 results in ^g/mL to avoid mis-interpreting C2 results as tough levels. We still report CsA trough results in ng/mL.

3.1.5. Methods of Analysis

Monitoring of CsA is critical for optimizing immunosuppression and organ survival while minimizing unwanted toxic side effects. Improvements in immunosuppressive regimens, along with demands for narrower and tighter control of CsA blood levels, have placed greater demand on clinical laboratories to provide timely and reliable drug concentrations. There are many methods currently available to measure CsA. Factors that need to be considered when selecting a CsA assay include metabolite cross-reactivity, cost of instrumentation and reagents, ease of operation, level of technical expertise required to perform testing, test volume, expected turnaround times, the current method being used when switching methods, and the history/preferences of the transplant physicians. For example, turnaround times can be a critical issue in an outpatient setting when it is desirable to have CsA test results available when patients are being seen by their physicians. Depending on the institution, this may require 2-4 h turnaround times for anywhere from 10 to 50 specimens that have been drawn a few hours before the scheduled clinic visit.

CsA can be measured by radioimmunoassay (RIA), semi-automated and automated non-isotopic immunoassays, and high-performance liquid chromatography (HPLC) with UV (HPLC-UV) or mass spectrometry detection systems (HPLC-MS). There are four companies manufacturing six different CsA assays currently being used in the USA. Assays for CsA and the percentage of laboratories using each method based on the College of American Pathologists Immunosuppressive Drugs Monitoring 1st Survey of 2006 are summarized in Table 3. The Cyclo-Trac SP RIA by Diasorin (Still water, MN, USA) is the least popular and is used by only 1% of all laboratories, most likely because of the manual format and need to handle radioisotopes. Interestingly, the Abbott monoclonal fluorescence polarization immunoassay (FPIA) (Abbott Park, IL, USA) is used by >70% of all laboratories. This is somewhat surprising because the

Table 3

Currently Used Methods to Measure Cyclosporine (CsA)

Table 3

Currently Used Methods to Measure Cyclosporine (CsA)

Method

Assay

Manufacturer

Laboratories Using Assay (%f

Radioimmunoassay

Cyclo-Trac SP

DiaSorin

1

Immunoassay

Semi-automated

Polyclonal FPIA

Abbott

2

Monoclonal FPIA

Abbott

71

CEDIA PLUS

Microgenics

8

Syva EMIT 2000

Dade-Behring

5

Automated

Dimension ACMIA

Dade-Behring

5

HPLC-UV

2

HPLC-MS

6

FPIA, fluorescence polarization immunoassay; CEDIA, cloned enzyme donor immunoassay; EMIT, enzyme-multiplied immunoassay technique; ACMIA, antibody-conjugated magnetic immunoassay; HPLC-UV, high-performance liquid chromatography with ultraviolet detection; HPLC-MS, highperformance liquid chromatography with mass spectrometry detection.

a Percentages are based on the College of American Pathologists Immunosuppressive Drug Monitoring 1st survey of 2006.

FPIA, fluorescence polarization immunoassay; CEDIA, cloned enzyme donor immunoassay; EMIT, enzyme-multiplied immunoassay technique; ACMIA, antibody-conjugated magnetic immunoassay; HPLC-UV, high-performance liquid chromatography with ultraviolet detection; HPLC-MS, highperformance liquid chromatography with mass spectrometry detection.

a Percentages are based on the College of American Pathologists Immunosuppressive Drug Monitoring 1st survey of 2006.

Abbott monoclonal FPIA has considerable cross-reactivity with CsA metabolites, and recommendations by numerous consensus panels specify that the analytical method should be specific for parent compound (40-42). HPLC methods to measure CsA are specific for parent compound and, because of this, are considered the "gold standard" for CsA quantitation. Yet, HPLC methods are used by only 8% of all laboratories and are primarily restricted to larger transplant centers. The lack of widespread acceptance of HPLC methods to measure CsA may reflect high initial equipment costs for MS detection systems and the need for specialized training for test performance. HPLC systems with UV detection are considerably less expensive and easier to operate but can suffer from a wide variety of chemical interferences depending on the specific protocol utilized. There are several excellent protocols to measure CsA using HPLC-MS and HPLC-MS/MS systems (72,73). Because sample requirements are the same for analysis of many of the immunosuppressants (CsA, tacrolimus, sirolimus, everolimus), simultaneous measurement of two or more immunosuppressive drugs in a single specimen can be performed using HPLC-MS (74). As therapeutic drug monitoring applications continue to emerge, the use of HPLC-MS will continue to increase and may become commonplace equipment in clinical laboratories in the not too distant future.

All the immunoassays, with the exception of the Dimension antibody conjugated magnetic immunoassay (ACMIA) (Dade Behring, Dearfield, IL, USA), are semi-automated because they require a whole blood pretreatment step. This typically involves preparing a whole blood hemolysate by adding an extraction reagent such as methanol to an aliquot of whole blood. The hemolysate is then centrifuged and the separated supernatant is analyzed by the FPIA or Syva enzyme-multiplied immunoassay (EMIT) (Dade Behring). The cloned enzyme donor immunoassay (CEDIA) PLUS (Microgenics Comp., Fremont, CA, USA) pretreatment step is simpler because a centrifugation step is not required after addition of the extraction reagent. Bayer (Bayer Health care, Tarrytown, NY, USA) has also developed a CsA assay with a simplified pretreatment

Table 4

Instrument Applications for Cyclosporine (CsA) Immunoassays

Table 4

Instrument Applications for Cyclosporine (CsA) Immunoassays

Immunoassay

Instrument Application

Manufacturer

Monoclonal FPIA

TDx, AxSYM

Abbott Laboratories

SYNCHRON LX, UniCel Dx Hitachi 902, 911, 912, 917, Modular P AU 400, 640, 2700, 5400 Aeroset

Microgenics Corp. Beckman Coulter Roche Diagnostics Olympus America Abbott Laboratories

Syva EMIT 2000

COBAS Miraa, INTEGRA 400, 800 Dimensión RxL Max, Xpand, Xpand Plus, V-Twin, Viva, Viva-E

Roche Diagnostics Dade-Behring

Dimension ACMIA

Dimensión RxL Max, Xpand, Xpand Plus, V-twin,Viva, Viva-E

Dade-Behring

FPIA, fluorescence polarization immunoassay; CEDIA, cloned enzyme donor immunoassay; EMIT, enzyme-multiplied immunoassay technique; ACMIA, antibody-conjugated magnetic immunoassay. a This instrument is no longer manufactured or supported by the company.

FPIA, fluorescence polarization immunoassay; CEDIA, cloned enzyme donor immunoassay; EMIT, enzyme-multiplied immunoassay technique; ACMIA, antibody-conjugated magnetic immunoassay. a This instrument is no longer manufactured or supported by the company.

step that is pending FDA approval for use on the ADVIA Centaur (75). The Dimension ACMIA does not require a pretreatment step allowing whole blood samples to be placed directly on the instrument. Instruments that currently have applications for the various CsA immunoassays are provided in Table 4.

3.1.6. Metabolite Cross-Reactivity

The Abbott polyclonal antibody-based FPIA is non-specific and has extensive cross-reactivity with CsA metabolites. The use of this assay has been declining over the years, and only about 2% of all laboratories currently use this assay (Table 3). CsA results using the Abbott polyclonal FPIA are approximately four times higher than those obtained using HPLC methods (76). Because of the magnitude of metabolite

Table 5

Cyclosporine (CsA) Metabolite Cross-Reactivity of Immunoassays

Percentage CsA Metabolite Cross-Reactivitya

Table 5

Cyclosporine (CsA) Metabolite Cross-Reactivity of Immunoassays

Percentage CsA Metabolite Cross-Reactivitya

Immunoassay

AMI

AM4n

AM9

AM19

Monoclonal FPIA

6-12

<6

14-27

<4

CEDIA PLUS

8

3G

18

2

Syva EMIT 2000

<5

8-13

<4

G

Dimension ACMIA

G

4

G

G

FPIA, fluorescence polarization immunoassay; CEDIA, cloned enzyme donor immunoassay; EMIT, enzyme-multiplied immunoassay technique; ACMIA, antibody-conjugated magnetic immunoassay.

a Each metabolite was evaluated at 1000 |xg/L except AMI, which was tested at 500 |xg/L in the CEDIA PLUS assay. Data are derived from references 77-81.

FPIA, fluorescence polarization immunoassay; CEDIA, cloned enzyme donor immunoassay; EMIT, enzyme-multiplied immunoassay technique; ACMIA, antibody-conjugated magnetic immunoassay.

a Each metabolite was evaluated at 1000 |xg/L except AMI, which was tested at 500 |xg/L in the CEDIA PLUS assay. Data are derived from references 77-81.

cross-reactivity and the poor correlation with clinical outcomes and toxicity, the use of this polyclonal assay should be discouraged. Cross-reactivity of the monoclonal immunoassays with CsA metabolites is summarized in Table 5. The Dimension ACMIA has the least overall metabolite cross-reactivity whereas the monoclonal CEDIA PLUS is reported to have the highest overall metabolite cross-reactivity. CsA metabolites, AMI and AM9, are typically present in the highest concentrations after transplantation (51) and cross-reacts the least in the Dimension ACMIA and Syva EMIT, and the most in the monoclonal FPIA (Table 5). The magnitude of metabolite cross-reactivity contributes to the degree of CsA overestimation when comparing immunoassays with HPLC. Mean CsA concentrations have been found to be approximately 12, 13, 17, 22, and 40% higher than HPLC when measured by the Dimension ACMIA, Syva EMIT, CEDIA PLUS, FPIA on the TDx, and FPIA on the AxSYM, respectively (77-81). Thus, it is important to consider metabolite cross-reactivity and the degree of CsA overestimation when selecting the "right" CsA immunoassay to support a solid organ transplant program.

3.1.7. Analytical Considerations

Consensus conference recommendations for CsA immunoassays are that the slope of the line should be 1.0 ± 0.1, with a y-intercept and Sy/x < 15 ^g/L, when compared with HPLC (41). None of the current immunoassays satisfy all these requirements (76-81). For instance, the Dimension ACMIA satisfies the slope and intercept requirements but exceeds the Sy/x limit, whereas the CEDIA PLUS and Syva EMIT satisfies only one requirement. The FPIA fails to satisfy any of the requirements. Between-day precision recommendations require a coefficient of variation (CV) of <10% at a CsA concentration of 50^g/L and a CV of <5% at 300^g/L (41,42). Most of the immunoassays satisfy the precision recommendation at 300 ^g/L, but it is important that each laboratory determine between-day precision studies at CsA concentrations around 50 ^g/L. This is particularly important because recent immunosuppressive drug regimens are designed to reduce CsA trough concentrations to minimize toxicity. Another potential problem is bias because of incorrect assay calibration. Results from the 2003 International Proficiency Testing Scheme have shown that the FPIA using the TDx and CEDIA PLUS overestimates CsA concentrations by 5-10%, whereas the Syva EMIT and Dimension ACMIA slightly underestimate target CsA concentrations by <5% (82). Lastly, for assays involving a manual extraction step, poor technique can significantly contribute to the overall imprecision of the assay. Careful attention to detail and good technique can minimize variations at this important preanalytical step. This holds true for all whole blood immunosuppressive drug assays requiring a manual extraction step (tacrolimus, sirolimus, and everolimus).

3.1.8. c2 Monitoring and Specimen Dilution

Therapeutic ranges for CsA are often organ-specific and can vary widely between transplant centers. They also differ based on various immunosuppressive drug combinations, the time after transplant, and during periods of toxicity and organ rejection. Trough whole blood CsA levels following kidney transplants are typically between 150-250 ^g/L shortly after transplant and are tapered down to <150 ^g/L during maintenance therapy. Recommended levels after liver and heart transplants are

250-350 ^g/L shortly after transplant and <150 ^g/L during maintenance therapy. These target ranges were determined using HPLC and will vary considerably when measured using immunoassay, depending on the amount of metabolite cross-reactivity.

For C2 monitoring, target concentrations vary between 600 and 1700 ^g/L depending on the type of graft and the time after transplantation (66). C2 concentrations often exceed the analytical range of most immunoassays because typical calibration curves are designed to measure trough CsA levels. The FPIA and Syva EMIT have analytical ranges up to 1500 and 500 ^g/L, respectively. The CEDIA PLUS and Dimension ACMIA have separate calibration curves for C2 monitoring, with an analytical range from 450 to 2000 and 350 to 2000 ^g/L, respectively. However, 28% of laboratories using the CEDIA PLUS reported using only the low-range calibration curve and would have to dilute samples above 450 ^g/L (83). Sample dilution can lead to major inaccuracies in test results, and dilution protocols need to be carefully validated before implementation (83,84). This is because CsA metabolites may not dilute in a linear fashion, and there may be differences in the amount of time needed for diluted samples to re-equilibrate, depending on the immunoassay and dilution protocol. Proficiency testing programs have demonstrated that laboratories produce widely varying results when challenged with samples with CsA concentrations outside the analytical range of immunoassays. For instance, at a CsA parent concentration of 2000 ^g/L, 125 laboratories participating in the survey reported CsA values ranging from 1082 to 3862 ^g/L (84). These findings indicate that laboratories need to develop carefully controlled validated dilution protocols. A validated dilution protocol for the monoclonal FPIA on the TDx has recently been described (85).

Another concern with C2 monitoring is metabolite concentrations and the need for therapeutic ranges that are assay-specific. This clearly is necessary when measuring trough CsA concentrations. A recent study monitoring C2 concentrations in kidney and liver transplant patients found equivalent CsA results when measured using the FPIA, CEDIA PLUS, and Syva EMIT (86). As expected, paired trough samples produced CsA concentrations that differed among the immunoassays. These data indicate that for C2 monitoring, assay-specific therapeutic ranges may not be necessary.

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