Tacrolimus

Tacrolimus (also known as FK-506) is a macrolide antibiotic with a molecular weight of 822 (Fig. 1) that was originally isolated from the fungus Streptomyces tsukubaensis (5). In the USA, tacrolimus (brand name Prograf) was approved for use in liver transplantation in 1994 and in kidney transplantation in 1997. It is approximately 100 times more potent than CsA and is associated with a decrease in acute and chronic rejection, and better long-term graft survival (87). In 2004, more than two-thirds of all kidney and liver transplant recipients, and approximately one-half of all heart transplant recipients, were receiving tacrolimus before hospital discharge (20). At the author's institution, approximately 3.5 times more tacrolimus tests are performed compared with CsA.

3.2.1. Pharmacokinetics

Tacrolimus is available for both oral and intravenous administration. Similar to CsA, oral absorption of tacrolimus from the gut is poor and highly variable, averaging

25% (88). Peak blood concentrations occur within 1.5-4h. Tacrolimus is primarily bound to albumin, aracid glycoprotein, and lipoproteins in the plasma. However, the majority of tacrolimus is found within erythrocytes (89).

Tacrolimus is metabolized using cytochrome P450 isoenzymes (CYP3A) located in the small intestine and liver. Similar to CsA, the bioavailability of tacrolimus is influenced by CYP3A and the multidrug efflux pump (P-glycoprotein) located in intestinal enterocytes. Biotransformation of tacrolimus occurs by demethylation, hydroxylation, and oxidative reactions (90). At least nine metabolites have been identified based on in vitro studies (91), and all, with the exception of 31-o-demethyl tacrolimus (M-II), have very little immunosuppressive activity. M-II has been shown in vitro to have the same immunosuppressive activity as parent compound (92). Metabolites represent 10-20% of whole blood tacrolimus concentrations (93). Tacrolimus is eliminated primarily by biliary excretion into the feces. Patients with hepatic dysfunction require dosage adjustments. Very little tacrolimus is found in urine, and blood concentrations are not altered in renal dysfunction.

3.2.2. Adverse Effects

Tacrolimus shares many dose-dependent side effects with CsA (94). These include nephrotoxicity, neurotoxicity, hepatotoxicity, hypertension, and glucose intolerance. Nephrotoxicity with tacrolimus may be less of a problem than with CsA, especially in renal transplantation (95). Diabetogenesis is approximately three times more common with tacrolimus than with CsA (96). Hyperkalemia, hyperuricemia, hyperlipidemia, hirsutism, and gingival hypertrophy are also observed following tacrolimus use, but less commonly than with CsA (97). Alopecia is also associated with tacrolimus use (94).

3.2.3. Drug Interactions

Because tacrolimus is metabolized mainly by the cytochrome P450 system, the majority of drug interactions described for CsA also apply to tacrolimus (88). St John's wort also decreases blood tacrolimus concentrations.

3.2.4. Preanalytic Variables

For quantitation of tacrolimus, EDTA-anticoagulated whole blood is the specimen of choice for the same reasons provided for CsA. Whole blood samples are stable for 1 week when shipped by mail without coolant (98,99), 1-2 weeks at room temperature (99,100), 2 weeks at refrigerator temperatures (100), and almost 1 year at —70°C (100).

Trough blood tacrolimus concentrations are almost exclusively used for routine monitoring and are believed to be a good indicator of total drug exposure (101). However, recent experience with CsA has challenged this notion, and alternative draw times 1-6 h after dosing have been proposed (102). Whereas some investigators have found a poor correlation between trough tacrolimus concentrations and total drug exposure, others have found good correlation (103,104). Overall, the findings suggest that trough tacrolimus concentrations are predictive of total drug exposure and that measuring tacrolimus at specified times after dosing may not result in dramatic improvements. Until this issue is fully resolved, trough levels will continue to be used for reasons of convenience and reproducibility.

3.2.5. Methods of Analysis

Monitoring of tacrolimus is an integral part of any organ transplant program because of variable dose-to-blood concentrations and the narrow therapeutic index. Tacrolimus can be measured using enzyme-linked immunosorbent assay (ELISA), semi-automated and automated immunoassay, and HPLC-MS (Table 6). The ELISA and semi-automated immunoassays require a manual whole blood pre-treatment step. The Dimension ACMIA does not require a pretreatment step allowing whole blood samples to be directly placed on the instrument. Sample extraction can be semi-automated using modern HPLC-MS systems (105).

The ELISA takes about 4h to complete, requires numerous manual steps, and is used by few clinical laboratories. The Abbott microparticle enzyme immunoassay (MEIA) II on the IMx instrument is currently used by 88% of the laboratories in the USA that participate in the College of American Pathologists immunosuppressive proficiency testing program (Table 6). The MEIA II has a reported detection limit of 2 ^g/L and replaced an earlier version (MEIA I) with a detection limit of 5 ^g/L. The tacrolimus Syva EMIT has applications for Dade Behring instrumentation, the COBAS Integra 400 (106), the Beckman Synchron LX20 PRO (107), and the Bayer ADVIA 1650 (108). However, the Syva EMIT is currently available only outside the USA. Microgenics has just released a CEDIA for tacrolimus in the USA that has applications for several Hitachi, Olympus, and Beckman instruments. Dade-Behring has just launched (July 2006) an ACMIA to measure tacrolimus using the Dimension family of analyzers and the V-Twin and Viva-E drug-testing analyzers. It uses the same monoclonal antibody used in the Syva EMIT to measure tacrolimus. Lastly, Abbott is developing a chemiluminescent immunoassay for use on their ARCHITECH system (109).

Table 6

Analytical Methods to Measure Tacrolimus

Table 6

Analytical Methods to Measure Tacrolimus

Method

Assay

Manufacturer

Laboratories Using Method (%)a

ELISA

Pro-Trac II

DiaSorin

< 3

Immunoassay

Semi-Automated

MEIA II

Abbott

88

Syva EMIT

Dade-Behring

_b

CEDIA

Microgenics

< 3

Automated

Dimension ACMIA

Dade-Behring

c

HPLC-MS

9

ELISA, enzyme-linked immunosorbent assay; MEIA, microparticle enzyme immunoassay; EMIT, enzyme-multiplied immunoassay technique; CEDIA, cloned enzyme donor immunoassay; ACMIA, antibody-conjugated magnetic immunoassay; HPLC-MS, high-performance liquid chromatography with mass spectrometry detection.

a Percentages are based on the College of American Pathologists Immunosuppressive Drugs Monitoring Survey of 2006.

b Currently available only outside the USA.

c This assay received Food and Drug Administration (FDA) clearance and was launched in July 2006.

ELISA, enzyme-linked immunosorbent assay; MEIA, microparticle enzyme immunoassay; EMIT, enzyme-multiplied immunoassay technique; CEDIA, cloned enzyme donor immunoassay; ACMIA, antibody-conjugated magnetic immunoassay; HPLC-MS, high-performance liquid chromatography with mass spectrometry detection.

a Percentages are based on the College of American Pathologists Immunosuppressive Drugs Monitoring Survey of 2006.

b Currently available only outside the USA.

c This assay received Food and Drug Administration (FDA) clearance and was launched in July 2006.

HPLC-MS methods are used by most of the laboratories not using the MEIA II. Tacrolimus cannot be measured by HPLC-UV because the molecule does not possess a chromophore. It is noteworthy that HPLC-MS is the only method that is specific for parent drug and meets the recommendations set forth in Consensus documents (42). There are numerous recently reported assays to quantitate tacrolimus by using HPLC-MS or HPLC-MS/MS with detection limits <0.5ng/mL (105,110). A major advantage of HPLC-MS over immunoassays is the ability to simultaneously measure other immunosuppressant drugs in the same whole blood sample, such as CsA, sirolimus, and everolimus (111).

3.2.6. Metabolite Cross-Reactivity

All the immunoassays have significant cross-reactivities with tacrolimus metabolites. The ELISA, MEIA II, and EMIT cross-react with M-II (31-o-demethyl), M-III (15-o-demethyl) and M-V (15,13-di-o-demethyl) metabolites of tacrolimus (112). The CEDIA has significant cross-reactivity with M-I (13-o-demethyl) but does not cross-react with M-II or M-III. Cross-reactivity of the CEDIA with M-V has not been examined (113). The ACMIA is expected to have metabolite cross-reactivity similar to the EMIT because both assays use the same monoclonal antibody. The extent of positive bias because of metabolite cross-reactivity is dependent on the transplant group studied. Metabolite cross-reactivity in patients with good liver function is typically not a problem because metabolite concentrations are relatively low compared with parent drug (114). However, metabolites tend to accumulate during reduced liver function and immediately after liver transplant, resulting in significant assay interference and falsely high blood tacrolimus concentrations (115). Overall, the MEIA II produces tacrolimus results that are 15-20% higher, the EMIT produces results 17% higher, and the CEDIA produces results 19% higher than those obtained by HPLC-MS, in kidney and liver transplant patients (107,112,113,116,117). Calibration error may also contribute to some of the overall positive bias.

3.2.7. Analytical Considerations

The recommended therapeutic range for whole blood tacrolimus concentrations after kidney and liver allograft transplants is 5-20 ^g/L when measured using HPLC-MS (118). When tacrolimus is used with other immunosuppressive agents such as sirolimus, the desired target concentration for tacrolimus can be considerably <5 ^g/L. In view of this, it is important for each laboratory to determine performance characteristics of their tacrolimus assay at concentrations <5 ^g/L and make transplant services aware of the lower limit of detection and the imprecision (%CV) at this concentration. The functional sensitivity (between-day CV <20%) of the MEIA II and CEDIA is reported to be around 2 ^g/L (112,116,119,120), whereas the detection limit of the EMIT is around 3 ^g/L (107). At our institution, we examined functional sensitivity of the MEIA II tacrolimus assay by measuring whole blood pools at various concentrations in duplicate during a 10-day period. As shown in Fig. 2, a 20% CV was observed at a tacrolimus concentration of approximately 2 ^g/L. In addition, we found that the MEIA II produced tacrolimus concentrations ranging from 0.8 to 1.7 ^g/L when testing samples from patients not receiving tacrolimus (n = 8). Homma et al. (121) also found false-positive results when measuring tacrolimus in whole blood samples

Tacrolimus (jjg/L)

Fig. 2. Functional sensitivity of the Abbott tacrolimus microparticle enzyme immunoassay (MEIA) II on the IMx instrument. Whole blood patient pools at varying tacrolimus concentrations were analyzed in duplicate on 10 separate days. The coefficient of variation (CV) is the standard deviation of the mean tacrolimus concentration divided by the mean. The value is multiplied by 100 and is expressed as a percentage (%).

Tacrolimus (jjg/L)

Fig. 2. Functional sensitivity of the Abbott tacrolimus microparticle enzyme immunoassay (MEIA) II on the IMx instrument. Whole blood patient pools at varying tacrolimus concentrations were analyzed in duplicate on 10 separate days. The coefficient of variation (CV) is the standard deviation of the mean tacrolimus concentration divided by the mean. The value is multiplied by 100 and is expressed as a percentage (%).

from patients not receiving tacrolimus using the MEIA. Based on our data, we use a cutoff of 2 ^g/L for tacrolimus and report values lower than this cutoff as <2 ^g/L.

The MEIA II has been shown to produce falsely elevated tacrolimus concentrations when the hematocrit is <25% (122,123). The EMIT for tacrolimus is not affected by changes in hematocrit values (123). Hematocrit bias in the MEIA II could result in therapeutic tacrolimus blood concentrations in under-immunosuppressed patients because of low hematocrit values. This would potentially be most problematic shortly after transplant when hematocrit values are typically at their lowest concentrations. This tacrolimus bias could also make it difficult to appropriately dose patients with widely fluctuating hematocrit values.

The reliability of the MEIA II at low whole blood tacrolimus concentrations has recently been questioned. At tacrolimus concentrations <9 ^g/L, the MEIA II exhibited greater between-day imprecision and a weaker correlation with results obtained by HPLC-MS/MS (124). Recovery experiments also demonstrated that the degree of over-estimation of tacrolimus using the MEIA II was more pronounced at lower drug concentrations (124). Poor precision at low tacrolimus concentrations was also noted in the College of American Pathologists longitudinal immunosuppressive drug study. The study found that the major source of imprecision was within-laboratory variation over time, and it was postulated that the variation might be due to changes in assay standardization or reagent lot-to-lot changes (125). Taken together, these performance variables are important to consider when selecting an assay to monitor whole blood tacrolimus concentrations.

Hair Loss Prevention

Hair Loss Prevention

The best start to preventing hair loss is understanding the basics of hair what it is, how it grows, what system malfunctions can cause it to stop growing. And this ebook will cover the bases for you. Note that the contents here are not presented from a medical practitioner, and that any and all dietary and medical planning should be made under the guidance of your own medical and health practitioners. This content only presents overviews of hair loss prevention research for educational purposes and does not replace medical advice from a professional physician.

Get My Free Ebook


Post a comment