Sirolimus

4.1.1. Pharmacokinetics

Sirolimus is available for both oral and intravenous administration. Its long half-life of approximately 60 h allows once-a-day dosing (130). Sirolimus is rapidly absorbed from the gastrointestinal tract, and peak blood concentrations occur 2 h after an oral dose (131). Oral bioavailability is low, ranging from 5 to 15% (132) and is considerably reduced (approximately fivefold) when administered within 4 h or concomitantly with CsA (133). There is considerable interpatient variability in total drug exposure that can vary by as much as 50% (133). Sirolimus is primarily found within erythrocytes (95%), with approximately 3 and 1% partitioning into the plasma and lympho-cytes/granulocytes, respectively (134). Almost all of the plasma sirolimus is bound to proteins, with lipoproteins being the major binding protein.

Similar to the calcineurin inhibitors, sirolimus is metabolized in the intestine and liver by cytochrome P450 enzymes (CYP3A) (135). The multidrug efflux pump P-glycoprotein in the gastrointestinal tract also controls metabolism by regulating bioavailability. Sirolimus is hydroxylated and demethylated to more than seven metabolites with the hydroxyl forms being the most abundant (136). Metabolites represent approximately 55% of whole blood sirolimus levels (136). The pharmacological activity of metabolites has not been fully investigated because of difficulties associated with their isolation. However, preliminary studies indicate that the immunosuppressive activity of metabolites is <30% of that observed for the parent compound (137). Sirolimus is eliminated primarily by biliary and fecal pathways, with small quantities appearing in urine (135). As with the calcineurin inhibitors, dosage adjustments are needed in patients with hepatic dysfunction.

4.1.2. Adverse Effects

The incidence of adverse effects is dose-related and includes metabolic, hemato-logical, and dermatological effects (138). Metabolic side effects include hypercholes-terolemia, hyper- and hypokalemia, hypophosphatemia, hyperlipidema, and increased liver function tests. Anemia can be problematic, with decreases in leukocyte, erythro-cytes, and platelet counts being the most common. Skin rashes, acne, and mouth ulcers are also observed in patients being switched to mTOR inhibitors. As with other immunosup-pressive drugs, there is an increased risk of infection and an association with lymphoma development. Interstitial pneumonitis is also associated with sirolimus therapy (139).

4.1.3. Drug Interactions

CYP3A inhibitors such as antifungal agents (itraconazole, ketoconazole), clarithromycin, erythromycin, and verapamil increase blood levels of sirolimus. CYP3A inducers such as carbamazepine, phenobarbital, phenytoin, and rapamycin may decrease sirolimus blood levels. Grapefruit juice can increase sirolimus by decreasing drug clearance. St John's wort can decrease sirolimus levels. As previously noted, the concomitant use of CsA can result in increased sirolimus concentrations (140). Although tacrolimus and sirolimus compete for sites on the same binding protein, the two drugs do not appear to have significant drug-drug interactions in clinical practice (104).

4.1.4. Preanalytic Variables

EDTA-anticoagulated whole blood is the recommended specimen matrix (132). This is because almost all of the sirolimus (~95%) is concentrated in erythrocytes, and plasma levels are too low for most analytical methods (134). Whole blood samples are stable for 10 days at ambient temperature (141), at least 1 week at 30-34°C (141, 142), 30 days at 4°C (143), and at least 2 months at -40°C (143). Whole blood samples can withstand three freeze-thaw cycles without altering measured sirolimus concentrations (141,142).

In contrast to the calcineurin inhibitors, there is good correlation between pre-dose sirolimus concentrations and total drug exposure based on area under the curve measurements (104,144). This also holds true when sirolimus is used in combination with CsA or tacrolimus (104,144). Thus, whole blood 24-h trough specimens are recommended when monitoring sirolimus (132).

4.1.5. Methods of Analysis

Therapeutic monitoring of sirolimus is critical because the administered dose is a poor predictor of total drug exposure because of individual patient variables. Because of the long drug half-life, daily monitoring of sirolimus is typically not necessary. Weekly monitoring of levels may be needed shortly after transplantation followed by monthly monitoring. Target concentrations for sirolimus range between 4 and 12 ^g/L when used in combination with a calcineurin inhibitor (145). Similar to tacrolimus, these relatively low whole blood concentrations can be a challenge analytically for some of the currently available methods of analysis. As combination immunosuppressant therapies continue to evolve, target concentrations for sirolimus may become lower, further challenging the analytical performance of some of the currently utilized assays.

Sirolimus can be measured by immunoassay and HPLC with UV or MS detection. According to the College of American Pathologist proficiency testing program (1st survey of 2006), more than 130 laboratories in the USA currently perform sirolimus testing. Approximately 60% of the laboratories measure whole blood sirolimus by the Abbott IMx MEIA that became commercially available in 2004. The original Abbott MEIA kit was only used experimentally to support early clinical studies (investigational use only) and was never available commercially for routine monitoring of sirolimus. The "investigational use only" Abbott immunoassay was discontinued in 2001. A CEDIA for sirolimus (Microgenics) has recently become commercially available for use on several Roche automated analyzers (Hitachi 911, 912, 917, and modular P). The Microgenics sirolimus immunoassay is currently not used by many laboratories in the USA. The majority of laboratories not using the Abbott MEIA (approximately 34%) measure sirolimus by HPLC-MS. The major advantage of HPLC-MS is increased sensitivity and specificity, despite the need for highly skilled personnel. A few laboratories measure sirolimus by HPLC-UV, although this method requires elaborate sample cleanup procedures and long chromatographic run times (146-148). This results in higher labor costs, making HPLC-UV methods unsuitable for laboratories supporting large transplant programs.

4.1.6. Metabolite Cross-Reactivity

Both of the currently available immunoassays have significant cross-reactivity with sirolimus metabolites. The MEIA method has 58 and 63% cross-reactivity with 41-o-demethyl-sirolimus and 7-o-demethyl-sirolimus, respectively (149). The CEDIA has 44% cross-reactivity with 11-hydyroxy-sirolimus and 73% cross-reactivity with 41-and 32-o-demethyl-sirolimus (150). This degree of metabolite cross-reactivity results in significant bias between assays. The MEIA produces whole-blood sirolimus concentrations that are 9-49% higher than those obtained by HPLC-UV and HPLC-MS, depending on the study and transplant group studied (149,151-155). One study found that the CEDIA method produces whole blood sirolimus levels with a mean positive bias of 20.4% compared with HPLC-MS (156). However, immunoassay metabolite cross-reactivity may be less of an issue from a clinical standpoint because the distribution of metabolites in whole blood are similar among patients and are relatively stable over long periods of time (157).

4.1.7. Analytical Considerations

The therapeutic window for sirolimus appears to be between 5 and 15 ^g/L when used in combination with CsA and between 12 and 20 ^g/L when used alone (130). Sirolimus levels slightly below the currently used therapeutic range can be a challenge for some of the HPLC-UV methods, with functional sensitivities (based on between-day

CVs of <20%) of 2-3 ^g/L (147,148). This is also true for the two currently available immunoassays. The MEIA method has a functional sensitivity that varies among laboratories, with values ranging from 1.3 to 3.0 ^g/L (149,151-155). Technical variations at the manual extraction step most likely contribute to the differences in functional sensitivity that were observed among laboratories evaluating the MEIA. One study found that the CEDIA has a functional sensitivity of 3.0^g/L (156). HPLC-MS methods have excellent sensitivity, with functional sensitivities <1 ^g/L (158,159). As previously mentioned, a further advantage of HPLC-MS methods is the ability to measure multiple immunosuppressants in the same whole blood sample. It is important that laboratories experimentally determine their own lower limit of detection based on long-term between day imprecision data (using whole blood samples) and not rely on package insert information or published data.

The sirolimus MEIA is prone to error that is dependent on hematocrit levels. There is an inverse relationship between hematocrit and measured sirolimus levels. At a sirolimus concentration of 5 ^g/L, results can be 20% higher for hematocrits of <35% and as much as 20% lower for hematocrits >45% (149,160). When the hematocrit is between 35 and 45%, MEIA bias is <10% at sirolimus concentrations ranging from 5 to 22 ^g/L. Incomplete extraction of sirolimus from erythrocyte-binding proteins is the most probable mechanism leading to the hematocrit interference. The CEDIA does not appear to be affected by variations in hematocrit between 20 and 60% (150); however, there are no independently published studies supporting the manufacturer's claim.

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