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Background: Sirolimus is an inhibitor of mammalian target of rapamycin, which exhibits large interindividual pharmacokinetic variability. We report sirolimus pharmacokinetic data collected as part of a concentration-controlled multicenter phase II clinical trial in pediatric patients with neurofibromatosis type 1. The purpose of this study was to explore the effect of growth on age-dependent changes in sirolimus clearance with a focus on cytochrome P450 3A (CYP3A) subfamily mediated metabolism., Methods: Predose blood samples were obtained at steady state from 18 patients with neurofibromatosis type 1. Sirolimus and its 5 CYP3A-dependent primary metabolites were quantified by HPLC-UV/MS. Concentration ratios of metabolites to sirolimus (metabolic ratio) were calculated as an index of metabolite formation., Results: Metabolic ratios of the main metabolites, 16-O-demethylsirolimus (16-O-DM) and 24-hydroxysirolimus (24OH), were significantly correlated with sirolimus clearance, whereas this was not the case for the other 3 metabolites (25-hydroxysirolimus, 46-hydroxysirolimus, and 39-O-demethylsirolimus). The ratios for the 16-O-DM and 24OH metabolites were lower in children than adults. No significant difference in allometrically scaled metabolic ratios of 16-O-DM and 24OH was observed between children and adults., Conclusions: This study suggests that the age-dependent changes in sirolimus clearance can be explained by size-related increases in CYP3A metabolic capacity, most likely due to liver and intestinal growth. These findings will help facilitate the development of age-appropriate dosing algorithms for sirolimus in infants and children.

This review summarizes the pharmacokinetics in children and youths of 2 commonly used immunosuppressive drugs, mycophenolate mofetil (MMF) and sirolimus (Sir), as presented at the IATDMCT 2007 conference. The review focuses on the developmental changes of drug disposition during childhood and adolescence. Regarding mycophenolate mofetil, the authors were unable to demonstrate age dependency of MMF in combination with cyclosporine. By contrast, there was an inverse relationship between age and the dose-normalized mycophenolate (MPA) area-under-the-time-concentration curve (AUC) in children who received concomitant tacrolimus (Tac). Dose-normalized MPA AUCs were higher than commonly observed in adult patients. It can be hypothesized that the age dependency is related to developmental changes in the expression of the UDP-glucuronosyltransferases. Sirolimus half-life and mean residence time (MRT) are shorter than in adults. Similar to that in adults, there is a profound drug-drug interaction between cyclosporine and Sir. In our own experience, Sir was started at 0.13 +/- 0.05 mg/kg/day. The average Sir AUC was 64.9 +/- 29.7 ng*h/mL. The median (range) AUC for each metabolite was as follows: 12-hydroxy-Sir, 7.6 (0.2-18.8); 46-hydroxy-Sir, 3.1 (0.0-12.4); 24-hydroxy-Sir, 4.3 (0.0-12.6); piperidine-hydroxy-Sir, 3.5 (0.0-8.3); 39-desmethyl-Sir, 3.6 (0.0-11.3); 16-desmethyl-Sir, 5.0 (0.1-9.9); and di-hydroxy-Sir, 4.3 (0.0-32.5) ng*h/mL. Of the total metabolite AUC, 77.5% was due to hydroxylated metabolites, while 39-O-desmethyl Sir (the main metabolite in adults) comprised only 8.4% of the metabolites. This is clinically relevant, as 39-O-desmethyl Sir shows 86% to 127% cross-reactivity with the Sir immunoassay. Metabolites reached a median AUC of 60% of that of Sir, but the range was 2.6% to 136%. The age dependency of Sir metabolite formation was confirmed in a human liver microsome model. On the basis of the age dependency of piperidine-hydroxy Sir, the authors postulate that the ontogeny of the drug disposition can be largely explained by developmental changes of the CYP2C8 expression. In conclusion, both Sir and MMF drug disposition vary in children and adolescents from adult patients, most likely because of developmental changes of biliary transporters and metabolic enzymes.

Everolimus is an immunosuppressant used as rejection prophylaxis in patients undergoing transplants. It requires blood concentration-guided dosing and is extensively metabolized. It was the goal to assess the crossreactivity of the major everolimus metabolites in the blood of patients undergoing kidney graft with the Innofluor Certican Assay (Seradyn, Inc., Indianapolis, IN), a clinical assay used to quantify the concentrations of everolimus in patients' blood samples. The three main hydroxy metabolites of everolimus (46-, 24-, and 25-hydroxy everolimus) and all other minor hydroxylated and demethylated metabolites were generated using pooled human liver microsomes and purified using semipreparative high-performance liquid chromatography with ultraviolet detection. Structures were confirmed using liquid chromatography-mass spectrometry/ion trap mass spectrometry and analysis of the fragmentation patterns. Blank blood samples were spiked with the isolated metabolites to determine the specific crossreactivity with the immunoassay. Crossreactivity testing with the immunoassay showed 1% or less for 46-hydroxy and 24-hydroxy everolimus and 6% or less crossreactivity for 25-hydroxy everolimus at therapeutically relevant concentrations. Crossreactivity testing of the minor metabolites showed crossreactivities of 16.3% for 45-hydroxy, 33.0% for 12-hydroxy, 18.3% for 11-hydroxy, 15.3% for 14-hydroxy, 38.7% for OH-piperidine I, 46.3% for OH-piperidine II, 43% for 39-O-desmethyl, 142% for 27-O-desmethyl, and 68% for 40-O-desethylhydroxy everolimus (sirolimus).

Everolimus is used as an immunosuppressant after organ transplantation. It is extensively metabolized, mainly by cytochrome P4503A enzymes, resulting in several hydroxylated and demethylated metabolites. The structures of these metabolites after in vitro metabolism of everolimus by human liver microsomes have recently been identified. It was the goal to elucidate the everolimus metabolite patterns in 128 trough blood samples from kidney graft patients using high-performance liquid chromatography (LC)-ion trap mass spectrometry (MS) in combination with analysis of the fragmentation patterns of the metabolites isolated from patient blood and comparison with the metabolites generated in vitro. After identification, concentrations of the metabolites were estimated using LC-MS. Relative to the everolimus concentrations in trough blood samples, metabolite concentrations were [median (range), n = 128] 46-hydroxy 44.1% (0-784%), 24-hydroxy 7.7% (0-85.6%), and 25-hydroxy 14.4% (0-155.4%); 11-Hydroxy, 12-hydroxy, 14-hydroxy, 49-hydroxy, two hydroxy-piperidine everolimus metabolites, 16-O-desmethyl, 16,39-O-didesmethyl, 16,27-O-didesmethyl, and 27,39-O-didesmethyl everolimus were also detected. However, when detectable, concentrations were consistently between the lower limit of detection (0.1 microg/L) and the lower limit of quantification (0.25 microg/L) of our LC-MS assay. In most trough blood samples, the total metabolite concentrations were between 50% and 100% of the everolimus concentrations. The clinical importance of everolimus metabolites in blood of patients including pharmacodynamics, toxicodynamics, and cross-reactivity with the antibodies of immunoassays used for therapeutic drug monitoring remains to be evaluated.

The pharmacokinetics and metabolic disposition of sirolimus (rapamycin, Rapamune), a macrocyclic immunosuppressive agent for the prevention of allograft rejection in organ transplantation, were investigated in 6 healthy male volunteers after a single nominal 40-mg oral dose of the C-radiolabeled drug, with the added aim of assessing the potential role of sirolimus metabolites in the clinical pharmacology of the parent drug. The absorption of parent drug and derived materials was rapid (tmax 1.3 +/- 0.5 hours, mean +/- SD), and the elimination of sirolimus was slow (t(1/2) 60 +/- 10 hours, mean +/- SD) in whole blood. The high whole blood to plasma (B/P) concentration ratio of sirolimus (142 +/- 39) was consistent with its extensive partitioning into formed blood elements. The markedly lower B/P value based on radioactivity (2.7 +/- 0.4) suggested that drug-derived products partitioned into formed blood elements to a much lesser extent. Based on AUC0-144h values, unchanged sirolimus represented an average 35% of total radioactivity in whole blood. Drug-derived products in whole blood were characterized by HPLC, LC/MS, and LC/MS/MS as 41-O-demethyl, 7-O-demethyl, and several hydroxy, dihydroxy, hydroxy-demethyl and didemethyl sirolimus metabolites. The percentage distribution of sirolimus metabolites in whole blood ranged from 3%-10% at 1 hour to 6%-17% at 24 hours after drug administration. Based on their low immunosuppressive activities and relative abundance in whole blood of humans after sirolimus administration, metabolites of sirolimus do not appear to play a major role in the clinical pharmacology of the parent drug. A majority of the administered radioactivity (91.0 +/- 8.0%) was recovered from feces, and only 2.2% +/- 0.9% was renally excreted.

In the presirolimus era, cyclosporine withdrawal in de novo renal transplant patients was associated with increased rates of rejections; thus, no improvement in long-term graft survival was achieved. However, those patients who did not reject exhibited longer graft survival than did those on cyclosporine. In recent years sirolimus has been introduced into clinical practice and, so far, 4 randomized cyclosporine withdrawal trials in de novo patients after renal transplantation have been published, together with 3 smaller studies directly comparing cyclosporine with sirolimus. The main finding in all these studies was better renal function after cyclosporine withdrawal. In the largest trial with the longest follow-up there was even a trend toward higher rates of graft survival at 3 years and fewer histopathological findings in protocol biopsies at that time. Whether this success in renal function and graft survival will project to prolonged patient survival remains to be determined. In summary, with the introduction of sirolimus as a potent immunosuppressive but nonnephrotoxic drug, a considerable improvement in graft and patient survival might be possible.

Sirolimus is a recently marketed immunosuppressant that, in common with cyclosporine and tacrolimus, exhibits a low average oral bioavailability (approximately 20%). Likewise, sirolimus is a substrate for the major drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) and the efflux transporter P-glycoprotein (P-gp), both of which are expressed in close proximity in epithelial cells lining the small intestine. Using CYP3A4-expressing Caco-2 cell monolayers, we examined the interplay between metabolism and transport on the intestinal first-pass extraction of sirolimus. Modified Caco-2 cells metabolized [14C]sirolimus to the same CYP3A4-mediated metabolites as human small intestinal and liver microsomes. [14C]Sirolimus also degraded to the known ring-opened product, seco-sirolimus. A ring-opened dihydro species (M2) was, surprisingly, the major product detected in cells at all sirolimus concentrations examined (2-100 micromol/L) and in incubations with human liver and intestinal homogenates but not in corresponding microsomes. M2 formation was NADPH-dependent but unaffected by prototypical CYP3A4 inhibitors. Although M2 was formed from purified seco-sirolimus (20 micromol/L) in the homogenates, it was not detected in cells when seco-sirolimus was added to the apical compartment because seco-sirolimus was essentially impermeable to the apical membrane. Sirolimus, seco-sirolimus (basolaterally dosed), and M2 were all secreted across the apical membrane, and secretion of each was inhibited by the P-gp inhibitor LY335979 (zosuquidar trihydrochloride). Along with CYP3A4-mediated metabolism and P-gp-mediated efflux, a novel elimination pathway was identified that may also contribute to the first-pass extraction, and hence low oral bioavailability, of sirolimus. This new insight into the intestinal elimination of sirolimus, which was not identified using traditional drug metabolism/transport screening methods, may represent another source for the limited absorption of sirolimus.

Sirolimus (rapamycin, Rapamune) is a potent immunosuppressive drug that received marketing approval from the US Food and Drug Administration on September 15, 1999. Research into defining its pharmacokinetic (PK) behavior, interaction with other agents, and metabolism is ongoing. It has been established that oral doses of both liquid and solid formulation are rapidly, though incompletely and variably, absorbed. Metabolism by the intestinal and hepatic CYP3A family of enzymes likely contributes to variability in absorption and low bioavailability. Sirolimus has a long terminal half-life, the AUC correlates well with trough and peak concentrations, and it exhibits a moderate degree of dose proportionality. There is significant interpatient variability in PK parameters of sirolimus, though it exhibits predictable PK behavior when used with prednisone and cyclosporine neoral. There is a decreased rejection risk with higher doses and target level attainment. Several species of sirolimus metabolites have been characterized, and are measurable in whole blood and tissue specimens. Many more species of sirolimus metabolites are detectable, but they are not quantifiable at this time. The total concentration of metabolites appears to be less than that of the parent drug when examined through the PK profile. A reference method for the quantitation of metabolites remains elusive because of a lack of proper standardization. The clinical significance of sirolimus metabolites remains to be proven.

An analytic technique using liquid chromatography (LC) coupled with electrospray-mass spectrometry (ESI-MS) has been developed for the simultaneous determination of the new immunosuppressant SDZ RAD (40-O-[2-hydroxy)ethylrapamycin) and cyclosporine (Cs), including their metabolites in blood. With the time-sparing, automated on-line extraction technique, the recovery of SDZ RAD averaged 95% and that of Cs, 94%. The calibration lines were linear from 0.5 to 100 microg/L (r2 = 0.99) for SDZ RAD and from 10 to 1,000 microg/L (r2 = 0.99) for Cs. The method has been tested on blood samples from renal transplant recipients taken between 1 and 5 hours after oral SDZ RAD and Cs administration. In blood, we found the following metabolites: Hydroxy-SDZ RAD, dihydroxy-SDZ RAD, demethyl-SDZ RAD, and the ring-opened form of SDZ RAD. The main metabolite of SDZ RAD in blood was hydroxy-SDZ RAD. This novel LC/ESI-MS technique provided an excellent method for simultaneous quantitative monitoring of SDZ RAD and Cs, including their relevant groups of metabolites in patients treated simultaneously with these immunosuppressants.