Kimberly L. Napoli

Immunosuppressive effects and safety of a sirolimus/cyclosporine combination regimen for renal transplantation

Background. Sirolimus, a novel immunosuppressant that inhibits cytokine-driven cell proliferation and maturation, prolongs allograft survival in animal models. After a phase I trial in stable renal transplant recipients documented that cyclosporine and sirolimus have few overlapping toxicities, we conducted an open-label, single-center, phase I/II dose-escalation trial to examine the safety and efficacy of this drug combination. Methods. Forty mismatched living-donor renal transplant recipients were sequentially assigned to receive escalating initial doses of sirolimus (0.5-7.0 mg/m 2 /day), in addition to courses of prednisone and a concentration-controlled regimen of cyclosporine. We conducted surveillance for drug-induced side effects among sirolimus-treated patients and compared their incidence of acute rejection episodes as well as mean laboratory values with those of a historical cohort of 65 consecutive, immediately precedent, demographically similar recipients treated with the same concentration-controlled regimen of cyclosporine and tapering doses of prednisone. Results. The addition of sirolimus reduced the overall incidence of acute allograft rejection episodes to 7.5% from 32% in the immediately precedent cyclosporine/prednisone-treated patients. At 18- to 47-month follow-up periods, both treatment groups displayed similar rates of patient and graft survival, as well as morbid complications. Although sirolimus-treated patients displayed comparatively lower platelet and white blood cell counts and higher levels of serum cholesterol and triglycerides, sirolimus did not augment the nephrotoxic or hypertensive proclivities of cyclosporine. The degree of change in the laboratory values was more directly associated with whole blood trough drug concentrations than with doses of sirolimus. Conclusions. Sirolimus potentiates the immunosuppressive effects of a cyclosporine-based regimen by reducing the rate of acute rejection episodes.

Therapeutic drug monitoring of sirolimus: correlations with efficacy and toxicity

We sought to examine the potential benefits of therapeutic drug monitoring of sirolimus, a potent immunosuppressive agent that displays a pleiotropic array of side effects. 
Methods. A high‐performance liquid chromatography (LC) procedure combined with ultraviolet detection (UV) was used to measure serial concentrations of parent compound sirolimus in 150 renal transplant recipients over a period of 4 yr. Drug concentrations in whole blood at trough time, as well as within pharmacokinetic profiles, were correlated with clinical events using contingency tables, logistic regression analysis, and receiver operating characteristic (ROC) curves. 
Results. The LC/UV method showed an excellent correlation with detection of LC‐resolved components by tandem mass spectrometry, demonstrating that the LC/UV method selectively detected parent compound. Sirolimus displayed the characteristics of a critical‐dose drug: Its concentration could not be predicted by a standard body or demographic measure, or by dose, and it showed high degrees of intra‐ and inter‐individual variability. However, there was a good correlation between trough and area‐under‐the‐curve measurements. There was a significant association between trough values expressed as either observed (<5 ng/mL) or dose‐corrected parameter (<1.7 ng/mL per mg administered drug) and the occurrence and severity of acute rejection episodes – despite the low overall incidence of 23 episodes among the cohort of 150 patients. Similarly, ROC functions showed a correlation of the occurrence of hypertriglyceridemia, thrombocytopenia, and leukopenia, but not hypercholesterolemia, with trough concentrations above 15 ng/mL. 
Conclusion. Due to its behavior as a critical‐dose drug, therapeutic monitoring to measure sirolimus concentrations by a LC/UV method may provide clinicians with a tool to optimize outcomes.

Simultaneous simple and fast quantification of three major immunosuppressants by liquid chromatography—tandem mass-spectrometry

OBJECTIVES The aim of the current study was to develop a simple, fast and universal method for quantification of any combination of the three major immunosuppressants sirolimus, tacrolimus and cyclosporin in whole blood, using a LC-tandem mass spectrometer (API-2000, SCIEX, Toronto, Canada). METHODS 250 microL whole blood was spiked with internal standard (ritonavir), and protein precipitated with 350 microL acetonitrile. The sample was centrifuged and 30 microL aliquot was injected onto the HPLC column, where it underwent an online extraction with ammonium acetate. After that the automatic switching valve was activated, changing the mobile phase to methanol and thereby eluting the analytes into the tandem mass spectrometer. The high selectivity of a tandem mass analyzer allows determination of any combination of the three drugs within a 5 min run. RESULTS Between-day precision was between 2.4% and 9.7% for all analytes at the concentrations tested. Accuracy ranged between 98.8% and 103.2% (n = 20). The method was linear over the measuring ranges of all analytes. Within-run precision was below %CV = 6% for all analytes. Good correlation with other analytical methods was observed. CONCLUSIONS The simplicity, universality and high throughput of the method make it suitable for application in a clinical laboratory. The method has been implemented in our laboratory for a routine use.

Effects of the pharmacokinetic interaction between orally administered sirolimus and cyclosporine on the synergistic prolongation of heart allograft survival in rats.

Oral administration, but not continuous intravenous infusion, of sirolimus (SRL) in combination with cyclosporine (CsA) produces a pharmacokinetic interaction, namely increases in the whole blood trough concentrations of SRL ([SRL(WB)]) and CsA ([CsA(WB)]). The effects of this pharmacokinetic interaction on the synergism between SRL and CsA was examined in Wistar Furth (RT1u) recipients of Buffalo (RT1b) heart allografts. A 14-day course of oral SRL produced dose-dependent prolongation of heart allografts: in untreated controls, 0.5 mg/kg SRL per day extended the mean survival time (MST) from 6.4+/-0.5 days to 12.3+/-3.8 days (P<0.05); SRL at 1.0 mg/kg per day prolonged the MST to 18.0+/-5.5 days (P<0.01); at 2.0 mg/kg SRL per day, MST was extended to 52.5+/-13.2 days (P<0.01); and 4.0 mg/kg SRL per day prolonged MST to 90.0+/-41.1 days (P<0.01). Comparison of the in vivo effects after oral versus continuous intravenous SRL administration suggested that the oral bioavailability of SRL is less than 10%. Combinations of oral SRL and CsA synergistically prolonged heart allograft survival, as documented by combination index values of 0.01-0.64 (combination index <1 indicates synergistic interaction). In rats treated with dual drug combinations, CsA increased the bioavailability of SRL by two- to elevenfold, and SRL increased the bioavailability of CsA by two- to threefold, thereby significantly decreasing the oral effective dose (ED) values for each drug. The ED50 for SRL alone is 2.4 mg/kg per day, which produces an average [SRL(WB)] of 13.2 ng/ml. The ED50 for CsA alone is 8.0 mg/kg per day, which produces an average [CsA(WB)] of 1642 ng/ml. However, when the two drugs are combined, the ED50 effect is achieved with only 0.34 mg/kg SRL per day ([SRL(WB)]=1.1 ng/ml) and 2.1 mg/kg CsA per day ([CsA(WB)] =326 ng/ml). Individually, 0.34 mg/kg SRL per day produces an ED9 with an average [SRL(WB)] of 0.6 ng/ml, and 2.1 mg/kg CsA per day produces an ED22 with an average [CsA(WB)] of 174 ng/ml. Thus, the pharmacokinetic interaction between oral SRL and CsA contributes to the in vivo synergism between the two drugs.

Relative tissue distributions of cyclosporine and sirolimus after concomitant peroral administration to the rat : Evidence for pharmacokinetic interactions

The authors sought to determine the effect of concomitant peroral (PO) administration of cyclosporine (CsA) and sirolimus (SRL, rapamycin) on the tissue distributions of CsA and SRL in the rat. Groups of four adult male Wistar-Furth rats were treated for 14 days with 2.5, 5.0, or 10.0 mg CsA/kg x day. Other groups of four adult male Wistar-Furth rats were treated for 14 days with a 1-to-6.25 weight-to-weight ratio of SRL to CsA at SRL doses of 0.4, 0.8, or 1.6 mg/kg x day. Concentrations of CsA and SRL in homogenates of heart, intestinal, kidney, liver, lung, muscle, spleen, and testes were compared to those in whole blood (WB). There was a large, dose-dependent, distinctive distribution of CsA among rat tissues, as has previously been well documented. At a constant molar dose ratio, concomitant oral administration of SRL produced an approximately two-fold increase in the concentrations of CsA in rat tissues, although SRL did not change the CsA tissue-to-WB partition coefficients. Concomitant oral CsA administration produced dose-dependent increases in SRL tissue concentrations and decreases in the SRL tissue-to-WB partition coefficients. The increases in tissue and WB concentrations on coadministration of both agents may be explained either by an increase in absorption caused by competition between the two agents for binding sites on P-glycoprotein in the gut, a reduced rate of metabolism, or to an as yet unidentified elimination mechanism. The dose-independent and unchanged CsA tissue-to-WB partition coefficients suggest that SRL does not affect the equilibrium of CsA between the central and tissue compartments, namely the tissue uptake or intracellular binding. Altered values of the SRL tissue-to-WB partition coefficients suggest that, under the conditions studied, CsA disturbs the equilibrium of SRL between the central and tissue compartments.

Is microparticle enzyme-linked immunoassay (MEIA) reliable for use in tacrolimus TDM? Comparison of MEIA to liquid chromatography with mass spectrometric detection using longitudinal trough samples from transplant recipients.

In the larger transplant centers where technical expertise is available, liquid chromatography with mass spectrometric detection (LC-MS) for tacrolimus therapeutic drug monitoring is replacing the popular microparticle enzyme-linked immunoassay (MEIA) as a cost-effective alternative technology. As more labs convert to LC-MS, the accuracy, precision, selectivity, and sensitivity of the tacrolimus MEIA are being challenged, using data from large populations of clinical samples. However, little attention has been paid to how the results of particular procedures may differ within and among individual patients and to how such differences may relate to patients’ characteristics or to relevant biochemical parameters. So, after validation of an LC/MS procedure and verification of an LC/MS/MS procedure, the author analyzed 552 serial trough blood tacrolimus samples, collected from 38 patients over a 3-month period, by controlled MEIA and LC-MS procedures. Corresponding hematocrit and serum albumin level data were obtained. In an attempt to investigate whether the observations of others who studied population-based data could be illustrated for individuals, longitudinal data from several patients were plotted to visually elucidate any relations between the tacrolimus concentration and biochemical data. Finally tacrolimus concentration, hematocrit, and serum albumin data were compared using data stratified by transplant type, in-/outpatient status, male/female gender, or period elapsed since transplant surgery. The validated/verified LC-MS procedures were shown to be much better controlled than the MEIA during the 3-month parallel comparison study period. The longitudinal data of several individuals who experienced wide changes in the biochemical parameters clearly illustrated the relation between the difference in tacrolimus concentrations determined by MEIA and LC-MS and hematocrit (and sometimes albumin). Differences between the MEIA- and LC-MS-determined tacrolimus concentration data were strongly correlated to transplant type, in-/outpatient status, gender, time elapsed since liver transplant surgery, hematocrit, and weakly to serum albumin levels. In summary, the LC-MS methods provide highly reliable and reproducible estimates of tacrolimus concentrations, whereas the performance of MEIA technology did not provide reliable long-term performance for longitudinal therapeutic drug monitoring of tacrolimus because it was effected by several inherent demographic factors and by factors that can change over time in transplant recipients.

The ability of pretransplant test-dose pharmacokinetic profiles to reduce early adverse events after renal transplantation

Pretransplant test-dose pharmacokinetic profiles were used to determine individual cyclosporine drug bioavailability and clearance rates in renal transplant patients. Assuming a linear relation between dose and area under the concentration curve (AUC), starting i.v. and p.o. CsA doses were computed from the test-dose results. Target values were 400 ng/ml steady-state concentration (Css) during continuous intravenous infusion, and 500 ng/ml average drug concentration (Cavss = AUC/dosing interval) after oral administration, based upon measurements with the specific monoclonal antibody 3H-tracer radioimmunoassay. The outcomes after dose individualization with a 1-(n = 32), 2-(n = 38), or 3-(n = 41) hr i.v. infusion test dose and a p.o. test dose (n = 111) were compared with 228 historical control patients who received a uniform protocol of CsA i.v. at 2.5 mg/kg/day and p.o. at 14 mg/kg/day. The observed Css after i.v. CsA was within 10% of the target concentration in 73% of recipients tested with the 3-hr protocol, a significantly greater fraction than achieved with either the uniform dose (14%), or the 1-(34%) and 2-(25%) hr protocols. Patients in the 3-hr protocol group showed reduced incidences of delayed graft function, early graft loss, and rejection episodes, and a lower mean serum creatinine value, particularly at 7 but also at 30 days posttransplantation. Administration of the predicted oral dose produced a peak concentration of greater than or equal to 700 ng/ml drug absorption in 60% of recipients at 3 days, 90% at 5 days, and 98% at 7 days. The test-dose method less effectively predicted the appropriate oral CsA dose to produce target Cssav and failed to reduce the 90-day rejection incidence. Despite its limitations with the more-complicated p.o. route, the test-dose method successfully predicts i.v. CsA doses, thereby reducing the incidence of early adverse events.

The FTY720 story.

The chemical 2-amino-2[2-(4-octylphenyl)ethyl]-1,3,propane diol is one of a class of small-molecule immunosuppressive agents. Better known as FTY720, this compound was chemically synthesized in an effort to minimize the toxic in vivo properties of a structurally related and highly potent immunosuppressive agent, myriocin. FTY720s mechanism of action, although not fully characterized, appears to be unique among immunosuppressants. Whereas the most well known biochemical characteristic of myriocin is its ability to inhibit serine palmitoyl transferase, the enzyme that initiates the biosynthetic pathway that leads to sphingosine, FTY720 is ineffective in this regard. In vivo, FTY720 induces a significant reduction in the number of circulating lymphocytes. It is thought to act by altering lymphocyte trafficking/homing patterns through modulation of cell surface adhesion receptors and ligands in a manner that has yet to be elucidated. Although much research has yet to be done to unravel the nature of the mechanism of action of FTY720, its efficacy has been sufficiently proven in numerous animal models, especially when administered in combination with cyclosporine. The agent is now progressing through human clinical trials, with the results of phase 1 clinical trials showing safety and tolerability in adult recipients of renal transplants. It is hoped that FTY720 will eventually prove to be an efficacious new weapon in the immunosuppressive armamentarium.

Cytochrome P450 3A9 catalyzes the metabolism of progesterone and other steroid hormones

The catalytic requirements and the role of P450 3A9, a female-specific isoform of CYP3A from rat brain, in the metabolism of several steroid hormones were studied using recombinant P450 3A9 protein. The optimal steroid hormone hydroxylase activities of P450 3A9 required cholate but not cytochrome b5. P450 3A9 was active in the hydroxylation reactions of testosterone, androstenedione, progesterone and dehydroepiandrosterone (DHEA). No activity of P450 3A9 toward cortisol was detectable under our reconstitution conditions. Among all the steroid hormones examined, female-specific P450 3A9 seemed to catalyze most efficiently the metabolism of progesterone, one of the major female hormones, to form three mono-hydroxylated products, 6β-, 16α-, and 21-hydroxyprogesterone. Our data also showed that P450 3A9 can catalyze the formation of a dihydroxy product, 4-pregnen-6β, 21-diol-3, 20-dione, from progesterone with a turnover number, 1.3 nmol/min/nmol P450. Based on the Vmax/Km values for P450 3A9 using either 21-hydroxprogesterone or 6β-hydroxyprogesterone as a substrate, 4-pregnen-6β, 21-diol-3, 20-dione may be formed either by 6β-hydroxylation of 21-hydroxprogesterone or 21-hydroxylation of 6β-hydroxyprogesterone. As a major isoform of CYP3A expressed in rat brain, the activities of P450 3A9 toward two major neurosteroids, progesterone and DHEA suggested a possible role for P450 3A9 in the metabolism of neurosteroids.

Conversion from liquid to solid rapamycin formulations in stable renal allograft transplant recipients

Sirolimus (rapamycin, RAPA, Rapamune™) is a potent immunosuppressive agent currently being investigated for prophylaxis against acute rejection episodes in renal transplant recipients. In the present study, stable renal allograft recipients under maintenance therapy with RAPA and cyclosporine (CsA) were converted from the original oil‐based liquid RAPA to a solid tablet formulation on a milligram‐to‐milligram basis, in order to evaluate the pharmacokinetics and safety of this new dosage form. Twelve‐hour pharmacokinetic (PK) profiles of both RAPA and CsA were conducted with the final liquid RAPA dose, and at 2, 4, and 8 weeks postconversion to the solid tablet. In addition, the parameters of the PK profiles for the solid formulation were compared with those for liquid RAPA, which were performed prior to this study. Area under the concentration–time curve (AUC) values for the liquid formulation and for the solid tablet at 2, 4, and 8 weeks postconversion were 256.5, 205.8, 226.1, and 224.4 ng·h/mL, respectively (p=NS). Time to maximum RAPA concentration was longer at 4 weeks postconversion, but similar at 2 and 8 weeks. There were no differences observed between the liquid and solid tablet trough concentrations. The only significant differences observed among the PK parameters of the solid tablet versus those of the liquid formulation were the lower Cmax values of the solid, namely 25.3, 24.9, and 26.7 ng/mL versus 37.1 ng/mL (p<0.05). In addition, the dose corrected Cmax was lower in the solid tablet PK profiles compared with the prior PK profiles for the liquid (7.7 versus 10.2 ng/mL, p<0.02). Cyclosporine AUC values did not change appreciably during the study. Conversion from the liquid to the solid formulation was neither associated with episodes of acute rejection, nor changes in laboratory values, during the 8‐week study. In summary, conversion from the liquid to the solid RAPA formulation resulted in similar PK profiles and appears to be both safe and well‐tolerated in renal transplant recipients. Copyright