Ala M. Alak

Measurement of tacrolimus (FK506) and its metabolites : A review of assay development and application in therapeutic drug monitoring and pharmacokinetic studies

Tacrolimus (FK506, Prograf) is a macrolide immunosuppressant used for the prevention of organ rejection after transplantation. Tacrolimus demonstrates considerable interindividual variation in its pharmacokinetic profile. This has caused difficulty in defining the optimum regimen and has highlighted the need for therapeutic drug monitoring. Several assay methods for the measurements of tacrolimus in biological specimens have been developed. These assay methods were used for therapeutic drug monitoring and/or pharmacokinetic studies. Two commercially available immunoassays, based on the same monoclonal antibody to tacrolimus, have been used for therapeutic drug monitoring of tacrolimus in whole blood. For pharmacokinetic studies, the assay methods were used to measure tacrolimus and its metabolites in very low concentrations in selected biological matrixes to determine the metabolic and pharmacokinetic profiles of this drug.

Coadministration of Tacrolimus and Mycophenolate Mofetil in Stable Kidney Transplant Patients: Pharmacokinetics and Tolerability

The tolerance and pharmacokinetics (PK) of tacrolimus (T) by the addition of mycophenolate mofetil (MMF) in stable kidney transplant patients (6/group) on long‐term tacrolimus‐based therapy were investigated. Patients received combination T and MMF therapy at three MMF doses: 1, 1.5, and 2 g/day administered twice daily. A 12‐hour blood PK profile for T was obtained prior to MMF dosing; concomitant 12‐hour profiles for T, mycophenolic acid (MPA), and mycophenolic acid glucuronide (MPAG) were obtained after 2 weeks of administration. Tolerance was monitored through 3 months. The intra‐ and intergroup PK of T were variable. The mean AUC0–12 of T for each group was increased after 2 weeks of concomitant MMF administration, but the increase was not statistically significant. Both drugs were well tolerated. Gastrointestinal events were of interest as such have been attributed to both T and MMF. Events reported were diarrhea, nausea, dyspepsia, and vomiting. Other common adverse events were headache, hypomagnesemia, and tremors. Most were mild, although a few were considered to be moderate. There was no apparent relationship between the incidence of any adverse event and MMF treatment group. In the present study, the coadministration of T and MMF did not significantly alter T pharmacokinetics.

Comparative Tacrolimus Pharmacokinetics: Normal versus Mildly Hepatically Impaired Subjects

Tacrolimus (FK506, Prograf®), marketed for the prophylaxis of organ rejection following allogenic liver or kidney transplantation, is virtually completely metabolized. The major metabolic pathways are P450 3A4‐mediated hydroxylation and demethylation. Since P450 hepatic drug‐metabolizing enzymes may be impaired din hepatic dysfunction, a study was conducted to characterize oral and intravenous tacrolimus pharmacokinetics in 6 patients with mild hepatic dysfunction and compared with parameters to those from normal subjects obtained in a separate study. Patients received two treatments: a single 0.020 mg/kg ideal body weight (IBW) IV dose infused over 4 hours and ∼0.12 mg/kg IBW orally; normal subjects were dosed at 0.02 mg/kg 4‐hour IV and 5 mg(0.065 mg/kg) PO. Mean blood pharmacokinetic parameters with mild hepatic dysfunction were as follows: clearance = 0.035 L/h/kg, terminal exponential volume of distribution = 2.59 L/kg, terminal exponential half‐life = 60.6 hours (IV), PO maximum blood concentration = 48.2 ng/mL, time of PO maximum blood concentration = 1.5 hours, and absolute bioavailability = 22.3%. The respective parameters in normal subjects were as follows: 0.040 L/h/kg, 1.91 L/kg, 34.2 hours (IV), 29.7 ng/mL, 1.6 hours, and 17.8%. Inasmuch as clearance and bioavailability were not substantially different from that in normal subjects, patients with mild hepatic impairment may initially be treated with conventional tacrolimus doses, with subsequent dosage adjustments based on response, toxicity, and therapeutic drug monitoring.

An HPLC/MS/MS assay for tacrolimus in patient blood samples Correlation with results of an ELISA assay

An HPLC/MS/MS assay for tacrolimus in whole blood using FR900520 as an internal standard was validated over the standard curve range of 0.100-10.040 ng ml-1. The calibration curve for tacrolimus in human blood gave a slope of 0.2481, an intercept of 0.007, and a correlation coefficient (r) of 0.9996, with no interference noted from human blood, analyte, or internal standard stock solutions. Use of EDTA or heparin as the preservative in blood resulted in no significant differences. Samples were stable for at least the time required to assay the maximum number of samples that could be placed in the automated system. The limit of sensitivity of the assay was set at the concentration of the lowest nonzero standard tested, i.e., 0.100 ng ml-1. However, validation of the assay to a limit of 0.010 ng ml-1 is currently underway. The within-run and between-run precision and accuracy of the method were determined for four quality control samples. The highest CV was seen at 0.1 ng ml-1 (17.6% within-run and 15.9% between-run), with other CV < 5%. The recovery ranged 79.6-81.3% for tacrolimus over the range 0.3-8.0 ng ml-1 and was 63.10 +/- 1.37% for FR900520. There was a linear correlation (r2 = 0.963) between assay results by HPLC/MS/MS and ELISA in whole blood from atopic dermatitis patients treated with topical tacrolimus ointment. The difference between the means +/- S.D. determined by HPLC/MS/MS (1.22 +/- 1.46 ng ml-1) and ELISA (1.12 +/- 1.29 ng ml-1) was significant by a paired t-test (P < 0.001) Similarly, there was a linear correlation (r2 = 0.841) between assay results by HPLC/MS/MS and IMx in whole blood from solid organ transplant patients treated with tacrolimus. The difference between the means was significantly higher (P < 0.001) for the IMx (15.80 +/- 8.37 ng ml-1) than the HPLC/MS/MS (13.42 +/- 6.87 ng ml-1).

A high-performance liquid chromatographic assay for the determination of amphotericin B serum concentrations after the administration of AmBisome, a liposomal amphotericin B formulation.

A sensitive and selective high-performance liquid chromatographic (HPLC) method has been developed for the determination of amphotericin B in human serum. After methanol deproteinization, amphotericin B and 3-nitrophenol (internal standard) are separated by reversed-phase chromatography and detected by ultraviolet absorbance. The analysis of human serum after the standard addition of amphotericin B (0.05-200.0 micrograms/mL) demonstrated excellent precision and accuracy over a five-day period. The HPLC assay uses two standard curve ranges. The high sensitivity curve range for low AmBisome dosage (1.0 mg/kg) is 0.05-20.0 micrograms/mL (curve 1), and the second curve range for the higher AmBisome dose regimens (2.5-5.0 mg/kg) is 0.5-200 micrograms/mL (curve 2). The intraday and interday coefficients of variations for standard curve 1 were 0.5-4.6% and 3.0-11.5%, respectively. The limit of quantitation was 0.05 microgram/mL. The intraday and interday coefficients of variation for standard curve 2 were 2.0-3.6 and 6.9-10.1, respectively. No interfering peak at the retention time for Amphotericin B and the internal standard were present in blank serums or serum samples spiked with fifteen potential co-administrated drugs with Amphotericin B treatment. The method was used to quantitate serum concentrations of amphotericin B in patients after the administration of AmBisome, a liposomal formulation of amphotericin B.

A highly sensitive enzyme-linked immunosorbent assay for the determination of tacrolimus in atopic dermatitis patients

A highly sensitive enzyme-linked immunosorbent assay method has been developed for the determination of tacrolimus in human blood samples. The assay is a modification of the previously published assay with improved sensitivity. Following extraction of tacrolimus with methanol and sulfosalicylic acid, the samples are incubated for 2 h at room temperature on a Nunc Maxisorb plate that has been treated for nonspecific binding by precoating with polyclonal antibody. The analysis of human blood following the standard addition of tacrolimus (0.02-5.0 ng/ml) demonstrated excellent precision and accuracy over a 6-day period. The interday and intraday co-efficients of variation were 6.0-28.9 and 3.9-15.2%, respectively. The limit of quantitation was 0.05 ng/ml. The method was used to quantitate blood concentration of tacrolimus in patients following the administration of tacrolimus ointment.

Quality assurance procedure for monitoring tacrolimus (FK506) concentrations in whole blood by IMx assay.

A Quality Assurance Program for the IMx assay for FK506 in whole blood samples was established to monitor the performance of the assay in clinical sites enrolled by Fujisawa USA, Inc. Forty investigative sites participating in the program were required to perform assay to establish intraassay variability, interassay variability, and performance on blinded samples. Only two of the sites were required to repeat part of the program. The intraassay and interassay results at the sites were in good agreement with the target values obtained at Fujisawa Research Laboratory. Most of the coefficients of variation (CV) were within +/- 15%, well within the acceptance range of +/- 30%. Only a few values were outside the acceptance window. For the blinded samples, the CVs were variable and depended on the concentration of FK506 in the sample. At lower blood FK506 concentrations (5-10 ng/ml), the mean CVs were often outside the acceptance window, and many individual values were not acceptable. At concentrations of 15-50 ng/ml, the CVs were generally acceptable. Thus individual sites can quickly learn to perform the FK506 IMx assay and achieve good within- and between-day results. The assay of lower blood concentrations of FK506 may show higher variability. Patients are usually monitored for clinical signs of rejection and toxicity in addition to blood FK506 concentrations.

In-vitro metabolic studies of tacrolimus using precision-cut rat and human liver slices

The objective of this study was to investigate the in-vitro metabolism of tacrolimus in liver slices from rats and humans. [14C]Tacrolimus (2 or 20 microM) was incubated with precision-cut human and rat liver slices in 12-well plates for up to 12 h. Concentrations of tacrolimus and metabolites were determined by high-performance liquid chromatography (HPLC) radiochromatography. The 13-O-demethylated tacrolimus metabolite (M-I) was the major oxidative metabolite in both rat and human liver slices. The other primary metabolites of tacrolimus (M-II, M-III, and M-IV) were not seen in either species. Unidentified peaks, which eluted early in the HPLC system, were probably due to secondary or conjugated metabolites. The eluate had no pharmacological activity. The finding that M-I was the major tacrolimus metabolite in both human and rat liver slice preparations is consistent with previous studies of rat and human liver microsomes.