Disposition and mass balance of [14C]vernakalant after single intravenous and oral doses in healthy volunteers.

Vernakalant hydrochloride is a novel antiarrhythmic drug for the rapid conversion of atrial fibrillation to sinus rhythm and prevention of relapse. In this open-label, parallel design study, 8 healthy men received single 240-mg doses of [(14)C]vernakalant hydrochloride, given as a 10-minute intravenous (IV) infusion on day 1, and as an oral gel capsule on day 22. Plasma, urine, and fecal samples were collected for 7 days after dosing to measure vernakalant and its metabolites and to estimate mass balance of total [(14)C] recovery. The disposition and metabolic profile of vernakalant after both IV and oral administration, depended on cytochrome P450 (CYP)2D6 genotype. Vernakalant underwent rapid and extensive distribution during infusion, which resulted in similar maximum plasma concentrations in extensive metabolizers (EMs) and poor metabolizers (PMs) for IV but not oral administration. Vernakalant was metabolized rapidly and extensively to a 4-O-demethylated metabolite with glucuronidation in EMs; direct glucuronidation predominated in PMs. Slower clearance in PMs contributed to 3- and 6-fold higher drug exposure (AUC(0-∞); IV and oral dosing, respectively). Urinary recovery of unchanged vernakalant was higher in PMs as well. Total [(14)C] was recovered predominantly in urine, while lower levels were recovered in feces. Mass balance was achieved, with a mean recovery of 99.7% of the IV dose and 98.7% of the oral dose, in EMs, and 89.2% and 88.2% of the IV and oral doses, respectively, in PMs. Vernakalant was well tolerated. The pharmacokinetics and metabolism of vernakalant depend on CYP2D6 genotype with more pronounced effects on exposure following oral administration; however, the differences between EMs and PMs are unlikely to be clinically significant following short-term IV infusion.

Steady-state pharmacokinetics of micafungin and voriconazole after separate and concomitant dosing in healthy adults.

We assessed the pharmacokinetics and interactions of steady-state micafungin (Mycamine) or placebo with steady-state voriconazole in 35 volunteers. The 90% confidence intervals around the least-squares mean ratios for micafungin pharmacokinetic parameters and placebo-corrected voriconazole pharmacokinetic parameters were within the 80%-to-125% limits, indicating an absence of drug interaction.

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 in 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) i.v. dose infused over 4 hours and approximately 0.12 mg/kg IBW orally; normal subjects were dosed at 0.02 mg/kg 4-hour i.v. and 5 mg (0.065 mg/kg) p.o. 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 (i.v.), p.o. maximum blood concentration = 48.2 ng/mL, time of p.o. 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 (i.v.), 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.

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.

Safety, tolerance, and pharmacokinetics of a small unilamellar liposomal formulation of amphotericin B (AmBisome) in neutropenic patients.

The safety, tolerance, and pharmacokinetics of a small unilamellar liposomal formulation of amphotericin B (AmBisome) administered for empirical antifungal therapy were evaluated for 36 persistently febrile neutropenic adults receiving cancer chemotherapy and bone marrow transplantation. The protocol was an open-label, sequential-dose-escalation, multidose pharmacokinetic study which enrolled a total of 8 to 12 patients in each of the four dosage cohorts. Each cohort received daily doses of either 1.0, 2.5, 5.0, or 7.5 mg of amphotericin B in the form of AmBisome/kg of body weight. The study population consisted of patients between the ages of 13 and 80 years with neutropenia (absolute neutrophil count, <500/mm3) who were eligible to receive empirical antifungal therapy. Patients were monitored for safety and tolerance by frequent laboratory examinations and the monitoring of infusion-related reactions. Efficacy was assessed by monitoring for the development of invasive fungal infection. The pharmacokinetic parameters of AmBisome were measured as those of amphotericin B by high-performance liquid chromatography. Noncompartmental methods were used to calculate pharmacokinetic parameters. AmBisome administered as a 1-h infusion in this population was well tolerated and was seldom associated with infusion-related toxicity. Infusion-related side effects occurred in 15 (5%) of all 331 infusions, and only two patients (5%) required premedication. Serum creatinine, potassium, and magnesium levels were not significantly changed from baseline in any of the dosage cohorts, and there was no net increase in serum transaminase levels. AmBisome followed a nonlinear dosage relationship that was consistent with reticuloendothelial uptake and redistribution. There were no breakthrough fungal infections during empirical therapy with AmBisome. AmBisome administered to febrile neutropenic patients in this study was well tolerated, was seldom associated with infusion-related toxicity, was characterized by nonlinear saturation kinetics, and was effective in preventing breakthrough fungal infections.

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.

Tacrolimus (FK506): validation of a sensitive enzyme-linked immunosorbent assay kit for and application to a clinical pharmacokinetic study.

Tacrolimus (FK506) is a macrolide immunosuppressant approved for the prophylaxis of organ rejection in liver transplant. Immunoassays of low intra- and interday variability and high sensitivity are necessary to adequately characterize terminal elimination phase concentrations in pharmacokinetic studies. A new ELISA kit for the quantitation of tacrolimus in human whole blood has been validated for use in pharmacokinetic studies. Methanol sample extracts were dried and reconstituted in a horseradish peroxidase (HPR)-FK506 conjugate solution. The reconstituted samples and mouse anti-FK506 were added to a microplate, precoated with secondary antibody, and incubated, FK506 and the HPR-FK506 conjugate competed to bind with anti-FK506, which was immobilized by binding to the secondary antibody. Unbound FK506 was washed away, and substrate was added for color development. Once the reaction was stopped with 2 N H2SO4, the plate was read at 450 nm. The linear range was 0.5-60 ng/ml, with a limit of quantitation of 0.5 ng/ml. Interday precision and accuracy were < or = 10.4% C.V. and < or = 3% R.E. for quality control samples. The lack of interference from endogenous compounds was established by parallelism and recoveries of FK506 from six lots of control matrix. Cross-reactivity against the metabolites and analogs were not performed because the kit monoclonal antibody was from the same source as Kobayashi et al (1). The utility and sensitivity of the kit present a good method for the quantitation of tacrolimus in blood from pharmacokinetic studies. The method is robust and has been used to assay tacrolimus in several thousand whole blood samples by multiple analysts.

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.

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).

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.

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.

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.

FK506 (tacrolimus) and its immunoreactive metabolites in whole blood of liver transplant patients and subjects with mild hepatic dysfunction.

PURPOSE: To determine the concentrations of FK506 and its metabolites in blood from liver transplant patients and subjects with hepatic dysfunction. METHODS: HPLC was combined with an enzyme-linked immunosorbent assay (ELISA) to determine the concentrations of FK506 and its immunoreactive metabolites in human whole blood. RESULTS: In four liver transplant patient, most of the immunoreactivity was seen in the HPLC fractions where unchanged FK506 eluted. FK506 accounted for about 95% or more of the total immunoreactivity in the first days of posttransplant. Immunoreactivity observed in the nonFK506 fractions ranged from 1.6% to 10.7% of the total immunoreactivity; about 30% of the nonFK506 immunoreactivity was due to M-III(15-O-demethyl FK506). Blood from subjects with mild hepatic dysfunction was examined at 1.5 and 6 hours after an oral and intravenous dose, respectively, by HPLC-ELISA. Regardless of the route of administration, more than 96% of the total immunoreactivity was recovered in the FK506 fraction. M-III was detected in the blood of 3 of 6 subjects after an oral dose, but in none of these after an intravenous dose. CONCLUSIONS: ELISA is an appropriate method for therapeutic drug monitoring of FK506.

Intraportal delivery of immunosuppression to intrahepatic islet allograft recipients.

Local delivery of immunosuppressive agents may dampen local alloreactive events with avoidance of systemic toxicity. We investigated the innovative strategy of intraportal (IPO) delivery of three immunosuppressive agents in streptozotocin diabetic rat recipients of islet allografts (Lewis to Wistar-Furth) transplanted intrahepatically. IPO budesonide (BUD, 240 or 360 micrograms/kg per day), a potent steroid, and cyclosporin (CyA, 2 or 4 mg/kg per day) did not prolong graft mean survival time [MST +/- standard deviation (SD)] as compared to nonimmunosuppressed recipients. Fourteen days of IPO FK 506 (0.16 mg/kg per day) significantly increased MST as compared with untreated controls (49 +/- 29 vs 7 +/- 1 days, P < 0.01) and was more effective than intravenous (IV) FK 506 (17 +/- 7 days, P < 0.01). When FK 506 was given for 28 days, the benefit of IPO over IV delivery was reaffirmed (MST 81 +/- 32 vs 34 +/- 4 days, P < 0.01). The potential for toxicity was lessened by lower mean systemic levels in the IPO group as compared to the IV group (1.3 +/- 0.6 vs 3.5 +/- 0.9 ng/mg, P < 0.02). The strategy of continuous IPO FK 506 was effective in the prevention of rejection of intrahepatic islet allografts.

NG-methyl-L-arginine, an inhibitor of nitric oxide formation, reverses IL-2-mediated hypotension in dogs.

The effects of NG-methyl-L-arginine (L-NMA), an inhibitor of nitric oxide formation, were studied in dogs treated with interleukin-2 (IL-2). The administration of IL-2 to dogs resulted in hypotension within 3 days of treatment. The development of hypotension correlated with accumulation in the serum of nitrate, which is a stable breakdown product of nitric oxide. Administration of L-NMA decreased serum nitrate levels and increased the mean arterial pressure. The antihypotensive effect was dose dependent with a maximum effect observed at a dose of 20 mg/kg. Administration of a continuous infusion of L-NMA (5 mg.kg-1.h-1) maintained the mean arterial pressure for 48 h with concurrent administration of IL-2. Evaluation of IL-2-induced lymphokine-activated killer cell proliferation and tumoricidal activity toward a canine glioblastoma target cell line was unaffected by L-NMA. These studies imply that L-NMA may effectively ameliorate the dose-limiting hypotension associated with administration of IL-2 without adversely affecting the antitumor effects.

Surface-active substrates for Raman and luminescence analysis.

This paper describes the development of active materials for optically enhanced Raman and fluorescence spectroscopy. The substrates for surface-enhanced Raman scattering investigated in this study involved silver-coated microspheres on glass plates. The effect of various experimental parameters, such as angle of incidence and excitation wavelength, were investigated. The substrate used for surface luminescence analysis consisted of a cellulose membrane coated with fumed silica microparticles, to enhance the sensitivity of analysis. Examples of analysis of benzo[a]pyrene and its derivatives are used to illustrate the efficacy of the analytical techniques.