A simple and rapid spectrofluorometric determination of pomalidomide in spiked plasma and urine. Application to degradation studies.

A rapid and accurate spectrofluorimetric method for the determination of pomalidomide was developed and validated based on the measurement of its native fluorescence without the need for any derivatization and separation for the first time. The fluorescence intensity of the drug in acetonitrile solution allowed precise detection at 460 nm after excitation at 296 nm. The calibration curve was linear in the concentration range 31.0-500.0 ng/ml. Limit of detection and limit of quantification were found to be 8.04 and 24.36 ng/ml, respectively. Sensitive results allowed the drug to be detected with good recovery (75.46-109.72%) in human plasma and urine using the developed method. The proposed method was validated in terms of linearity, sensitivity, precision, accuracy, recovery, and stability parameters. Pomalidomide was subjected to degradation under various stress conditions (hydrolytic, oxidative and thermal) to demonstrate that the method was stable, indicating and identifying possible degradation products. In addition, the drug was exposed to electrochemical degradation using the chronoamperometry technique for the first time. Characterization of pomalidomide degradation products obtained because of oxidative degradation and electrochemical degradation was carried out using attenuated total reflection Fourier transform infrared spectroscopy, mass spectrometry and high performance liquid chromatography - mass spectrometry methods and possible structures were proposed.

Comparison of the predictive ability of several second-order multivariate methods in the simultaneous determination of two therapeutic drugs in human urine.

The aim of this paper is to study the applicability of second-order multivariate methods in the simultaneous determination of two therapeutic drugs in human urine samples. The studied drugs, irinotecan and thalidomide, are used in the treatment of malignant tumours. Irinotecan (CPT-11) is used to treat colon cancer; recent studies have shown the benefits of using thalidomide in combination with CPT-11 in the treatment of this disease. CPT-11 is highly fluorescent, but the native fluorescence of thalidomide is very weak. The second-order methods assayed were parallel factor analysis (PARAFAC), unfolded partial least-squares (U-PLS) and multidimensional partial least-squares (N-PLS), both combined with the residual bilinearization procedure (RBL). The excitation-emission matrices (EEMs) of the samples were recorded as analytical signal. The accuracy and precision of the algorithms were evaluated through the root mean square error of prediction (RMSEP) and the elliptical joint confidence region test (EJCR), obtaining better results with PARAFAC, which was successfully applied to the determination of thalidomide and CPT-11 in human urine samples, after a previous liquid-liquid extraction with chloroform.

Pharmacokinetics, metabolism and excretion of [(14)C]-lenalidomide following oral administration in healthy male subjects.

PURPOSE: Assessment of the absorption, metabolism and excretion of [(14)C]-lenalidomide in healthy male subjects following a single oral dose. METHODS: Six healthy male subjects were administered a single 25 mg oral suspension dose of [(14)C]-lenalidomide. Blood (plasma), semen and excreta were collected. Mass balance assessments were done by radioactivity measurements. Metabolite profiling and quantitation were accomplished using liquid chromatography with mass spectrometric and radiochemical detection. RESULTS: [(14)C]-Lenalidomide was rapidly absorbed (T (max) 0.77-1.0 h), and the levels declined with a terminal half-life of approximately 3 h, with similar profiles for total blood and plasma radioactivity as well as plasma lenalidomide. The whole blood to plasma radioactivity exposure levels were comparable, suggesting equal distribution between plasma and blood cells. On average, 94% of the administered radioactivity was recovered within 10 days, with >88% recovered within 24 h. Urinary excretion was the primary route of elimination (90% of radioactive dose), with minor amounts excreted in feces (4%). Semen contained a small amount of the radioactive dose (0.0062%). Lenalidomide was the primary radioactive component in plasma (92% of the [(14)C]-area under the concentration-time curve) and urine (>90% of the radioactivity in urine). The remaining radioactivity was composed of primarily two metabolites: 5-hydroxy-lenalidomide and N-acetyl-lenalidomide, each accounting for less than 5% of the total radioactivity as well as lenalidomide levels in plasma and excreta. CONCLUSIONS: In summary, following oral administration, lenalidomide is highly absorbed and bioavailable, metabolized minimally, and eliminated predominantly via urinary excretion in the unchanged form in humans.

On-line preconcentration and enantioseparation of thalidomide racemates by CEC with the hyphenation of octyl and norvancomycin monoliths.

A method was developed for simultaneous preconcentration and chiral separation of thalidomide enantiomers in human urine by CEC in combination with self-concentration and solvent gradient effects. A 4 cm long octyl (C8) monolithic column was hyphenated with a 15 cm long norvancomycin (NVC)-bonded monolithic column via a fluorinated ethylene-propylene interface. Sample solution was injected into the C8 monolithic column, the two thalidomide enantiomers were first preconcentrated on the C8 monolithic column, and then separated with a further concentration on the NVC-bonded monolithic column by CEC. Injection of 34.8 mm plug of sample solution gave 278- and 298-fold enhancement in sensitivity, and detection limits of 90 and 94 microg/L for the two thalidomide enantiomers. Peak areas of the two isomers were linear in a range of 0.5-50 mg/L. The precision for five replicate injections of 10 mg/L were 0.8-0.9 and 1.1-2.3% for the migration time and peak height, respectively. The developed method was applied to the determination of racemic thalidomide in spiked human urine samples.

Plasma pharmacokinetics and urinary excretion of thalidomide after oral dosing in healthy male volunteers.

The plasma pharmacokinetics and urinary excretion of thalidomide have been evaluated in eight healthy male volunteers receiving a single oral dose of 200 mg. Concentrations of thalidomide were determined by a new HPLC assay. Plasma concentration vs. time data were well fit by a one-compartment model. The mean (+/- SD) peak concentration, 1.15 +/- 0.2 microgram/ml, was achieved at 4.39 +/- 1.27 hr. Absorption and elimination half-lives were 1.70 +/- 1.05 hr and 8.70 +/- 4.11 hr, respectively, with a lag time of 0.41 +/- 0.17 hr observed in six subjects. The apparent volume of distribution and total body clearance rate, based on assumed complete bioavailability, were 120.69 +/- 45.36 liters and 10.41 +/- 2.04 liters/hr. The urinary excretion of thalidomide accounted for only 0.6 +/- 0.22% of the total dose administered over 24 hr, and the renal clearance rate was 0.08 +/- 0.03 liter/hr. This suggests that the major route of elimination of thalidomide is nonrenal.

Determination of the thalidomide analogues 2-(2,6-dioxopiperidine-3-yl)phthalimidine (EM 12), 2-(2,6-dioxopiperidine-4-yl)phthalimidine (EM 16) and their metabolites in biological samples.

A rapid and sensitive method for quantitative analysis of the thalidomide analogues EM 12 and EM 16 and their metabolites has been developed. Following an optional extraction, samples were analysed by reversed-phase high-performance liquid chromatography with ion depression. The recovery of the extraction procedures was 65-80%. The method has been applied to pharmacokinetic studies in small laboratory animals and in vitro model experiments.

The enantiomers of the teratogenic thalidomide analogue EM 12. 2. Chemical stability, stereoselectivity of metabolism and renal excretion in the marmoset monkey.

The rate of metabolism as well as the metabolic profiles of the enantiomers and the racemate of 2-(2,6-dioxopiperidine-3-yl)-phthalimidine (EM 12), a teratogenic thalidomide analogue, have been investigated in vitro and in the marmoset monkey in vivo. The half-life of racemic EM 12 and of the two enantiomers in vitro (phosphate buffer pH 7.4, 37 degrees C) were all in the same range (12 h). Two major hydrolysis products were found which were formed via amide cleavage of the piperidinedione ring of the molecule (EM 27 and EM 356). Their concentrations were similar. In contrast, EM 356 was the main metabolite of EM 12 present in the urine of marmoset monkeys following single i.p. or p.o. doses of 5 mg/kg b.w. About twice as much EM 356 was produced after administration of R-EM 12 than after administration of S-EM 12. The concentration ratio of the metabolites obtained after p.o. and i.p. administration of the substances were in the same range. Separation of the EM 12 enantiomers present in urine suggests that considerable racemisation took place in vivo, although at a slower rate than in vitro: about 25% of the respective optical antipodes were present 5 h after administration of the R-enantiomer and 7.5 h after administration of the S-enantiomer (compared to 1.5 h in vitro). Our results indicate that racemic EM 12 as well as its enantiomers are chemically and metabolically more stable than thalidomide; however, extensive racemisation occurs both in vivo and in vitro. The metabolism and renal excretion of the enantiomers of EM 12 in the marmoset monkey were shown to be stereoselective.

Pharmacokinetics and metabolism of taglutimide (K-2004) in the rat.

Absorption, blood level course, renal, fecal, and biliary elimination and the metabolisation of 2-(bicyclo[2,2,1]-heptane-2-endo-3-endo-dicarboximido)-glutarimide (taglutimide, K-2004), a new hypnotic-sedative substance, were studied in the rat, and the pharmacokinetic constants were calculated. After oral administration of 10 mg per animal, which corresponds to the 7- to 10-fold human dose, a fast and practically total absorption and a quick and complete excretion was observed. Within 1 h 82% of the dose given were absorbed, and 12 h after the dosing 88% had been excreted again. The elimination was effected nearly exclusively (over 95%) via the urine. Besides 10 defined metabolites only 10% of the drug appeared unchanged.