Direct chiral LC-MS/MS analysis of fexofenadine enantiomers in plasma and urine with application in a maternal-fetal pharmacokinetic study.

This study shows the development and validation of two enantioselective LC-MS/MS methods for the determination of fexofenadine in biological matrices including the elution order determination. Plasma (200 µL) or urine (50 µL) aliquots were added to the internal standard solution [(S)-(-)-metoprolol] and extracted in the acid medium with chloroform. Resolution of the (R)-(+)- and (S)-(-)-fexofenadine enantiomers was performed in a Chirobiotic V column. The methods showed linearity at the range of 0.025-100 ng/mL plasma and 0.02-10 µg/mL urine for each fexofenadine enantiomer. These methods were applied to the maternal-fetal pharmacokinetics of fexofenadine enantiomers in plasma and urine of parturient women (n = 8) treated with a single oral 60 mg dose of racemic fexofenadine. Enantiomeric ratio in plasma (AUC0-∞(R)-(+)/(S)-(-)) was close to 1.5, nevertheless in urine was closed to unity. The transplacental transfer was approximately 18% for both fexofenadine enantiomers. The enantioselective methods can also be useful in future clinical studies of chiral discrimination of drug transporters.

D-Optimal mixture design optimization of an HPLC method for simultaneous determination of commonly used antihistaminic parent molecules and their active metabolites in human serum and urine.

This study describes a specific, precise, sensitive and accurate method for simultaneous determination of hydroxyzine, loratadine, terfenadine, rupatadine and their main active metabolites cetirizine, desloratadine and fexofenadine, in serum and urine using meclizine as an internal standard. Solid-phase extraction method for sample clean-up and preconcentration of analytes was carried out using Phenomenex Strata-X-C and Strata X polymeric cartridges. Chromatographic analysis was performed on a Phenomenex cyano (150 × 4.6 mm i.d., 5 µm) analytical column. A D-optimal mixture design methodology was used to evaluate the effect of changes in mobile phase compositions on dependent variables and optimization of the response of interest. The mixture design experiments were performed and results were analyzed. The region of ideal mobile phase composition consisting of acetonitrile-methanol-ammonium acetate buffer (40 mm; pH 3.8 adjusted with acetic acid): 18:36:46% v/v/v was identified by a graphical optimization technique using an overlay plot. While using this optimized condition all analytes were baseline resolved in <10 min. Solvent mixtures were delivered at 1.5 mL/min flow rate and analytes peaks were detected at 222 nm. The proposed bioanalytical method was validated according to US Food and Drug Administration guidelines. The proposed method was sensitive with detection limits of 0.06-0.15 µg/mL in serum and urine samples. Relative standard deviation for inter- and intra-day precision data was found to be <7%. The proposed method may find application in the determination of selected antihistaminic drugs in biological fluids.

Effects of one-time apple juice ingestion on the pharmacokinetics of fexofenadine enantiomers.

PURPOSE: We examined the effect of a single apple juice intake on the pharmacokinetics of fexofenadine enantiomers in healthy Japanese subjects. METHODS: In a randomized two phase, open-label crossover study, 14 subjects received 60 mg of racemic fexofenadine simultaneously with water or apple juice. For the uptake studies, oocytes expressing organic anion-transporting polypeptide 2B1 (OATP2B1) were incubated with 100 µM (R)- and (S)-fexofenadine in the presence or absence of 10 % apple juice. RESULTS: One-time ingestion of apple juice significantly decreased the area under the plasma concentration-time curve (AUC0-24) for (R)- and (S)-fexofenadine by 49 and 59 %, respectively, and prolonged the time to reach the maximum plasma concentration (t max) of both enantiomers (P < 0.001). Although apple juice greatly reduced the amount of (R)- and (S)-fexofenadine excretion into urine (Ae0-24) by 54 and 58 %, respectively, the renal clearances of both enantiomers were unchanged between the control and apple juice phases. For in vitro uptake studies, the uptake of both fexofenadine enantiomers into OATP2B1 complementary RNA (cRNA)-injected oocytes was significantly higher than that into water-injected oocytes, and this effect was greater for (R)-fexofenadine. In addition, apple juice significantly decreased the uptake of both enantiomers into OATP2B1 cRNA-injected oocytes. CONCLUSIONS: These results suggest that OATP2B1 plays an important role in the stereoselective pharmacokinetics of fexofenadine and that one-time apple juice ingestion probably inhibits intestinal OATP2B1-mediated transport of both enantiomers. In addition, this study demonstrates that the OATP2B1 inhibition effect does not require repeated ingestion or a large volume of apple juice.

Effects of verapamil on the pharmacokinetics and hepatobiliary disposition of fexofenadine in pigs.

The pharmacokinetics (PK) of fexofenadine (FEX) in pigs were investigated with the focus on exploring the interplay between hepatic transport and metabolism when administered intravenously (iv) alone or with verapamil. The in vivo pig model enabled simultaneous sampling from plasma (pre-liver, post-liver and peripheral), bile and urine. Each animal was administered FEX 35mg iv alone or with verapamil 35mg. Plasma, bile and urine were analyzed with liquid chromatography-tandem mass spectrometry. Non-compartmental analysis (NCA) was used to estimate traditional PK parameters. In addition, a physiologically based pharmacokinetic (PBPK) model consisting of 11 compartments (6 tissues +5 sample sites) was applied for mechanistic elucidation and estimation of individual PK parameters. FEX had a terminal half-life of 1.7h and a liver extraction of 3%. The fraction of the administered dose of unchanged FEX excreted into the bile was 25% and the bile exposure was more than 100 times higher than the portal vein total plasma exposure, indicating carrier-mediated (CM) disposition processes in the liver. 23% of the administered dose of FEX was excreted unchanged in the urine. An increase in FEX plasma exposure (+50%) and a decrease in renal clearance (-61%) were detected by NCA as a direct effect of concomitant administration of verapamil. However, analysis of the PBPK model also revealed that biliary clearance was significantly inhibited (-53%) by verapamil. In addition, PBPK analysis established that metabolism and CM uptake were important factors in the disposition of FEX in the liver. In conclusion, this study demonstrated that CM transport of FEX in both liver and kidneys was inhibited by a single dose of verapamil.

Effects of the P-glycoprotein inducer carbamazepine on fexofenadine pharmacokinetics.

The aim of this study was to evaluate the possible effects of carbamazepine, a P-glycoprotein inducer, on fexofenadine pharmacokinetics. Twelve healthy Japanese volunteers (nine males and three females) were enrolled in this study after giving written informed consent. This randomized open-label study consisted of two phases (control and 7-day treatment) with a 2-week washout period. In the control phase, volunteers received 60 mg fexofenadine hydrochloride after an overnight fast. In the treatment phase, carbamazepine was dosed 100 mg three times daily (for a total daily dose of 300 mg) for 7 days, and on Day 7, a single 60-mg dose of fexofenadine was coadministered with a 100-mg dose of carbamazepine. The plasma concentrations and urinary excretion of fexofenadine were measured for 24 hours after dosing. Carbamazepine pretreatment significantly altered fexofenadine pharmacokinetics, decreasing the mean (+/- standard deviation) peak plasma concentration from 176.6 (+/- 82.1) ng/mL to 103.2 (+/- 33.6) ng/mL (P < 0.01) and the area under the plasma concentration-time curve from 1058.4 (+/- 528.7) ng/h/mL to 604.8 (+/- 255.9) ng/h/mL (P < 0.01) without changing the elimination half-life. Relatively, carbamazepine significantly reduced the amount of fexofenadine excreted into the urine from 8.1 (+/- 2.1) mg to 4.5 (+/- 1.4) mg (P < 0.001), although the renal clearance of fexofenadine remained constant between the two study phases. Thus, this study indicates that carbamazepine significantly decreases fexofenadine plasma concentrations, probably as a result of P-glycoprotein induction in the small intestine. Carbamazepine treatment, therefore, is of moderate clinical significance for patients receiving fexofenadine.

Laser ablation electrospray ionization for atmospheric pressure, in vivo, and imaging mass spectrometry.

Mass spectrometric analysis of biomolecules under ambient conditions promises to enable the in vivo investigation of diverse biochemical changes in organisms with high specificity. Here we report on a novel combination of infrared laser ablation with electrospray ionization (LAESI) as an ambient ion source for mass spectrometry. As a result of the interactions between the ablation plume and the spray, LAESI accomplishes electrospray-like ionization. Without any sample preparation or pretreatment, this technique was capable of detecting a variety of molecular classes and size ranges (up to 66 kDa) with a detection limit of 8 and 25 fmol for verapamil and reserpine, respectively, and quantitation capabilities with a four-decade dynamic range. We demonstrated the utility of LAESI in a broad variety of applications ranging from plant biology to clinical analysis. Proteins, lipids, and metabolites were identified, and antihistamine excretion was followed via the direct analysis of bodily fluids (urine, blood, and serum). We also performed in vivo spatial profiling (on leaf, stem, and root) of metabolites in a French marigold (Tagetes patula) seedling.

Lack of dose-dependent effects of itraconazole on the pharmacokinetic interaction with fexofenadine.

The aim of this study was to determine the inhibitory effect of itraconazole at different coadministered doses on fexofenadine pharmacokinetics. In a randomized four-phase crossover study, 11 healthy volunteers were administered a 60-mg fexofenadine hydrochloride tablet alone on one occasion (control phase) and with three different doses of 50, 100, and 200 mg of itraconazole simultaneously on the other three occasions (itraconazole phase). Although the elimination half-life and the renal clearance of fexofenadine remained relatively constant, a single administration of itraconazole with fexofenadine significantly increased mean area under the plasma concentration-time curve (AUC(0-infinity)) of fexofenadine (1701/3554, 4308, and 4107 ng h/ml for control; 50 mg, 100 mg, and 200 mg of itraconazole, respectively). Although mean itraconazole AUC(0-48) from 50 mg to 200 mg increased dose dependently from 214 to 772 ng h/ml (p = 0.003), no significant difference was noted in the three parameters, AUC (p = 0.423), C(max) (p = 0.636), and renal clearance (p = 0.495), of fexofenadine among the three doses of itraconazole. Itraconazole exposure at a lower dose (50 mg) compared with the clinical dose (200 mg once or twice daily) had the maximal effect on fexofenadine pharmacokinetics, even though itraconazole plasma concentrations gradually increased after higher doses. These findings suggest that the interaction may occur at the gut wall before reaching the portal vein circulation, and the inhibitory effect must be saturated by substantial local concentrations of itraconazole in the gut lumen after 50-mg dosing.

Effects of itraconazole and diltiazem on the pharmacokinetics of fexofenadine, a substrate of P-glycoprotein.

AIMS: Fexofenadine is a substrate of several drug transporters including P-glycoprotein. Our objective was to evaluate the possible effects of two P-glycoprotein inhibitors, itraconazole and diltiazem, on the pharmacokinetics of fexofenadine, a putative probe of P-glycoprotein activity in vivo, and compare the inhibitory effect between the two in healthy volunteers. METHODS: In a randomized three-phase crossover study, eight healthy volunteers were given oral doses of 100 mg itraconazole twice daily, 100 mg diltiazem twice daily or a placebo capsule twice daily (control) for 5 days. On the morning of day 5 each subject was given 120 mg fexofenadine, and plasma concentrations and urinary excretion of fexofenadine were measured up to 48 h after dosing. RESULTS: Itraconazole pretreatment significantly increased mean (+/-SD) peak plasma concentration (Cmax) of fexofenadine from 699 (+/-366) ng ml-1 to 1346 (+/-561) ng ml-1 (95% CI of differences 253, 1040; P<0.005) and the area under the plasma concentration-time curve [AUC0,infinity] from 4133 (+/-1776) ng ml-1 h to 11287 (+/-4552) ng ml-1 h (95% CI 3731, 10575; P<0.0001). Elimination half-life and renal clearance in the itraconazole phase were not altered significantly compared with those in the control phase. In contrast, diltiazem pretreatment did not affect Cmax (704+/-316 ng ml-1, 95% CI -145, 155), AUC0, infinity (4433+/-1565 ng ml-1 h, 95% CI -1353, 754), or other pharmacokinetic parameters of fexofenadine. CONCLUSIONS: Although some drug transporters other than P-glycoprotein are thought to play an important role in fexofenadine pharmacokinetics, itraconazole pretreatment increased fexofenadine exposure, probably due to the reduced first-pass effect by inhibiting the P-glycoprotein activity. As diltiazem pretreatment did not alter fexofenadine pharmacokinetics, therapeutic doses of diltiazem are unlikely to affect the P-glycoprotein activity in vivo.

Different effects of three transporting inhibitors, verapamil, cimetidine, and probenecid, on fexofenadine pharmacokinetics.

OBJECTIVE: Fexofenadine is a substrate of P-glycoprotein and organic anion transporting polypeptides. The aim of this study was to compare the inhibitory effects of different transporting inhibitors on fexofenadine pharmacokinetics. METHODS: Twelve male volunteers took a single oral 120-mg dose of fexofenadine. Thereafter three 6-day courses of either 240 mg verapamil, an inhibitor of P-glycoprotein, 800 mg cimetidine, an inhibitor of organic cation transporters, or 2000 mg probenecid, an inhibitor of organic anion transporting polypeptides, were administered on a daily basis in a randomized fashion with the same dose of fexofenadine on day 6. Plasma and urine concentrations of fexofenadine were monitored up to 48 hours after dosing. RESULTS: Verapamil treatment significantly increased the peak plasma concentration by 2.9-fold (95% confidence interval [CI], 2.4- to 4.0-fold) and the area under the plasma concentration-time curve from time 0 to infinity [AUC(0-infinity)] of fexofenadine by 2.5-fold (95% CI, 2.0- to 3.3-fold). No changes in any plasma pharmacokinetic parameters of fexofenadine were found during cimetidine treatment. AUC(0-infinity) was slightly but significantly increased during probenecid treatment by 1.5-fold (95% CI, 1.1- to 2.4-fold). Renal clearance of fexofenadine was significantly decreased during cimetidine treatment to 61% (95% CI, 50%-98%) and during probenecid treatment to 27% (95% CI, 20%-58%) but not during verapamil treatment. CONCLUSION: This study suggests that verapamil increases fexofenadine exposure probably because of an increase in bioavailability through P-glycoprotein inhibition and that probenecid slightly increases the area under the plasma concentration-time curve of fexofenadine as a result of a pronounced reduction in renal clearance. However, it may be difficult to explain these interactions by simple inhibitory mechanisms on target transporters.

MDR1 gene polymorphisms and disposition of the P-glycoprotein substrate fexofenadine.

AIMS: The C3435T polymorphism in the human MDR1 gene is associated with lower intestinal P-glycoprotein expression, reduced protein function in peripheral blood cells and higher plasma concentrations of the P-glycoprotein substrate digoxin. Using fexofenadine, a known P-glycoprotein substrate, the hypothesis was tested whether this polymorphism also affects the disposition of other drugs in humans. METHODS: Ten Caucasian subjects homozygous for the wild-type allele at position 3435 (CC) and 10 individuals homozygous for T at position 3435 participated in this study. A single oral dose of 180 mg fexofenadine HCl was administered. Plasma and urine concentrations of fexofenadine were measured up to 72 h using a sensitive LC/MS method. In addition, P-glycoprotein function was assessed using efflux of the P-glycoprotein substrate rhodamine 123 from CD56+ cells. Results Fexofenadine plasma concentrations varied considerably among the study population. However, fexofenadine disposition was not significantly different between the CC and TT groups (e.g. AUC(0,infinity) CC vs TT: 3567.1+/-1535.5 vs 3910.1+/-1894.8 ng ml-1 h, NS; 95% CI on the difference -1364.9, 2050.9). In contrast, P-glycoprotein function was significantly decreased in CD56+ cells of the TT compared with the CC group (rhodamine fluorescence CC vs TT: 45.6+/-7.2% vs 61.1+/-12.3%, P<0.05; 95% CI on the difference 5.6, 25.5). Conclusions In spite of MDR1 genotype-dependent differences in P-glycoprotein function in peripheral blood cells, there was no association of the C3435T polymorphism with the disposition of the P-glycoprotein substrate fexofenadine in this German Caucasian study population. These data indicate that other mechanisms including uptake transporter function are likely to play a role in fexofenadine disposition.

Determination of fexofenadine in human plasma and urine by liquid chromatography-mass spectrometry.

A sensitive method was developed to determine fexofenadine in human plasma and urine by HPLC-electrospray mass spectrometry with MDL 026042 as internal standard. Extraction was carried out on C18 solid-phase extraction cartridges. The mobile phases used for HPLC were: (A) 12 mM ammonium acetate in water and (B) acetonitrile. Chromatographic separation was achieved on a LUNA CN column (10 cm x 2.0 mm I.D., particle size 3 microm) using a linear gradient from 40% B to 60% B in 10 min. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH+ ions, m/z 502.3 for fexofenadine and m/z 530.3 for the internal standard. The limit of quantification achieved with this method was 0.5 ng/ml in plasma and 1.0 ng in 50 microl of urine. The method described was successfully applied to the determination of fexofenadine in human plasma and urine in pharmacokinetic studies.

Investigation of the stereoselective metabolism of the chiral H1-antihistaminic drug terfenadine by high-performance liquid chromatography.

The enantiomers of the racemic H1-antihistaminic drug terfenadine (1) have been resolved by fractional crystallization of the diastereomeric salts with optically active 2-chlorotartranilic acid. The enantiomeric excess of both terfenadine enantiomers was determined using an achiral and a chiral HPLC system after formation of diastereomers with S-(+)-naphthylethylisocyanate. To investigate the metabolism of terfenadine after oral administration, an achiral HPLC system, equipped with a conventional reversed-phase column, was used to quantify the main metabolite MDL 16.455 (2) in human serum and urine. The determination of the enantiomeric composition of 2 was achieved using an Ultron ES-OVM column as chiral stationary phase. Metabolite 2, extracted from human blood plasma, was found to be enriched in the R-enantiomer, but was excreted in urine as racemate. The results of a study including six volunteers are presented.

Direct enantiomeric separation of terfenadine and its major acid metabolite by high-performance liquid chromatography, and the lack of stereoselective terfenadine enantiomer biotransformation in man.

Direct enantiomeric separation of terfenadine and its major acid metabolite was achieved by using two different chiral stationary phase columns with two different mobile phase systems. Further, the enantiomeric composition of the human urinary acid metabolite has been determined, indicating a non-stereoselective biotransformation in man.

Determination of the metabolites of terfenadine in human urine by thermospray liquid chromatography-mass spectrometry.

Thermospray liquid chromatography-mass spectrometry (TSP LC-MS) was used to determine human urinary metabolites of terfenadine after oral administration of terfenadine tablets. In addition to the two previously identified major metabolites, azacyclonol (MDL 4829) and the 'acid' metabolite (MDL 16,455), three additional metabolites were also detected. One of the additional metabolites was identified as the 'alcohol' metabolite (MDL 17,523) and the other two were proposed to be an 'aldehyde' and a 'ketone-acid' metabolites from their TSP mass spectra. The results of this study demonstrated that TSP LC-MS is a useful technique for the study of terfenadine biotransformation.