Identification of a novel CYP2C19-mediated metabolic pathway of S-citalopram in vitro.

Systemic exposure of the antidepressant S-citalopram (escitalopram, SCIT) differs several-fold according to variable cytochrome P450 2C19 activity, demonstrating the importance of this enzyme for the metabolic clearance of SCIT in vivo. However, previous studies have indicated that the involvement of CYP2C19 in formation of the metabolite N-desmethyl S-citalopram (SDCIT) is limited. Therefore, the purpose of the present in vitro study was to investigate to what extent the CYP2C19-mediated clearance of SCIT was due to a metabolic pathway different from N-desmethylation and to identify the product(s) of this possible alternative metabolic reaction. CYP2C19-mediated metabolism of SCIT was investigated using recombinant Supersomes expressing human CYP2C19. Initial experiments showed that approximately half of the CYP2C19-mediated clearance of SCIT was accounted for by the N-desmethylation pathway. Subsequent experiments identified that, in addition to SDCIT, the propionic acid metabolite of SCIT (SCIT PROP) was formed by CYP2C19 in vitro. Formation of SCIT PROP accounted for 35% of total CYP2C19-mediated clearance of SCIT (calculated as the ratio between metabolite formation rate and substrate concentration at low substrate concentration). Moreover, analysis of samples from six CYP2C19-genotyped patients treated with SCIT indicated that differences in serum concentrations of SCIT between CYP2C19 genotypes may be due to a combined effect on SCIT PROP and SDCIT formation. Identification of SCIT PROP as a metabolic pathway catalyzed by CYP2C19 might explain why impaired CYP2C19 activity has a substantially larger effect on SCIT exposure than estimated from in vitro data based solely on formation of SDCIT.

Significantly altered systemic exposure to atorvastatin acid following gastric bypass surgery in morbidly obese patients.

The impact of gastric bypass on atorvastatin pharmacokinetics was investigated in 12 morbidly obese patients being treated with 20-80 mg atorvastatin each morning. Eight-hour pharmacokinetic investigations were performed the day before the surgery and at a median of 5 weeks (range 3-6 weeks) after the surgery. Gastric bypass surgery produced a variable effect on individual systemic exposure to atorvastatin acid (area under the plasma concentration vs. time curve from 0 to 8 h postdose (AUC(0-8))), ranging from a threefold decrease to a twofold increase (median ratio = 1.1, P = 0.99). Patients with the highest systemic exposure to atorvastatin before surgery showed reduced exposure after surgery (n = 3, median ratio = 0.4, range = 0.3-0.5, P < 0.01), whereas those with lower systemic exposure before surgery showed a median 1.2-fold increase in atorvastatin AUC(0-8) (n = 9, range = 0.8-2.3, P = 0.03) after surgery. This study indicates that the presurgical first-pass metabolic capacity influences the effect of gastric bypass on atorvastatin bioavailability. Because individual first-pass metabolic capacity is not readily assessable clinically, retitration up to the lowest effective dose should be performed after the surgery.

Identification of unknown quaternary ammonium compounds in corneal epithelium and aqueous humor.

Bovine corneal epithelium and bovine aqueous humor are investigated for their content of quaternary ammonium compounds. In total, four compounds are found. Three of these are identified. For the fourth compound, a proposal for its structure is made on the basis of tandem mass spectrometry fragmentation spectra. The compounds investigated have m/z values of 146, 160, and 174. The compounds with m/z 146 are confirmed as acetylcholine (in corneal epithelium) and (3-carboxypropyl)-trimethylammonium (in both corneal epithelium and aqueous humor). The compound with m/z 174 is identified as butyrylcholine (in corneal epithelium). The compound with m/z 160 is probably acetyl-g-homocholine (in both corneal epithelium and aqueous humor). For both butyrylcholine and acetyl-g-homocholine, it is the first time the presence of these compounds in corneal epithelium or aqueous humor (or both) is described. Both acetylcholine and butyrylcholine are unstable compounds, which are probably susceptible to enzymatic degradation by acetylcholine-esterase and butytrylcholine- esterase, respectively.

Determination of atorvastatin and metabolites in human plasma with solid-phase extraction followed by LC-tandem MS.

The aim of the present study was to develop a chromatographic method for the analysis of atorvastatin, o- and p-hydroxyatorvastatin (acid and lactone forms) in human plasma after administration of atorvastatin at the lowest registered dose (10 mg) in clinical studies. Sample preparation was performed by solid-phase extraction and was followed by separation of the analytes on an HPLC system with a linear gradient and a mobile phase consisting of acetonitrile, water and formic acid. Detection was achieved by tandem mass spectrometry operated in the electrospray positive ion mode. Validation of the method for the compounds for which reference compounds were available (acid forms of atorvastatin, o- and p-hydroxyatorvastatin) showed linearity within the concentration range (0.2-30 ng/ml for atorvastatin acid and p-hydroxyatorvastatin acid, and 0.5-30 ng/ml for o-hydroxyatorvastatin acid) (r2 > or = 0.99, n = 5 for all analytes). Accuracy and precision (evaluated at 0.5, 3 and 30 ng/ml for atorvastatin, p-hydroxyatorvastatin and 1, 3 and 30 ng/ml for o-hydroxyatorvastatin) were both satisfactory. The detection limit was 0.06 ng/ml for atorvastatin and p-hydroxyatorvastatin, and 0.15 ng/ml for o-hydroxyatorvastatin. The method has been successfully applied in a clinical study where atorvastatin, o- and p-hydroxyatorvastatin (both acid and lactone forms) could be detected in a 24-h sampling interval after administration of the lowest registered dose of atorvastatin (10 mg) for one week.

Sample preparation and determination of gabapentin in venous and capillary blood using liquid chromatography-tandem mass spectrometry.

An analytical method for the determination of gabapentin in serum obtained from venous blood samples has been developed using high-performance liquid chromatography (HPLC)-tandem mass spectrometry. In addition, a comparative study between capillary plasma samples and venous serum samples was carried out. This demonstrates the potential for the use of the described analytical system using very small amounts of blood. As internal standard (S)-(+)-alpha-amino-cyclohexane-propionic acid hydrate was used. Gabapentin and the internal standard are structural isomers, but have different m/z values for the fragments after collision induced dissolution. Gabapentin has 172-->154 and 172-->136 transitions and amino-cyclohexane-propionic acid hydrate has a 172-->126 transition which can be detected in tandem MS. Analysis of gabapentin was carried out on a C8 HPLC column using an isocratic mobile phase consisting of ammonium acetate (pH 3.0; 5mM)-methanol (96:4, v/v). The analytical method was validated for venous serum samples. Limit of detection was 1.6ng/ml and lower limit of quantification was 7.5ng/ml. R.S.D. values and bias values were within the range of acceptance for all concentration levels. The method developed for venous serum samples is being used in a gabapentin monitoring study using population pharmacokinetic modeling.

Sample preparation and determination of acetylcholine in corneal epithelium cells using liquid chromatography-tandem mass spectrometry.

A sample preparation method with subsequent liquid chromatography (LC)-mass spectrometry (MS)-MS analysis for acetylcholine in corneal epithelium is developed. The sample preparation is developed with a focus on compatibility with the LC-MS-MS system and the stability of acetylcholine because acetylcholine esterase is present in the tissue. It appears that both acetylcholine as well as the internal standard (IS) used (acetyl-beta-methylcholine) have fragments at m/z values in the tandem MS spectrum, which correspond with the m/z values of fragments of endogenous substances. Acetylcholine and (3-carboxypropyl)triethylammonium both have 146-->87 and 146-->60 transitions. Acetyl-beta-methylcholine and an unknown compound both have 160-->101 and 160-->60 transitions. This makes it necessary to use a chromatographic step, which has a baseline separation between these endogenous compounds, acetylcholine, and the IS. The analytical procedure has linearity from 1 ng/mL (30 pg/mg corneal epithelium tissue) to at least 250 ng/mL (7.55 ng/mg corneal epithelium tissue). The limits of detection and quantitation are 15 and 45 pg on column, respectively. Relative standard deviation and bias values are within the range of acceptance for all concentration levels.

Evaluation of essential parameters in the chromatographic determination of cyclosporin A and metabolites using a D-optimal design.

The extensive use of routine monitoring of cyclosporin A (INN, ciclosporin) whole blood levels of patients undergoing such therapy has resulted in a wide variety of chromatographic conditions for analysing this drug. The aim of this study was to evaluate the importance of essential parameters in the chromatographic determination of cyclosporin A and its main metabolites, AM1, AM9 and AM4N. A D-optimal design was used to evaluate the effect of type and amount of organic modifier, temperature, flow rate, pH and gradient steepness. The optimal chromatographic conditions were determined by multi-linear regression. In the final chromatographic method separation of the compounds was carried out on a reversed phase C(8) column maintained at 80 degrees C. The mobile phase consisted of a linear gradient with two mobile phases containing acetonitrile and water. The flow rate was set at 0.8 ml/min. UV detection was carried out at 214 nm. Validation of the analytical method showed linearity over the range 25-1000 ng/ml (r>0.997). The detection limits of cyclosporin A, AM1, AM9 and AM4N were 1.3 pmol on column. The within-day and between-day relative standard deviations were <15% for cyclosporin A at all concentrations and for the metabolites at 250 and 1000 ng/ml, and <21% for the metabolites at limit of quantification (25 ng/ml).

Intake of grapefruit juice alters the metabolic pattern of cyclosporin A in renal transplant recipients.

OBJECTIVE: The aim of the present study was to investigate the effect of grapefruit juice on the pharmacokinetics of cyclosporin A (CsA), as Sandimmun Neoral, and its main metabolites, M1, M9 and M4N, in renal transplant recipients. METHODS: Ten renal transplant recipients, on CsA-based immunosuppressive therapy, were included in this open, randomized crossover study. Patients were given their individualized morning dose of CsA, administered with either 250 ml water or 250 ml grapefruit juice and 12-hour CsA pharmacokinetic investigations were performed. The 2 investigation days were separated by at least 7 days. RESULTS: Administration of CsA with grapefruit juice compared with water significantly increased the area under the whole blood concentration versus time curve in the interval from 0-12 hours (AUC(0-12)) of CsA, by an average of 25 +/- 19% (p = 0.002). Intake of grapefruit juice did not have any significant influence on maximum whole blood concentration (Cmax) or time to Cmax (tmax) of CsA. AUC(0-12) and Cmax of M9 decreased significantly with intake of grapefruit juice, on average 22 +/- 11% (p = 0.0007) and 36 +/- 6% (p = 0.0001), respectively. AUC(0-12) of M1, however, was on average 13 +/- 14% (p = 0.02) higher upon co-administration of CsA with grapefruit juice as compared with water. The level of M4N was below the limit of quantification in most samples, and an effect of co-administration of CsA with grapefruit juice could not be determined for this metabolite. CONCLUSION: The present study shows that co-administration of grapefruit juice with CsA compared with water affects the formation and/or elimination of the 2 metabolites M1 and M9 differently. In addition, administration of CsA with grapefruit juice compared with water induced a moderate, but significant increase in systemic exposure of CsA in renal transplant recipients.