Gerard Hopfgartner

Pharmacokinetics and pharmacodynamics of the endothelin-receptor antagonist bosentan in healthy human subjects

Bosentan (Ro 47‐0203) is a potent and mixed ETA‐ and ETB‐receptor antagonist. Its activity has been studied in a variety of preclinical disease models.

Exact mass measurement of product ions for the structural elucidation of drug metabolites with a tandem quadrupole orthogonal-acceleration time-of-flight mass spectrometer

We herein report upon an approach whereby the interpretation of tandem mass spectrometry spectra can be both expedited and simplified via the accurate mass assignment of product ions utilizing a tandem quadrupole time-of-flight mass spectrometer (QqTOF). The applicability of the QqTOF in the drug metabolism laboratory is illustrated by the elucidation and differentiation of the dissociative pathways for Bosentan and its hydroxylated and demethylated metabolites. Target analyte fragmentation mechanisms were readily achieved by the measurement of product ions with a mass accuracy <5 ppm, possible by single-point internal recalibration using the residual precursor ion as calibrant. Differentiation of both precursor and product ions from nominally isobaric matrix species derived from biological extracts is demonstrated by operation of the QqTOF at resolutions of ∼8000 (m/ΔFWHM).

P‐Glycoprotein and Surfactants: Effect on Intestinal Talinolol Absorption

Surfactants used in pharmaceutical formulations can modulate drug absorption by multiple mechanisms including inhibition of intestinal P‐glycoprotein (P‐gp). Our objective was to analyze the effect of 2 surfactants with different affinity for P‐gp in vitro on the intestinal absorption and bioavailability of the P‐gp substrate talinolol in humans.

Determination of carnitine and acylcarnitines in urine by high-performance liquid chromatography–electrospray ionization ion trap tandem mass spectrometry

A high-performance liquid chromatography-mass spectrometry method has been developed for the simultaneous determination of native carnitine and eight acylcarnitines in urine. The procedure uses a solid-phase extraction on a cation-exchange column and the separation is performed without derivatization within 17 min on a reversed-phase C8 column in the presence of a volatile ion-pairing reagent. The detector was an ion trap mass spectrometer and quantification was carried out in the MS-MS mode. Validation was done for aqueous standards at ranges between 0.75 and 200 micromol/l, depending on the compound. Carnitine was quantified in urine and comparison with a radioenzymatic assay gave a satisfactory correlation (R2 = 0.981). The assay could be successfully applied to the diagnostic of pathological acylcarnitines profile of metabolic disorders in urines of patients suffering from different organic acidurias.

Multiple-dose pharmacokinetics, safety, and tolerability of bosentan, an endothelin receptor antagonist, in healthy male volunteers

The multiple‐dose pharmacokinetics, safety, and tolerability of oral bosentan, a selective endothelin receptor antagonist, were investigated in healthy male volunteers. In study A, an ascending‐dose, double‐blind, placebo‐controlled trial, doses of 100, 200, 500, and 1000 mg bosentan or placebo were given once daily for 8 days as tablets (100 and 500 mg dose strength). In study B, a double‐blind, placebo‐controlled trial, 500 mg tablets of bosentan or placebo tablets were given once daily for 8 days with two additional single intravenous dose administrations of 250 mg bosentan 48 hours before the first and 24 hours after the last oral dose. The drug was very well tolerated. No effects on pulse rate, ECGs, or clinical laboratory tests were observed. Marginal effects on blood pressure were seen in subjects only when standing. The oral bioavailability of bosentan was 43% to 48%, with a small interindividual variability of 20%. Doses above 500 mg did not lead to significant further increases in plasma levels of bosentan. From the first to the last day of the oral treatment phase, plasma concentrations of bosentan decreased by 30% to 40% due to a 2‐fold increase in plasma clearance. Absorption and plasma protein binding did not change. The 24‐hour urinary excretion of 6β‐hydroxycortisol was increased in parallel by approximately 1.7‐fold, indicating induction of cytochrome P450 3A isozymes. The two metabolites of bosentan reached plasma concentrations well below those of bosentan and will most likely not contribute to the pharmacological activity.

Metabolic Profiles of Minimally Absorbed Orlistat in Obese/Overweight Volunteers

To determine the metabolic profile of minimally absorbed orlistat in obese/overweight patients, an open‐label, single‐dose study was performed in eight obese/overweight volunteers between 23 and 68 years of age. Each subject received a single oral dose of 360 mg orlistat containing approximately 400 μCi of 14C‐labeled orlistat. Serial blood samples were collected at specified times over 10 hours after administration of orlistat for determination of total radioactivity, unchanged orlistat, and major metabolites in the plasma. Urine samples were collected over 24 hours and analyzed to evaluate the urinary recovery of total radioactivity and the profile of orlistat metabolites in the urine. In addition, all fecal samples were collected and analyzed for total radioactivity. Urinary and fecal recovery of the administered dose of total radioactivity were 1.13 ± 0.50% (24‐hour data only) and 96.4 ± 18.1% (n = 7), respectively. Maximum observed concentration (Cmax) and time to Cmax (tmax) values of plasma total radioactivity were 150 ± 51 ng·eq/mL and 6.8 ± 1.5 hrs, respectively. All these parameters obtained in obese/overweight subjects were similar to those reported previously in healthy subjects. On the basis of the area under the concentration‐time curve from 0 to 10 hours (AUC0–10), two major metabolites comprise a total of ∼42% of the total radioactivity in plasma. The primary metabolite (M1) has a short half‐life (∼2 hours), whereas the secondary metabolite (M3) disappeared at a slower rate. No strikingly apparent difference in the urinary metabolic profile was observed between two gender groups. It is concluded that the disposition of orlistat appears to be similar between normal and obese/overweight subjects. Of the minimal fraction of the dose that was absorbed systemically, the presence of two major metabolites accounts for ∼42%.

Self-assembly of multinuclear coordination species with chiral bipyridine ligands: silver complexes of 5,6-CHIRAGEN (o,m,p-xylidene) ligands and equilibrium behaviour in solution

The complexation reactions between Ag- and a series of enantiopure ligands belonging to the CHIRAGEN (from CHIRAlity GENerator) family (L1, L2, L3, based on (-)-5,6-pinene bipyridine) have been studied in solution. It has been shown that the length of the bridge plays a fundamental role in the self-assembly processes leading to different compounds: mononuclear complexes (with L3), mixtures of polynuclear complexes (with L2) and circular helicates (with L 1). Although the absolute configuration of the chiral centres in all three ligands is the same, the metal-centred chirality of L3 (delta) is inverted with respect to that in the other two complexes with L1 and L2 (delta). The metal configuration is thus opposite in the mononuclear complex with respect to the polynuclear species. Detailed thermodynamic studies were carried out for the Ag+ and L1 ligand system by 1H and 109Ag NMR spectroscopy (as a function of concentration, temperature and pressure). At low temperature and high pressure, the [Ag6L1(6)]6+ hexanuclear circular helicate forms a tetranuclear circular helicate [Ag4L1(4)]4+: 2[Ag6L1(6)]6+ <=> 3 [Ag4L1(4)]4+. The thermodynamics parameters, obtained by temperature and pressure variation, have the following values: K298 = (8.7 +/- 0.7) x 10(-5) mol x kg(-1), deltaHo = -15.65 +/- 0.8 kJ x mol(-1), deltaSo = -130.2 +/- 3 J x mol(-1) x K(-1) and deltaVo(256 K)= -160 +/- 12 cm3 x mol(-1). The reaction volume calculated according to Connollys method indicates that the calculated structure of [Ag4L1(4)]4+ is plausible. Both the signs and large magnitudes of deltaSo and deltaVo are counterintuitive, yet can be understood by modelling methods.

Ion spray-tandem mass spectrometry of supramolecular coordination complexes

Double-helical [M2L2]n+, triple-helical [M2L3]n+, and toroidal [M3L3]n+ (M = Cu, Co, Fe, Ni, La, Eu, Gd, Tb, or Lu) supramolecular complexes have been fully characterized by ion spray mass spectrometry (IS-MS). The IS-MS spectra from pure acetonitrile solutions reflect the nature of the cations present in solution with conservation of the charge state and allow an efficient qualitative speciation of the compounds. The mass spectrometry results can be correlated with other powerful techniques (nuclear magnetic resonance and electronic spectroscopy) for the characterization of supramolecular complexes in solution, Structural information is obtained by collision-induced dissociation, which strongly depends on the metal ions used in the supramolecular complexes and on the various connectivities and topologies of the ligands. When the ligand contains 3,5dimethoxybenzyl groups bound to the benzimidazole rings, the partial fragmentation of the complexes is associated with a decrease of the total charge of the complexes and the appearance of the characteristic fragment at m/z 151 that corresponds to the 3,5-dimethoxybenzyl cation. A detailed analysis of the fragmentation pathways of these supramolecular complexes suggests that the metal-nitrogen coordination bonds are very strong in the gas phase.

High-throughput quantification of drugs and their metabolites in biosamples by LC-MS/MS and CE-MS/MS: Possibilities and limitations

Off-line solid phase extraction with C18 disk plates and turbulent flow chromatography were evaluated versus on-line solid phase extraction using column-switching HPLC as sample preparation techniques for high-throughput analysis of pharmaceutical compounds and their metabolites by LC-MS/MS. Turbulent flow chromatography was found to be very straightforward in its applicaton, but the LOQs were more than fivefold higher compared with off-line or other on-line solid phase extraction methods. Solid phase extraction (SPE) on disk was found to be fast and sufficient efficient to minimize matrix effects and therefore an apprach to provide sensitive and reliable LC-MS/MS methods. Column-switching HPLC with microbore columns (0.5 mm i.d.) were used for fast analysis of a parent drug and four of its metabolites utilizing steep gradients in 1 minute. The application of CZE-MS/MS for bionalysis of pharamaceutical compounds is also discussed.

Dinuclear metal complexes of Cd(II), Zn(II) and Fe(II) with triple-helical structure and predetermined chirality

Abstract The complex formation of members of the so-called chiragen ligand family (stereoselective linked bis-[4,5]-pineno-2,2′-bipyridines) with Cd(II), Zn(II) and Fe(II) was investigated. Using spectrophotometric titrations and electrospray MS it was determined that the major species formed in solution are complexes with an M:L=2:3 stoichiometry. Based on circular dichroism (CD) and NMR measurements, a triple-helical structure with enantiomerically pure homochiral configuration at the metal centers is proposed for these dinuclear complexes. This is one of the first examples of a spontaneous self-assembly process leading to a dinuclear triple-helicate with pronouced preference for the formation of one of the possible stereoisomers.