Julie Ducharme

Dextromethorphan as an in vivo probe for the simultaneous determination of CYP2D6 and CYP3A activity

Dextromethorphan (DM) is O-demethylated into dextrorphan (DEX) in humans by the cytochrome P450 designated as CYP2D6 and N-demethylated into 3-methoxymorphinan (3MM) via CYP3As. Clinically, DM has been successfully used as an index of CYP2D6 and this paper describes analytical and clinical data that will help evaluate the use of DM hydrobromide as a probe of CYP3A activity. DM and its three demethylated metabolites were measured in a 4-h spot urine sample using a HPLC method employing solid-phase extraction (C(18)), analysis on a phenyl column [mobile phase, methanol-acetonitrile-phosphate buffer (10 mM, pH 3.5, 20:25:55, v/v)] and fluorescence detection (excitation at lambda=228 nm, no emission cut-off filter). The urinary molar ratio DM-DEX was used to assess CYP2D6 activity while DM-3MM was used for CYP3As. The DM-3MM ratios were sensitive to the co-administration of selective CYP3A inhibitors grapefruit juice and erythromycin. In addition, in healthy volunteers and cancer patients, the N-demethylation of DM correlated with the CYP3A-mediated metabolism of verapamil and tamoxifen. DM appears to be a promising way to simultaneously phenotype patients for CYP2D6 and CYP3As.

Influence of extreme obesity on the body disposition and neuromuscular blocking effect of atracurium

The pharmacokinetics and pharmacodynamics of atracurium, a nondepolarizing neuromuscular blocking agent, were compared between morbidly obese patients and nonobese patients. Atracurium besylate (0.2 mg/kg) was administered intravenously as a bolus to patients who had received anesthesia. The force of contraction of the adductor pollicis was measured and plasma samples were collected for a 2‐hour period. The concentrations of atracurium and its major end product, laudanosine, were determined by use of a chromatographic method. The pharmacokinetic‐pharmacodynamic relationship was characterized by use of several models. No difference was observed between obese patients and nonobese patients in atracurium elimination half‐life (19.8 ± 0.7 versus 19.7 ± 0.7 minutes), volume of distribution at steady state (8.6 ± 0.7 versus 8.5 ± 0.7 L), and total clearance (444 ± 29 versus 404 ± 25 ml/min). However, if values were expressed on a total body weight basis, there was a difference between obese and nonobese patients in the volume of distribution at steady state (0.067 versus 0.141 L/kg) and total clearance (3.5 ± 0.2 versus 6.6 ± 0.5 ml/min/kg). Although atracurium concentrations were consistently higher in obese patients than in nonobese patients, there was no difference in the time of recovery from neuromuscular blockade between the two groups. Consequently, the median effective concentration was higher in obese than in nonobese patients (470 ± 46 versus 312 ± 33 ng/ml).

Importance of Early Blood Sampling on Vecuronium Pharmacokinetic and Pharmacodynamic Parameters

SummaryThe effect of early blood sampling on the description of the vecuronium pharmacokinetic-pharmacodynamic relationship was studied following a bolus injection. Sample collection every 10 sec during the first 2 min showed a high concentration peak at 30 to 40 sec, accounting for an important proportion of the total area under the plasma concentration-time curve (AUC). Neglecting it, using only blood samples drawn at 1 and 2 min (limited sampling), led to a significant overestimation of noncompartmentally derived values of mean residence time, clearance, volume of distribution at steady-state and rate of transfer of vecuronium into the effect compartment. Compartmental pharmacokinetics could not be applied to concentration-time curves constructed with early samples, but limited sampling data were fitted to a 2-compartment model. Derived compartmental pharmacokinetic and pharmacokinetic-pharmacodynamic parameters were similar to those obtained noncompartmentally with complete sampling every 10 sec, because back-extrapolation to time zero contributed to the increase in the AUC. However, compartmental analysis does not provide an accurate description of concentration changes following injection.

Pharmacokinetics and pharmacodynamics of atracurium obtained with arterial and venous blood samples

To determine the influence of sampling site on atracurium pharmacokinetic‐pharmacodynamic relationships, blood was drawn simultaneously from the radial artery and a peripheral vein during a 20‐minute period after injection of atracurium, 0.2 mg/kg, in eight patients. Atracurium and laúdanosme concentrations were measured by HPLC. Neuromuscular blockade was measured at the adductor pollicis, after stimulation of the ulnar nerve. Venous levels were lower than corresponding arterial values for up to 20 minutes, and this difference was marked for the early samples. Neuromuscular blockade was maximum after 5 to 7 minutes, much later than the peak venous concentration (1 to 3 minutes). Nonparametric analysis yielded (mean ± SEM) a rate constant, concentration for 50% blockade, and slope of the effect‐concentration relationship of 0.092 ±0.01 min−1, 379 ± 27 ng/ml, and 7.3 ± 1.67, respectively, when based on arterial samples. The values were statistically different (0.135 ± 0.011 min−1, 235 ± 42 ng/ml, and 3.41 ± 0.37, respectively) when venous levels were used (p < 0.05). It is concluded that forearm venous levels do not correspond to adductor pollicis neuromuscular blockade and the kinetics and kinetic‐dynamic relationship for atracurium are heavily dependent on sampling site.

The N-dechloroethylation of ifosfamide: using stereochemistry to obtain an accurate picture of a clinically relevant metabolic pathway.

Abstract The cumulative urinary excretions of the enantiomers of ifosfamide [(R)-IFF, (S)-IFF)] and their 2-N-dechloroethylated (2-DCE-IFF) and 3-N-dechloroethylated (3-DCE-IFF) metabolites were determined in 11 adult cancer patients who received a single 3-h infusion of IFF (3 g/m2) with mesna uroprotection. The urine samples were analyzed for the compounds of interest using an enantioselective gas chromatographic- mass spectrometric assay. The results indicated an enantioselective excretion of the parent and N-dechloroethylated metabolites: the urinary recovery of (R)-IFF was significantly greater than that of (S)-IFF (1.73± 0.45 vs 1.43±0.41 mmol, P<0.0001); the excretion of (S)-2-DCE-IFF (0.75±0.53 mmol) was greater than that of (R)-2-DCE-IFF (0.42± 0.22 mmol, P=0.071) while the excretion of (R)-3-DCE-IFF (1.64± 0.76 mmol) was greater than that of (S)-3-DCE-IFF (0.77±0.59 mmol, P=0.012). The study also revealed two distinct metabolic patterns in which the urinary recoveries of (R)-2-DCE-IFF and (R)-3-DCE-IFF were linked as were those of (S)-2-DCE-IFF and (S)-3-DCE-IFF. The results suggest that at least two enzymes are involved in the N-dechloroethylation of IFF. The data also demonstrate the importance of following the metabolic fate of (R)-IFF and (S)-IFF and of determining the relative urinary excretion of all dechloroethylated metabolites.

Stereoselective pharmacokinetics of ifosfamide and its 2- and 3-N-dechloroethylated metabolites in female cancer patients

The pharmacokinetics of the R and S enantiomers of ifosfamide (IFF) and of its 2- and 3-N-dechloroethylated metabolites (2-DCE-IFF and 3-DCE-IFF) were investigated in 14 cancer patients treated with a 3-h infusion of (R,S)-IFF (3 g/m2) with mesna uroprotection. An enantioselective gas chromato-graphic-mass spectrometric (GC-MS) assay was used to determine the concentrations in plasma and urine. The AUCs of (R)-IFF were significantly larger than those of (S)-IFF (2480 ± 200 vs 1960 ± 150 µM. h). The terminal half-lives (7.57 ± 0.99 h) and mean residence times (11.17 ± 1.10 h) of (R)-IFF were significantly longer than those of (S)-IFF, 6.03 ± 0.82 h and 9.37 ± 0.88 h, respectively. The mean volume of distribution at steady state of (R)-IFF (25.68 ± 0.80 1/m2) was slightly smaller than that of (S)-IFF (27.35 ± 0.89 1/m2). While the renal clearances of (R)-IFF and (S)-IFF were similar, the nonrenal clearance was significantly lower for (R)-IFF (30.20 ± 2.70 vs 41.40 ± 3.55 ml/m2 per min) as was total clearance (41.52 µ 2.90 vs 52.37 µ 3.75 ml/m2 per min). The AUC values for all of the DCE metabolites from (S)-IFF were significantly greater than those from (R)-IFF with 47% of the measured AUC accounted for by DCE from (S)-IFF compared to only 20% for (R)-IFF. Therefore, the enantioselective difference in IFF elimination can be partially explained by differences in N-dechloroethylation.

Using chirality as a unique probe of pharmacological properties

Abstract The development of enantioselective chromatographic techniques has made it feasible to routinely follow the metabolism and disposition of the separate enantiomers of a chiral drug. These studies are a source of data about in vivo pharmacological processes. The key question is recognition of the fundamental information contained within the results and the application of this data to the development of a deeper understanding of the clinical consequences of stereochemistry. This manuscript presents two examples of how chirality can be used as a unique probe of basic pharmacological properties.

Use of bioconjugation with cytochrome P450 enzymes

Bioconjugation, defined as chemical modification of biomolecules, is widely employed in biological and biophysical studies. It can expand functional diversity and enable applications ranging from biocatalysis, biosensing and even therapy. This review summarizes how chemical modifications of cytochrome P450 enzymes (P450s or CYPs) have contributed to improving our understanding of these enzymes. Genetic modifications of P450s have also proven very useful but are not covered in this review. Bioconjugation has served to gain structural information and investigate the mechanism of P450s via photoaffinity labeling, mechanism-based inhibition (MBI) and fluorescence studies. P450 surface acetylation and protein cross-linking have contributed to the investigation of protein complexes formation involving P450 and its redox partner or other P450 enzymes. Finally, covalent immobilization on polymer surfaces or electrodes has benefited the areas of biocatalysis and biosensor design. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Enzymes Beat Chemists in the Formation of an Unnatural Bond

A fundamental difference? A heme protein known to catalyze electron transfers was engineered into an enzyme that catalyzes the formation of C-Si bonds with >99 % ee. The new enzyme uses diazoesters as carbene donors for the insertion of various silanes into the Si-H bond. This is the first reported organosilicon-producing enzyme.