Kurt-Jürgen Hoffmann

The Direct Thrombin Inhibitor Melagatran and Its Oral Prodrug H 376/95: Intestinal Absorption Properties, Biochemical and Pharmacodynamic Effects

UNLABELLED Suboptimal gastrointestinal absorption is a problem for many direct thrombin inhibitors. The studies presented herein describe the new oral direct thrombin inhibitor H 376/95, a prodrug with two protecting residues added to the direct thrombin inhibitor melagatran. Absorption properties in vitro: H 376/95 is uncharged at intestinal pH while melagatran is charged. H 376/95 is 170 times more lipophilic (octanol water partition coefficient) than melagatran. As a result, the permeability coefficient across cultured epithelial Caco-2 cells is 80 times higher for H 376/95 than for melagtran. Pharmacokinetic studies in healthy volunteers: H 376/95 is converted to melagatran in man. Oral bioavailability, measured as melagatran in plasma, is about 20% after oral administration of H 376/95, which is 2.7-5.5 times higher than after oral administration of melagatran. The variability in the area under the drug plasma concentration vs. time curve (AUC) is much smaller with oral H 376/95 (coefficient of variation 20%) than with oral melagatran (coefficient of variation 38%). Pharmacodynamic properties: H 376/95 is inactive towards human alpha-thrombin compared with melagatran [inhibition constant (K(i)) ratio, 185 times], a potential advantage for patients with silent gastrointestinal bleeding. In an experimental thrombosis model in the rat, oral H 376/95 was more effective than the subcutaneous low molecular weight heparin dalteparin in preventing thrombosis. CONCLUSION By the use of the prodrug principle, H 376/95 endows the direct thrombin inhibitor melagatran with pharmacokinetic properties required for oral administration without compromising the promising pharmacodynamic properties of melagatran.

INFLUENCE OF ERYTHROMYCIN ON THE PHARMACOKINETICS OF XIMELAGATRAN MAY INVOLVE INHIBITION OF P-GLYCOPROTEIN-MEDIATED EXCRETION

A pharmacokinetic interaction between erythromycin and ximelagatran, an oral direct thrombin inhibitor, was demonstrated in this study in healthy volunteers. To investigate possible interaction mechanisms, the effects of erythromycin on active transport mediated by P-glycoprotein (P-gp) in vitro in Caco-2 and P-gp-over-expressing Madin-Darby canine kidney-human multidrug resistance-1 cell preparations and on biliary excretion of melagatran in rats were studied. In healthy volunteers (seven males and nine females; mean age 24 years) receiving a single dose of ximelagatran 36 mg on day 1, erythromycin 500 mg t.i.d. on days 2 to 5, and a single dose of ximelagatran 36 mg plus erythromycin 500 mg on day 6, the least-squares mean estimates (90% confidence intervals) for the ratio of ximelagatran with erythromycin to ximelagatran given alone were 1.82 (1.64–2.01) for the area under the concentration-time curve and 1.74 (1.52–2.00) for the maximum plasma concentration of melagatran, the active form of ximelagatran. Neither the slope nor the intercept of the melagatran plasma concentration-effect relationship for activated partial thromboplastin time statistically significantly differed as a function of whether or not erythromycin was administered with ximelagatran. Ximelagatran was well tolerated regardless of whether it was administered with erythromycin. Erythromycin inhibited P-gp-mediated transport of both ximelagatran and melagatran in vitro and decreased the biliary excretion of melagatran in the rat. These results indicate that the mechanism of the pharmacokinetic interaction between oral ximelagatran and erythromycin may involve inhibition of transport proteins, possibly P-gp, resulting in decreased melagatran biliary excretion and increased bioavailability of melagatran.

Effects of artemisinin antimalarials on Cytochrome P450 enzymes in vitro using recombinant enzymes and human liver microsomes: potential implications for combination therapies.

Abstract 1. Cytochrome P450 enzyme system is the most important contributor to oxidative metabolism of drugs. Modification, and more specifically inhibition, of this system is an important determinant of several drug–drug interactions (DDIs). 2. Effects of the antimalarial agent artemisinin and its structural analogues, artemether, artesunate and dihydroartemisinin, on seven of the major human liver CYP isoforms (CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6 and 3A4) were evaluated using recombinant enzymes (fluorometric assay) and human liver microsomes (LC–MS/MS analysis). Inhibitory potency (IC50) and mechanisms of inhibition were evaluated using nonlinear regression analysis. In vitro–in vivo extrapolation using the [I]/Ki ratio was applied to predict the risk of DDI in vivo. 3. All compounds tested inhibited the enzymatic activity of CYPs, mostly through a mixed type of inhibition, with CYP1A2, 2B6, 2C19 and 3A4 being affected. A high risk of interaction in vivo was predicted if artemisinin is coadministrated with CYP1A2 or 2C19 substrates. 4. With respect to CYP1A2 inhibition in vivo by artemisinin compounds, our findings are in line with previously published data. However, reported risks of interaction may be overpredicted and should be interpreted with caution.

A rapid and selective HPLC-UV method for the quantitation of efavirenz in plasma from patients on concurrent HIV/AIDS and tuberculosis treatments

Owing to heterogeneity in therapeutic response, efavirenz is of research and clinical interest. There is a need to quantitate it using noncostly and selective methods. A method for efavirenz quantitation in plasma containing HIV and tuberculosis drugs was developed. Chromatographic separation was carried out using a C18 column. The mobile phase consisted of 0.1% formic acid and acetonitrile, and was pumped at a flow rate of 0.3 mL/min. Efavirenz and ritonavir (internal standard) were monitored at 247 nm. Plasma proteins were precipitated by centrifugation. The analysis time was 6 min. The response was linear (r = 0.9997). The accuracy ranged between 98 and 115% (intraday) and between 99 and 117% (interday). The precision ranged from 1.670 to 4.087% (intraday) and from 3.447 to 13.347% (interday). Recovery ranged from 98 to 132%. Stability ranged between 99 and 123%. The selectivity was proven by analysis of drugs used for the management of HIV/AIDS and tuberculosis. Plasma sample analysis showed an efavirenz retention time of 5.57 min and a peak plasma concentration of 2.4 µg/mL occurring at 2 h. This method is rapid and selective, and thus suitable for monitoring efavirenz in patients with HIV/AIDS alone or co-infected with tuberculosis in a less resourced setting.

A high-throughput LC-MS/MS assay for quantification of artesunate and its metabolite dihydroartemisinin in human plasma and saliva.

AIM Saliva is an alternative sampling matrix to plasma, offering a noninvasive technique, but requires a highly sensitive bioanalytical method. MATERIALS & METHODS An API 3000 triple quadrupole mass spectrometer with an electrospray ionization source operated in the positive ion mode was used for the analysis. RESULTS A high-throughput LC-MS/MS method using SPE for the quantification of artesunate and dihydroartemisinin in plasma and saliva has been optimized and validated according to US FDA guidelines. For both analytes the LLOQ was determined to 5 ng/ml and the calibration range was 5-1000 ng/ml for artesunate and 5-2000 ng/ml for dihydroartemisinin. CONCLUSION For the first time, a bioanalytical method for determination of artesunate and dihydroartemisinin in human saliva has been described, showing possible applicability in clinical saliva samples in addition to plasma samples.

Biliary Excretion of Ximelagatran and Its Metabolites and the Influence of Erythromycin Following Intraintestinal Administration to Healthy Volunteers

The biliary excretion of the oral thrombin inhibitor ximelagatran and its metabolites was investigated by using duodenal aspiration in healthy volunteers following intraintestinal dosing. In the first investigation, radiolabeled [14C]ximelagatran was administered, enabling quantification of the biliary excretion and identification of metabolites in the bile. In the second study, the effect of erythromycin on the biliary clearance of ximelagatran and its metabolites was investigated to clarify the reported ximelagatran‐erythromycin interaction. Approximately 4% of the intraintestinal dose was excreted into bile with ximelagatran and its active form, melagatran, being the most abundant compounds. Four novel ximelagatran metabolites were identified in bile (<0.1% of dose). Erythromycin changed the pharmacokinetics of ximelagatran and its metabolites, with an elevated ximelagatran (78% increase), OH‐melagatran (89% increase), and melagatran (86% increase) plasma exposure and higher peak plasma concentrations of the compounds being measured. In parallel, the biliary clearance was moderately reduced. The results suggest that inhibition of hepatobiliary transport is a likely mechanism for the interaction between erythromycin and ximelagatran. Furthermore, the study demonstrated the value of direct bile sampling in humans for the identification of primary biliary metabolites.

LC–MS/MS quantitation of antimalarial drug piperaquine and metabolites in human plasma

PURPOSE This study aimed to develop a sensitive, quantitative assay for the antimalarial piperaquine (PQ) and its metabolites M1 and M2 in human plasma. RESULTS Analytes were gradiently separated on a C18 column and detected with a Sciex API 4000 MS/MS with an ESI source operated in the positive ion mode with deuterated PQ as internal standard. The response was linear in the range 3.9-2508nM with a runtime of 7.0min per sample. The method was applied to clinical samples from healthy volunteers. CONCLUSION This LC-MS/MS method for the simultaneous quantitation of PQ and two of its metabolites in plasma may prove helpful for assessment of metabolite safety issues in vivo.