Pharmacokinetics and pharmacodynamics of Ro 44-3888 after single ascending oral doses of sibrafiban, an oral platelet aggregation inhibitor, in healthy male volunteers.

AIMS: This study constituted the first administration of the oral platelet inhibitor, sibrafiban, to humans. The aim was to investigate the pharmacokinetics and pharmacodynamics of Ro 44-3888, the active principle of sibrafiban, after single ascending oral doses of sibrafiban. Particular emphasis was placed on intersubject variability of the pharmacokinetic and pharmacodynamic parameters of Ro 44-3888. METHODS: The study consisted of three parts. Part I was an open ascending-dose study to determine target effect ranges of sibrafiban. Part II, a double-blind, placebo-controlled, parallel-group study, addressed the intersubject variability of pharmacokinetic and pharmacodynamic parameters of the active principle at a sibrafiban dose achieving an intermediate effect. Part III was a double-blind, placebo-controlled, ascending-dose design covering the complete plasma concentration vs pharmacodynamic response curve of sibrafiban. RESULTS: At sibrafiban doses between 5 mg and 12 mg, the pharmacokinetics of free Ro 44-3888 in plasma were linear whereas those of total Ro 44-3888 were non-linear because of the saturable binding to the glycoprotein IIb-IIIa receptor. Saturation of the GP IIb-IIIa receptor was reached at plasma concentrations of 15.9 ng ml-1. At sibrafiban doses up to 2 mg, ADP-induced platelet aggregation was inhibited by 50%, whereas the inhibition of TRAP-induced platelet aggregation was about 20-30%. At the higher doses, ADP-induced platelet aggregation was almost completely inhibited while a clear dose-response could be observed with TRAP-induced inhibition of platelet aggregation at sibrafiban doses of 5 to 12 mg. Ivy bleeding time increased very steeply with dose with a significant prolongation observed at doses of 5 to 7 mg of sibrafiban (5-7 min, >30 min in one case). At a sibrafiban dose of 12 mg, the stopping criterion for dose escalation (prolongation of the Ivy bleeding time >30 min in three out of four subjects per dose group) was reached. The interindividual coefficients of variation of the integrated pharmacokinetic and pharmacodynamic parameters (AUC and AUE) were below 20%, thus lying well within the pre-set level of acceptance. CONCLUSIONS: With a low intersubject variability of its pharmacokinetic and pharmacodynamic parameters, linear pharmacokinetics and pharmacodynamic effects closely related to its plasma concentrations, Ro 44-3888 has good pharmacological prerequisites for a well controllable therapy of secondary prevention of arterial thrombosis in patients with acute coronary syndrome.

Determination of the oral platelet aggregation inhibitor Sibrafiban in rat, dog, and human plasma utilising HPLC-column switching combined with turbo ion spray single quadrupole mass spectrometry.

A sensitive and selective HPLC-column switching method with single quadrupole mass spectrometric detection was developed for the simultaneous determination of the oral platelet aggregation inhibitor Sibrafiban (double protected prodrug), its prodrug and the active metabolite in rat, dog, and human plasma. The three analytes together with their tri-deuterated internal standards were isolated from plasma by protein precipitation (0.5 M perchloric acid). The de-proteinated samples were injected onto a standard-bore trapping column (4.0 mm i.d., LC-ABZ) of an HPLC-column switching system. Polar plasma components were removed by flushing the trapping column with ammonium formate (pH 3.6; 5 mM). Enriched compounds (including the analytes of interest) were backflushed onto a narrow-bore analytical column (2.1 mm i.d., Inertsil ODS-2) and separated by gradient elution (formic acid/ methanol). The whole effluent (200 microl/min) from the analytical column was passed to the turbo ion spray interface without splitting. Selected ion monitoring (SIM) was used for mass spectrometric detection. The limit of quantification for all three analytes was 1 ng/ml, using a 250-microl specimen of plasma. The mean precision and inaccuracy for the three analytes in all species were < 6 and < 5%, respectively. The practicability of the new analytical method was demonstrated by the analysis of about 500 rat and dog plasma and about 14,000 human plasma samples. The new method represents a successful example for the application of LC single MS with ionspray ionisation to the analysis of small molecule drugs in biological matrices from toxicokinetic studies and large clinical trials.

Oral platelet aggregation inhibitor Ro 48-3657: determination of the active metabolite and its prodrug in plasma and urine by high-performance liquid chromatography using automated column switching.

A sensitive and highly automated high-performance liquid chromatography (HPLC) column-switching method has been developed for the simultaneous determination of the active metabolite III and its prodrug II, both derivatives of the oral platelet inhibitor Ro 48-3657 (I), in plasma and urine of man and dog. Plasma samples were deproteinated with perchloric acid (0.5 M), while urine samples could be processed directly after dilution with phosphate buffer. The prepared samples were injected onto a pre-column of a HPLC column switching system. Polar plasma or urine components were removed by flushing the precolumn with phosphate buffer (0.1 M, pH 3.5). Retained compounds (including II and III) were backflushed onto the analytical column, separated by gradient elution and detected by means of UV detection at 240 nm. The limit of quantification for both compounds was 1 ng/ml (500 microl of plasma) and 25 ng/ml (50 microl of urine) for plasma and urine, respectively. The practicability of the new method was demonstrated by the analysis of about 6000 plasma and 1300 urine samples from various toxicokinetic studies in dogs and phase 1 studies in man.

Pharmacokinetics of retinoids in women after meal consumption or vitamin A supplementation.

These studies were conducted to evaluate the pharmacokinetics of several retinoids after meal consumption or vitamin A supplementation to establish a reference for future assessment of teratogenic risks of retinoid therapeutic agents. In the first study, 36 healthy young female volunteers consumed single meals containing vitamin A amounts ranging from 1,305 to 169,474 IU. In the second study, 24 other female volunteers took vitamin A supplements at a dose level of 5,000, 10,000, or 25,000 IU/day for 60 days. Plasma concentrations of tretinoin, isotretinoin, 4-oxo-tretinoin, and 4-oxo-isotretinoin in samples collected during the studies were analyzed using a high-performance liquid chromatography method with ultraviolet detection. Pharmacokinetic parameters for the retinoids were calculated using model-independent methods. Plasma concentrations of tretinoin were not altered by meal consumption or vitamin A supplementation. Plasma levels of 4-oxo-tretinoin were below the assay detection limit (0.3 ng/mL) in the majority of samples collected throughout the studies. Linear relationships between dose and maximum concentration (Cmax) and dose and area under the concentration-time curve (AUC) for isotretinoin and 4-oxo-isotretinoin were derived from data from the meal study. For the most bioavailable formulation used in the supplement study, daily ingestion of 5,000 IU of vitamin A caused increases of 141 +/- 53% and 171 +/- 77% from baseline in the 24-hour AUCs of isotretinoin and 4-oxo-isotretinoin, respectively. Dose-related increases in systemic exposure to retinoids were observed after ingestion of vitamin A by means of a meal or a supplement. Findings from these studies can be used as a basis for future safety evaluations of retinoid compounds.

Flumazenil kinetics in the elderly.

In an open design, randomised, two-way cross-over study, a single 2 mg i.v. dose and a single 30 mg oral dose of flumazenil were each administered to a group of healthy young (n = 6) and elderly (n = 12) volunteers (male: female 2/1). Plasma samples were collected at intervals and intact drug was assayed. Both the i.v. and oral doses of flumazenil were very well tolerated by both age groups and no severe or unexpected adverse effects were observed. The main complaints were dizziness and headache, mainly after oral dosing, probably due to the higher Cmax and AUC following this route of administration. After 2 mg i.v. the disposition parameters in the two age groups (elderly/young) were very similar: volume of distribution (Vss): 0.88/0.90 l.kg-1; total body clearance (ClPL): 0.86/0.99 l.min-1; terminal elimination half-life (t1/2 beta): 1.02/0.91 h. After the 30 mg oral dose the mean Cmax of 87.6 ng.ml-1 (elderly) and 78.4 ng.ml-1 (young) were generally reached within 0.5 to 1 h. In 26% (elderly) and 23% (young), the absolute bioavailability of flumazenil was very similar. It is concluded that the absorption and disposition parameters of flumazenil were not significantly affected by aging.

Combined and alternating ganciclovir and foscarnet in acute and maintenance therapy of human immunodeficiency virus-related cytomegalovirus encephalitis refractory to ganciclovir alone. A case report and review of the literature.

Cytomegalovirus (CMV) causes life-threatening disseminated infections and in particular vision-threatening infections of the retina in patients with the acquired immunodeficiency syndrome. Ganciclovir currently represents the most frequently used therapy for CMV retinitis. However, cases of ganciclovir-resistant CMV strains have been described, in which foscarnet seems to be an effective alternative. Both drugs have serious toxicities, and relapses frequently occur during maintenance therapy. In a patient with CMV encephalitis, we administered a 3-week combination ganciclovir/foscarnet induction therapy (ganciclovir 5 mg/kg every 12 h; foscarnet 60 mg/kg every 8 h), followed by an alternating maintenance administration of both drugs every other day (ganciclovir 5 mg/kg, foscarnet 120 mg/kg) to reduce toxicity and resistance. This regimen was tolerated well and seemed to be more effective than ganciclovir alone in a patient with CMV encephalitis.

Determination of the catechol-O-methyltransferase inhibitor Ro 40-7592 in human plasma by high-performance liquid chromatography with coulometric detection.

A sensitive and specific high-performance liquid chromatographic method has been developed to measure the catechol-O-methyl-transferase (COMT) inhibitor 3,4-dihydroxy-4'-methyl-5-nitrobenzophenone (Ro 40-7592) in human plasma. The compound and the internal standard were extracted from plasma at pH 2 with n-butyl chloride-ethyl acetate (95:5, v/v). The extract was chromatographed on a reversed-phase column (Hypersil ODS, 5 microns) using a mixture of phosphate buffer (0.05 M, pH 2), methanol and tetrahydrofuran (45:55:5, v/v/v) as the mobile phase. Long-retained components were removed from the system by means of a simple column-switching system. Quantification of the catechol-O-methyltransferase inhibitor was performed by means of coulometric detection (0.1 V). The limit of quantification was about 1 ng/ml, using a 1-ml specimen of plasma. The recovery from human plasma was greater than 88%. The mean inter-assay precision was 5.3% in the range 2.5-1000 ng/ml. Linearity of the standard curve was obtained in the concentration range 2.5-500 ng/ml. The catechol-O-methyltransferase inhibitor was stable in human plasma when stored for six months at -20 degrees C and for 24 h at room temperature. The practicability of the new method was demonstrated by the analysis of more than 400 plasma samples from a tolerance study performed in human volunteers.

Determination of the partial benzodiazepine receptor agonist Ro 16-6028 in plasma by capillary gas chromatography with nitrogen-selective detection after conversion into the ethyl ester derivative.

A highly sensitive capillary gas chromatographic method was developed to determine plasma levels of a novel partial benzodiazepine receptor agonist in man following the very low therapeutic doses required for anxiolysis. The compound was isolated from plasma by liquid-liquid extraction at basic pH, converted into the ethyl ester analogue by a two-step procedure, separated from plasma constituents by capillary gas chromatography and quantified by means of nitrogen-selective detection. Because of the thermolabile tert.-butyl ester function, the agonist could not be gas chromatographed without degradation. Formation of the far more stable ethyl ester analogue was achieved by treatment with hydrogen chloride in ethanol, followed by an ethylation step with diazoethane. The high sensitivity of the new method (about 100 pg/ml, using 1-ml plasma specimens) allowed the monitoring of plasma levels of the agonist for up to 8 h (about three elimination half-lives) after a single 0.1-mg oral dose to human volunteers. The practicability of the procedure was demonstrated by the analysis of more than 600 plasma samples from clinical studies performed with human volunteers.

Determination of a new non-benzodiazepine anxiolytic and its O-demethyl metabolite in plasma by high-performance liquid chromatography using automated column-switching.

An highly sensitive and fully automated high-performance liquid chromatographic assay was developed for the determination of a novel non-benzodiazepine anxiolytic (I) [(R)-2-(methoxymethyl)-1-[(7-oxo-8-phenyl-7H-thieno[2,3-a]quinolizin+ ++- 10-yl)carbonyl]pyrrolidine] and its O-demethyl metabolite (II) in plasma, using column-switching for direct injection of plasma samples. After dilution in internal standard solution, the sample was injected onto a pre-column (17 mm x 4.6 mm) dry-packed with pellicular C18 reversed-phase material. Polar plasma components were removed by flushing the pre-column with water-acetonitrile (90:10, v/v). Retained substances, including I and II, were backflushed onto an analytical column, separated by gradient elution and detected by means of fluorescence detection (excitation, 304 nm; emission, 475 nm). After washing the analytical column and re-equilibrating the pre-column, the system was ready for the next injection. The limit of quantification for I and II was 0.25 and 0.5 ng/ml, respectively, using a 350-microliter specimen of plasma. The practicability of the new method was demonstrated by analysis of more than 300 plasma samples from a tolerance study performed with human volunteers. Owing to its high sensitivity, the method can be used to calculate pharmacokinetic parameters of compounds I and II in man after a single oral dose of about 1 mg of I.

Experimental model for the investigation of kinetic and/or dynamic interactions between drugs and ethanol in humans.

This study was performed to establish an experimental method for the investigation of interactions between ethanol and drugs under predictable and controlled conditions. The model was tested with flumazenil (Ro 15-1788), a short-acting benzodiazepine antagonist with an elimination half-life of 1 h. Six healthy volunteers (5 males, 1 female) were administered ethanol by intravenous infusion with stepwise changing rates. The infusion rates were adapted to each subject on the basis of individual disposition parameters of ethanol, which were derived from preceding short-term infusions of 120 min duration (1.0 mg/kg in males, 0.8 mg/kg in the female). This two-step procedure led to individual ethanol plasma levels between 1.47 +/- 0.04 and 1.71 +/- 0.03 g/L, which were reached after 2.5 h and thereafter maintained over another 6 h. Within the period of constant ethanol levels, single doses of flumazenil and placebo, respectively, were injected intravenously as a bolus (2 min) in a double-blind fashion according to a randomized two-way crossover design. Three subjects received a dose of 0.10 mg/kg of flumazenil, and the remaining three subjects received a dose of 0.20 mg/kg. Evaluation of the plasma concentration time curves of flumazenil did not reveal evidence of an effect of ethanol on the pharmacokinetics of this drug.

Highly sensitive assay of benzodiazepine antagonist in plasma by capillary gas chromatography with nitrogen-selective detection.

A selective and highly sensitive capillary gas chromatographic method was developed for the determination of a benzodiazepine antagonist in human plasma. The analytical procedure involved extraction of the compound and its internal standard from basified plasma with n-butyl chloride-dichloromethane and chromatography of the extract on a DB-5 fused-silica column (30 m X 0.25 mm I.D.), applying automated splitless injection and nitrogen- phosphorus detection. The limit of quantification was about 50 pg/ml, using a 1-ml plasma specimen. The mean inter-assay precision was 2.6% in the concentration range 0.5-10 ng/ml. The method was shown to be specific with respect to various benzodiazepines and their main metabolites. The practicability of the method was demonstrated by the analysis of more than 300 plasma samples from a dose proportionality study performed with human volunteers. Owing to its high sensitivity, the new method can be used to obtain pharmacokinetic parameters of the benzodiazepine antagonist in man after doses near the envisaged therapeutic intravenous dose of less than 1 mg.

Determination of an aldosterone antagonist in urine by high-performance liquid chromatography using automated column switching.

An automated high-performance liquid chromatographic assay for the determination of an aldosterone antagonist (I) is described using column switching for direct injection of urine samples. After dilution with buffered internal standard solution, the sample was injected onto a clean-up column (17 X 4.6 mm I.D.), dry-packed with C18 reversed-phase material (particle size 30 micron). Polar urine components were removed by flushing the clean-up column with water. Retained substances, including I and the internal standard, were desorbed by backflush elution onto a 5-micron ODS-silica analytical column (125 X 4 mm I.D.), separated with water-methanol-tetrahydrofuran, and detected at 295 nm. After backflushing the analytical column and re-equilibrating the clean-up column, the system was ready for the next injection. The limit of quantification was ca. 100 ng/ml, using a 100-microliter specimen of diluted urine. The mean inter-assay precision of the method up to 25.6 micrograms/ml was 2%. Practicability and accuracy of the new method were demonstrated by the application to excretion studies performed with human volunteers.

A new approach for dealing with the stability of drugs in biological fluids.

The purpose of this study was to develop a procedure for investigating the stability of drugs in biological fluids based on sound experimental design and to use a statistical procedure which would allow conclusions to be made concerning stability with an acceptable degree of certainty (95%). The experimental procedure involved replicate analysis of the drug in stored and freshly prepared samples on the same day. The relative difference in response between these two sets of samples and a 90% confidence interval for the true change in response was calculated. This confidence interval enabled us to detect a pharmacokinetically relevant degradation. It is argued that this approach is superior to the stability test procedures based on the t test. The application of the method to the assessment of the stability of three different drugs in biological fluids is described.

Determination of the benzodiazepine antagonist Ro 15-1788 in plasma by high-performance liquid chromatography with UV detection.

A sensitive and specific HPLC-assay was developed for the determination of the benzodiazepine antagonist ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-[1,5-a] [1,4]benzodiazepine-3-carboxylate (Ro 15-1788; envisaged INN: flumazepil) in plasma. Ro 15-1788 and the internal standard were extracted from plasma at a basic pH, separated from coextracted plasma components on a reversed-phase column and quantitated by means of a UV detector. Ro 15-1788 was extracted quantitatively from plasma in the concentration range 25.6 to 300 ng/ml. The sensitivity limit was about 10 ng/ml plasma using a 1-ml specimen. The method was shown to be specific with respect to several benzodiazepines and their main metabolites. The method was successfully applied to pharmacokinetic studies in man after oral and intravenous administration of Ro 15-1788.

Determination of pyrimethamine in human plasma after administration of fansidar of fansidar-mefloquine by means of high-performance liquid chromatography with fluorescence detection.

A sensitive, rapid and selective high-performance liquid chromatographic (HPLC) method has been developed to measure plasma levels of pyrimethamine in human subjects dosed with the antimalarials Fansidar or Fansidar and mefloquine. The drug was extracted from plasma at basic pH with n-butyl chloride-dichloromethane (96:4, v/v) and quantified on a normal-phase HPLC column with fluorescence detection (excitation 290 nm, emission 345 nm). Pyrimethamine was almost quantitatively extracted from plasma in the concentration range 20-200 ng/ml. The sensitivity limit was about 10 ng/ml of plasma, using a 0.5-ml specimen. The method was shown to be specific with respect to the other two components in the antimalarial combinations, namely sulfadoxine and mefloquine, and their metabolites. The assay was applied to pharmacokinetic studies of pyrimethamine in man following the oral administration of Fansidar of Fansidar and mefloquine.

Sensitive gas-liquid chromatographic method for the determination of oxiconazole in plasma.

A specific and highly sensitive gas-liquid chromatographic method was developed for the determination of oxiconazole in rat, dog, and human plasma. The compound and its internal standard were extracted from plasma at basic pH with n-hexane-isoamyl alcohol (98:2, v/v), gas chromatographed on 3% SP-2250/Supelcoport (80-100 mesh) and quantified by means of an electron-capture detector. Oxiconazole was extracted almost quantitatively from plasma in the concentration range 10-5000 ng/ml. The sensitivity limit was about 1 ng/ml, using a 1-ml specimen. The method was applied to 13-week tolerance studies in dogs and rats in order to follow oxiconazole concentrations in plasma after oral administration of the compound. The assay was sensitive enough to measure precisely the small amounts of unchanged compound in plasma after intravaginal application of labelled oxiconazole to human volunteers.

[Comparative studies on the stability of aqueous drug solutions in the isothermal and the non-isothermal short-time test as well as in the long-time test. Part 2: The stability of aqueous tetracaine solutions in the non-isothermal short-time test (author's transl)]. / Vergleichende Untersuchungen zu Stabilität wässriger Arzneistofflösungen im isothermen und nichtisothermen Kurzzeittest sowie im Langzeitversuch. Teil 2: Stabilität wässriger Tetracainiösungen im nichtisothermen Kurzzeittest.

A study of the hydrolytic degradation of tetracaine solutions at various pH values demonstrates that the results from non-isothermal stability testing with logarithmic rise in temperature are in good agreement with the activation energies determined, under analogous conditions, by means of the isothermal short-time test and long-time test. The range of the maximum of stability is more clearly evinced by the non-isothermal short-time test than by the isothermal stability test. The comparison of the two methods reveals that the deviation of the reaction rate constants is greater in the non-isothermal test, which is due to the calculation required for the logarithmic rise in temperature. The results obtained with tetracaine evidence that the non-isothermal stability test is an appropriate method for the rapid determination of stability parameters (e.g. stability maximum, hydrolysis velocities) in the frame-work of testing potential drugs for stability and in the optimization of prescriptions.

[Enzymatic breakdown of linoleic acid hydroperoxides to volatile carbonyl compounds by isomerase from barley (author's transl)]. / Enzymatischer Abbau von Linolsäurehydroperoxiden zu flüchtigen Carbonylverbindungen durch Isomerase aus Gerste.

Barley protein fractions with active isomerase, purified by means of gelchromatography were incubated at room temperature with linoleic acid hydroperoxides (LHPO), containing 9-hydroperoxy-10-trans,12-cis-octadecadienoic acid (9-LHPO) and 13-hydroperoxy-9-cis,11-trans-octadecadienoic acid (13-LHPO) in the ratio of about 1:1. The volatile compounds resulting from the reaction have been isolated, concentrated and investigated by means of gas- and radio-gaschromatography. In the case of incomplete LHPO-breakdown remaining hydroperoxides and nonvolatile breakdown products have been separated before gaschromatographic analysis. In addition to hexanal as main product, traces of 2-tr-heptenal and 2-tr-octenal were found; about 6% of the converted hydroperoxides were transformed to carbonyl compounds. By numerous additional experiments it was confirmed that the volatile compounds are formed by enzymatic catalysis.

[On characterization of lipoxygenase from barley (author's transl)]. / Zur Charakterisierung der Lipoxygenase aus Gerste.

Lipoxygenase from brewing barley of Nordbaden has optimum activity at alkaline pH and predominantly forms 9-hydroperoxy-10-trans, 12-cis-octadecadienoic acid (9-LHPO) from linoleic acid. While at pH 7 90% 9-LHPO is produced, its proportions drops during incubation at more alkaline pH (pH 7,75) to 70%. This positional specifity is not caused by isoenzymes. While linoleic acid methylester is converted only to a lesser degree, trilinolein is not accepted as substrate.