An enzyme inhibition assay for the quantitative determination of the new bisphosphonate zoledronate in plasma.

Zoledronate, 2-(imidazole-1-yl)-1-hydroxyethane-1,1-bisphosphonic acid is a new 3rd generation bisphosphonate. A specific enzyme inhibition assay was developed for the determination of zoledronate in plasma of animals and man. The multistep synthesis of cholesterol and some of its precursors (lanosterol, squalene) from 14C-labeled mevalonic acid lactone is catalyzed by the S12 fraction of homogenized rat liver in the presence of ATP, NADH and Mg2+. After hydrolysis of the reaction mixture with KOH, lipophilic reaction products were extracted with hexane and the overall yield determined by radiometry. Addition of zoledronate inhibited the formation of cholesterol and its precursors in a dose dependent manner. The described method is suitable to specifically and quantitatively measure concentrations of zoledronate down to 25 ng ml-1 in human and animal (dog, rat) plasma with acceptable reproducibility and accuracy.

Quantitative determination of CGP 61755, a protease inhibitor, in plasma and urine by high-performance liquid chromatography and fluorescence detection.

A liquid chromatographic assay for the determination of CGP 61755 (I) in plasma and urine is described. A similar method for CGP 53437, another HIV-1 protease inhibitor, has been developed and reported previously. After a deproteinization step, a liquid-liquid extraction is performed. Compound I and the internal standard CGP 55749 (II) are hydrolyzed and the primary amine group derivatized using fluorescamine. Chromatography is achieved by isocratic elution with a mobile phase of 30 mM borax buffer (pH 9)-acetonitrile (58:42, v/v). The derivatives of the compounds I and II fluoresce at 480 nm, on excitation at 395 nm and the retention times under these conditions were approximately 6 and 8 min, respectively. The limit of quantitation (LOQ) which is the lowest concentration of the analyte that can be measured with a coefficient of variation and a deviation from theory of less than 20%, was 15 ng/ml plasma and 20 ng/ml urine. The analyte is stable for at least four months in human plasma and sixteen months in dog plasma samples. Different human plasma sources and three different species (rat, rabbit and dog) were tested and no interference between analyte and plasma constituents was observed.

Determination of a new antimalarial drug, benflumetol, in blood plasma by high-performance liquid chromatography.

A rapid and selective high-performance liquid chromatographic assay for determination of a new antimalarial drug (benflumetol, BFL) is described. After extraction with hexane-diethyl ether (70:30, v/v) from plasma, BFL was analysed using a C18 Partisil 10 ODS-3 reversed-phase stainless steel column and a mobile phase of acetonitrile-0.1 M ammonium acetate (90:10, v/v) adjusted to pH 4.9 with ultraviolet detection at 335 nm. The mean recovery of BFL over a concentration range of 50-400 ng/ml was 96.8 +/- 5.2%. The within-day and day-to-day coefficients of variation were 1.8-4.0 and 1.8-4.2%, respectively. The minimum detectable concentration in plasma for BFL was 5 ng/ml with a C.V. of less than 10%. This method was found to be suitable for clinical pharmacokinetic studies.

Automated quantitative determination of a new polyamine biosynthesis inhibitor (CGP 48 664) and a potential metabolite in human and animal plasma by high-performance liquid chromatography.

A specific and sensitive liquid chromatographic assay for the determination of 4-amidino-1-indanone-2'-amidinohydrazone (CGP 48 664, I) and a potential metabolite, 2-(4-carbamoyl-2,3-dihydro-1H-inden-1-yliden) hydrazine carboximidamide (CGP 53 391, II), in human and animal plasma was developed. CGP 51 467, a structural analog, was added to the plasma samples (up to 1 ml) as an internal standard. After mixing, the samples were processed automatically using an ASPEC solid-phase extraction system. The final extracts were chromatographed on a 5 microns Purospher RP-18 HPLC column. Chromatography was performed using a gradient system and UV detection. The described HPLC method is suitable for specific and quantitative measurement of concentrations of I, as well as its potential metabolite II down to 5-10 ng/ml in human and animal (dog, rat) plasma with acceptable reproducibility and accuracy.

The influence of food on the disposition of the antiepileptic oxcarbazepine and its major metabolites in healthy volunteers.

The effect of food on the pharmacokinetics of the antiepileptic oxcarbazepine (OXC) was investigated in healthy volunteers. Six healthy male volunteers were treated with single peroral doses of 600 mg of oxcarbazepine (Trileptal) after overnight fasting or a fat- and protein-rich breakfast. Mean (+/- SD) areas under the plasma concentration-time curves (AUC) of the major component in plasma, the active monohydroxy metabolite (MHD), which is responsible for the therapeutic effect in man, were 672 (25) mumol L-1 h when given to the fasted volunteers and 780 (31) mumol L-1 h (p = 0.042) when given after a substantial breakfast. Mean (+/- SD) maximum concentrations (Cmax) were 25.5 (4.8) mumol L-1 when given to the fasted volunteers and 31.4 (5.3) mumol L-1 (p = 0.025) when given after breakfast. Thus, the average AUC was increased by 16% and Cmax by 23% when oxcarbazepine was given with food. The times at which Cmax was reached (tmax) as well as the terminal half-lives were not influenced by concomitant intake of food. The tolerability was the same whether oxcarbazepine was given before or after food in healthy volunteers. The slight effect of food on the kinetics of oxcarbazepine should be of little therapeutic consequence.

Quantitative determination of CGP 53,437, a new HIV protease inhibitor, in plasma by high-performance liquid chromatography and fluorescence detection.

A specific and sensitive liquid chromatographic assay for CGP 53,437 (I), a potent HIV protease inhibitor, is described. The method is based on a deproteinization step, followed by a liquid-liquid extraction with diisopropyl ether. Then a deprotection step of the primary amine and derivatization using fluorescamine is performed. Chromatography is achieved by isocratic elution with a mobile phase of 63 mM borax buffer (pH 9)-acetonitrile (58:42, v/v). The flow-rate of the mobile phase is 1 ml/min. The derivatives of compound I and its internal standard CGP 54,451, II, fluoresce at 480 nm on excitation at 395 nm. The limit of quantitation which is the lowest concentration of the analyte that can be measured with a coefficient of variation and a deviation from theory of less than 20%, was 5 nmol/l plasma. The analyte is stable for at least seven months in spiked human plasma samples. It is also stable after freezing and thawing cycles. Different human plasma sources and plasma samples from three different species (dog, marmoset, and rat) were tested and no interferences from plasma constituents was observed.

Urinary excretion of aminohydroxypropylidene bisphosphonate in cancer patients after single intravenous infusions.

The effect of the rate of infusion of disodium aminohydroxypropylidene bisphosphonate (APD; CGP 23339A), an inhibitor of bone resorption, on urinary excretion of this agent was studied in a randomized open-label study in 20 cancer patients. Ten patients received 60 mg of APD over 4 h, and the remaining 10 patients received the same dose over 24 h. Urine collected during specified intervals for 72 h after the start of the infusion was analyzed by high-performance liquid chromatography for unchanged APD. Mild and transient adverse experiences were observed in 12 (60%) patients; the most common were headache, fever, and phlebitis at the infusion site. No clinically significant laboratory abnormalities were observed, and none of the experiences were serious enough to require discontinuation of treatment. Cumulative urinary excretion of APD was a linear function of time, increasing rapidly after both the 4- and 24-h infusions were started. The mean (+/- standard deviation) cumulative urinary excretion of APD was 51.1 +/- 13.0% of the dose in the 20 patients, 55.0 +/- 15.0% in the 10 patients given the 4-h infusion, and 47.2 +/- 9.9% in the 10 patients receiving the 24-h infusion. Thus, the rate of infusion of the 60-mg dose did not influence retention of APD at 72 h after the start of therapy. Similarly, the presence or absence of bone metastases did not influence cumulative urinary excretion or the retention of APD.

Influence of food on the disposition of the monoamine oxidase-A inhibitor brofaromine in healthy volunteers.

The effect of food on the bioavailability of brofaromine hydrochloride was investigated in a randomized cross-over study. Eight healthy male volunteers were given single peroral doses of 75 mg brofaromine hydrochloride after overnight fasting or a fat- and protein-rich breakfast. Mean (+/- SD) areas under the plasma concentration-time curves (AUC) were 9.66 (2.35) mumol l-1 h when given to the fasted volunteers and 11.82 (3.78) mumol l-1 h (p = 0.0413) when given after a substantial breakfast. Mean (+/- SD) maximum plasma concentrations (Cmax) were 0.71 (0.13) mumol l-1 when given to the fasted volunteers and 0.85 (0.22) mumol l-1 (p > 0.05) when given after breakfast. Thus, both the average AUC and Cmax were increased by approximately 20 per cent when brofaromine hydrochloride was given with food. The times when Cmax was reached (tmax) as well as the elimination half-lives were not influenced by concomitant intake of food. The tolerability was the same whether brofaromine was given before or after food in healthy volunteers. The slight effect of food on the bioavailability of brofaromine should be of little therapeutic consequence because of the observed wide inter-subject variability of the plasma levels.

Determination of the R-(-) and S-(+) enantiomers of the monohydroxylated metabolite of oxcarbazepine in human plasma by enantioselective high-performance liquid chromatography.

An enantioselective liquid chromatographic assay for the simultaneous determination of the S-(+) and R-(-) enantiomers of the monohydroxylated metabolite of oxcarbazepine in human plasma is described. The metabolite is the active principle. The method is based on the extraction of plasma with diethyl ether-dichloromethane (2:1, v/v), separation of the organic phase, evaporation of the solvent and dissolution of the residue in the mobile phase. The two enantiomers were resolved on a Chiralcel OD (250 mm x 4.6 mm I.D.) high-performance liquid chromatographic column. The separation was achieved by isocratic elution with n-hexane-2-propanol (77:23, v/v). The flow-rate of the mobile phase was 1.0 ml/min and the two enantiomers were detected by ultraviolet absorbance at 210 nm. The analytical method is suitable for the quantitative and simultaneous determination of the two enantiomers in plasma at concentrations down to 0.4 mumol/l after administration of oxcarbazepine.

Determination of 4-hydroxyandrostenedione in plasma and urine by extractive alkylation and electron-capture gas chromatography.

A sensitive and specific quantitative assay has been developed for the determination of 4-hydroxyandrostenedione (4-OHA), a potent aromatase inhibitor used in the treatment of estrogen-dependent breast cancer. This steroid has a high first-pass metabolism and is extensively metabolized, mainly by glucuronidation. Plasma levels of unchanged 4-OHA are very low, even after high peroral doses. The analytical method is based on the addition of 17 alpha-ethinylestradiol (internal standard), liquid-liquid extraction from biological material followed by extractive alkylation with pentafluorobenzyl bromide and quantitation by gas chromatography. The method has been validated for sensitivity, accuracy and precision and was found to be suitable for application to pharmacokinetic and bioavailability studies of peroral formulations of 4-OHA.

Burst suppression EEG with baclofen overdose.

The article reports the case history of a patient with baclofen intoxication and burst suppression activity in the EEG several hours after baclofen ingestion. With symptomatic treatment the patient recovered within 5 days and the EEG became normal, again.

Determination of the new monoamine oxidase inhibitor brofaromine and its major metabolite in biological material by gas chromatography with electron-capture detection.

A sensitive gas chromatographic assay for the simultaneous determination of brofaromine [4-(7-bromo-5-methoxy-2-benzofuranyl)piperidine hydrochloride], a new monoamine oxidase-A inhibitor, and its major metabolite was developed and validated. After addition of 4-(5-bromo-2-benzofuranyl)piperidine as internal standard, the compounds were isolated from biological fluids by liquid-liquid extraction at basic pH. After derivatization with heptafluorobutyric anhydride the compounds were chromatographed using a packed column (OV-17) and an electron-capture detector. The limit of quantitation was ca. 0.03 nmol per sample (10 ng) for both compounds. analysis of spiked samples demonstrated the good accuracy and precision of the method, which is suitable for use in pharmacokinetic and bioavailability studies. The method was applied to samples from an experiment in a healthy volunteer treated with a single oral dose of 75 mg of brofaromine hydrochloride. Plasma profiles before and after enzymic hydrolysis showed that about one-third of the total brofaromine in plasma and practically all of the major metabolite (O-desmethylbrofaromine) were present in the conjugated form.

Pharmacokinetics of diclofenac and five metabolites after single doses in healthy volunteers and after repeated doses in patients.

1. The kinetics of diclofenac (I) and five of its metabolites (II-VI) were investigated in three healthy volunteers and in six patients. Compounds I-VI were measured by capillary column gas chromatography in plasma and urine. 2. After a single 100 mg dose of diclofenac sodium to volunteers, the drug was absorbed rapidly and showed peak plasma levels of 10-12 nmol/g. The maximum concentrations of five metabolites were comparatively low (0.36-2.94 nmol/g). The mono- and dihydroxy metabolites (II-V) had apparent terminal half-lives similar to that of I (1-3 h), but the hydroxymethoxy metabolite (VI) had a half-life of about 80 h. Renal elimination of VI within 96 h was about 1% of dose and that of I-VI was 36% (free plus conjugated). 3. Following daily treatment with 2 x 75 mg of an experimental sustained release formulation to patients for 6-10 months, steady-state trough concentrations of I-V in plasma were low (average values: 0.23-0.57 nmol/g). The mean trough concentration of VI was comparatively higher at 3.69 +/- 0.91 nmol/g presumably reflecting its accumulation. Despite this it is unlikely to contribute to the drug's therapeutic activity, since it has been shown in laboratory tests to be devoid of anti-inflammatory activity.

Pre-dosing plasma concentrations and beta-adrenoceptor blocking effects during repeated once daily dosing with 160 mg sustained-release propranolol (Inderal LA) and 16/260 oxprenolol Oros to healthy volunteers.

Plasma drug concentrations and attenuations in sub-maximal exercise tachycardia were measured in six healthy subjects 24 h after the third and fourth doses of Inderal LA and 16/260 oxprenolol Oros administered once daily. Both drugs significantly reduced exercise heart rates relative to the placebo response, at both observation times. The mean percentage reductions on the third and fourth days were, respectively, 15.7 and 11.6% for Inderal LA compared with 18.1 and 12.2% for 16/260 oxprenolol Oros. The differences between formulations were not statistically significant. The mean plasma concentrations at 24 h on the third and fourth day were 119 and 101 ng/g for oxprenolol and 16.2 and 15.8 ng/g for propranolol.

Correlated nerve conduction, somatosensory evoked potential and neuropathological studies in clioquinol and 2,5-hexanedione neurotoxicity in the baboon.

Observations have been made on 10 baboons receiving a high-dose regimen of clioquinol administered orally, 6 receiving a low-dose regimen and 6 treated with 2,5-hexanedione. The results were compared with those obtained from 10 control animals. Motor and sensory nerve conduction velocity was markedly reduced in the hexanedione-treated animals but only very minor abnormalities were detected in the clioquinol-treated baboons. Cervical and Rolandic somatosensory evoked potentials to lower and upper limb stimulation were delayed in both the high-dose clioquinol-treated and the hexanedione-treated animals, particularly in the latter. Histopathological studies in the low-dose clioquinol-treated group showed no abnormalities. In the high-dose group; axonal degeneration was confined to the spinal cord, cerebellar vermis and optic tract. It was most marked in the rostral portions of the dorsal spinal columns and the caudal parts of the direct and crossed corticospinal tracts. Occasional dorsal column fibres had degenerated back to the root entry zone in the cord. The distribution was that of a selective central distal axonopathy. There appeared to be no correlation with estimated blood levels of unaltered clioquinol. In hexanedione-treated animals there was also degeneration in the distal optic tracts and peripheral nerves in a pattern of central-peripheral distal axonopathy.

Determination of pirprofen in biological material by gas-liquid chromatography with nitrogen-specific and electron-capture detection.

Pirprofen, 2-[3-chloro-4-(3- pyrrolin -1-yl)phenyl]propionic acid, is an analgesic and anti-inflammatory agent. Most of the pirprofen is present in plasma in the unchanged form (80-90%), the remainder in the form of the pyrrole derivative. However, pyrrole is also formed during sample manipulation by oxidation of pirprofen. Thus, a method based on conversion of pirprofen to pyrrole by oxidation with 2,3- dicyano -5,6- dichlorobenzoquinone and subsequent measurement of the total pyrrole concentration was developed. The method is based on the introduction of an internal standard, extraction and esterification followed by oxidation. The methyl ester of the pyrrole is then determined by gas-liquid chromatography. A nitrogen-specific detector is generally used. However, for small sample sizes (0.1 ml of plasma), an electron-capture detector may be utilized. With this detector measurements of concentrations as low as 0.02 nmol/g (5 ng/g) are still possible. The kinetics of the degradation of pirprofen to its pyrrole derivative were investigated. Plasma levels of pirprofen after a single oral dose of 400 mg in a healthy volunteer were measured by a method described previously as well as by the new method.

Estimation of the individual metabolic state and its consideration in optimal pharmacotherapy.

The efficacy and safety of long-term therapy depends on the dose regimen. The early recognition of individual pharmacokinetic defects is a professional task of the clinical pharmacologist. The application of test compounds has been used to differentiate between slow and fast metabolizers. Modern techniques for identifying and quantitating drug metabolites facilitate the determination of the individual metabolic state without resorting to compounds foreign to the particular therapy. This paper exemplifies this principle by examining the metabolism and pharmacokinetics of carbamazepine, oxprenolol, hydralazine, maprotiline and diclofenac sodium. The systematic collection and analysis of representative samples of data is shown to be a prerequisite for the conclusive assessment and interpretation of the individual metabolic state.