Multicenter studies on the pharmacokinetic profile of sustained-release oral diltiazem (300 mg) after once a day repeated administration: influence of age.

The influence of age on the pharmacokinetics of the oral sustained release diltiazem Mono-Tildiem LP 300 mg was investigated in 12 middle-aged (40-64 years), 12 elderly (65-80 years) patients and compared to a control group of 54 young healthy volunteers (18-36 years). Each subject received daily a single dose of diltiazem slow release (300 mg) in the morning for 5 consecutive days. On the fifth day of treatment, the pharmacokinetic parameters of diltiazem and of 2 of its circulating metabolites (N-monodemethyldiltiazem and deacetyldiltiazem) were evaluated. The mean diltiazem Cmax was 199.3 +/- 117.8 ng/ml, 254.8 +/- 85.2 ng/ml and 154.5 +/- 63.2 ng/ml in middle-aged, elderly, and young healthy subjects, respectively. Mean plasma Cmin concentration was also higher in elderly subjects than in middle-aged and young subjects: 129.7 +/- 77.9 ng/ml versus 66.8 +/- 56.8 and 66.3 +/- 32.3 ng/ml, respectively. The AUC0-24 showed the same trend: 4,042 +/- 1,136 ng/ml.h in elderly, 2,995 +/- 1,905 ng/ml.h in middle-aged, and 2,564 +/- 1,205 ng/ml.h in young subjects. These parameters were statistically higher (p < 0.01) in the elderly subjects than those obtained in younger people. No statistical difference was observed between young volunteers and middle-aged patients. The Tmax did not differ significantly with age (5.1 +/- 4.4, 6.8 +/- 2.8, 6.1 +/- 3.5 hours, respectively). The ratios between the AUC of each metabolite and that of the parent compound did not vary with the age. These results suggest that in elderly people (> 65 y) the bioavailability of diltiazem is increased, probably due to a reduction of the first-pass effect. Based on the pharmacokinetic results, although safety data did not show any specific trend with age, a precautionary reduction of the dose at the start of the treatment seems advisable.

Stereoselective determination of unchanged and glucuroconjugated eliprodil, a new anti-ischaemic drug, in human plasma and urine by precolumn derivatization and column-switching high-performance liquid chromatography with fluorescence detection.

An HPLC method was developed and validated for the determination in human plasma and urine of the enantiomers of eliprodil, (+/-)-alpha-(4-chlorophenyl)-4[(4-fluorophenyl) methyl]piperidine-1-ethanol hydrochloride, a new anti-ischaemic agent administered as a racemate. Both enantiomers are present in human plasma in unchanged and glucuroconjugated form, whereas only the glucuroconjugated form is excreted into urine; as a consequence, such metabolites in human plasma and urine should be submitted to enzymatic deconjugation with beta-glucuronidase (Escherichia coli) before being extracted. The general method involves a liquid-liquid extraction of eliprodil and internal standard from alkalinized plasma or urine with n-hexane, evaporation of the organic phase and derivatization with (S)-(+)-naphthylethyl isocyanate to give carbamate diastereoisomeric derivatives of (S)-(+)- and (R)-(-)-eliprodil and internal standard; after evaporation of the derivatizing mixture and dissolution of the residue in a small volume of phosphate buffer-acetonitrile (60:40, v/v), an aliquot is injected into a column-switching HPLC system. The derivatized sample extract is purified on a precolumn filled with C8-bonded silica material, which is flushed with acetonitrile-water, then diastereoisomers of eliprodil and the internal standard are automatically transferred by the mobile phase to the analytical column. The analytical column is a C8 type, specially deactivated for basic compounds, the mobile phase is 0.025 M phosphate buffer (pH 2.6)-methanol-acetonitrile (42:2:56) at a flow-rate of 1.2 ml min-1 and fluorimetric detector operating at lambda ex = 275 nm and lambda em = 336 nm is used. The retention times, under these conditions, are about 16 and 17 min for (S)-(+)- and (R)-(-)-eliprodil diastereoisomers, respectively, and about 19 min for the first-eluted diastereoisomer of the internal standard. During the analysis time, the precolumn, reset in a different path from that of the analytical column, is back-flushed with different solvents, then re-equilibrated with acetonitrile-water before the next injection. Linearity in plasma, for unchanged eliprodil enantiomers, was assessed in the range 0.15-10 ng ml-1 and for total eliprodil enantiomers (unchanged + conjugated) in the range 0.75-500 ng ml-1; the limit of quantitation (LOQ) is 0.15 ng ml-1 for each unchanged enantiomer and 0.75 ng ml-1 for each total enantiomer. Linearity was also assessed in urine for total (conjugated) eliprodil enantiomers in the range 50-25 000 ng ml-1; the LOQ is 50 ng ml-1 for each enantiomer. The intra- and inter-day precision and accuracy of the method were investigated in plasma and urine and found to be satisfactory for pharmacokinetic studies. The method has been extensively used in pharamcokinetic studies in man treated with a 20-mg dose of eliprodil racemate and some results of this application are reported.

Stereospecific determination of amisulpride, a new benzamide derivative, in human plasma and urine by automated solid-phase extraction and liquid chromatography on a chiral column. application to pharmacokinetics.

Amisulpride, a drug belonging to the benzamide series, demonstrates antischizophrenic and antidepressant (antidysthymic) properties in man. For the pharmacokinetic studies of the racemic drug in man, a method of determination based on solid-phase extraction (SPE) from plasma and HPLC on a stereoselective column was developed. For this aim, one millilitre of plasma, after the addition of the internal standard, tiapride or metoclopramide, is diluted with a borate buffer at pH 9, then automatically loaded onto a SPE C18 100-mg column. The column is washed with different solvents, then eluted with 0.5 ml of methanol. After evaporation of the eluted fraction, the residue is reconstituted in 0.25 ml of eluent mixture. An aliquot is injected onto the HPLC column, a Chiralpak AS, equilibrated with an eluent mixture constituted by n-hexane-ethanol, (67:33, v/v) containing 0.2% (v/v) of diethylamine (DEA) or n-heptane-ethanol, (70:29.8, v/v) containing 0.2% of DEA and connected to a UV detector set at 280 nm or to a fluorimetric detector set at lambda ex = 280 nm and lambda cm = 370 nm. The limit of quantitation (LOQ) in human plasma is 2.5 ng ml-1 for both S-(-)- and R-(+)-amisulpride isomers with both detection methods. The method has been demonstrated to be linear in the range 2.5-320 ng ml-1 for both R-(+)- and S-(-)-amisulpride in human plasma with both UV and fluorescence detection. Absolute recovery of S-(-)- and R-(+)-amisulpride enantiomers from human plasma, as well as selectivity, precision and accuracy have been demonstrated to be satisfactory for pharmacokinetics in man and equivalent for both the proposed methods that have been cross-validated on real dosed human plasma samples. The methods have been used for clinical pharmacokinetic studies allowing pharmacokinetic parameters for amisulpride enantiomers in agreement with those obtained for the racemate to be obtained. After dilution with water, urinary samples from subjects treated with amisulpride racemate can be analysed according to the method used for plasma.

Determination of amisulpride, a new benzamide derivative, in human plasma and urine by liquid-liquid extraction or solid-phase extraction in combination with high-performance liquid chromatography and fluorescence detection. application to pharmacokinetics.

Amisulpride (SOLIAN) belongs to the benzamide series and shows antischizophrenic and antidepressant (anti-dysthymic) properties in man. Two methods suitable for pharmacokinetic investigations are proposed for the determination of amisulpride in human plasma. For the liquid-liquid extraction (LLE) based method, the plasma, added with the internal standard (an amisulpride analogue) is alkalinised with NaOH and extracted with a diethyl ether-chloroform mixture. The organic phase is removed, evaporated to dryness and redissolved in an acidic phosphate-acetonitrile mixture that, after a back-washing step with n-hexane, is injected onto the HPLC column (C18 BDS type) connected with a fluorimetric detector. The second method is based on an automatic solid-phase extraction (SPE) performed on an ASPEC device. The plasma sample, diluted with a pH 9 borate buffer, is loaded onto a disposable SPE C18 100-mg column. The analytes of interest (amisulpride and internal standard), after two washing steps with different solvents, are recovered in pure methanol; after evaporation to dryness, the residue is dissolved in an acidic phosphate buffer and injected onto the chromatographic apparatus already described. The limit of quantitation (LOQ) is 0.5 ng ml-1 for both methods; a linear correlation between concentrations and detector response has been demonstrated in the range 0.5-640 ng ml-1 for LLE, which is the most used method; for SPE methods, less used, linearity has been assessed in the plasma range of 0.5-160 ng ml-1.

Determination of diltiazem and its main metabolites in human plasma by automated solid-phase extraction and high-performance liquid chromatography: a new method overcoming instability of the compounds and interference problems.

An automated sample preparation method, based on solid-phase extraction (SPE) was developed on an ASPEC-Gilson device and combined with HPLC for the determination of diltiazem and three of its metabolites in human plasma (N-desacetylmonodesmethyldiltiazem, N-monodesmethyldiltiazem, O-desacetyldiltiazem). A 1-ml volume of plasma is diluted with 0.5 ml of 0.1 M ammonium dihydrogen phosphate and the sample is automatically loaded onto a SPE silica (C18) column (100 mg); the column is flushed with two different solvents, then eluted with 0.5 ml of a 0.1 M ammonium dihydrogen phosphate-acetonitrile mixture (20:80, v/v) containing 0.06% of triethylamine. The eluate is evaporated to dryness and the residue reconstituted with a suitable solvent and injected onto a C8 silica column connected to a UV detector (lambda = 238 nm). This method overcomes problems caused by the partial instability of diltiazem and metabolites in human plasma during analysis. There is no chromatographic interference from endogenous compounds. The limits of quantitation (LOQ) are 2.5 and 2 ng ml-1 for diltiazem and the metabolites in human plasma, respectively. Linearity between concentrations and detector response for diltiazem and metabolites ranged from 10-200 and 5-100 ng ml-1 in human plasma, respectively. The method has been validated.

Determination of alpidem, an imidazopyridine anxiolytic, and its metabolites by column-switching high-performance liquid chromatography with fluorescence detection.

Alpidem, 6-chloro-2-(4-chlorophenyl)-N,N-dipropylimidazo[1,2-a]pyridine- 3-acetamide, is an anxiolytic imidazopyridine that undergoes a first-pass elimination after oral administration to humans; it is actively metabolized and three circulating metabolites have been identified in plasma due to N-dealkylation, oxidation or a combination of both processes. For the determination of the unchanged drug and its metabolites in human plasma, a column-switching HPLC method was developed. The method, based on solid-phase extraction (performed on-line), involves the automatic injection of plasma samples (200 microliters) on to a precolumn filled with C18 material, clean-up of the sample with water in order to remove protein and salts and transfer of the analytes to the analytical column (after valve switching) by means of the mobile phase. All the processes were performed in the presence of an internal standard, a compound chemically related to alpidem. During the analytical chromatography, the precolumn was flushed with different solvents and after regeneration with water, it was ready for further injections. The analytical column was a C8 type and the mobile phase was acetonitrile-methanol-phosphate buffer solution (45:15:45, v/v/v) at a flow-rate of 1.5 ml min-1. The column was connected to a fluorimetric detector operating at excitation and emission wavelengths of 255 and 423 nm, respectively. The limits of quantitation of alpidem and three metabolites were 2.5 and 1.5 ng ml-1, respectively, in human plasma.

Determination of mizolastine, a new antihistaminic drug, in human plasma by liquid-liquid extraction, solid-phase extraction and column-switching techniques in combination with high-performance liquid chromatography.

For the determination of mizolastine (2-[[[1-[(4-fluorophenyl)methyl]-1H-benzimidazol-2-yl]-4- piperidinyl]methylamino]-4(3H)-pyrimidinone, SL 85.0324), a new antihistaminic drug, in human plasma, three methods were developed based on liquid-liquid extraction, solid-phase extraction and column-switching in combination with high-performance liquid chromatography with ultraviolet detection. The liquid-liquid extraction method included a back-extraction step that preconcentrates the drug into a small aqueous volume, resulting in very high sensitivity (0.5 ng/ml of plasma); it can be used in conventional bioanalytical laboratories that do not have sophisticated automatic devices. The solid-phase extraction method is performed by using a robotic system (Benchmate). It is completely automated from the initial sampling to the final injection into the chromatograph. It has a good sensitivity (1 ng/ml of plasma), but requires an expensive apparatus and skilled analysts. The column-switching method is based on a solid-phase extraction performed on-line with chromatographic analysis; it is not completely automatic, because some operations are performed manually. The device required for valve switching is not expensive and can be managed by a simple integrator or a personal computer; it is very easy to use and affords a sensitivity (2.5 ng/ml of plasma) that generally satisfies the needs of pharmacokinetic investigations of mizolastine. The conditions were similar for all the three methods: a C8 type column, an eluent of phosphate buffer and acetonitrile, and a spectrophotometric ultraviolet detector operated at 285 nm.

Determination of zolpidem, a new sleep-inducing agent, and its metabolites in biological fluids: pharmacokinetics, drug metabolism and overdosing investigations in humans.

For the determination of zolpidem, a new sleep inducer, and its metabolites in human plasma and urine, three methods were developed that are suitable for pharmacokinetics, drug metabolism and overdosing investigations. The methods used for pharmacokinetic and drug metabolism studies are based on column-switching high-performance liquid chromatography; they do not require any sample manipulation because the plasma or diluted urine is injected into a pre-column where clean-up and preconcentration take place. The analytes are transferred by valve-switching to the C18 analytical column for chromatography. To investigate overdose cases, urine samples only are used: the method is simple, because the diluted urine can be injected directly into the analytical column (phenyl type). This allows the identification and quantification of the principal urinary metabolite of zolpidem, the unchanged drug being practically undetectable. All the methods use fluorescence detection, which affords high sensitivity and selectivity. It is necessary to use a method capable of the determination of metabolites even if these are apparently pharmacologically inactive, because in different physiopathological populations the qualitative and quantitative metabolic profiles of zolpidem could be different. The method designed for the investigation of (accidental or deliberate) overdose cases is, as required on such occasions, simple and rapid, with good selectivity with respect to commonly prescribed psychotropic drugs.

Bioavailability of diltiazem as a function of the administered dose.

Diltiazem undergoes extensive first-pass metabolism; extrapolation from single to repeated administration thus underestimates plasma concentration values. In order to validate the hypothesis of a partially saturable first-pass effect, four single doses of diltiazem (10, 20, 40, and 120 mg) were administered at weekly intervals to eight healthy volunteers. Results showed that: (a) the inter-subject variability was highest at the lowest dose at the highest dose; (b) bioavailability was almost nil in 3 of 8 of the subjects after the administration of the 10 mg dose; (c) the mean bioavailability increased with the dose from 11.8 +/- 2.5 per cent after 10 mg to 28.2 per cent after 120 mg; (d) the elimination half-life was dose-related; (e) the renal excretion of diltiazem increased with the administered dose from 1.0 +/- 0.3 per cent after 10 mg to 3.0 +/- 0.5 per cent after 120 mg; (f) the greatest amounts of circulating metabolites were present after the lowest doses. These results are consistent with a partially saturable first-pass effect for diltiazem.

Therapeutic effects of fengabine, a new GABAergic agent, in depressed outpatients: a double-blind study versus clomipramine.

The results of a double-blind clinical trial of fengabine vs clomipramine in depressed outpatients are reported. Fengabine, a new GABAergic agent, seems to be as effective as the reference drug, with a faster onset of action and a more marked effect on cognitive disturbances and retardation. The new drug is free of any significant anticholinergic or cardiovascular effect, and it is not sedative.