Micro-extraction of chlordiazepoxide and its primary metabolites, desmethylchlordiazepoxide and demoxepam, from plasma and their measurement by liquid chromatography.

We have developed a micro-extraction procedure for the analysis of chlordiazepoxide and its two unique metabolites, demoxepam and desmethylchlordiazepoxide, in plasma, using liquid chromatography. The method is both reliable and sensitive for the quantitation of low plasma concentrations of these three compounds. The extraction procedure allows rapid sample processing, which, together with the small sample volume (100 microL), makes it ideal for routine sample handling. The limit of detection for the three compounds ranged from 0.075 to 0.125 mg/L and recovery of the three different benzodiazepines ranged from 87 to 94%. Within- and between-assay coefficients of variation ranged from 3.1-4.5% and from 4.7 to 7.6%, respectively.

Chronic treatment with ethanol or chlordiazepoxide alters the metabolism of chlordiazepoxide.

Although chronic ethanol administration in C57BL/6J mice did not cause an induction of ethanol metabolism, it altered the metabolism of chlordiazepoxide (CDP). Significantly lower blood levels of CDP, but higher levels of N-desmethyl CDP (NDCDP), were observed in ethanol-dependent mice compared to pair-fed controls during the first hour after CDP injection. Mice treated chronically with CDP showed significantly lower blood levels of CDP and NDCDP than pair-fed controls after a test dose of CDP. In response to an injection of ethanol, the CDP-dependent mice had lower blood alcohol levels (BAL) than the pair-fed controls, but the rate of fall of BAL was not different in the two groups. Thus, chronic CDP treatment affected the absorption and distribution of ethanol. These results provide a metabolic basis for the manifestations of CDP tolerance and ethanol cross-tolerance that have been reported in CDP-dependent mice.

DNA damage induced in rats by oral administration of chlordiazepoxide plus sodium nitrite or of N-nitrosochlordiazepoxide.

Chlordiazepoxide (CDE) reacts in acidic conditions with NaNO2 yielding N-nitrosochlordiazepoxide (NO-CDE), previously shown to exert genotoxic effects in some in vitro systems. The possible intragastric nitrosation of CDE to NO-CDE has been investigated in rats given by gavage high single doses of this benzodiazepine along with NaNO2. Liver DNA fragmentation, as revealed by both DNA alkaline elution and a more sensitive viscometric method, was found to occur consistently and to be essentially independent of the molar ratio drug/nitrite or of gastric pH. The significant increase in the frequency of DNA lesions observed in rats treated for 15 successive days indicates that DNA repair did not keep pace with the accumulation of the damage. Oral administration of single doses of NO-CDE induced similar dose-dependent amounts of DNA fragmentation in liver, gastric mucosa, and brain. Due to the demonstrated absence of carcinogenic activity in rodents, the present results should be interpreted solely as indicating that NO-CDE is intrinsically capable of producing DNA lesions in vivo, an effect by itself not sufficient to induce tumor growth.

HPLC analysis of the nitrosation products of chlordiazepoxide.

Products originated from Chlordiazepoxide (I) hydrochloride/sodium nitrite interaction were analyzed by HPLC. The studied reactions were carried out in diluted hydrochloric acid solutions at pH values ranging between 0.5-5.0. Depending on the reaction pH values and molar ratios it was possible to find and assess variable amounts of the N-nitrosochlordiazepoxide (II), the dihydroquinazoline (III) and the lactam (IV). The highest degree of N-nitrosation was found at pH 3.5. At this pH value the yields of (II) were respectively 54.8% and 18.3% when the drug (I)/nitrite molar ratios were correspondingly 0.41 and 0.25. When the reaction was performed in concentrations which is possible to find in the gastric juice of patients taking (I) together with nitrite-rich foods the yield of (II) at pH 3.5 was 2.5%. Since in the meantime the genotoxicity of (II) was proved, "in vivo" formation of N-nitrosochlordiazepoxide (II) represents a potential risk not to be underestimated.

Toxicity in a case of acute and massive overdose of chlordiazepoxide and its correlation to blood concentration.

In previous studies no correlation between blood concentration and toxicity was found in acute chlordiazepoxide overdose. We describe a case of acute overdosage with 5.2 g chlordiazepoxide, in which toxicity correlated to blood concentration of the second metabolite of chlordiazepoxide, to demoxepam. Therefore, it is recommended to determine not only chlordiazepoxide, but also its active metabolites in cases of overdose. This can easily been achieved using the described method, HPLC with photodiode array detection.

Peptide derivatives as prodrugs.

The results quoted here suggest strongly that peptides as prodrugs are a real possibility for future forms of therapy. Pharmacokinetics and bioavailability are certainly altered by such modifications, usually in a positive sense. The possibilities in utilizing active transport permeases to direct drugs to the desired receptor are an obvious reality, and will undoubtedly lead to new methods for treating bacterial, fungal or even viral infections, and for improved ways of presenting anti-tumour agents. The number of patents appearing in this field is indicative of the interest shown in the pharmaceutical industry.

Involvement of the N4-oxide group in the phototoxicity of chlordiazepoxide in the rat.

To verify whether or not N4-oxide function is involved in the phototoxicity of chlordiazepoxide (CDZ, Librium), photopharmacology of reduced chlordiazepoxide (RCDZ) lacking the N4-oxide group was carried out and compared to that of CDZ. From the distribution of the 2 compounds in the skin and their UV-spectra in the wavelength region of the UV lamp, doses were calculated to allow the comparison of the photopharmacological effects. Contrary to what has been described for CDZ, no difference was found for RCDZ between irradiated and non-irradiated rats. The discussion leads to the conclusion that the N4-oxide group is responsible for systemic effects reported for phototoxic CDZ.

Effect of ketoconazole on hepatic oxidative drug metabolism.

Several clinical reports have suggested (but not demonstrated) that ketoconazole, a broad-spectrum antifungal drug, may inhibit hepatic oxidative drug metabolism in man. We recently demonstrated that ketoconazole inhibits caffeine and aminopyrine oxidation in the rat; we now study the influence of ketoconazole on theophylline and chlordiazepoxide kinetics in man. These studies were performed before and after varying doses of ketoconazole within the currently accepted therapeutic range. Ketoconazole had no effect on theophylline clearance, whereas the drug impaired chlordiazepoxide clearance from plasma. After a single dose of ketoconazole, there was a 20% decrease in clearance and a 26% decrease in volume of distribution without evidence of inhibition of drug metabolism. These changes apparently were not related to ketoconazole dose. After repetitive dosing with ketoconazole, chlordiazepoxide clearance decreased by 38% and was associated with reduced concentrations of its first oxidative metabolite, N-desmethylchlordiazepoxide. We conclude that ketoconazole inhibits at least one subset of the hepatic mixed-function oxidase system, but is not as general an inhibitor of hepatic oxidative drug metabolism as cimetidine appears to be. For some coadministered drugs, ketoconazole may also have an effect on other kinetic parameters such as volume of distribution. Therefore, we caution that clinically important drug interactions may occur with the concurrent use of ketoconazole.

Pharmacokinetic studies on Ro 15-1788, a benzodiazepine receptor ligand, in the brain of the rat.

Methods for determining Ro 15-1788 in brain tissue were developed using gas chromatography with nitrogen-phosphorus detection, and using reverse-phase high performance liquid chromatography. Application of the methods to pharmacokinetic studies in the rat found the elimination half-life of Ro 15-1788 from rat brain to be 16 min. Ro 15-1788 was undetectable in rat plasma at the time points studied. Concentrations of Ro 15-1788 in the brain were reduced if chlordiazepoxide was coadministered.

Methods for the determination of lorazepam and chlordiazepoxide and metabolites in brain tissue. A comparison with plasma concentrations in the rat.

Rapid and sensitive methods are described for determining lorazepam and for determining chlordiazepoxide and its metabolites in brain tissue of the rat. Lorazepam was determined by means of solvent extraction and electron-capture gas-liquid chromatography and concentrations as low as 5 ng/g tissue could be measured. High-performance liquid chromatography with UV detection was used to determine chlordiazepoxide and its metabolites and was sensitive to 0.1 micrograms/g tissue. The methods were used to investigate the brain and plasma pharmacokinetics of these compounds in animals that had been chronically treated with lorazepam or chlordiazepoxide. In both experiments brain and plasma levels of all compounds assayed were found to correlate highly.

Alcohol-chlordiazepoxide interaction.

The effects of chlordiazepoxide (CDP) or its N-demethyl metabolite (NDCDP) on ethanol-induced sleep time were investigated. The results indicate that CDP or NDCDP produced a supra-additive effect on the duration of sleep time induced by ethanol. These effects were not due to an alteration in the rate of elimination of blood ethanol levels. Mice which were administered CDP/ethanol had significantly higher blood and brain CDP levels than mice injected with CDP alone. The increase in CDP concentrations could be partly responsible for the supra-additive prolongation of ethanol sleep time. Our results also indicate that NDCDP and/or its metabolites were largely responsible for the supra-additive effect, because mice injected with CDP/ethanol or NDCDP/ethanol (ethanol 4 g/kg: CDP or NDCDP, 10 mg/kg) showed comparable increases in sleep time, and the blood and brain levels of NDCDP were comparable in these two groups.

Lack of tolerance and rapid recovery of cimetidine-inhibited chlordiazepoxide (Librium) elimination.

Cimetidine has been shown to inhibit oxidative metabolism of several drugs while sparing the glucuronidation pathways of drug metabolism. We studied the time-course of inhibition and recovery of cimetidine-inhibited chlordiazepoxide elimination in 7 healthy subjects. Chlordiazepoxide elimination was studied after cimetidine treatment for 1 and 30 days, and after withdrawing cimetidine for 48 h. The plasma clearance of chlordiazepoxide was reduced by 54% (p less than 0.001) after 24 h of cimetidine, by 57% (p less than 0.001) after 30 days of cimetidine and returned to normal after cimetidine was stopped for 48 h. In the absence of changes in volume of distribution, these changes resulted in proportional increases in the elimination half-life (t 1/2 beta) after 24 h and 30 days cimetidine treatment, and returned to pretreatment values after stopping cimetidine. In addition, the impaired chlordiazepoxide elimination was accompanied by inhibition of generation and subsequent elimination of N-desmethylchlordiazepoxide, the first metabolite of chlordiazepoxide metabolism. This study demonstrates a rapid inhibitory effect on chlordiazepoxide elimination, an absence of tolerance to this effect and a rapid reversal of this effect upon stopping cimetidine. These findings may have important therapeutic implications for patients receiving both drugs simultaneously.

Differential pulse amperometric detection of drugs in plasma using a dropping mercury electrode as a high-performance liquid chromatographic detector.

High-performance liquid chromatographic separation prior to reductive electrochemical determination at its dropping mercury electrode imparts specificity and sensitivity not attainable by conventional polarographic analysis of drugs and their metabolites. The utility of this novel approach is demonstrated by the analysis of chlordiazepoxide and its N-desmethyl metabolite in plasma which previously required thin-layer chromatographic separation prior to polarographic measurement. A mobile phase of methanol-isopropanol--0.0075 M acetate buffer, pH 3.5 (53:5:42), is used with the detector operated in the differential pulse mode at Ep = -0.820 V vs. Ag/AgCl. The response was linear (r = 0.998) in the concentration range of 0.05--2.0 micrograms/ml plasma for each component. The minimum detectability for each component under these conditions is 5.0 ng injected at a current range of 0.5 microamperemeter full scale. Techniques for oxygen removal and hydrodynamic consideration for the pumping system are presented.

Simultaneous assay of diazepam, chlordiazepoxide, N-desmethyldiazepam, N-desmethylchloridazepoxide, and demoxepam in serum by high performance, liquid chromatography.

A method is presented for simultaneously determining diazepam and chlordiazepoxide along with their respective major active serum metabolites N-desmethyldiazepam, and N-desmethylchlordiazepoxide and demoxepam. The drugs are extracted from one ml of buffered serum using chloroform containing 5-(p-methylphenyl)-5-phenylhydantoin as an internal standard. The elution is accomplished using a reversed-phase column with a mobile phase consisting of an acetonitrile/methanol/acetate buffer pH 5.0 (200/225/500) at a flow rate of 2.0 ml/min. Absorbance is monitored at 240 nm using a variable wavelength detector. Each chromatographic separation requires approximately 15 minutes at ambient temperature. Of more than twenty drugs tested for possible interference with this procedure, only methaqualone interferes with the internal standard, and phenytoin with demoxepam.

The screening and quantitation of diazepam, flurazepam, chloridazepoxide, and their metabolites in blood and plasma by electron-capture gas chromatography and high pressure liquid chromatography.

A combined GLC-ECD and HPLC procedure has been developed for the analysis of the most commonly encountered benzodiazepine drugs and has been applied to both plasma and postmortem blood samples. There is no doubt that since their introduction the use of these sensitive analytical methods have resulted in an increase in the incidence of detection of these drugs in both clinical and forensic toxicology cases.

Quantitation of chlordiazepoxide and its metabolites in biological fluids by thin-layer chromatography.

Chlordiazepoxide and its 4 major metabolites were assayed after separation by thin-layer chromatography following extraction from biological fluids. The compounds become intensely fluorescent in the presence of red, fuming nitric acid. The resulting compounds are quantitated with a spectrodensitometer with a fluorescent attachment. The sensitivity varies between 0.05 and 0.1 microgram. The coefficient of variation is 1.4% for assays in urine and 6.4% in serum.

Factors influencing blood concentrations of chlordiazepoxide: a use of multiple regression analysis.

Three groups of male and female subjects aged 24-74 years received 25, 100, or 200 mg of chlordiazepoxide hydrochloride by mouth as a single dose or as two divided doses. The relation of plasma or whole blood concentrations for chlordiazepoxide (CDX) and its metabolite, desmethylchlordiazepoxide (DMCDX), to time since the last dose, weight, age, and sex were determined by simple and multiple regression analyses. Both CDX and DMCDX levels were negatively correlated with weight. Concentrations of CDX decreased, while those of DMCDX increased, with the time since the last dose. Lower levels of both drugs were associated with female sex, and lower levels of DMCDX were noted with increasing age. In the largest sample group, age and weight were more important variables than sex in accounting for CDX and DMCDX. Sex was of significance, and more important than time or age in explaining the variance of CDX in one series of observations. Multiple regression analysis is a useful approach to assessing interrelated factors influencing blood levels of drugs, especially when combined with a consideration of the interactive components of variance. Age and sex, in addition to weight and time, may be important factors that deserve further attention.