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Diazepam (DZP) is one of the most commonly prescribed drugs for treating status epilepticus (SE). A simple, sensitive and selective LC/MS/MS method with a wide linear calibration range was developed to quantify DZP and its major metabolites, N-desmethyldiazepam (DMDZP), temazepam (TZP), and oxazepam (OZP), in rat cerebrospinal fluid (CSF). The method was used to simultaneously determine the concentrations of all analytes in a small sample volume (as little as 25 microL) of rat CSF. The lower limits of quantification (LLOQ) of the method are 0.04 ng/mL for DZP and 0.1 ng/mL for its metabolites. The calibration range is 0.04-200 ng/mL for DZP and 0.1-200 ng/ml for the metabolites. All intra- and inter-assay coefficients of variation (%CV) and mean percent errors of the method are less than 12%. This method successfully addresses the need to determine low therapeutic drug concentrations in small physiological samples, namely rat CSF. Moreover, it can be used to investigate the distribution of the drug and its metabolites among blood plasma, brain tissue, and CSF in pharmacokinetic and pharmacodynamic studies in a variety of laboratory animals. With respect to animal experiments involving assays in CSF, this method addresses two of the three criteria of Russell and Bruch (Principles of Humane Experimental Techniques, 1959, Methuen and Co., London) for minimizing animal use, namely refinement and reduction.

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PURPOSE: To determine whether repeat boluses of diazepam (DZP) lead to significant accumulation in the central nervous system and/or peripheral compartments, as repeat intravenous boluses of diazepam are commonly used in the treatment of status epilepticus (SE). METHODS: In a rat model that permits simultaneous serum and cerebrospinal fluid (CSF) sampling, we characterized the pharmacokinetics of DZP and its metabolite, desmethyldiazepam, in CSF and blood using HPLC. DZP was administered by intraperitoneal injection as either a single dose (20 or 30 mg/kg) or repeat doses (10 or 20 mg/kg x 3, 1 h apart). RESULTS: After a single intraperitoneal dose, DZP was rapidly absorbed with a time to maximum concentration of 10 min. The serum concentrations then declined biexponentially. DZP rapidly entered the CSF, the CSF to serum ratio reached equilibrium within 10 min, and was equivalent to the ratio of free to total serum concentration. Repeated DZP dosing resulted in a threefold decrease in volume of distribution and clearance (p < 0.001). This was reflected in the CSF concentration data; however, after the third dose, the ratio of CSF to serum concentration, also increased greatly, representing further persistence of DZP in the CSF compartment. CONCLUSIONS: Repeat dosing of DZP leads to substantial accumulation, and high, persistent serum and CSF concentrations, which may explain the toxic effects of repeat DZP dosing. Repeat dosing of DZP using a tapering protocol, however, may increase the effectiveness of DZP in treating SE by preventing relapses without substantially increasing toxicity.

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The compartmental distribution of diazepam (DZ) and nordiazepam (ND) and their metabolites was studied in DZ and ND dependent dogs. The levels of DZ, and ND and their metabolites were determined during the last week of stabilization in the extraneuronal brain space, in brain tissue, in plasma and in CSF. In these studies dependent dogs were anesthetized with pentobarbital and microdialysis probes were inserted bilaterally into the parietal cortex and perfused with artificial cerebrospinal fluid. Microdialysis probes were also used to determine the unbound parent drugs and their metabolites in plasma. The brain-plasma distribution of total ND and oxazepam (OX) is about equal in ND dependent dogs but in DZ dependent dogs total ND and OX are about 2-fold higher in brain than in plasma. The levels of DZ, ND, and OX in the extraneuronal brain space are similar to their unbound levels in plasma. These data suggest that the concentration of free benzodiazepines in plasma is a good approximation of the concentration in the vicinity of the membrane receptors in the dependent dogs.

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In 11 neurological patients, levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) were determined in cerebrospinal fluid (CSF) before and 1, 3, 5, and 8 min after intravenous injection of diazepam (2 or 5 mg). GABA levels increased progressively after intravenous injection of 5 but not 2 mg of the benzodiazepine, the differences from preinjection values being significant at 3, 5, and 8 min. Furthermore, when relative CSF GABA alterations determined after injection of diazepam were compared to those determined in sequential CSF aliquots of 10 patients without diazepam injection, mean GABA increases after diazepam were significantly different from controls in all CSF fractions. The data suggest that, in addition to its well-known effects on postsynaptic GABA function, diazepam may exert effects on endogenous GABA concentrations and/or on GABA release in the human CNS as reflected by elevation of GABA levels in human CSF.

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Both placental and blood-lumbar CSF transfer of diazepam (5 mg orally) and its two metabolites, N-desmethyldiazepam and unconjugated oxazepam, was measured (by GLC) in 15 patients undergoing Caesarean section under spinal analgesia. Differing from our earlier studies with atropine (Virtanen et al. 1982; Kanto et al. 1981 & 1987), a reasonably fast penetration of diazepam and its two metabolites through the two biological membranes was found. Diazepam, N-desmethyldiazepam and to a lesser extent unconjugated oxazepam accumulated on the foetal side of the placenta, apparently due to a higher degree of plasma protein binding in the foetus. No accumulation was found in CSF, probably due to the lack of binding proteins in this tissue compartment. Concerning atropine, lumbar CSF with an incomplete drug penetration was found to be a "deeper" compartment than amniotic fluid, but in the present study with diazepam there was no clear difference between these two tissue compartments.

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A five-compartment open model was used to simulate the blood concentration profiles of diazepam and its metabolite, desmethyldiazepam, following single- and multiple-dose administrations of diazepam. The parameter estimates for diazepam were previously reported literature values. The parameters estimates for the metabolite were calculated from literature values of blood concentrations of desmethyldiazepam following the administration of clorazepate. The five-compartment open model suggests that approximately 50% of the administered diazepam is biotransformed to desmethyldiazepam, and that the elimination profile of the metabolite is not altered by the presence of the drug. The model may also be readily adapted to predict the concentrations of diazepam and desmethyldiazepam in cerebrospinal fluid following the administration of diazepam by simply correcting the blood or plasma concentrations of the drug and metabolite for the degree of plasma protein binding.

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Six samples of ventricular cerebral spinal fluid from an external ventricular drain, and peripheral venous blood were taken during the course of parenteral diazepam administration in man. The diazepam level was measured by gas phase chromatography. The levels of diazepam in the CSF were low compared with the plasms concentrations and around the same level as the free circulating fraction. The drug appears in the CSF relatively late after intravenous administration. It would appear that the passage of the drug from the blood into the CSF is slow and not very marked. Therefore it is not the main cause for the pharmocodynamic effects which are the result of the drug passing the blood barrier in the more restricted sense.

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1 Salivary and plasma diazepam and nordiazepam concentrations were measured in 51 paired samples from four experimental situations. In seven of the patients CSF samples were estimated. 2 Correlation of 0.89 (P less than 0.001) was observed between salivary and plasma diazepam and 0.81 (P less than 0.001) between salivary and plasma nordiazepam. 3 Mean salivary diazepam was 1.6% (+/- 0.3%) of the plasma diazepam. It was found to vary markedly in an acute dosage study. Mean salivary nordiazepam was 2.9% (+/- 1%) of the plasma measure and was dependent on salivary flow rate. 4 CSF diazepam was in equilibrium with unbound plasma diazepam and salivary diazepam. 5 Mean protein binding of diazepam in vitro was 99.3% with no variations as a function of concentration. 6 The results suggest salivary diazepam and nordiazepam measures to be of value in epidemiological studies. However, they do not predict accurately the plasma total or unbound drug concentration from a salivary sample in an individual.

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Five dogs received a single 1.0 mg/kg dose of diazepam (DZ) IV. Concentrations of DZ and its major metabolite desmethyldiazepam (DMDZ) were simultaneously measured in plasma and cisternal cerebrospinal fluid (CSF) for up to 8 h after the dose by electron-capture gas-liquid chromatography. DZ was rapidly eliminated from plasma (half-life 0.3--1.3 h); DZ disappearance was mirrored by formation of DMDZ, which in turn was eliminated slowly, Both DZ and DMDZ rapidly penetrated CSF and concentrations in CSF declined parallel with those in plasma. Despite rapid uptake, the extent of CSF transfer of DZ and DMDZ was limited by plasma protein binding. Mean CSF:plasma concentrtion ratios for DZ (range 0.023--0.137) and DMDZ (range 0.047--0.119) were highly correlated with the unbound fraction in plasma (r = 0.95 and 0.80, respectively). Thus DZ and DMDZ concentrations in CSF, presumed to reflect concentrations at the site of action, are determined by unbound plasma concentrations. The intensity of pharmacologic action is more likely to correlate with unbound than with total plasma concentrations.

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