Postmortem Brain and Blood Reference Concentrations of Alprazolam, Bromazepam, Chlordiazepoxide, Diazepam, and their Metabolites and a Review of the Literature.

To interpret postmortem toxicology results, reference concentrations for non-toxic and toxic levels are needed. Usually, measurements are performed in blood, but because of postmortem redistribution phenomena this may not be optimal. Rather, measurement in the target organ of psychoactive drugs, the brain, might be considered. Here we present reference concentrations of femoral blood and brain tissue of selected benzodiazepines (BZDs). Using LC-MS/MS, we quantified alprazolam, bromazepam, chlordiazepoxide, diazepam, and the metabolites desmethyldiazepam, oxazepam and temazepam in postmortem femoral blood and brain tissue in 104 cases. BZDs were judged to be unrelated to the cause of death in 88 cases and contributing to death in 16 cases. No cases were found with cause of death solely attributed to BZD poisoning. All BZDs investigated tended to have higher concentrations in brain than in blood with median brain-blood ratios ranging from 1.1 to 2.3. A positive correlation between brain and blood concentrations was found with R(2) values from 0.51 to 0.95. Our reported femoral blood concentrations concur with literature values, but sparse information on brain concentration was available. Drug-metabolite ratios were similar in brain and blood for most compounds. Duplicate measurements of brain samples showed that the pre-analytical variation in brain (5.9%) was relatively low, supporting the notion that brain tissue is a suitable postmortem specimen. The reported concentrations in both brain and blood can be used as reference values when evaluating postmortem cases.

Monoterpenes affect chlorodiazepoxide-micelle interaction through micellar dipole potential modifications.

The ability of several natural terpenes to affect benzodiazepine (BZD)-micelle interaction through the membrane dipolar organization was investigated. The acid-base equilibrium of chlorodiazepoxide (CDX) and the spectroscopic behavior of the electrochromic dye merocyanine were tested in the presence and in the absence of Triton X-100 micelles (used to mimic a membrane environment) containing or not cineole, menthol, geraniol or camphor. CDX's apparent pK increased in the environment of terpene-containing micelles compared with pure Triton X-100 micelles. Decrements in electric potentials (between -111 and -128 mV with respect to pure detergent) were calculated from Boltzmann equation. This result suggested, that in the presence of terpenes, the tendency of CDXH(+) to remain in the membrane phase increased. The dielectric constant (D) of the microenvironment sensed by merocyanine within Triton X-100 micelles, determined from lambda(max,2) of merocyanine monomer, was D=9 and increased in the presence of all the terpenes assayed (D congruent with 11). The decrease in merocyanine partitioning (A(peak1)/A(peak2) increased) also reflected an increment in the negative dipole potential. The present results suggest that terpenes contributed to the whole dipolar arrangement of the micelle with a dipole moment vector which had an intense component oriented parallel to the intrinsic dipole of the Triton X-100 molecules in the micelles. This led to a more negative environment of the interface region where CDX was located, and increased the net polarity of the deepest micelle regions sensed by merocyanine.

Distribution of venlafaxine in three postmortem cases.

Venlafaxine (V) is a second-generation antidepressant approved for use in the United States in 1993. It is a derivative of phenethylamine and is structurally unrelated to first- and other second-generation antidepressants. Nevertheless, its mechanism of action is similar to other antidepressants; it inhibits the reuptake of presynaptic norepinephrine and serotonin. Its major routes of elimination involve O and N demethylation. O-Desmethylvenlafaxine (ODV) is biologically active. Therapeutic concentrations of V and ODV are approximately 0.2 and 0.4 mg/L, respectively. Three cases of drug intoxication involving V are presented. V and ODV were identified by gas chromatography-nitrogen-phosphorus detection after alkaline extraction of the biological specimen. On an HP-5 column, V and ODV elute after bupropion and fluoxetine, but prior to the first-generation antidepressants, sertraline, amoxapine, and trazodone. V and ODV were confirmed by full scan electron impact gas chromatography-mass spectrometry. The heart-blood V and ODV concentrations (mg/L) in the three cases were 6.6 and 31; 84 and 15; and 44 and 50, respectively. In Case 1, acetaminophen and diphenhydramine were found in the heart blood at 140 and 2.6 mg/L respectively. In Case 2, amitriptyline, nortriptyline, and chlordiazepoxide were found in the blood at 2.8, 0.5 and 3.3 mg/L, respectively. In each case, the manner of death was suicide.

A constitutively active mutant thyrotropin-releasing hormone receptor is chronically down-regulated in pituitary cells: evidence using chlordiazepoxide as a negative antagonist.

A carboxyl-terminus truncated mutant of the guanine nucleotide-binding (G) protein-coupled TRH receptor (TRH-R) was previously shown to exhibit constitutive, i.e. TRH-independent, activity (C335Stop TRH-R). Chlordiazepoxide (CDE), a known competitive inhibitor of TRH binding to wild-type (WT) TRH-Rs, is shown to compete for binding to C335Stop TRH-Rs also. More importantly, CDE is shown to be a negative antagonist of C335Stop TRH-Rs. CDE rapidly caused the basal rate of inositol phosphate second messenger (IP) formation to decrease in AtT-20 pituitary cells stably expressing C335Stop TRH-Rs (AtT-C335Stop cells), but not in cells expressing WT TRH-Rs (AtT-WT cells). Similar observations were made in HeLa cells transiently expressing C335Stop or WT TRH-Rs. CDE inhibition of IP formation was shown to be specific for TRH-Rs using GH4C1 cells expressing both TRH-Rs and receptors for bombesin. In these cells, CDE inhibited TRH-stimulated IP formation, but had no effect on bombesin-stimulated IP formation. The effects of chronic administration of CDE were studied. Preincubation of AtT-C335Stop cells, but not AtT-WT cells, with CDE for several hours caused an increase in cell surface receptor number (up-regulation) that led to increased TRH stimulation of inositol phosphate formation and elevation of intracellular free Ca2+. Preincubation with CDE did not affect methyl-TRH binding affinity or TRH potency in cells expressing AtT-C335Stop or in AtT-WT cells. We conclude that CDE is a negative antagonist of C335Stop TRH-Rs and that constitutively active C335Stop TRH-Rs are down-regulated in AtT-20 pituitary cells in the absence of agonist.

Dual action of the cyclodiene insecticide dieldrin on the gamma-aminobutyric acid receptor-chloride channel complex of rat dorsal root ganglion neurons.

The gamma-aminobutyric acidA (GABAA) receptor-chloride channel complex is known to be the target site of dieldrin, a cyclodiene insecticide, and lindane. In order to elucidate the mechanisms of dieldrin interaction with the GABA system, whole-cell patch clamp experiments were performed with rat dorsal root ganglion neurons in primary culture. When co-applied with GABA, dieldrin exerted a dual effect on the GABA-induced chloride current. The chloride current induced by 10 microM GABA was greatly enhanced by the first 20-sec co-application with 1 microM dieldrin, but the enhancement subsided during repeated co-applications, and the current was eventually suppressed below the control level. No recovery occurred after a prolonged washing with dieldrin-free solution. Desensitization of the chloride current was accelerated by dieldrin. However, when the period of co-application was limited to 2 sec, which was short enought to avoid desensitization, no suppression of current was observed during repeated co-applications and recovery occurred after washing. The desensitization and suppression occurred with an EC50 of 92 nM, whereas the enhancement required a higher EC50 at 754 nM. The GABA-induced chloride current comprised two components, one with a high sensitivity to dieldrin suppression with an EC50 of 5 nM and the other with a lower sensitivity with an EC50 of 92 nM. Dieldrin exerted its inhibitory effect on the GABA-induced current regardless of the presence or absence of pentobarbital and chlordiazepoxide. However, its effect was attenuated by the presence of picrotoxin. Furthermore, dieldrin suppressed the GABA-induced chloride current in a noncompetitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral-type mitochondrial binding sites for benzodiazepines in GH3 pituitary cells.

Benzodiazepines (BZs) interact with two classes of high affinity binding sites, equilibrium dissociation constants in the nanomolar range, a neuronal or central-type and a non-neuronal or peripheral-type. The peripheral-type binding site has been shown to be present on the outer mitochondrial membrane and appears to be involved in regulation of cholesterol transport in steroid hormone-producing endocrine cells. In rat pituitary GH3 cells, BZs bind to receptors for thyrotropin-releasing hormone (TRH) and via interaction at a different site block Ca2+ influx through voltage-sensitive channels. These, however, are low affinity interactions occurring at micromolar BZ concentrations. Here, using [3H]Ro 5-4864, we report that GH3 cells also have high affinity peripheral-type BZ binding sites. Apparent equilibrium dissociation constants of 7.8 +/- 1.7 nM and 9.3 +/- 4.5 nM for [3H]Ro 5-4864 were measured with intact cells and isolated mitochondria, respectively. As predicted from studies of these sites in other cells, the order of potencies of BZs to displace [3H]Ro 5-4864 was Ro 5-4864 greater than diazepam (DZP) much greater than clonazepam (CIZP); chlordiazepoxide (CDE) did not affect binding. Nifedipine, a dihydropyridine antagonist of Ca2+ channels that has been shown to displace BZs from peripheral-type sites in other cells, was shown to be a competitive inhibitor of [3H]Ro 5-4864 binding with a half-effective concentration in the micromolar range. Ro 5-4864, however, had no effect on Ca2+ influx or efflux in mitochondria isolated from GH3 cells. Hence, GH3 cells exhibit mitochondrial, peripheral-type BZ binding sites but the role of these putative receptors in these neuroendocrine cells, which do not produce steroid hormones, is unclear.

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.

Genotoxicity of N-nitrosochlordiazepoxide in cultured mammalian cells.

Chlordiazepoxide, a benzodiazepine derivative commonly used for the treatment of anxiety, was found to react with sodium nitrite in HCl aqueous solution yielding, at pH ranging from 0.5 to 5,N-nitrosochlordiazepoxide (NO-CDE). In the absence of a metabolic activation system, a dose-dependent frequency of DNA single-strand breaks was revealed by the alkaline elution technique in V79 cells exposed to subtoxic NO-CDE concentrations ranging from 33 to 330 microM. DNA lesions were only partially repaired within 48 hr, and their promutagenic character was demonstrated by the induction of 6-thioguanine resistance in the same cells. The genotoxicity of NO-CDE was confirmed by results obtained in metabolically competent primary cultures of both rat and human hepatocytes, which displayed similar dose-related amounts of DNA fragmentation and of DNA repair synthesis after treatment with concentrations ranging from 33 to 1000 microM. In conclusions similar to those which might occur in the stomach of a patient taking chlordiazepoxide the concentration of NO-CDE in the reaction mixture (50 microM) was of the same order as the concentrations found to induce a genotoxic effect in cultured mammalian cells.

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.

Evidence for tight coupling of receptor occupancy by thyrotropin-releasing hormone to phospholipase C-mediated phosphoinositide hydrolysis in rat pituitary cells: use of chlordiazepoxide as a competitive antagonist.

Chlordiazepoxide (CDE) has been shown to antagonize the effects of TRH to stimulate the hydrolysis of phosphoinositides and elevate cytoplasmic free calcium in rat pituitary tumor (GH3) cells. Herein, we show that CDE inhibits TRH stimulation of PRL secretion and that the effect of CDE to antagonize TRH action is caused by its ability to compete with TRH for binding to receptors on GH3 cells. We also use CDE to explore whether continued receptor occupancy is required for prolonged stimulation of cellular responses. CDE had no effect on basal PRL secretion, but caused a dose-dependent inhibition of TRH-induced PRL secretion. CDE decreased the affinity of TRH binding to intact GH3 cells without affecting the maximum binding capacity. As shown previously, CDE had no effect on phosphoinositide metabolism, which was monitored because it appears to be a mechanism for signal transduction by TRH, and when added simultaneously with TRH, caused a dose-dependent inhibition of TRH-induced phosphoinositide metabolism. When CDE was added to cells 2.5 or 5 min after TRH, CDE rapidly terminated the stimulation by TRH of phosphoinositide hydrolysis, shown as inhibition of the continued formation of inositol phosphates and inositol, and of the decrease in phosphoinositides. Lastly, when cells were stimulated with 50 nM TRH, then exposed to 100 microM CDE, and finally to 1000 nM TRH, inositol phosphate formation was stimulated, then inhibited, and then restimulated. These data demonstrate that CDE acts as a competitive antagonist of TRH action on GH3 cells by competing with TRH for binding to its receptor and that continued stimulation by TRH of phospholipase C-mediated hydrolysis of phosphoinositides is tightly coupled to receptor occupancy.

Benzodiazepine interactions with central thyroid-releasing hormone binding sites: characterization and physiological significance.

Thyrotropin-releasing hormone (TRH) and several TRH analogs were examined in the [3H]-3-Me-His2-TRH ([3H]MeTRH) receptor-binding assay in rat amygdala, striatal and cortical membranes. The benzodiazepine, chlordiazepoxide, as reported in the literature was found to displace [3H]MeTRH with an IC50 value of 3.6 X 10(-7) M in amygdala membranes. Midazolam was, however, identified as being 6-fold more active than chlordiazepoxide with an IC50 value of 6.3 X 10(-8) M. The effect of these benzodiazepines on [3H]MeTRH binding did not appear to be related to their anxiolytic activity because the novel pyrazoloquinoline nonsedating anxiolytic, CGS 9896 was without effect on [3H]MeTRH binding at concentrations up to 1 X 10(-5) M. Chlordiazepoxide had similar activity in cortical membranes whereas midazolam was some 5 times less active in this preparation than in amygdala. Both compounds were weak displacers of [3H]MeTRH binding in striatal membranes, being at least two orders of magnitude less potent than in amygdala. In contrast TRH and its analogs, RX 77368 and DN-1417, were approximately 2 to 8 times more active in striatum than amygdala membranes. TRH and DN-1417 were less active in cortical membranes whereas RX 77368 was some three times more active than in striatum and amygdala. In three test procedures indicative of TRH agonist activity; thyroid-stimulating hormone release, reversal of pentobarbital sleeping time in mice and elevation of cerebellar cyclic GMP levels, the benzodiazepines were found to be devoid of activity, whereas TRH and related compounds produced their expected responses.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Evidence of specific benzodiazepine binding to myometrial membrane preparations from human pregnant uterus.

The muscle relaxant effect of benzodiazepines (BDZ) is widely held to be mediated at central as well as at peripheral level. In this study we report that crude membrane preparations from the myometrium of pregnant women possess binding sites for [3H]-RO 5-4864, specific ligand for the peripheral type of BDZ receptor. Scatchard analysis shows a high affinity binding site (KD = 3.1 +/- 1.2 nM) and a class of low affinity binding sites (KD greater than 30 nM). Displacement experiments show that various BDZ are differently effective in inhibiting [3H]-RO 5-4864 binding: RO 5-4864 greater than diazepam greater than flunitrazepam greater than lorazepam greater than chlordiazepoxide. In conclusion, these data seem to demonstrate that human myometrium possesses specific binding sites of 'peripheral type' for BDZ. It may be suggested that these binding sites are involved in mediating the myometrial relaxant effect exerted by BDZ. Thus, peripherally active BDZ could be used as tocolytic agents, avoiding BDZ central effects.

Lack of effect of nizatidine on hepatic drug metabolism in man.

The effect of nizatidine, a new H2-receptor antagonist, on the hepatic metabolism of three probe drugs was studied in normal volunteers. The drugs studied were chlordiazepoxide and theophylline which are metabolized in part by N-demethylation by the hepatic microsomal cytochrome P-450 system and lorazepam which is conjugated to lorazepam glucuronide. A 7 day course of nizatidine did not interfere with the disposition of any of these therapeutic agents in man.

Solubilization of peripheral benzodiazepine-binding sites from rat kidney.

The ability of a variety of detergents to solubilize peripheral benzodiazepine-binding sites from rat kidney was tested. Of all the detergents tested, only digitonin was found to be suitable for solubilization. This detergent solubilized 21% of the binding activity; 47% was inactivated, and 32% remained in the pellet. Specific binding of [3H]Ro 5-4864 to membrane-bound and solubilized peripheral benzodiazepine-binding sites was saturable, yielding a linear Scatchard plot (r = 0.96). KD values obtained for the membrane-bound and solubilized peripheral benzodiazepine binding sites were 3.9 +/- 0.4 nM and 5.4 +/- 0.4 nM, respectively. Respective Bmax values were 4.6 +/- 0.5 and 1.9 +/- 0.2 pmol/mg of protein. The KD value for the solubilized material obtained from kinetic experiments was 5.3 +/- 0.6 nM. The potency of PK 11195, Ro 5-4864, diazepam, flurazepam, chlordiazepoxide, Ro 15-1788, methyl-beta-carboline-3-carboxylate, and clonazepam to displace bound [3H]Ro 5-4864 from peripheral binding sites was similar in the membrane-bound and the soluble states. Most of the binding activity of the solubilized binding sites was destroyed by heating at 60 degrees C for 30 min or by treatment with 2 M guanidinium chloride or 4 M urea. More than 95% of the binding activity of the solubilized binding sites was retained after 18 hr at 4 degrees C, and more than 60% was retained after 4 days at the same temperature. These results indicate that the binding characteristics of peripheral benzodiazepine-binding sites extant in the membrane-bound state are retained after solubilization.

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.

Psychotropic drugs in the elderly. Selection of the appropriate agent.

Caution is required when prescribing psychotropic drugs to elderly patients, because of altered pharmacokinetics. Benzodiazepines with a relatively short half-life, such as temazepam (Restoril), alprazolam (Xanax), and lorazepam (Ativan), are better hypnotics for the elderly than those with a long half-life. Lorazepam and clorazepate dipotassium (Tranxene) are appropriate anxiolytics for elderly patients. Antipsychotics have anticholinergic and extrapyramidal symptoms in varying degrees. Thiothixene (Navane) and haloperidol (Haldol) in low doses are the drugs of choice despite their extrapyramidal symptoms. Trazodone (Desyrel) is a good antidepressant for the elderly because of its minimal anticholinergic effects, although it is quite sedating. Desipramine (Norpramin) is the least sedating and has the fewest anticholinergic effects of the tricyclic antidepressants and therefore may be better tolerated. Monoamine oxidase inhibitors and lithium should be used with great caution.