Metabolic profiling of the Uncaria hook alkaloid geissoschizine methyl ether in rat and human liver microsomes using high-performance liquid chromatography with tandem mass spectrometry.

Geissoschizine methyl ether (GM) is an indole alkaloid found in Uncaria hook, which is a galenical constituent of yokukansan, a traditional Japanese medicine. GM has been identified as the active component responsible for anti-aggressive effects. In this study, the metabolic profiling of GM in rat and human liver microsomes was investigated. Thirteen metabolites of GM were elucidated and identified using a high-performance liquid chromatography with tandem mass spectrometry method, and their molecular structures were proposed on the basis of the characteristics of their precursor ions, product ions, and chromatographic retention times. There were no differences in the metabolites between the rat and human liver microsomes. Among the 13 identified metabolites, there were two demethylation metabolites, one dehydrogenation metabolite, three methylation metabolites, three oxidation metabolites, two water-adduct metabolites, one di-demethylation metabolite, and one water-adduct metabolite followed by oxidation. The metabolic pathways of GM were proposed on the basis of this study. This study will be helpful in understanding the metabolic routes of GM and related Uncaria hook alkaloids, and provide useful information on the pharmacokinetics and pharmacodynamics. This is the first report that describes the separation and identification of GM metabolites in rat and human liver microsomes.

Driving Impairment Cases Involving Etizolam and Flubromazolam.

This study describes 12 cases of drivers stopped for impaired driving, where a designer benzodiazepine was detected, specifically etizolam or flubromazolam. Etizolam was detected in three cases, with blood concentrations ranging from 40 to 330 ng/mL. Two of these cases had low concentrations of methamphetamine and/or amphetamine, and in the third case tetrahydrocannabinol (THC) was detected. Flubromazolam was detected in nine cases; in all cases, at least one other drug was detected, with THC being the most prevalent. The mean blood concentration of flubromazolam was 16.3 ng/mL and had a median concentration of 17.0 ng/mL, ranging from 7.0 to 31 ng/mL. The low concentrations of designer benzodiazepines that produce pharmacological effects may allow many of these drugs to go undetected using routine testing in laboratories; therefore, it is necessary to include these novel compounds within validated analytical methods to reduce the chance of reporting false negative results. The prevalence in which laboratories are detecting the presence of novel benzodiazepines in impaired drivers illustrates the increased threat to public safety. These case studies demonstrate the importance of investigating agencies and forensic laboratories to be vigilant in monitoring the emerging novel psychoactive substances in their region.

Drug metabolism by the gastrointestinal mucosa.

Circumstantial evidence for first-pass metabolism across the gastrointestinal mucosa includes reduced bioavailability after oral administration, despite complete or good absorption. There may also be route-dependent variation in the pattern of metabolism with the latter occurring to a greater extent after oral administration than after parenteral injection. However, direct proof that first-pass metabolism takes place across the gastrointestinal mucosa relies upon cannulation of either the portal or mesenteric venous tree. Such studies are not possible in most patients because of the potential hazards involved and the attendant ethical considerations. Additional information has come from the study of enzyme activity in biopsies of intestinal mucosa and experiments performed on isolated loops of intestine in various animal species. Although the former have identified the fact that enzyme activity may vary along the length of the intestine and the latter have provided quantitative information on what can occur in vivo, these data cannot be extrapolated to intact man. Both phase I (preconjugation) and phase II (conjugation) reactions have been described. However, except for oxidative deamination, e.g. tyramine and hydrolysis of esters such as pivampicillin and aspirin, phase I reactions appear to be quantitatively unimportant. In contrast, synthetic reactions are much more active. Sulphate conjugation, in particular, is important for the beta-adrenoceptor stimulants isoprenaline (isoproterenol), isoetharine and rimiterol, as well as for steroid hormones. Glucuronidation has also been demonstrated to occur in man for a small number of drugs. N-Acetylation is an important pathway and, as in the liver, there is evidence of polymorphism. Metabolism of hydralazine, isoniazid, p-aminosalicylic acid as well as certain sulphonamides by intestinal N-acetyl transferase has been demonstrated, but in all probability affects other drugs as well. Little is known concerning the physiological factors which alter the activity of the gastrointestinal drug-metabolising enzymes. However, significant drug-drug interactions have been demonstrated to occur at this site-particularly for drugs which undergo sulphate conjugation.

Formation of nitroso compounds and mutagens from tranquilizers by drug/nitrite interaction.

The formation of nitroso compounds and mutagens by drug/nitrite interaction was screened for 14 tranquilizers. The drug (0.05 M) was reacted with nitrite (0.5 M) at pH 3-3.5. After 4 h at 37 degrees C, nitroso compound formation was observed for flupentixol, chlordiazepoxide, spiperone, thiothixene, and chlorprothixene in more than 40% yield. Mutagenicity was found in the reaction products of opipramol, chlordiazepoxide, bromazepam, thiothixene, and carpipramine by the Ames assay using Salmonella typhimurium TA98 and TA100 as tester strains.

Drug prescribing in hepatobiliary disease.

Liver disease in man is associated with a variety of pathophysiological processes which may influence the disposition of drugs in several ways. Interpretation of the observed pharmacokinetic changes in liver disease requires an understanding of the relationship between systemic drug clearance (Cls), volume of distribution (Vd) and the elimination half-life [t1/2(beta)], i.e. t1/2(beta) = 0.693 . Vd/Cls. Half-life will be a measure of the fluctuation in drug level one may expect with continued administration of a drug while clearance will determine the dose required to achieve a particular steady state level. Liver disease may affect clearance and volume of distribution and so produce changes in half-life; in addition, alterations in plasma binding of drugs may occur and so influence free (unbound) drug levels. It is also possible that the end organ response, particularly in the case of sedative drugs, may be affected by liver disease. Other factors such as age, nutrition, smoking, and concomitant drug therapy may also influence drug elimination in patients with liver disease. At the present time, caution should be exercised in prescribing drugs to patients with liver disease and the dose should be titrated to the clinical response. The development of liver 'function' tests using model or marker drugs may offer some help to the prescriber in the future and enable a less empirical approach.

Behavioural and biochemical evidence of the interaction of the putative antipsychotic agent, BMY 14802 with the 5-HT1A receptor.

The behavioural and biochemical profile of the sigma ligand and putative antipsychotic agent, BMY 14802 (alpha-(4-fluorophenyl)-4-(5-fluoro-2- pyrimidinyl)-1-piperazine butanol) has been determined in the mouse and rat. In mice, pretreatment with BMY 14802 attenuated both amphetamine-induced hyperactivity and conditioned avoidance responding, consistent with its previously reported antipsychotic potential. In common with 5-HT1A receptor agonists or partial agonists, BMY 14802 induced (a) a dose-dependent hypothermia in mice; (b) aspects of the 5-HT behavioural syndrome in rats, (c) antagonised mescaline-induced head twitches in mice and (d) generalised to the 8-hydroxy-2-(di-n-propylamino)tetralin discriminative stimulus over the dose range of 3-15 mg/kg. BMY 14802 had appreciable affinity for the 5-HT1A receptor (pIC50 = 6.7 compared to 7.3 for sigma binding) and antagonised forskolin-stimulated adenylate cyclase activity with a pEC50 of 6.2, consistent with an agonist action at this receptor. The results support the involvement of 5-HT1A receptors, but not the sigma binding site, in the behavioural profile of BMY 14802.

Secretion of drugs by the parotid glands of rats and human beings.

The following drugs have been demonstrated to be secreted by the parotid glands of rats and human beings: amobarbital, chlorpromazine, codeine, glutethimide, meprobamate, pentobarbital, phenobarbital, and secobarbital. Methadone could not be detected in the parotid saliva of either rats or human beings, and morphine has been demonstrated only in parotid saliva of rats.

Blockade of apomorphine's discriminative stimulus properties: relation to neuroleptic activity in neuropharmacological and biochemical assays.

Using a food-reinforced two-lever operant conditioning procedure, rats were trained to discriminate 0.16 mg/kg apomorphine from saline. Eight neuroleptics of the phenothiazine, butyrophenone or diphenylbutylamine type were investigated for thsir ability to antagonize the discriminative stimulus properties of apomorphine. The same drugs were also assayed for in vivo antagonism of apomorphine-induced stereotyped behavior as well as for in vitro inhibition of stereospecific 3-H-haloperidol binding in rat striatal tissue preparations. The data are consistent with the hypothesis that apomorphine exerts its discriminative stimulus properties by a mechanism similar to that underlying its stereotypogenic action. The loci involved in these two phenomena are likely to be distinct.

Determination of trace lithium in human erythrocytes by electrothermal atomic-absorption spectrometry with pyrocoated graphite tubes and integrated platform.

Electrothermal graphite-furnace atomic-absorption spectroscopy with pyrocoated graphite tubes, integrated platform and matrix modification was used to determine submicromolar concentrations of trace lithium in human red blood cells. Matrix-matched samples were used to establish calibration curves for concentrations up to 0.58 microM (addition-calibration method) with satisfactory linearity (r2 > 0.99) and intra- and inter-day variability (CV < 11.4%). The median concentration of trace lithium in the cells of 40 healthy Caucasian volunteers devoid of medical or psychiatric history was 0.23 microM (inter-quartile range 0.20-0.30). The levels of trace lithium in the red blood cells correlated (r2 = 0.83) with plasma concentrations (median 0.13 microM, inter-quartile range 0.11-0.19) measured in the same blood sample. Dietary factors (e.g. consumption of lithium-containing mineral water) affected both levels. The red blood cell/plasma lithium ratio had a median value of 1.57 (inter-quartile range 1.16-2.07), implying that trace lithium is accumulated in erythrocytes. This contrasts with most reports of red blood cell/plasma ratio, measured during therapeutic treatment with lithium, for which the average value is 0.5-0.8, albeit for much higher concentrations of lithium (approx. 500-800 microM). The proposed analytical method has the required sensitivity and accuracy for determination of trace lithium in red blood cells and makes it possible to perform epidemiological studies to assess human exposure to environmental lithium in diet and beverages, and inter-individual variations in trans-membrane and renal lithium kinetics at the submicromolar level.

Factors affecting the binding of tricyclic tranquillizers and antidepressants to human serum albumin.

The linear free energy-related model for structure activity relations developed by Hansch & Fujita (1964) has been used to correlate the binding of tricylic tranquillizers and antidepressants to human serum albumin (HSA) with hydrophobic and electronic parameters. The parameters chosen being the chromatographic parameter (Rm) and the affinity of charge transfer complex formation (kc). The relative importance of these factors has been assessed by linear and multiple linear regression analysis. Results show that the major factor in binding is electronic with only a minor contribution from the hydrophobic parameter.

Potential interference of cyclobenzaprine and norcyclobenzaprine with HPLC measurement of amitriptyline and nortriptyline: resolution by GC-MS analysis.

Cyclobenzaprine and its major metabolite, norcyclobenzaprine, differ from amitriptyline and nortriptyline only by the presence of a double bond in the cycloheptane ring. Three patients developed sufficient levels of cyclobenzaprine and norcyclobenzaprine because of either rapid or long-term ingestion of cyclobenzaprine to cause positive interferences in both a Syva EMIT assay and a high-performance liquid chromatographic (HPLC) assay for identification and quantitation of tricyclic antidepressants in serum. Cyclobenzaprine coeluted with amitriptyline, and norcyclobenzaprine eluted slightly earlier than, but was poorly resolved from, nortriptyline in this HPLC assay. We found that cyclobenzaprine could be distinguished from amitriptyline and that norcyclobenzaprine could be distinguished from nortriptyline on the basis of gas chromatographic retention times upon gas chromatographic-mass spectrometric analyses after derivatization with trifluoroacetic anhydride. The compounds were also distinguishable by mass spectrometric criteria.

Tranquilizers and sedative/hypnotics: appropriate use in the elderly.

In treating the elderly patient, it is generally true that the fewer pharmacologic agents needed, the less the potential for drug interactions and puzzling toxic reactions. Nevertheless, sedative/hypnotics and anxiolytic drugs (mostly the benzodiazepines) are safe and effective therapy for the older patient, when the appropriate indications and dosages are observed. On the other hand, the toxic potential of nonbenzodiazepines (eg, barbiturates and pseudobarbiturates) and of even the relatively safe alternatives (eg, chloral hydrate, diphenhydramine, and hydroxyzine) render them less useful for the treatment of sleep disturbances. A final nonpharmacologic note: the physician should bear in mind that a balanced medical and psychological approach to sleep disturbance and anxiety in the elderly often yields success. This includes establishing a bedtime routine and using behavioral techniques, such as relaxation training, biofeedback, and even self-hypnosis. Patients can often be referred to a center where these approaches are employed.

Hydrolysis of haloperidol decanoate in vitro by cultured cells.

[14C]Haloperidol decanoate was hydrolysed by partially purified carboxylesterase but not in plasma, blood, lymph and lymphatic liquid. These fluids inhibited the enzyme-mediated hydrolysis of the ester. Within the same incubation period as above, the ester was found hydrolysed to various extents in cell cultures of isolated rat liver cells, of human and rat lymphocytes and of established cell lines (BGM cells, WI-38 cells and L6 cells). Thus, the hydrolysis of the ester was demonstrated in vitro with use of viable cell cultures instead of enzyme preparation. From the time course study on the metabolism of haloperidol decanoate in cell cultures, it was concluded that haloperidol decanoate was first concentrated in the cells and hydrolysed to haloperidol. Based on these results, the metabolic sequences in vivo leading to the formation of active principle haloperidol after intramuscular administration of its decanoate were discussed.

[Genotoxic effects of metabolic derivatives of the new drug phosphabenzide]. / Genotoksicheskie éffekty metabolicheskikh proizvodnykh novogo lekarstvennogo preparata fosfabenzida.

Genotoxic action of four possible metabolites of the new tranquilizer phosphabenzide (acetylphosphabenzide, diphenylphosphinylacetic acid, phosphabenzide hydrazone with pyruvic acid, bis-1,2-(diphenylphosphinylacetyl)hydrazine) has been studied. These metabolites belong to slightly toxic phosphororganic compounds. The Ames Salmonella/microsomes tests performed on strains TA100 and TA98 showed that of these compounds only acetylphosphabenzide possessed mutagenic action. Metabolic activation of liver microsomes decreased the mutagenic effect. The mechanism of action of acetylphosphabenzide is likely to involve the formation of acetylhydrazine, capable of producing active electrophiles attacking DNA.

[Use of gas chromatography--mass spectrometry system in toxicology]. / Aplikace systému plynové chromatografie - hmotové spektrometrie v toxikologii

In the presented paper experiences with the practical usage of gas chromatography - mass spectrometry system are given. Cases of the identification of forbidden doping drugs and some significant groups of medicaments are described. The possibility of the identification of drug metabolites and interfering substances is pointed as well. Examples of chemical ionization usage are given from literature papers. This work points out the advantages of both methods for the identification of drugs in biological material. The priority and possibility of both procedures are evaluated critically.

[Assessment of CYP2D6 activity as a form of optimizing antidepressant therapy]. / Badanie aktywnosci CYP2D6 jako forma optymalizacji terapii lekami przeciwdepresyjnymi.

Cytochrome 2D6 catalyzes oxidation processes of many antidepressants (TCAs, SSRIs, maprotyline, mianserine, nefazodon, trazodon, venlafaxine). CYP2D6 is characterized by genetically determined polymorphism which may lead to serious clinical consequences. Based on CYP2D6 activity four phenotypes are distinguished: poor metabolism (PM), intermediate (IM), extensive (normal) EM and ultrarapid (UM). In case of PM and IM increased plasma concentration of a drug and adverse events or toxicity may appear. Decreased plasma level and lack of clinical effect may be connected with the ultrarapid phenotype. CYP2D6 activity may be assessed by phenotyping or genotyping . Model drugs such as sparteine, debrisoquine, dextromethorphan and metoprolol are used in the phenotyping method. Based on the metabolic ratio of model drug the phenotype status is established. Genotyping consists in an assessment of genotype i.e. an identification of alleles coding the CYP2D6 protein. The environmental factors may modify the CYP2D6 activity and have influence on phenotyping but not genotyping results. The knowledge of CYP2D6 phenotype is of special value when drugs characterized by a narrow therapeutic index are used and in polymedicated and older patients.

Interactions of phenothiazine drugs with bile salts: micellization and binding studies.

An evaluation of the interactions of phenothiazine tranquilizer drugs (promazine hydrochloride; PMZ and promethazine hydrochloride; PMT) with bile salts viz., sodium cholate (NaC) and sodium deoxycholate (NaDC) in aqueous medium, investigated through different physicochemical measurements is presented in this work. The mixed micellization behavior and surface properties of the phenothiazine-bile salt systems have been analyzed by conductivity and surface tension measurements. Application of different theoretical approaches to all the phenothiazine-bile salt mixtures shows a non-ideal behavior. Further, the spectroscopic techniques such as UV-visible and steady state fluorescence have been employed to study the binding of phenothiazines with bile salts. The stoichiometric ratios, binding constants (K), and free energy change (ΔG) for the phenothiazine-bile salt complexes were estimated from the Benesi-Hildebrand (B-H) double reciprocal plots obtained by using the changes in spectral intensities of phenothiazines on addition of bile salts. The results are discussed in the light of use of bile salts as promising drug delivery agents for phenothiazines and hence improve their bioavailabilty.