Modular real-time PCR screening assay for common European animal families.

A screening assay based on real-time PCR and melt curve analysis was developed to detect DNA from nine common European animal families/species and human. The assay consists of a 10-cycle universal pre-amplification followed by specific nested PCR and was designed to exploit the different melting temperatures (T m) of family/species-specific 12S ribosomal ribonucleic acid and cytochrome b fragments, which are amplified in duplex reactions. Case-related modular application is possible. Beyond determination of the animal family and discrimination from human DNA, evaluation of the melt curve in some cases additionally allows for species determination (e.g. cat vs. lynx). The method presents a quick, flexible and sample-saving approach to assess non-human DNA at low expenses, and it is especially useful in resolution of DNA mixtures.

Fatal doxepin intoxication--suicide or slow gradual intoxication?

The differentiation of intoxication courses is one of the most difficult challenges for forensic pathologists and toxicologists. The case of a 52-year-old female inpatient of a psychiatric clinic with multiple medications who died from doxepin intoxication is reported. Concentrations of doxepin metabolites and isomers, pharmacokinetic modelling and genotyping of the doxepin-metabolizing cytochrome P450 (CYP) enzymes led to the following conclusion: the lethal doxepin concentration of 2100 ng/mL was more likely to have been reached due to drug interactions and genetic peculiarities leading to a reduction of the metabolic capacity and not by an acute (suicidal) overdose.

Accidental clozapine intoxication in a toddler: clinical and pharmacokinetic lessons learnt.

WHAT IS KNOWN AND OBJECTIVE: Clozapine, a second generation antipsychotic which is relatively safe in overdose, has been used as an effective treatment alternative to traditional antipsychotics. The therapeutic use in children remains controversial. However, in accordance with the increasing prescription in adults, the accidental ingestion in childhood becomes more frequent. We report the youngest case of accidental clozapine ingestion. CASE SUMMARY: A 13-month-old girl presented with acute respiratory insufficiency and coma of unknown origin. The medical history, laboratory and radiological assessment did not link to aetiology until an almost spontaneous arousal after 22 h pointed towards intoxication. The initial standard drug screening using immunoassay had been negative. Hence, liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) was performed, and clozapine was detected with a serum concentration of 736 ng/mL. WHAT IS NEW AND CONCLUSION: This case illustrates the diagnostic and forensic pitfalls in a coma of unknown origin due to the limits of toxicological screening immunoassays. LC-MS/MS analysis by an established method showed clozapine metabolites (norclozapine and clozapine-N-oxide) are detectable for longer period, especially in urine, when compared with clozapine. The clinical course is presented in unique correlation with plasma and urine concentrations of clozapine and its metabolites. The elimination pattern of clozapine in toddlers is similar to adults, and the toxic dose was found to be lower when compared with school-age children and adults.

Disposition of L-738,167, a potent and long-acting fibrinogen receptor antagonist, in dogs. Dose-dependent pharmacokinetics.

L-738,167 is a potent and long-acting fibrinogen receptor antagonist and may be useful for treatment of chronic thrombotic occlusive disorders. The purposes of this study were to characterize the metabolism and disposition of L-738,167, and to investigate factors affecting its pharmacokinetic behaviors in dogs, one of the animal models used in pharmacological and toxicological studies. In vitro and in vivo experiments indicated that L-738,167 was not metabolized to any appreciable extent in dogs. Biliary excretion was found to be the major route (approximately 75%) of drug elimination. Following 1 and 3 micrograms/kg iv doses, blood pharmacokinetics of L-738,167 were linear. Total blood clearance (CLB) was much lower than hepatic blood flow, and the apparent volume of distribution at steady-state (Vdss,B) was comparable with blood volume. Blood pharmacokinetics in the dose range of 3-250 micrograms/kg were dose-dependent; both CLB and Vdss,B for L-738,167 increased markedly with increasing doses. However, the terminal half-life (t1/2) was dose-independent, with a mean value of approximately 4 days. L-738,167 was found to bind negligibly to dog plasma proteins. Determinations of whole blood (WB), platelet-rich plasma, and platelet-poor plasma concentrations after several intravenous doses of [3H]L-738,167 revealed significant concentration-dependent binding of the compound to platelets. Kinetic analysis of the platelet binding indicated that L-738,167 was bound to dog platelets with high affinity (apparent Kd approximately 1 nM platelet-poor plasma concentration) and relatively low capacity (approximately 70 nM WB concentration). Findings are consistent with the binding kinetics of L-738,167 to glycoprotein IIb/IIIa (GP IIb/IIIa) receptor, supporting that GP IIb/IIIa was the primary binding component on the platelets. It was concluded that the dose-dependent pharmacokinetics of L-738,167 were the consequence of the concentration-dependent drug-platelet binding. Due to this extensive platelet binding, L-738,167, when given in therapeutic doses or lower, resided primarily in the vascular compartment-the site of pharmacological action. At doses exceeding the receptor binding capacity, the excess amount or the unbound drug was eliminated rapidly. In all cases, the equally long t1/2 of L-738,167 was also a consequence of the high-affinity binding to platelets, in good agreement with its prolonged pharmacodynamic profile.

Microsomal metabolism of the 5-lipoxygenase inhibitor L-739,010: evidence for furan bioactivation.

The novel 5-lipoxygenase inhibitor [1S,5R]-3-cyano-1-(3-furyl)-6-(6-[3-(3 alpha-hydroxy-6,8-dioxabicyclo[3.2.1]octanyl)]pyridin-2-yl- methoxyl)naphthalene (L-739,010), when administered to rats and rhesus monkeys, was found to produce metabolites which appeared to be covalently bound to plasma proteins. Incubation of [14C]L-739,010 with rat liver microsomes did not yield appreciable amounts of soluble metabolites but resulted in covalent binding to microsomal proteins. The covalent binding was NADPH-dependent and was enhanced by 1.5- and 2-fold in liver microsomes from rats, pretreated with phenobarbital and dexamethasone, respectively. Addition of triacetyloleandomycin and diethyldithiocarbamate to the incubation mixture inhibited the covalent binding by 60% and 46%, respectively. These findings suggest that the cytochrome P450 3A family of enzymes play an important role in the bioactivation of L-739,010. The presence of GSH attenuated the covalent binding by 50%, while methoxylamine, an aldehyde trapping agent, blocked the covalent binding completely and, concurrently, produced several soluble metabolic adducts. Subsequently, major methoxylamine adducts were identified by LC-MS/MS and NMR as O-methyloximes of the ring-opened furan moiety of L-739,010. Incubation of L-739,010 with methoxylamine and hepatic microsomes from dog, rhesus monkey, and human produced similar metabolic adducts as those formed by rat liver microsomes. Therefore, under these experimental conditions, the furan moiety, which undergoes oxidative cleavage to the highly reactive 2-butene-1,4-dialdehyde, represents the major site of L-739,010 biotransformation. This putative reactive intermediate could react with microsomal proteins in vitro and physiological proteins in vivo. Since furan bioactivation is believed to be responsible for the toxicity of many furan-containing compounds, the furan moiety of L-739,010 may be regarded as undesirable.