Cytotoxicity evaluation of haloperidol, clozapine and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells

Abstract Schizophrenia is an illness that affects 26 million people worldwide. However, conventional antipsychotics present side effects and toxicity, highlighting the need for new antipsychotics. We aimed to evaluate the cytotoxicity of haloperidol (HAL), clozapine (CLO), and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells. The neutral red uptake assay and the MTT assay were performed to evaluate cell viability and mitochondrial activity, morphological changes were assessed, and intracellular reactive oxygen species (ROS) detection was performed. HAL and CLO (0.1 µM) showed a decrease in cell viability in the neutral red uptake assay and in the MTT assay. In addition, cell detachment, content decrease, rounding and cell death were also observed at 0.1 µM for both antipsychotics. An increase in ROS was observed for HAL (0.001, 0.01 and 1 µM) and CLO (0.01 and 1 µM). PT-31 did not alter cell viability in any of the assays, although it increased ROS at 0.01 and 1 µM. HAL and CLO present cytotoxicity at 0.1 µM, possibly through apoptosis and necrosis. In contrast, PT-31 does not present cytotoxicity to NIH-3T3 cells. Further studies must be performed for a better understanding of these mechanisms and the potential risk of conventional antipsychotics

Pharmacogenetic excitation of the median raphe region affects social and depressive-like behavior and core body temperature in male mice.

AIMS: Median raphe region (MRR) is an important bottom-up regulatory center for various behaviors as well as vegetative functions, but detailed descriptions and links between the two are still largely unexplored. METHODS: Pharmacogenetics was used to study the role of MRR in social (sociability, social interaction, resident intruder test) and emotional behavior (forced swim test) parallel with some vegetative changes (biotelemetry: core body temperature). Additionally, to validate pharmacogenetics, the effect of clozapine-N-oxide (CNO), the ligand of the artificial receptor, was studied by measuring (i) serum and brainstem concentrations of CNO and clozapine; (ii) MRR stimulation induced neurotransmitter release in hippocampus; (iii) CNO induced changes in body temperature and locomotor activity. KEY FINDINGS: MRR stimulation decreased locomotion, increased friendly social behavior in the resident intruder test and enhanced depressive-like behavior. The latter was accompanied by diminished decrease in core body temperature. Thirty minutes after CNO injection clozapine was predominant in the brainstem. Nonetheless, peripheral CNO injection was able to induce glutamate release in the hippocampus. CNO had no immediate (<30 min) or chronic (repeated injections) effect on the body temperature or locomotion. SIGNIFICANCE: We confirmed the role of MRR in locomotion, social and depressive-like behavior. Most interestingly, only depressive-like behavior was accompanied by changed body temperature regulation, which was also observed in human depressive disorders previously. This indicates clinical relevance of our findings. Despite low penetration, CNO acts centrally, but does not influence the examined basic parameters, being suitable for repeated behavioral testing.

Analysis of clozapine in its tablets using two novel spectrophotometric reactions targeting its tertiary amino group.

Two simple spectrophotometric methodologies have been proposed and validated for the measurement of an atypical antipsychotic drug Clozapine (CLZ). Method A depends on interaction of CLZ with N-bromosuccinimide(NBS) resulting in formation of a yellowish orange colored product, measured at 320 nm. The linearity range was 5.0-70.0 µg/mL. Method B depends on condensation of the same drug with acetic acid mixed anhydride reagent producing a purple colored product, measured at 319 nm. The linearity range was 8.0-24.0 µg/mL. All parameters affecting the reaction condition (volume of both reagent, temperature, time and the different diluting solvents) were optimized. Both methods were successfully applied to assay CLZ in its pure form and tablets giving mean percentage recoveries of (98.87 ± 1.8 and 100 ± 1.7) for method A, and corresponding values of (98.6 ± 0.96 and 99.5 ± 1) for method B. Besides, the study of reactions stoichiometry was performed and the reaction mechanisms were proposed.

Quantification and assessment of detection capability in imaging mass spectrometry using a revised mimetic tissue model.

Aim: A revised method of preparing the mimetic tissue model for quantitative imaging mass spectrometry (IMS) is evaluated. Concepts of assessing detection capability are adapted from other imaging or mass spectrometry (MS)-based technologies to improve upon the reliability of IMS quantification. Materials & methods: The mimetic tissue model is prepared by serially freezing spiked-tissue homogenates into a cylindrical mold to create a plug of tissue with a stepped concentration gradient of matrix-matched standards. Weighted least squares (WLS) linear regression is applied due to the heteroscedastisity (change in variance with intensity) of most MS data. Results & conclusions: Imaging poses several caveats for quantification which are unique compared with other MS-based methods. Aspects of the design, construction, application, and evaluation of the matrix-matched standard curve for the mimetic tissue model are discussed. In addition, the criticality of the ion distribution in the design of a purposeful liquid chromatography coupled to mass spectrometry (LC-MS) validation is reviewed.

Multicenter Validation Study of Quantitative Imaging Mass Spectrometry.

The aim of this study was to assess potential sources of variability in quantitative imaging mass spectrometry (IMS) across multiple sites, analysts, and instruments. A sample from rat liver perfused with clozapine was distributed to three sites for analysis by three analysts using a predefined protocol to standardize the sample preparation, acquisition, and data analysis parameters. In addition, two commonly used approaches to IMS quantification, the mimetic tissue model and dilution series, were used to quantify clozapine and its major metabolite norclozapine in isolated perfused rat liver. The quantification was evaluated in terms of precision and accuracy with comparison to liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The results of this study showed that, across three analysts with six replicates each, both quantitative IMS methods achieved relative standard deviations in the low teens and accuracies of around 80% compared to LC-MS/MS quantification of adjacent tissue sections. The utility of a homogeneously coated stable-isotopically labeled standard (SIL) for normalization was appraised in terms of its potential to improve precision and accuracy of quantification as well as qualitatively reduce variability in the sample tissue images. SIL normalization had a larger influence on the dilution series, where the use of the internal standard was necessary to achieve accuracy and precision comparable to the non-normalized mimetic tissue model data. Normalization to the internal standard appeared most effective when the intensity ratio of the analyte to internal standard was approximately one, and thus precludes this method as a universal normalization approach for all ions in the acquisition.

Pharmacokinetic and pharmacodynamic actions of clozapine-N-oxide, clozapine, and compound 21 in DREADD-based chemogenetics in mice.

Muscarinic Designer Receptors Exclusively Activated by Designer Drugs (DREADD) gated by clozapine-N-oxide (CNO) allow selective G-protein cascade activation in genetically specified cell-types in vivo. Here we compare the pharmacokinetics, off-target effects and efficacy of CNO, clozapine (CLZ) and compound 21 (Cmpd-21) at the inhibitory DREADD human Gi-coupled M4 muscarinic receptor (hM4Di). The half maximal effective concentration (EC50) of CLZ was substantially lower (0.42 nM) than CNO (8.1 nM); Cmpd-21 was intermediate (2.95 nM). CNO was back-converted to CLZ in mice, and CLZ accumulated in brain tissue. However, CNO itself also entered the brain, and free cerebrospinal fluid (CSF) levels were within the range to activate hM4Di directly, while free (CSF) CLZ levels remained below the detection limit. Furthermore, directly injected CLZ was strongly converted to its pharmacologically active metabolite, norclozapine. Cmpd-21 showed a superior brain penetration and long-lasting presence. Although we identified a wide range of CNO and Cmpd-21 off-targets, there was hardly any nonspecific behavioural effects among the parameters assessed by the 5-choice-serial-reaction-time task. Our results suggest that CNO (3-5 mg/kg) and Cmpd-21 (0.4-1 mg/kg) are suitable DREADD agonists, effective at latest 15 min after intraperitoneal application, but both require between-subject controls for unspecific effects.

Rapid degradation of clozapine by heterogeneous photocatalysis. Comparison with direct photolysis, kinetics, identification of transformation products and scavenger study.

In this study TiO2-mediated photocatalytic degradation of the persistent drug clozapine under the simulated solar radiation was studied for the first time. The experiments were conducted both in the ultrapure and river water, which enabled the assessment of the organic matrix impact. The direct and indirect photolysis experiments were conducted for a comparison. Influence of the catalyst loading on the efficiency of the process was also assessed, and the highest catalyst loading (300 mg L-1) was found to be the most effective. The TiO2 photocatalysis was extremely effective for clozapine degradation - the decomposition was almost 300 times faster in comparison to the direct photolysis (t1/2 = 1.7 min, neither clozapine, nor the intermediates were detected after 20 min of irradiation), and presence of the organic matrix did not negatively affect the process. Nevertheless the photocatalytic process turned out to be highly sensitive to act of the ROS scavengers. Thirteen transformation products (TPs) were found and their structures were elucidated by the means of high resolution mass spectrometry. Properties - toxicity, biodegradability, BCF and BAF - of TPs and the parent molecule were estimated with the use of computational methods. Identified TPs were found as generally less toxic and more biodegradable than clozapine.

Effect of sulfobutyl ether-ß-cyclodextrin and propylene glycol alginate on the solubility of clozapine.

Clozapine (CLZ) is an atypical antipsychotic medication used in the treatment of schizophrenia and is poorly soluble in water (0.05 mM). In this study, we have investigated the effect of ß-cyclodextrin (CD) and its derivatives on the solubility of CLZ. The solubility of the CLZ was measured to generate a phase solubility diagram, and the interaction between CLZ and sulfobutyl ether-ß-cyclodextrin (SBE-ß-CD) in aqueous solution was observed by 1H- and 2D rotating-frame Overhauser enhancement spectroscopy (ROESY)-NMR methods. Moreover, the synergistic effect of SBE-ß-CD and water-soluble polymers, including polyvinylpyrrolidone, hydroxypropyl methylcellulose, carboxymethylcellulose sodium salt, polyvinyl alcohol, sodium alginate, and propylene glycol alginate (PGA), on the solubility of CLZ was investigated. The results show that the solubility of CLZ with 1 w/v% PGA was 7.6 mM, which was almost four times greater than that of CLZ without PGA in a 15 mM SBE-ß-CD solution. In contrast, the solubility of CLZ with 1 w/v % PGA in an aqueous solution decreased by one-third relative to that of CLZ in a 15 mM SBE-ß-CD solution. 2D ROESY-NMR indicated that a CLZ/SBE-ß-CD/PGA ternary complex formed. It was found that the combination of PGA and SBE-ß-CD enhanced the solubility of CLZ.

Toward Higher Sensitivity in Quantitative MALDI Imaging Mass Spectrometry of CNS Drugs Using a Nonpolar Matrix.

Tissue-specific ion suppression is an unavoidable matrix effect in MALDI mass spectrometry imaging (MALDI-MSI), the negative impact of which on precision and accuracy in quantitative MALDI-MSI can be reduced to some extent by applying isotope internal standards for normalization and matrix-matched calibration routines. The detection sensitivity still suffers, however, often resulting in significant loss of signal for the investigated analytes. An MSI application considerably affected by this phenomenon is the quantitative spatial analysis of central nervous system (CNS) drugs. Most of these drugs are low molecular weight, lipophilic compounds, which exhibit inefficient desorption and ionization during MALDI using conventional polar acidic matrices (CHCA, DHB). Here, we present the application of the (2-[(2 E)-3-(4- tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile) matrix for high sensitivity imaging of CNS drugs in mouse brain sections. Since DCTB is usually described as an electron-transfer matrix, we provide a rationale (i.e., computational calculations of gas-phase proton affinity and ionization energy) for an additional proton-transfer ionization mechanism with this matrix. Furthermore, we compare the extent of signal suppression for five different CNS drugs when employing DCTB versus CHCA matrices. The results showed that the signal suppression was not only several times lower with DCTB than with CHCA but also depended on the specific tissue investigated. Finally, we present the application of DCTB and ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry to quantitative MALDI imaging of the anesthetic drug xylazine in mouse brain sections based on a linear matrix-matched calibration curve. DCTB afforded up to 100-fold signal intensity improvement over CHCA when comparing representative single MSI pixels and >440-fold improvement for the averaged mass spectrum of the adjacent tissue sections.

Simulation of phase I metabolism reactions of clozapine by HLM and photocatalytic methods with the use of UHPLC-ESI-MS/MS.

In this study the comparison of human liver microsomes in in vitro incubation as well as ZnO- and TiO2 -assisted photocatalytic degradation of clozapine as a mimicking method of phase I metabolism transformation was performed. Based on reversed-phase UHPLC separation and high-resolution MS/MS data, eight transformation products were identified and seven of them were found to be hepatic metabolites of the parent compound. The multivariate chemometric comparison of the obtained results shows ZnO-assisted photocatalysis to be a more suitable approach to phase I metabolism simulation. The photocatalytic experiments demonstrated that the disappearance of clozapine followed pseudo-zero order kinetics.

Correlación de las concentraciones plasmáticas de clozapina con el síndrome metabólico / Correlation of plasma concentrations of clozapine with the metabolic syndrome

La clozapina ha demostrado ser el antipsicóticomás efectivo para la esquizofrenia resistente, perotambién está vinculada con un mayor riesgo de producir alteraciones metabólicas en los pacientes. Se realizó un estudio prospectivo de pacientes de la Policlínica del Hospital Vilardebó donde se correlacionaron las siguientes variables: dosis diaria de clozapina, concentración plasmática de clozapina y su principal metabolito norclozapinaa predosis, cociente metabólico clozapina/norclozapina, sexo, edad, estatus fumador, duración del tratamiento con clozapina, marca comercialde clozapina utilizada, comedicación con ácido valproico, comedicación con antipsicóticos, comedicación con litio y consumo de café enrelación con el síndrome metabólico (definido porla Asociación Latinoamericana de Diabetes). Laúnica covariable significativa como dependiente del síndrome metabólico fue la concentración plasmática en valle de clozapina. El modelo que se encontró para dicha explicación es el siguiente: P(SM)=Exp(-1.69+0.00358*CZP)/((1+Exp(-1.69+0.00358*CZP)). A mayores concentraciones de clozapina el riesgo de síndrome metabólico aumenta. Esto puede tener implicancias en la clínica, ya que a pacientes con altas concentraciones de clozapina se deberían buscar estrategias para disminuir el impacto metabólico que pueda existir.

Clozapine has proven to be the most effective antipsychotic for resistant schizophrenia, but it is also linked to an increased risk of producing metabolic alterations in patients. A prospective study of patients from the Vilardebó Hospital Polyclinic where the following variables were correlated: daily dose of clozapine, plasma concentration of clozapine and its main metabolite norclozapinaa predose, metabolic ratio clozapine / norclozapine, sex, age, smoking status, duration of the treatment with clozapine, trademark of clozapine used, comedication with valproic acid, comedication with antipsychotics, comedication with lithium and consumption of coffee related to the metabolic syndrome (defined by the Latin American Diabetes Association). The only significant covariate as dependent on the metabolic syndrome was the plasma concentration in clozapine. The model found for this explanation is as follows: P (SM) = Exp (-1.69 + 0.00358 * CZP) / ((1 + Exp (-1.69 + 0.00358 * CZP)) At higher concentrations of clozapine the risk of Metabolic syndrome increases This may have clinical implications, since patients with high concentrations of clozapine should seek strategies to reduce the metabolic impact that may exist.

Molecular processes in an electrochemical clozapine sensor.

Selectivity presents a crucial challenge in direct electrochemical sensing. One example is schizophrenia treatment monitoring of the redox-active antipsychotic clozapine. To accurately assess efficacy, differentiation from its metabolite norclozapine-similar in structure and redox potential-is critical. Here, the authors leverage biomaterials integration to study, and effect changes in, diffusion and electron transfer kinetics of these compounds. Specifically, the authors employ a catechol-modified chitosan film, which the authors have previously presented as the first electrochemical detection mechanism capable of quantifying clozapine directly in clinical serum. A key finding in our present work is differing dynamics between clozapine and norclozapine once the authors interface the electrodes with chitosan-based biomaterial films. These additional dimensions of redox information can thus enable selective sensing of largely analogous small molecules.

Rapid determination of some psychotropic drugs in complex matrices by tandem dispersive liquid-liquid microextraction followed by high performance liquid chromatography.

Simple and rapid determinations of some psychotropic drugs in some pharmaceutical wastewater and human plasma samples were successfully accomplished via the tandem dispersive liquid-liquid microextraction combined with high performance liquid chromatography-ultraviolet detection (TDLLME-HPLC-UV). TDLLME of the three psychotropic drugs clozapine, chlorpromazine, and thioridazine was easily performed through two consecutive dispersive liquid-liquid microextractions. By performing this convenient method, proper sample preconcentrations and clean-ups were achieved in just about 7min. In order to achieve the best extraction efficiency, the effective parameters involved were optimized. The optimal experimental conditions consisted of 100µL of CCl4 (as the extraction organic solvent), and the pH values of 13 and 2 for the donor and acceptor phases, respectively. Under these optimum experimental conditions, the proposed TDLLME-HPLC-UV technique provided a good linearity in the range of 5-3000ngmL-1 for the three psychotropic drugs with the correlation of determinations (R2s) higher than 0.996. The limits of quantification (LOQs) and limits of detection (LODs) obtained were 5.0ngmL-1 and 1.0-1.5ngmL-1, respectively. Also the proper enrichment factors (EFs) of 96, 99, and 88 for clozapine, chlorpromazine, and thioridazine, respectively, and good extraction repeatabilities (relative standard deviations below 9.3%, n=5) were obtained.

In Vitro Effects of Concomitant Use of Herbal Preparations on Cytochrome P450s Involved in Clozapine Metabolism.

Herbal supplements are increasingly used in psychiatric practice. Our epidemiological study has identified several herbal preparations associated with adverse outcomes of antipsychotic therapy. In this study, we evaluated the in vitro effects of four herbal preparations-Radix Rehmanniae (RR), Fructus Schisandrae (FS), Radix Bupleuri (RB) and Fructus Gardeniae (FG)-on cytochrome P450s (CYPs) involved in the metabolism of clozapine in human liver microsomes (HLMs) and recombinant human cytochrome P450 enzymes (rCYPs). N-desmethylclozapine and clozapine N-oxide, two major metabolites of clozapine, were measured using high-performance liquid chromatography (HPLC). FG, RR and RB showed negligible inhibitory effects in both in vitro systems, with estimated half-maximal inhibitory concentrations (IC50) and apparent inhibitory constant values (Ki) greater than 1 mg/mL (raw material), suggesting that minimal metabolic interaction occurs when these preparations are used concomitantly with clozapine. The FS extract affected CYP activity with varying potency; its effect on CYP 3A4-catalyzed clozapine oxidation was relatively strong (Ki: 0.11 mg/mL). Overall, the weak-to-moderate inhibitory effect of FS on in vitro clozapine metabolism indicated its potential role in herb-drug interaction in practice.

Bioconcentration of two basic pharmaceuticals, verapamil and clozapine, in fish.

The present study examined the bioconcentration of 2 basic pharmaceuticals: verapamil (a calcium channel blocker) and clozapine (an antipsychotic compound) in 2 fresh water fishes, fathead minnow and channel catfish. In 4 separate bioconcentration factor (BCF) experiments (2 chemicals × 1 exposure concentration × 2 fishes), fathead minnow and channel catfish were exposed to 190 µg/L and 419 µg/L of verapamil (500 µg/L nominal) or 28.5 µg/L and 40 µg/L of clozapine (50 µg/L nominal), respectively. Bioconcentration factor experiments with fathead consisted of 28 d uptake and 14 d depuration, whereas tests conducted on catfish involved a minimized test design, with 7 d each of uptake and depuration. Fish (n = 4-5) were sampled during exposure and depuration to collect different tissues: muscle, liver, gills, kidneys, heart (verapamil tests only), brain (clozapine tests only), and blood plasma (catfish tests only). Verapamil and clozapine concentrations in various tissues of fathead and catfish were analyzed using liquid chromatography-mass spectrometry. In general, higher accumulation rates of the test compounds were observed in tissues with higher perfusion rates. Accumulation was also high in tissues relevant to pharmacological targets in mammals (i.e. heart in verapamil test and brain in the clozapine test). Tissue-specific BCFs (wet wt basis) for verapamil and clozapine ranged from 0.7 to 75 and from 31 to 1226, respectively. Tissue-specific concentration data were used to examine tissue-blood partition coefficients.

Imaging MALDI MS of Dosed Brain Tissues Utilizing an Alternative Analyte Pre-extraction Approach.

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry has been adopted in the pharmaceutical industry as a useful tool to detect xenobiotic distribution within tissues. A unique sample preparation approach for MALDI imaging has been described here for the extraction and detection of cobimetinib and clozapine, which were previously undetectable in mouse and rat brain using a single matrix application step. Employing a combination of a buffer wash and a cyclohexane pre-extraction step prior to standard matrix application, the xenobiotics were successfully extracted and detected with an 8 to 20-fold gain in sensitivity. This alternative approach for sample preparation could serve as an advantageous option when encountering difficult to detect analytes.

Prediction of brain clozapine and norclozapine concentrations in humans from a scaled pharmacokinetic model for rat brain and plasma pharmacokinetics.

BACKGROUND: Clozapine is highly effective in treatment-resistant schizophrenia, although, there remains significant variability in the response to this drug. To better understand this variability, the objective of this study was to predict brain extracellular fluid (ECF) concentrations and receptor occupancy of clozapine and norclozapine in human central nervous system by translating plasma and brain ECF pharmacokinetic (PK) relationships in the rat and coupling these with known human disposition of clozapine in the plasma. METHODS: Unbound concentrations of clozapine and norclozapine were measured in rat brain ECF using quantitative microdialysis after subcutaneous administration of a 10 mg/kg single dose of clozapine or norclozapine. These data were linked with plasma concentrations obtained in the same rats to develop a plasma-brain ECF compartmental model. Parameters describing brain ECF disposition were then allometrically scaled and linked with published human plasma PK to predict human ECF concentrations. Subsequently, prediction of human receptor occupancy at several CNS receptors was based on an effect model that related the predicted ECF concentrations to published concentration-driven receptor occupancy parameters. RESULTS: A one compartment model with first order absorption and elimination best described clozapine and norclozapine plasma concentrations in rats. A delay in the transfer of clozapine and norclozapine from plasma to the brain ECF compartment was captured using a transit compartment model approach. Human clozapine and norclozapine concentrations in brain ECF were simulated, and from these the median percentage of receptor occupancy of dopamine-2, serotonin-2A, muscarinic-1, alpha-1 adrenergic, alpha-2 adrenergic and histamine-1 for clozapine, and dopamine-2 for norclozapine were consistent with values reported in the literature. CONCLUSIONS: A PK model that relates clozapine and norclozapine disposition in rat plasma and brain, including blood-brain barrier transport, was developed. Using allometry and published human plasma PK, the model was successfully translated to predict clozapine and norclozapine concentrations and accordant receptor occupancy of both agents in human brain. These predicted exposure and occupancy measures at several receptors that bind clozapine may be employed to extend our understanding of clozapine's complex behavioral effects in humans.

Determination of clozapine in hair and nail: the role of keratinous biological materials in the identification of a bloated cadaver case.

Keratinous biological materials, such as hair and nails, offer a substantially longer retrospective window of detection compared to other body fluids. Little research on drug analysis in nails is currently being conducted. In this study, the hair and nails from a bloated cadaver was analyzed. The study showed that the forensic toxicology results of keratinous biological materials could provide valuable clues for solving cases. In this study, a method was developed for the extraction and analysis of clozapine from hair and nails. The keratinous bio-samples were washed and then pulverized using a freeze mill. After ultrasonic bath extraction, the supernatants were analyzed by ultra-performance liquid chromatography tandem mass spectrometer (UPLC-MS/MS). The method presented in this study proved to be reliable, specific, selective and sensitive with high precision and accuracy. Clozapine was found in both hair and nails from a long term user's remains, even after serious decomposition. The mean concentration of clozapine in the hair was 322.9 pg/mg and 138.3 pg/mg in the nails. Toxicological results helped police narrow the scope of the investigation and improved the efficiency of the breaking of the case. The findings of the present study demonstrated that the method can be used in forensic investigation. Toxicological results increased the efficiency of cadaver identification and the solving of the case. The study demonstrated that hair and nail analysis could provide vital clues for solving cases and showed the value of keratinous biological materials in the forensics field.

[The problems of expert evaluation of the results of forensic chemical investigations of clozapine in the biological material].

The objective of the present review was to analyse the problems of expert evaluation of the results of forensic chemical investigations of clozapine in the biological material. Such an analysis is needed because many topical aspects of the quantitative evaluation of toxic clozapine concentrations remain unclear. The treatment with clozapine is associated with its accumulation in blood in concentrations up to 2 mg/l in the absence of any toxic effect allegedly due to the development of tolerability of this agent. In the tolerant patients the ratio of the main clozapine metabolite, norclozapine, to clozapine itself in the serum amounts to 0.6-0.9. This value falls down to 0.3-0.4 in case of acute intoxication. In the case of identification of other pharmaceutical products narcotic drugs together with clozapine their influence on the activity of enzymes responsible for clozapine biotransformation should be taken into consideration. The concomitant intake of clozapine and alcohol may be dangerous for the clozapine-intolerant subjects. It is concluded that the above observations must be borne in mind in the assessment of the results of forensic chemical analysis.

[The detection of clozapine in the tissues of an exhumed corpse].

The authors report the case of finding clozapine in the tissues of an exhumed corpse. The circumstances surrounding the death and the results of primary examination of the corpse are described; in addition, the results of repeated expertise are presented. The data obtained in this study are compared with the relevant literature publications. Special attention is given to the peculiarities of interpretation of the results of forensic chemical expertise essential for the formulation of the scientifically-sound conclusion.