Toxicological findings in three cases of suicidal asphyxiation with helium.

INTRODUCTION: Toxicological findings in deaths by asphyxiation due to a pure inert gas like helium are rare. We present three suicide cases of asphyxial death attributed to anoxia caused by inhalation of helium in a plastic bag positioned over the head. METHODS: In one case, lung tissue, brain tissue and heart blood were obtained during standard autopsy procedures. In two cases, samples were obtained differently: heart blood, femoral blood, brain tissue, lung tissue and/or air from the lungs were directly sealed into headspace vials during autopsy. Air from the lungs was collected using a syringe and transferred into an aluminum gas sampling bag which was heat sealed as soon as possible. Semi-quantitative gas analyses were performed using headspace gas chromatography-thermal conductivity detection (HS-GC/TCD) with a molsieve column capable of separating permanent gasses. Nitrogen was used as carrier gas. RESULTS: In the first case no helium was detected in lung tissue, brain tissue and heart blood. In the second case the presence of helium was detected in lung tissue (approximately 5% helium in gaseous phase) but not in femoral blood. In the third case the presence of helium was detected in air from the lungs (0.05%), lung tissue (0.4%), brain tissue (0.1%) and heart blood (0.04%). CONCLUSIONS: Helium is easily lost if sampling is not performed properly. The presented cases suggest that quick sample collection of various matrices during autopsy is suitable to detect gasses like helium in postmortem cases. Use of HS-GC/TCD enables to detect an inert gas like helium.

Prevalence of medicinal drugs in suspected impaired drivers and a comparison with the use in the general Dutch population.

The aim of this study was to investigate the prevalence of psychotropic medicines in drivers suspected of driving under the influence of medicinal and illicit drugs in The Netherlands and to compare the prevalence of selected impairing medicines with the use of these medicines in the general Dutch population. In total, 3038 blood samples of suspected impaired drivers in The Netherlands have been analyzed for the presence of medicinal and illicit drugs between January 2009 and December 2012. In 94% (2842/3038) of the cases medicinal and/or illicit drugs were detected. Medicinal drugs were found in 33% of the blood samples, with the highest prevalence for anxiolytics. In 86% of the cases illicit drug-positive results were obtained, with the highest prevalence for cannabis. At least in 56% of the blood samples poly-drug use was determined, including medicinal and/or illicit drugs. The highest prevalence of poly-medicine use was found for combinations including anxiolytic and hypnotic drugs. In general, the prevalence of driving impairing medicines in suspected impaired drivers is higher than the use of these medicines in the general Dutch population, due to a positive selection bias in the first population. Differences between both populations may be explained by the used methodological approach (e.g., classification criteria of analytical findings, sample selection bias) and abuse of certain medicinal drugs (e.g., diazepam). Negative effects of medicinal drugs on driving performance determine largely the prevalence in the population of suspected impaired drivers. The degree of impairment depends on different factors, including pharmacokinetic properties of the drug and pharmacodynamic aspects. More research is needed to study the prevalence of all prescribed driving impairing medicines and to investigate if providing additional information to medicinal drug users on driving impairing medicines would lower the prevalence of medicinal drug positive drivers.

Toxicological findings in cases of sexual assault in the Netherlands.

Reports on cases of alleged drug-facilitated sexual assault (DFSA) have increased since the mid-1990s. The aim of this study was to identify the extent and types of drugs found in cases of alleged sexual assault (DFSA) in the Netherlands. In total, 135 cases of alleged DFSA were identified. Most of the victims were women (94%), and the mean age of the victims was 25 years. Blood and urine samples were tested for the presence of alcohol, drugs (drugs of abuse and prescription drugs), or both. In 27% of the cases, no alcohol and/or drugs were found. With increasing time delay, more cases were found to be negative. Alcohol is the most commonly found drug followed by nonopiate analgesics, illicit drugs, and benzodiazepines. In some cases, the absence of alcohol and drugs may represent false-negative results owing to the time delay between alleged sexual assault and sampling.

A case of extreme agitation and death after the use of mephedrone in The Netherlands.

A 36-year old man, having injured himself severely by smashing windows in a rage of fury, was arrested by the police. He died despite resuscitation attempts. The forensic autopsy showed many superficial skin lacerations, bruises and minor brain swelling, but there was no definitive cause of death. Toxicological analysis showed a high concentration of mephedrone in femoral blood (5.1mg/L) and traces of cocaine, MDMA and oxazepam. The remaining dose of mephedrone in the stomach contents was estimated at 113 mg. Tablets that were found in the house of the deceased also contained mephedrone. We attribute this man's death to a fatal oral intake of mephedrone, which probably led to a state of excited delirium. This was aggravated by blood loss from multiple wounds.

Post-mortem cases involving amphetamine-based drugs in The Netherlands. Comparison with driving under the influence cases.

In this study we reviewed the post-mortem cases in the years 1999-2004 that were presented at the Netherlands Forensic Institute. The concentrations of amphetamine-based drugs in femoral blood from cases of suspected unnatural death were compared with concentrations in whole blood from non-fatal cases of driving under the influence (DUI cases) and with literature. Furthermore, the combinations with other drugs and/or alcohol were investigated. Amphetamine-based drugs were present in 70 post-mortem cases and 467 DUI cases. The most detected amphetamine-based drug was MDMA, followed by amphetamine. The presence of MDA could usually be explained by metabolism of MDMA. Methamphetamine and MDEA were rarely present. Frequently, the amphetamine-based drugs were taken in combination with alcohol and/or other non-amphetamine-based drugs such as cocaine or cannabinoids. The 70 post-mortem cases were divided into 38 amphetamine-based drug caused (i.e. the amphetamine-based drug directly caused or contributed to the death) and 32 amphetamine-based drug related deaths (i.e. death was not directly caused by the amphetamine-based drug). In the latter category, other (poly)drug intoxications and death by violence or drowning were the most frequent causes of death. In 30 cases, MDMA caused death directly. The range in blood concentrations of MDMA in these cases was substantial, i.e. 0.41-84 mg/L with a median concentration of 3.7 mg/L (n=30). MDMA blood concentrations in the MDMA related deaths (n=20) and in the DUI cases (n=360) varied up to 3.7 and 4.0 mg/L, respectively. Seven victims died from the direct effects of amphetamine; the blood concentration of amphetamine ranged from 0.24 to 11.3 mg/L, with a median concentration of 1.7 mg/L (n=7). The median concentrations of amphetamine in the amphetamine related deaths (n=13) and the DUI cases (n=208) were much lower, i.e. 0.28 and 0.22 mg/L, respectively. Amphetamine blood concentrations up to 6.0 and 2.3 mg/L were seen in the drug related deaths and DUI cases, respectively. The most frequently encountered amphetamine-based drugs in the investigated deaths were MDMA and amphetamine. The majority of MDMA- and amphetamine-caused deaths, i.e. 90% of these deaths, occurred with blood concentrations above 1.5 and 0.80 mg/L, respectively. MDMA and amphetamine blood concentrations in drug related deaths and DUI cases, however, overlap the range of fatal concentrations. Therefore, MDMA or amphetamine concentrations should never be used alone to establish the cause of death.

Possible (enzymatic) routes and biological sites for metabolic reduction of BNP7787, a new protector against cisplatin-induced side-effects.

Disodium 2,2'-dithio-bis-ethane sulfonate (BNP7787) is under investigation as a potential new chemoprotector against cisplatin-induced nephrotoxicity. The selective protection of BNP7787 appears to arise from the preferential uptake of the drug in the kidneys, where BNP7787 would undergo intracellular conversion into mesna (2-mercapto ethane sulfonate), which in turn can prevent cisplatin induced toxicities. In the present study, we have investigated whether the reduction of BNP7787 into the reactive compound mesna is restricted to the kidney or whether it can also occur in other organs, cells and physiological compartments, including the cytosolic fraction of the renal cortex, plasma, red blood cells (RBCs), liver and small intestine from rats and several tumors (OVCAR-3, MRI-H-207 and WARD). We also determined whether the endogenous thiols glutathione (GSH) and cysteine and the enzyme systems glutaredoxin and thioredoxin, which are all present in the kidney, can be involved in the BNP7787 reduction. UV detection and micro-HPLC with dual electrochemical detection were used to analyze the various incubation mixtures. Our observations are that, in contrast to plasma, a very large reductive conversion of BNP7787 to mesna was measured in RBC lysate. Intact RBCs, however, did not take up BNP7787. Although BNP7787 could be reduced in cytosol of liver and several tumors, this reduction will not be relevant in vivo, since these tissues do not take up large amounts of BNP7787. Kidney cortex cytosol was, similar to the small intestine cytosol, able to substantially reduce BNP7787 to mesna. The ability to reduce BNP7787 in the presence of the endogenous thiols GSH and cysteine, the glutaredoxin system as well as the thioredoxin system, could at least in part explain the high BNP7787 reductive activity of the kidney cortex cytosol. In conclusion, the high reduction of BNP7787 into mesna in the kidney as well as our earlier observation that the distribution of BNP7787 and mesna was mainly restricted to rat kidney are strong arguments in favor of selective protection of the kidney by BNP7787.

Pharmacokinetics and preliminary clinical data of the novel chemoprotectant BNP7787 and cisplatin and their metabolites.

INTRODUCTION: BNP7787 (disodium 2,2'-dithio-bis-ethane sulfonate) is currently undergoing development as a chemoprotective agent to prevent common and serious cisplatin-induced side effects. In the kidneys, intestine, and liver, BNP7787 is believed to undergo intracellular conversion into 2-mercaptoethane sulfonate (mesna), which can locally inactivate toxic platinum species. Methods and objectives In a phase I trial, 25 patients with advanced solid tumors received a 1-hour intravenous infusion of 75 mg/m(2) cisplatin immediately preceded by a 15-minute intravenous infusion of BNP7787 every 3 weeks. For pharmacokinetic investigation of BNP7787 and mesna and a possible mutual pharmacokinetic interaction between BNP7787 and cisplatin, cisplatin and BNP7787 were also administered as single agents in 14 of 25 patients. The dose of BNP7787 was escalated from 4.1 to 41 g/m(2). Patients were also monitored for tumor response and possible side effects from BNP7787. RESULTS: The maximum plasma concentration of mesna was reached approximately 1.7 hours after the start of the BNP7787 infusion. The maximum plasma concentration and area under the curve to infinity (AUC( infinity )) of BNP7787 and mesna increased linearly with the dose. The mean volume of distribution of BNP7787 (+/-SD) was approximately 0.26 +/- 0.08 L/kg. The mean normalized AUC( infinity ) of mesna was only approximately 8% of the normalized AUC( infinity ) of BNP7787. The pharmacokinetic profile of mesna was unaffected by cisplatin and its metabolites. None of the dose levels of BNP7787 (4.1-41 g/m(2)) administered appeared to influence the pharmacokinetic profile of total platinum, unbound platinum, or monohydrated cisplatin. The observed effects regarding a possible mutual interaction between BNP7787 and intact cisplatin were minor, and none were statistically significant at BNP7787 dose levels of 18.4 to 41 g/m(2). The confidence intervals for the pharmacokinetic parameters of BNP7787 and intact cisplatin, however, were relatively broad. Overall, BNP7787 was well tolerated at all dose levels (4.1-41.0 g/m(2)). The most frequently reported event related to BNP7787 was local intravenous site discomfort; the majority of events were mild (grade 1). Side effects of BNP7787 at the highest dose level of 41 g/m(2) were more prominent and included nausea and vomiting, as well as a warm feeling or flushing (grade 2 or lower). Partial tumor responses and stable disease were measured in 12 of 25 patients. CONCLUSION: BNP7787 was relatively nontoxic at doses up to 41 g/m(2). The combination of BNP7787 with cisplatin did not alter the pharmacokinetic profiles of mesna or the cisplatin metabolites. At the higher dose levels of BNP7787 (18.4 to 41 g/m(2)), there appeared to be no mutual interaction between BNP7787 and intact cisplatin, which needs to be confirmed in a larger number of patients. The absence of a mutual interaction between BNP7787 and intact cisplatin is consistent with the observation that several patients had objective tumor responses with BNP7787 and cisplatin administration.

Pharmacokinetics of BNP7787 and its metabolite mesna in plasma and ascites: a case report.

PURPOSE: BNP7787 (2',2'-dithio-bis-ethane sulfonate sodium) is a novel protector against cisplatin-induced toxicities. The pharmacokinetics of BNP7787 and its metabolite mesna were investigated in plasma and ascites of a cancer patient. We also evaluated potential pharmacokinetic interactions between BNP7787 and cisplatin. METHODS: BNP7787 and mesna were measured as mesna in deproteinized plasma and ascites using high-performance liquid chromatography with an electrochemical detector provided with a wall-jet gold electrode. RESULTS: After the i.v. administration of 41 g/m(2) BNP7787, BNP7787 and mesna had a half-life of 1.5 and 3.4 h, respectively. The AUC( infinity ) of mesna was approximately 8% of the AUC( infinity ) of BNP7787. Coadministration of cisplatin did not appear to influence the plasma concentration-time curves of BNP7787 and mesna. In ascites, approximately 0.02% of the BNP7787 dose was present as mesna, whereas approximately 4% of the dose was present as BNP7787 at the time of the maximum concentration. CONCLUSIONS: It can be concluded that the presence of ascites did not have a major impact on the pharmacokinetics of BNP7787 and coadministration of cisplatin did not influence the pharmacokinetics of BNP7787 and mesna.

The chemical reactivity of BNP7787 and its metabolite mesna with the cytostatic agent cisplatin: comparison with the nucleophiles thiosulfate, DDTC, glutathione and its disulfide GSSG.

PURPOSE: BNP7787 is a new chemoprotective agent presently under clinical investigation to protect against cisplatin-induced toxicities, especially nephrotoxicity and neurotoxicity. In the kidneys BNP7787 is postulated to undergo selective conversion into mesna, which can locally detoxify cisplatin. The reactivity of cisplatin with this new chemoprotective agent and with its metabolite mesna was investigated at clinically observed plasma concentrations and compared with the nucleophiles thiosulfate (TS) and DDTC, and with the endogenous compounds glutathione (GSH) and oxidized glutathione (GSSG). METHODS: Reaction kinetics experiments were performed at 37 degrees C and pH 7.4 in the presence of a high chloride concentration (0.15 M). The degradation of cisplatin was measured over time using HPLC with off-line flameless atomic absorption spectrophotometry. RESULTS: The degradation half-lives of cisplatin (13.5 microM) with 17.2 m M BNP7787, 340 microM mesna and 17.2 m M mesna were 124 min, about 790 min and 73 min, respectively. Cisplatin reacted at least 9.5 times more slowly with 17.2 mM BNP7787 and 5.5 times more slowly with 17.2 mM mesna than with 17.2 mM of the modulating agents DDTC or TS (i.e. half-lives 11 and 13 min, respectively). The half-lives of cisplatin with 17.2 m M GSH and GSSG (i.e. 122 and 115 min, respectively) were comparable with the half-life obtained with BNP7787. The thiol mesna was shown to be a stronger nucleophile than its corresponding disulfide BNP7787. CONCLUSIONS: The much slower relative reactivity of BNP7787, the short residence of BNP7787 (approximately 2 h) and the much lower concentration of mesna in the circulation following BNP7787 administration precludes chemical inactivation of cisplatin in the circulation, and thus the antitumor activity of cisplatin is maintained.

Simultaneous determination of BNP7787 and its metabolite mesna in plasma and tissue by micro-HPLC with a dual electrochemical detector.

Sensitive, accurate, and precise assays are described to determine BNP7787 (disodium 2,2'-dithio-bis-ethane sulfonate) and its metabolite mesna (sodium 2-mercaptoethane sulfonate) simultaneously in plasma and tissue by micro-high-performance liquid chromatography (HPLC) with dual electrochemical detection. After separation of BNP7787 and mesna by micro-HPLC, the disulfide BNP7787 was reduced to mesna by a reactor cell with a glassy carbon working electrode (-1.6 V versus Hy-REF). At the second electrode, which consisted of a gold wall-jet electrode, the mesna generated from BNP7787 and the mesna already present in the samples were detected (+0.85 V versus Ag/AgCl). The lower limit of quantification (LLQ) of both compounds was 3 microM in plasma and 20 nmol/g in tissue. The dynamic range of the assay in plasma was 3-120 microM for mesna and 15-1200 microM for BNP7787. In tissue, the dynamic range was 20-2000 nmol/g for both compounds. The recovery of mesna from plasma and tissue ranged from 61.4 to 90.5% and 82.7 to 90.2%, respectively, and seemed to be concentration dependent. The recovery of BNP7787 from plasma and tissue was complete (i.e., 101.5 and 96.4%, respectively). The within- and between-day accuracy and precision for the plasma and tissue assay were within 14 and 7%, respectively. The utility of the assay was shown by determination of the stability of mesna and BNP7787 in a kidney sample of a rat and by analysis of plasma samples obtained from a patient receiving 18.4 g/m(2) BNP7787 as a 15-min intravenous infusion.

Quantification of mesna and total mesna in kidney tissue by high-performance liquid chromatography with electrochemical detection.

A sensitive and selective assay for the determination of mesna and total mesna in tissue was developed and validated. After a simple homogenization, extraction and deproteinization step, mesna could be measured immediately by HPLC with an electrochemical detector provided with a sensitive wall-jet gold electrode. Total mesna (i.e., free mesna and mesna present in mesna disulfides and mixed mesna disulfides) could be measured after pre-column reduction with sodium borohydride to free mesna. The lower limit of quantification of mesna and total mesna was for both compounds 10 nmol/g. The assays for mesna and total mesna in tissue were linear over the ranges of 10-3000 and 10-10000 nmol/g, respectively. The within-day and between-day precisions of both methods were better than 9%. The within-day and between-day accuracy of the mesna assay ranged from 103.7 to 113.6%, whereas the accuracies of the total mesna assay ranged from 97.8 to 106.7%. Mesna in an EDTA containing tissue homogenate or in deproteinized tissue homogenate stored at -80 degrees C was stable for at least 12 weeks. Total mesna was stable under all conditions measured. The developed assays will be applied for the determination of the distribution of mesna and total mesna in tissues of the rat after administration of mesna or BNP7787.

Simultaneous determination of intact cisplatin and its metabolite monohydrated cisplatin in human plasma.

Cisplatin is a cytotoxic platinum compound, used in the treatment of several solid tumors. Cisplatin and to a greater extent its hydrolysis product monohydrated cisplatin are responsible for side-effects like nephrotoxicity. A sensitive, accurate and precise method was developed to simultaneously determine cisplatin and monohydrated cisplatin in plasma. The compounds were separated by high-performance liquid chromatography and quantified by off-line furnace atomic absorption spectrophotometry. The linear ranges for cisplatin and monohydrated cisplatin in deproteinized plasma were 60-600 and 87.5-700 nM, respectively. From plasma, the mean recovery of cisplatin was 83.2% and that of monohydrated cisplatin 79.1%. The lower limits of quantification of cisplatin and monohydrated cisplatin in deproteinized plasma were 60 and 87.5 nM, respectively. Over the whole calibration range, the within- and between-day accuracy of intact cisplatin ranged from 100.7 to 111.4 and 94.8-102.0%, respectively. The within- and between-day accuracy of monohydrated cisplatin ranged from 107.1 to 113.3 and 101.4-104.9%, respectively. The within-day and between-day precision of cisplatin ranged from 3.4 to 11.5 and 7.3-10.3%, respectively. For monohydrated cisplatin, the within-day and between-day precision ranged from 3.7 to 6.2 and 5.6-7.9%, respectively. Currently, the developed assay has been implemented in pharmacokinetic studies of patients treated with cisplatin alone or in combination with other drugs.