Pentobarbital Concentrations Research Articles

Unexpected epileptogenic effect of lethal doses of pentobarbital: a case report

Background: Barbiturate poisoning is rare but potentially fatal. Case Report: We reported a case of barbiturate poisoning in a 28-year-old woman who recovered from lethal pentobarbital deliberate self-poisoning. The initial blood pentobarbital concentration was 61 mg/L, corresponding to a potentially lethal dose. Despite the ingestion of a high dose of pentobarbital, the electroencephalogram revealed an unattended pattern compatible with possible nonconvulsive status epilepticus. Following resuscitation maneuvers, appropriate care, and antiseizure medication, the patient awakened after 7 days. The evolution was excellent without neurological deficits at 2 months.Conclusion: Despite the expected and known effects of high-dose pentobarbital in reducing and suppressing cortical activity in the brain, the present case demonstrates that lethal dose of pentobarbital may have an epileptogenic effect. Our hypothesis was that the mechanism of the origin of such a picture is a relatively abrupt decrease in toxic doses of pentobarbital, resulting in a withdrawal phenomenon.

Effect of pentobarbital as a euthanasia agent on equine in vitro embryo production

Postmortem and pre-euthanasia oocyte retrieval provides the last opportunity to preserve the genetic material in mares. Pentobarbital (PB) is the most common euthanasia agent; however, its effect on the developmental competence of oocytes has not been determined. Here, we evaluated the concentration of PB in equine follicular fluid (FF) and investigated its effect on the developmental competence of oocytes using a bovine IVF model to overcome the low availability of equine oocytes. The concentration of PB was measured by gas-chromatography/mass-spectrometry in FF collected from mare ovaries immediately after euthanasia (n = 10), 24 h post-euthanasia (n = 10), and from the ovaries collected by ovariectomy (negative control; n = 10). The serum concentration of PB was also evaluated as a positive control. PB was detected in all FF samples with an average concentration of 56.5 μg/ml. Next, bovine cumulus-oocyte complexes (COC) were held in holding media with PB for 6 h at 60 μg/ml (H60, n = 196), 164 μg/ml (H164, n = 215) or without PB (control; n = 212). After holding, the oocytes were matured and fertilized in vitro, followed by in vitro culture to the blastocyst stage. The cumulus expansion grade, cleavage rate, blastocyst rate, embryo kinetic rate and the blastocyst cell numbers were compared among the experimental groups of bovine COC. Higher rates of Grade 1 cumulus expansion were found in controls (54%, 32–76%; median, min-max) in comparison to H60 and H164 (24%,11–33% and 13%, 8–44%; P < 0.001). The cleavage rate was higher in the controls than in H164 (64% vs. 44%; P < 0.01). Blastocyst rates (blastocyst/cleaved oocytes) and total cell number were not different among the groups (control 29%, H60 25%, and H164 24%). In a preliminary study, equine oocytes (n = 28) were exposed to PB in vitro for 6 h followed by intracytoplasmic sperm injection (ICSI) and in vitro embryo production. Exposed oocytes showed a numerically lower maturation rate (43% Vs 52%; P > 0.05) in comparison to the laboratory-established rate during the same timepoints. Overall, we showed that PB reaches the FF immediately after euthanasia, exposing oocytes to this drug. This exposure affected cumulus expansion and cleavage rates in a bovine model, suggesting initial damage caused by PB that may not completely impede the formation of embryos, although lower overall embryo numbers might be obtained.

Pharmacokinetic Modeling of Optimized Midazolam and Pentobarbital Dosing Used in Treatment Protocols of Refractory Status Epilepticus.

To model bolus dosing, infusion rate, and weaning rate on theoretical serum concentration of midazolam and pentobarbital used in the treatment of refractory status epilepticus (RSE). One- and two-compartment in silico pharmacokinetic models of midazolam and pentobarbital. Not applicable. Not applicable. We compared the model variables used in midazolam and pentobarbital protocols for standard RSE. Standard RSE treatment protocols result in steady-state serum concentrations that are 6.2-9.0-fold higher for the one-compartment model and 2.3-4.7-fold higher for the two-compartment model. In the model, not including bolus doses delays the achievement of serum steady-state concentration by 0.5 and 2.7 hours for midazolam and pentobarbital, respectively. Abrupt discontinuation of these medications reduces modeled medication exposure by 1.1 and 6.4 hours, respectively. Our in silico pharmacokinetic modeling of standard midazolam and pentobarbital dosing protocols for RSE suggests potential variables to optimize in future clinical studies.

Characteristics and Sodium Pentobarbital Concentrations of Equine Mortality Compost Piles in the Upper Midwest

Recent changes in rendering availability for chemically-euthanized animals have created a need to explore composting as an equine mortality management method. The objectives of this study were to evaluate equine mortality composting in the Upper Midwest during fall and spring, document sodium pentobarbital concentrations throughout the process, and determine nutrient content of finished compost. During each season, four horses were euthanized by intravenous administration of sodium pentobarbital. Carcasses were positioned at the center of a woodchip and shavings base and covered with a 2:1 mixture of horse stall waste and cattle feedlot waste. Data loggers were placed at 46- and 91cm depths to record pile temperatures every 8 hours. At turning (44–50 days) and trial conclusion (181–216 days), compost piles were sampled for sodium pentobarbital and nutrient composition. Piles contained large bones after 6 months of composting. Sodium pentobarbital remained detectable at trial conclusion (<0.002–1.49 mg kg1 dry matter); however, composting reduced the estimated quantities of sodium pentobarbital by ≥94% (P ≤ .001). Compost from each season met ideal land application values for organic matter (45%–48%), pH (7.3–7.6), and electrical conductivity (3.3–3.4 mm hos cm−1). Low NPK and high C:N ratio (20–30) indicate compost could partially replace synthetic fertilizers when land applied. These findings suggest equine mortality composting is an effective management practice during fall and spring in the Upper Midwest. While remaining sodium pentobarbital residues were minimal, further research is needed to determine environmental implications of composting chemically-euthanized equines.

Pentobarbital may protect against neurogenic inflammation after surgery via inhibition of substance P release from peripheral nerves of rats

The inflammatory response related to surgery is considered surgical inflammation. Most anesthetic agents directly or indirectly suppress the immune response. However, the intravenous anesthetics pentobarbital and ketamine were reported to inhibit the lipopolysaccharide-induced inflammatory response such as cytokines formation. Neurogenic inflammation is inflammation originating from the local release of inflammatory mediators, such as substance P (SP), by primary afferent neurons after noxious stimuli like surgery. Thus, in this study, we examined whether pentobarbital and ketamine suppress SP release from cultured dorsal root ganglion (DRG) neurons. DRG cells were dissected from male Wistar rats. Released SP was measured by radioimmunoassay. We demonstrated that higher concentrations of pentobarbital (100–1,000 μM) significantly inhibited capsaicin (100 nM)-induced, but not high K+ (50 mM)-induced, SP release from DRG cells, although a high concentration of ketamine (1 mM) did not. This study revealed that pentobarbital functions between the activation of vanilloid receptor subtype 1 (TRPV1) receptors, to which capsaicin selectively binds, and the opening of voltage-operated Ca2+ channels (VOCC) in the nerve endings. Therefore, the anti-inflammatory action of pentobarbital is mediated through different mechanisms than those of ketamine. Thus, the inhibitory effect of pentobarbital on SP release from peripheral terminals may protect against neurogenic inflammation after surgery.

Increasing incidence of barbiturate intoxication in avian scavengers and mammals in Spain

Pharmaceuticals are considered emerging contaminants in terms of impacts on wildlife. One chemical group of concern is euthanasia agents used in veterinary medicine. Here we present data on the occurrence of barbiturate intoxication using samples collected from 2004 to 2020 of suspected wildlife and domestic animal poisoning cases in Spain (n = 3210). Barbiturate intoxication was seen in 3.4% (45/1334) of the total number of confirmed intoxicated animals. Barbiturates were detected in 0.2% (1/448) of baits containing detectable poisons. The most frequently detected barbiturate was pentobarbital (42/45, 93.3%), but we also detected phenobarbital, barbital, and thiopental (2.2% prevalence for each). Avian scavengers were most frequently affected by barbiturate intoxication (n = 36), especially Eurasian griffon vultures (Gyps fulvus) (n = 28). Median pentobarbital concentrations detected in intoxicated griffon vultures was 27.3 mg kg−1 in gastric content and 38.1 mg kg−1 in liver, which highlights the acute effect of the chemical soon after ingestion. At least two large intoxication events affecting griffon vultures were related to the consumption of carcasses from euthanized livestock. We also found phenobarbital in a prepared bait linked to the intoxication of one Eurasian buzzard (Buteo buteo). This study highlights the need for stronger regulation of barbiturates to avoid secondary intoxications due to improper disposal of euthanized livestock.

88 Exploring equine mortality composting

Composting is an environmentally friendly, economical, and bio secure method for livestock mortality disposal. Recent changes in availability of rendering for chemically-euthanized animals has created a need to explore composting. The objectives of this study were to demonstrate successful equine mortality composting in the winter and document sodium pentobarbital concentrations throughout the process. The authors hypothesized composting would degrade carcasses and reduce pentobarbital concentrations. On 26 September 2019, 4 horses (451 ± 36 kg) were euthanized for terminal medical reasons by intravenous administration of sodium pentobarbital (88 ± 2 mg/kg; Fatal-Plus, Vortech Pharmaceuticals, LTD, Dearborn, MI). Carcasses were transported immediately to a private composting site. Four separate compost piles were constructed on a 6.1- by 27.4-m concrete pad. Each pile consisted of 46 cm of playground woodchips, 8 cm of dry pine shavings, a horse carcass, and 61 cm of a 2:1 mixture of horse stall waste and cattle feedlot waste. Data loggers were placed at 46- and 91-cm depths to record pile temperatures every 8 h. Compost piles were topped with manure mixture at d 9 to cover cracks, turned at d 50 when pile temperatures were ≤54.4°C, and concluded on d 216 following approximately 5 mo. of curing (≤43.3°C). On d 50 and 216 piles were cut into 3 mirrored cross sections and subsampled (n = 36) to form one composite sample per cross section. Samples were analyzed for sodium pentobarbital (on an “as is” basis) and nutrient content. Carcass degradation was scored on a subjective scale (1 = discernable carcass, 5 = few large brittle bones). Pentobarbital concentrations were compared using a 2-way ANOVA with cross section and day as fixed effects. P-values < 0.05 were considered significant. Three piles received a carcass degradation score of 2 at d 50 with soft tissue, hair, and hide present. By d 216, all piles received scores of 3 (n = 2) or 4 (n = 2) with some hide and large bones remaining. Pentobarbital concentrations declined between d 50 and d 216; however, remained detectable at d 216. On average, pentobarbital concentrations tended to be higher (9.05 mg/kg) at the center of the compost pile on d 50 (P < 0.05); however, on d 216 concentrations were similar (0.432 to 0.735 mg/kg; P > 0.05) among cross sections. While pentobarbital concentrations at d 216 were minimal, further research is needed to determine environmental implications of composting chemically-euthanized equines. These findings suggest mortality composting is an effective method for disposing of equine carcasses.

Nrf2 Antioxidative System is Involved in Cytochrome P450 Gene Expression and Activity: A Delay in Pentobarbital Metabolism in Nrf2-Deficient Mice.

NF-E2-related factor 2 (Nrf2) is a transcriptional regulator of biologic defense proteins, such as antioxidant proteins and phase II detoxification enzymes. Cytochrome P450 (P450) enzymes have been shown to regulate phase I metabolism of various drugs and are partially regulated by Nrf2; however, the influence of Nrf2 on drug pharmacokinetics is not known. Here, we showed that Nrf2 depletion prolonged the effect of pentobarbital, a sleep-promoting drug. Pretreatment with phenobarbital, a P450 inducer, shortens the sleeping time associated with pentobarbital-induced sedation in wild-type (WT) mice; however, this effect was not observed in Nrf2-/- mice. Furthermore, the blood pentobarbital concentration was higher in Nrf2-/- mice than in WT mice at 30-60 minutes, and the phenobarbital-induced enhancement of its clearance was attenuated in Nrf2-/- mice compared with WT mice. Total P450 content was decreased in Nrf2-/- mouse livers, and the phenobarbital-induced increase in P450 content was lower in Nrf2-/- mice than WT mice. Cyp1a2, Cyp2a5, Cyp2c29, and Cyp2e1 gene expression levels under physiologic conditions and Cyp1a2, Cyp2a5, and Cyp2b10 gene expression levels under phenobarbital-treated conditions were lower in Nrf2-/- mice compared with WT mice. Additionally, pentobarbital metabolism in liver microsomes was attenuated by Nrf2 depletion. Taken together, these findings suggested that Nrf2 influenced pentobarbital pharmacokinetics through the regulation of drug metabolism and P450 gene expression. Thus, Nrf2-mediated regulation of P450 may contribute to the biologic defense against increased reactive oxygen species production. SIGNIFICANCE STATEMENT: NF-E2-related factor 2 (Nrf2) plays a critical role in the cellular defense against oxidative stress. Nrf2-/- mice with reduced ability to eliminate reactive oxygen species (ROS) showed a significant delay in emergence from pentobarbital-induced sleep, which was associated with decreased P450 activities and gene expression. Our findings provide that Nrf2 dysfunction or ROS that exceed a threshold level of the eliminating ability of the Nrf2 system may reduce P450 activity.

Pentobarbital suicide attempt in a young pregnant veterinary assistant

We describe a case of pentobarbital (Dolethal®) acute intoxication in a 20 weeks pregnant woman and discuss the intoxication consequences in the mother and in the foetus. We report the case of a 24-year-old pregnant veterinary assistant who attempt to kill herself by ingestion of 9 g of pentobarbital. At the EMS arrival, the patient is in cardio respiratory arrest and an external cardiac massage is undertaken. It permits to restore the cardio respiratory activity without the use of adrenaline or external electrical shock. The patient is then intubated and transferred directly to medical intensive care unit for further management. At the admission, a hypotension and a deep hypothermia at 33.3 °C are observed. The neurological examination also reveals reactive miosis, hypotonia and a Glasgow Coma Scale (GCS) score of 3. A complete blood biological investigation including a screening by LC-MS-MS of pharmaceuticals and drugs of abuse has been performed at the admission. It has been completed by a regular measurement of pentobarbital concentrations by HPLC-DAD in sera samples taken respectively at 3, 11, 30, 50 and 70 ho after the supposed ingestion of pentobarbital. As there are few data in the literature regarding the pentobarbital toxicokinetics in pregnant women, we have conducted a non-compartmental analysis using the PKSolver software to determine the toxicokinetics parameters including elimination half-life (t 1/2 ), total plasma clearance (Cl), volume of distribution (V D ) and area under the curve (AUC). Para clinical exams (an electroencephalogram [EEG]) (on day 1) and multiple foetal ultrasounds (at the admission and on day 2) have also been performed to evaluate the gravity of the intoxication in the mother and in the foetus. At the beginning of her care, the patient shows no alteration of her biological parameters except of hypercapnic acidosis (pH: 7.28, pCO2: 50 mmHg). The toxicological analyses don’t reveal the presence of any drug apart from pentobarbital. The pentobarbital serum concentration measured 3 h after pentobarbital ingestion is very high (68.3 mg/L) and a 6 h continuous veinoveinous haemodiafiltration (CCVHDF) has been quickly set up. The serum pentobarbital concentration measured at the end of the CCVHDF (i.e. 11 h after the pentobarbital ingestion) has been determined at 33.6 mg/L (t 1/2 = 8 h). After the haemodialysis stopping, pentobarbital sera concentrations then slowly decrease and are respectively equal to 28.9, 15.9 and 6.7 mg/L at 30, 50 and 70 h after the overdose (t 1/2 = 19.2 h). The non-compartmental analyse has permit to estimate the pentobarbital toxicokinetics parameters in our pregnant patient. Thus V D , Cl and AUC have been respectively calculated at 126.5 L, 76.7 mL/min and 1782.5 mg/L*h. The decrease in pentobarbital concentrations has been correlated with a progressive improvement of the patient consciousness state (evaluated at the admission by an EEG and by the GCS score and then regularly monitored by the GCS score) and with a recovery of spontaneous foetal movements on day 2. As pentobarbital is exclusively reserved for veterinary use in France, few cases of voluntary acute intoxication have been described in recent years and, to our knowledge, no case has been reported in pregnant woman. In our patient, the initial pentobarbital concentration is very high. The rapid setting up of CVVHDF has allowed the pentobarbital elimination half-life to be divided by 2 and to decrease the intoxication duration. Thus foetal movements, that were absent at admission, have been recovered on day 2. During the end course of the pregnancy, no malformation or any development problem have been observed in the foetus. However, as barbiturates cross the placental barrier and distribute in foetal tissues and especially in the brain, close post-natal monitoring is recommended to evaluate the possible later consequences of this in utero exposure.

\u22069-Tetrahydrocannabinol, a major marijuana component, enhances the anesthetic effect of pentobarbital through the CB1 receptor

Purpose∆9-Tetrahydrocannabinol (∆9-THC) and cannabidiol (CBD), major psychoactive constituents of marijuana, induce potentiation of pentobarbital-induced sleep in mice. We have elucidated the mechanism of enhancement of the anesthetic effect of pentobarbital by cannabinoids.MethodsWe carried out pharmacological experiment and cannabinoid1 (CB1) receptor binding assay using CB1 antagonists to clarify whether the CB1 receptor is involved in the synergism or not. The affinities of cannabinoids for the CB1 receptor in the mouse brain synaptic membrane were evaluated using a specific CB1 ligand, [3H]CP55940.ResultsAlthough the potentiating effect of ∆9-THC on pentobarbital-induced sleep was attenuated by co-administration of CB1 receptor antagonists, such as SR141716A and AM251, at a dose of 2 mg/kg, intravenously (i.v.) to mice, the CBD-enhanced pentobarbital-induced sleep was not inhibited by SR141716A. The inhibitory constant (Ki) values of ∆9-THC and CBD were 6.62 and 2010 nM, respectively, showing a high affinity of ∆9-THC and a low affinity of CBD for the CB1 receptor, respectively. A high concentration of pentobarbital (1 mM) did not affect specific [3H]CP55940 binding on the mouse brain synaptic membrane.ConclusionsThese results suggest that binding of ∆9-THC to the CB1 receptor is involved in the synergism with pentobarbital, and that potentiating effect of CBD with pentobarbital may differ from that of ∆9-THC. We successfully demonstrated that ∆9-THC enhanced the anesthetic effect of pentobarbital through the CB1 receptor.

Suicide kit purchased on the Internet: A case report of self-poisoning with pentobarbital

Popularization of assisted-suicide and increase of online drug sellers could lead to resurgence of self-poisoning by a forgotten drug: pentobarbital. We report a case of self-poisoning with pentobarbital purchased on a Chinese website. A 43-year old woman with a background history of borderline personality disorder ingested at home a “suicide kit” (pentobarbital sodium) purchased beforehand on the Internet, a few minutes before calling the French national emergency telephone number. Upon admission, she was comatose, Glasgow Coma Score (GCS) 3/15, symmetrical slight myosis, generalized hypotonia and hyporeflexia. Blood pressure was 96/66 mmHg, heart rate was 93/min, and body temperature was 33.3 °C. Blood gas analysis showed a metabolic acidosis and severe hypoxemia, related to an aspiration pneumonia (arterial pH 7.30, pCO 2 40.7 mmHg, pO 2 63.3 mmHg with F i O 2 = 1). Hemodynamic instability rapidly appeared and norepinephrine infusion was started (up to 0.32 μg/kg/min). At day 2, clinical examination revealed signs consistent with brain death, i.e. bilateral mydriasis with absence of pupillary and oculo-cardiac reflexes. Electroencephalographic study found a severe diffuse slowing compatible with a sedative state. Plasma pentobarbital concentration was 19.9 μg/mL. At day 3, brain-stem reflexes reappeared, although pentobarbital concentration was still beyond lethal range, at 18.1 μg/mL. Neurological evolution was then progressively favourable. GCS was 5/15 at day 5, corresponding to a concentration of 9.4 μg/mL, and 15/15 at day 6 allowing extubation of the patient. She was finally discharged to the psychiatry department at day 10. Pentobarbital is a short-acting barbiturate, now rarely used in suicide attempt because it is no longer prescribed for medical purposes in most countries. Only a few cases have been published in the last decade, mostly among veterinarians because this drug is used as euthanasia agent. Pentobarbital is indeed the drug of choice for assisted-suicide. Pentobarbital is a weak acid with high lipid-solubility. After oral absorption, it is readily absorbed with an onset of action of 10 to 60 minutes. The degree of protein binding is about 45 to 70% and the volume of distribution is 0.5 to 1L/Kg. The plasma half-life for pentobarbital is 15 to 50 hours and appears to be dose-dependent. Renal excretion is negligible (< 1%). Pentobarbital is metabolized by the liver and excreted in the urine as inactive metabolites. Consciousness impairment, respiratory and circulatory depression are the main effects of acute poisoning. At worst, severe poisoning can mimic clinical and EEG features of brain death. Therapeutic blood levels are 1–3 μg/mL; levels are considered toxic at 5 μg/mL or more and reported lethal blood concentrations range from 10 to 169 μg/mL. Management of pentobarbital poisoning is mainly based on supportive care. Invasive mechanical ventilation, fluid loading and vasopressors are required in most severe cases. Several procedures to enhance elimination have been reported. Forced diuresis with urinary alkalinisation is not effective for short-acting barbiturates. Hemodialysis, hemoperfusion or continuous venovenous hemodiafiltration have been proposed as therapeutic options but improvement in supportive therapies make these techniques obsolete. Pentobarbital is readily available without prescription on the Internet. Although there is little public awareness about this drug, current mediatisation of assisted-suicide could lead to an increased interest from suicide attempters. Supportive care is the cornerstone of medical management.

Correction for Inhibition Leads to an Allosteric Co-Agonist Model for Pentobarbital Modulation and Activation of α1β3γ2L GABAA Receptors.

BackgroundPentobarbital, like propofol and etomidate, produces important general anesthetic effects through GABAA receptors. Photolabeling also indicates that pentobarbital binds to some of the same sites where propofol and etomidate act. Quantitative allosteric co-agonist models for propofol and etomidate account for modulatory and agonist effects in GABAA receptors and have proven valuable in establishing drug site characteristics and for functional analysis of mutants. We therefore sought to establish an allosteric co-agonist model for pentobarbital activation and modulation of α1β3γ2L receptors, using a novel approach to first correct pentobarbital activation data for inhibitory effects in the same concentration range.MethodsUsing oocyte-expressed α1β3γ2L GABAA receptors and two-microelectrode voltage-clamp, we quantified modulation of GABA responses by a low pentobarbital concentration and direct effects of high pentobarbital concentrations, the latter displaying mixed agonist and inhibitory effects. We then isolated and quantified pentobarbital inhibition in activated receptors using a novel single-sweep “notch” approach, and used these results to correct steady-state direct activation for inhibition.ResultsCombining results for GABA modulation and corrected direct activation, we estimated receptor open probability and optimized parameters for a Monod-Wyman-Changeux allosteric co-agonist model. Inhibition by pentobarbital was consistent with two sites with IC50s near 1 mM, while co-agonist model parameters suggest two allosteric pentobarbital agonist sites characterized by KPB ≈ 5 mM and high efficacy. The results also indicate that pentobarbital may be a more efficacious agonist than GABA.ConclusionsOur novel approach to quantifying both inhibitory and co-agonist effects of pentobarbital provides a basis for future structure-function analyses of GABAA receptor mutations in putative pentobarbital binding sites.

Allosteric Modulation of GABA-A Receptors by Different Drugs

Understanding how drugs allosterically modulate GABA-A receptor (GABAAR) channel function remains a challenge. Anesthetic drugs like etomidate, propofol and pentobarbital bind at transmembrane domain (TMD) inter-subunit interfaces at different locations. Benzodiazepines bind at the αγ interface in the extracellular domain (ECD) and neurosteroids are believed to bind within the α-subunit TMD helix bundle. A common feature of these drugs is that they potentiate GABA-induced currents. Here, we examined whether structural mechanisms underlying their functional effects are distinct. We monitored motions induced individually by these drugs at each inter-subunit TMD interface and also tested whether modulation caused by pairs of drugs binding to distinct sites are additive or super-additive. We individually introduced a cysteine in the M1 helices of each subunit at α1I227, β2L223 and γ2LI238 to probe the different inter-subunit interfaces. We expressed wild-type and mutant α1β2γ2L GABAARs in Xenopus oocytes and measured rates of modification of the substituted cysteines in the absence and presence of GABA and different allosteric modulators: the anesthetics pentobarbital (PB) and etomidate, the neurosteroid THDOC and the benzodiazepine flurazepam. GABA activation significantly increased MTSEA modification of αI227C, βL223C and γI238C. PB activation increased modification of αI227C and γI238C but not βL223C. Modulating concentrations of PB or etomidate had no effects. THDOC significantly increased the rate of modification of βL223C whereas flurazepam increased γI238C modification. We found that potentiation of EC10 GABA currents by combining modulating concentrations of THDOC and flurazepam was the sum of the potentiation produced by the two drugs individually. Combining PB and THDOC produced potentiation more than the sum of the potentiation produced by the two drugs individually. Our data suggests that drugs binding to different sites act via distinct mechanisms. Further analysis is underway to determine if these mechanisms are independent or coupled.

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Introduction: Pentobarbital (PB) is a barbiturate used as a treatment for status epilepticus and alternate agent for sedation. PB contains 40% propylene glycol (PG) and could result in lactic acidosis (LA). A previous report in an adult suggested probable PG-induced LA following PB continuous infusion (CI); however this has not been reported in children. A 3 year-old male was admitted for new-onset seizures. He was initially treated with IV lorazepam and midazolam and loaded with levetiracetam and fosphenytoin, which continued on as maintenance therapy. He was later intubated for respiratory depression and transferred to the PICU. Despite repeat loading doses and increased maintenance doses of his antiepileptics, he continued to have clinical and electrographic subclinical seizures. A midazolam CI was initiated but failed to control his seizures. On hospital day (HD) 3, he was started on PB due to continual subclinical seizures. He was initiated with a 10 mg/kg loading dose followed by a 5 mg/kg/dose IV q 12 hr. The seizures continued, and he was initiated on a PB CI on HD 5 to achieve burst suppression. His CI was titrated to a peak dose of 10 mg/kg/hour. He also received maintenance antiepileptics including phenobarbital, fosphenytoin, levetiracetam, lacosamide, topiramate, and valproic acid. On HD 15, he developed hypotension with a mean arterial pressure (MAP) in the 40s and had a venous LA of 3.01 (nl 0.4-2.0 mmol/L). He received epinephrine and his PB was decreased to 8 mg/kg/hour. His PB serum concentration was 68 mcg/mL (20-40 mcg/ml). The cumulative PG dose from all medications was calculated on HD 15 and was 1398 mg/kg. Over the next few weeks, he continued to have subclinical seizures and required an increase in PB back to 10 mg/kg/hour. On HD 37, he developed hypotension and LA of 6.28, with an osmolar gap of 20.4 mOsm/kg. He received a Lactated Ringer's bolus, sodium bicarbonate IV, dopamine CI, and PB decreased to 5 mg/kg/hr. His pentobarbital concentration was 79 mcg/mL and cumulative PG dose on HD 37 was 4045.8 mg/kg. After discussion with his parents, his PB was tapered off, and the decision was made to treat clinical seizures only. He was discharged home on levetiracetam, valproic acid, and diazepam. The World Health Organization recommends a maximum of 25 mg/kg of PG. On HD 15 and 37, our patient received more than this threshold. The Naranjo probability scale supports a high-probable drug-related adverse event in our patient. Practitioners should be aware of this potential adverse effect with medications containing large amounts of PG. Routine monitoring of osmolar gap should be performed for patients with prolonged use or higher PB doses.

Spectroscopic Investigations of Pentobarbital Interaction with Transthyretin

Transthyretin (TTR) aggregation has been characterized to be responsible for several amyloid diseases. Fourier transform infrared (FTIR) spectroscopy, fluorescence, and atomic force microscopy (AFM) are used to investigate secondary structure changes in transthyretin, induced upon thermal denaturation and interaction with pentobarbital. Spectral analysis revealed a strong static quenching of the intrinsic fluorescence of TTR by pentobarbital with a binding constant (K) estimated at2.092×103 M-1. Fourier self-deconvolution (FSD) technique is used to evaluates intensity changes in the spectra of the component bands in the amide I and amide II regions due to the changes in pentobarbital concentration in the protein complex. The increases of the relative intensities of the peaks at 1614 cm−1and 1507 cm−1are due to the increase of pentobarbital concentrations which is linked to the formation of oligomers in the protein.

Comparative effects of pentobarbital on spontaneous and evoked transmitter release from inhibitory and excitatory nerve terminals in rat CA3 neurons

Pentobarbital (PB) modulates GABA(A) receptor-mediated postsynaptic responses through various mechanisms, and can directly activate the channel at higher doses. These channels exist both pre- and postsynaptically, and on the soma outside the synapse. PB also inhibits voltage-dependent Na⁺ and Ca²⁺ channels to decrease excitatory synaptic transmission. Just how these different sites of action combine to contribute to the overall effects of PB on inhibitory and excitatory synaptic transmission is less clear. To compare these pre- and postsynaptic actions of PB, we used a 'synaptic bouton' preparation of isolated rat hippocampal CA3 pyramidal neurons where we could measure in single neurons the effects of PB on spontaneous and single bouton evoked GABAergic inhibitory and glutamatergic excitatory postsynaptic currents (sIPSCs, sEPSCs, eIPSCs and eEPSCs), respectively. Low (sedative) concentrations (3-10 μM) of PB increased the frequency and amplitude of sIPSCs and sEPSCs, and also presynaptically increased the amplitude of both eIPSCs and eEPSCs. There was no change in current kinetics at this low concentration. At higher concentrations (30-300 μM), PB decreased the frequency, and increased the amplitude of sIPSCs, and presynaptically decreased the amplitude of eIPSCs. The current decay phase of sIPSCs and eIPSCs was increased. An increase in both frequency and amplitude was seen for sEPSCs, while the eIPSCs was also decreased by a bicuculline-sensitive presynaptic effect. The results confirm the multiple sites of action of PB on inhibitory and excitatory transmission and demonstrate that the most sensitive site of action is on transmitter release, via effects on presynaptic GABA(A) receptors. At low concentrations, however, both glutamate and GABA release is similarly enhanced, making the final effects on neuronal excitability difficult to predict and dependent on the particular systems involved and/or on subtle differences in susceptibility amongst individuals. At higher concentrations, release of both transmitters is decreased, while the postsynaptic effects to increase IPSPs and decrease EPSCs would be expected to both results in reduced neuronal excitability.

Packing of the Extracellular Domain Hydrophobic Core Has Evolved to Facilitate Pentameric Ligand-gated Ion Channel Function

Protein function depends on conformational flexibility and folding stability. Loose packing of hydrophobic cores is not infrequent in proteins, as the enhanced flexibility likely contributes to their biological function. Here, using experimental and computational approaches, we show that eukaryotic pentameric ligand-gated ion channels are characterized by loose packing of their extracellular domain β-sandwich cores, and that loose packing contributes to their ability to rapidly switch from closed to open channel states in the presence of ligand. Functional analyses of GABA(A) receptors show that increasing the β-core packing disrupted GABA-mediated currents, with impaired GABA efficacy and slowed GABA current activation and desensitization. We propose that loose packing of the hydrophobic β-core developed as an evolutionary strategy aimed to facilitate the allosteric mechanisms of eukaryotic pentameric ligand-gated ion channels.

Allosteric modulators induce distinct movements at the GABA-binding site interface of the GABA-A receptor

Benzodiazepines (BZDs) and barbiturates exert their CNS actions by binding to GABA-A receptors (GABARs). The structural mechanisms by which these drugs allosterically modulate GABAR function, to either enhance or inhibit GABA-gated current, are poorly understood. Here, we used the substituted cysteine accessibility method to examine and compare structural movements in the GABA-binding site interface triggered by a BZD positive (flurazepam), zero (flumazenil) and negative (3-carbomethoxy-4-ethyl-6,7-dimethoxy-β-carboline, DMCM) modulator as well as the barbiturate pentobarbital. Ten residues located throughout the GABA-binding site interface were individually mutated to cysteine. Wild-type and mutant α1β2γ2 GABARs were expressed in Xenopus laevis oocytes and functionally characterized using two-electrode voltage clamp. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive methanethiosulfonate (MTS) reagents in the absence and presence of BZD-site ligands and pentobarbital. Flurazepam and DMCM each accelerated the rate of reaction at α1R131C and slowed the rate of reaction at α1E122C, whereas flumazenil had no effect indicating that simple occupation of the BZD binding site is not sufficient to cause movements near these positions. Therefore, BZD-induced movements at these residues are likely associated with the ability of the BZD to modulate GABAR function (BZD efficacy). Low, modulating concentrations of pentobarbital accelerated the rate of reaction at α1S68C and β2P206C, slowed the rate of reaction at α1E122C and had no effect at α1R131C. These findings indicate that pentobarbital and BZDs induce different movements in the receptor, providing evidence that the structural mechanisms underlying their allosteric modulation of GABAR function are distinct.

Pentobarbital inhibition of human recombinant α1A P/Q‐type voltage‐gated calcium channels involves slow, open channel block

Pre-synaptic neurotransmitter release is largely dependent on Ca(2+) entry through P/Q-type (Ca(V)2.1) voltage-gated Ca(2+) channels (PQCCs) at most mammalian, central, fast synapses. Barbiturates are clinical depressants and inhibit pre-synaptic Ca(2+) entry. PQCC barbiturate pharmacology is generally unclear, specifically in man. The pharmacology of the barbiturate pentobarbital (PB) in human recombinant alpha(1A) PQCCs has been characterized. PB effects on macroscopic Ca(2+)(I(Ca)) and Ba(2+)(I(Ba)) currents were studied using whole-cell patch clamp recording in HEK-293 cells heterologously expressing (alpha(1A))(human)(beta(2a)alpha(2)delta-1)(rabbit) PQCCs. PB reversibly depressed peak current (I(peak)) and enhanced apparent inactivation (fractional current at 800 ms, r(800)) in a concentration-dependent fashion irrespective of charge carrier (50% inhibitory concentration: I(peak), 656 microM; r(800), 104 microM). Rate of mono-exponential I(Ba) decay was linearly dependent on PB concentration. PB reduced channel availability by deepening non-steady-state inactivation curves without altering voltage dependence, slowed recovery from activity-induced unavailable states and produced use-dependent block. PB (100 microM) induced use-dependent block during physiological, high frequency pulse trains and overall depressed PQCC activity by two-fold. The results support a PB pharmacological mechanism involving a modulated receptor with preferential slow, bimolecular, open channel block (K(d)= 15 microM). Clinical PB concentrations (<200 microM) inhibit PQCC during high frequency activation that reduces computed neurotransmitter release by 16-fold and is comparable to the magnitude of Ca(2+)-dependent facilitation, G-protein modulation and intrinsic inactivation that play critical roles in PQCC modulation underlying synaptic plasticity. The results are consistent with the hypothesis that PB inhibition of PQCCs contributes to central nervous system depression underlying anticonvulsant therapy and general anaesthesia.

Barbiturates Require the N Terminus and First Transmembrane Domain of the δ Subunit for Enhancement of α1β3δ GABAA Receptor Currents

GABA(A) receptors are composed predominantly of alphabetagamma receptors, which mediate primarily synaptic inhibition, and alphabetadelta receptors, which mediate primarily extrasynaptic inhibition. At saturating GABA concentrations, the barbiturate pentobarbital substantially increased the amplitude and desensitization of the alpha1beta3delta receptor but not the alpha1beta3gamma2L receptor currents. To explore the structural domains of the delta subunit that are involved in pentobarbital potentiation and increased desensitization of alpha1beta3delta currents, chimeric cDNAs were constructed by progressive replacement of gamma2L subunit sequence with a delta subunit sequence or a delta subunit sequence with a gamma2L subunit sequence, and HEK293T cells were co-transfected with alpha1 and beta3 subunits or alpha1 and beta3 subunits and a gamma2L, delta, or chimeric subunit. Currents evoked by a saturating concentration of GABA or by co-application of GABA and pentobarbital were recorded using the patch clamp technique. By comparing the extent of enhancement and changes in kinetic properties produced by pentobarbital among chimeric and wild type receptors, we concluded that although potentiation of alpha1beta3delta currents by pentobarbital required the delta subunit sequence from the N terminus to proline 241 in the first transmembrane domain (M1), increasing desensitization of alpha1beta3delta currents required a delta subunit sequence from the N terminus to isoleucine 235 in M1. These findings suggest that the delta subunit N terminus and N-terminal portion of the M1 domain are, at least in part, involved in transduction of the allosteric effect of pentobarbital to enhance alpha1beta3delta currents and that this effect involves a distinct but overlapping structural domain from that involved in altering desensitization.