Effect of reducing isoflurane level on glucosamine uptake in the mouse brain during magnetic resonance imaging studies.

Anesthesia is often required during magnetic resonance imaging (MRI) examinations in animal studies. Anesthetic drugs differ in their capacity to interfere with homeostatic mechanisms responsible for glucose metabolism in the brain, which may create a constraint in the study design. Recent studies suggest that the chemical exchange saturation transfer (CEST) MRI scanning technique can detect localized metabolic changes in rodent brains induced by the uptake of glucose or its analogs; however, most of these studies do not account for the impact of anesthesia type on the brain metabolism. Herein, we aimed to evaluate the effect of reduced isoflurane levels on the preclinical imaging of glucosamine (GlcN) uptake in healthy mouse brains to establish optimal conditions for future brain imaging studies using the CEST MRI technique. The commonly used anesthesia protocol for longitudinal MRI examinations using 1.5% isoflurane level was compared to that using a mixture of low isoflurane (0.8%) level combined with midazolam (2 mg/kg, SC). Magnetization transfer ratio asymmetry (MTRasym) and area under the curve (AUC) analyses were used to characterize GlcN signals in the brain. The results indicated that mice injected with GlcN and anesthetized with 1.5% isoflurane exhibited low and insignificant changes in the MTRasym and AUC signals in the frontal cortex, whereas mice administered with 0.8% isoflurane combined with midazolam demonstrated a significant increase in these signals in the frontal cortex. This study highlights the diverse GlcN metabolic changes observed in mouse brains under variable levels of isoflurane anesthesia using the CEST MRI method. The results suggest that it is feasible to maintain anesthesia with low-dose isoflurane by integrating midazolam, which may enable the investigation of GlcN uptake in the brain. Thus, reducing isoflurane levels may support studies into mouse brain metabolism using the CEST MRI method and should be considered in future studies.

Efficacy of Lacosamide and Rufinamide as Adjuncts to Midazolam-Ketamine Treatment Against Cholinergic-Induced Status Epilepticus in Rats.

Benzodiazepine pharmacoresistance develops when treatment of status epilepticus (SE) is delayed. This response may result from gamma-aminobutyric acid A receptors (GABAAR) internalization that follows prolonged SE; this receptor trafficking results in fewer GABAAR in the synapse to restore inhibition. Increase in synaptic N-methyl-D-aspartate receptors (NMDAR) also occurs in rodent models of SE. Lacosamide, a third-generation antiseizure medication (ASM), acts on the slow inactivation of voltage-gated sodium channels. Another ASM, rufinamide, similarly acts on sodium channels by extending the duration of time spent in the inactivation stage. Combination therapy of the benzodiazepine midazolam, NMDAR antagonist ketamine, and ASMs lacosamide (or rufinamide) was investigated for efficacy against soman (GD)-induced SE and neuropathology. Adult male rats implanted with telemetry transmitters for monitoring electroencephalographic (EEG) activity were exposed to a seizure-inducing dose of GD and treated with an admix of atropine sulfate and HI-6 1 minute later and with midazolam monotherapy or combination therapy 40 minutes after EEG seizure onset. Rats were monitored continuously for seizure activity for two weeks, after which brains were processed for assessment of neurodegeneration, neuronal loss, and neuroinflammatory responses. Simultaneous administration of midazolam, ketamine, and lacosamide (or rufinamide) was more protective against GD-induced SE compared with midazolam monotherapy. In general, lacosamide triple therapy had more positive outcomes on measures of epileptogenesis, EEG power integral, and the number of brain regions protected from neuropathology compared with rats treated with rufinamide triple therapy. Overall, both drugs were well tolerated in these combination models. SIGNIFICANCE STATEMENT: We currently report on improved efficacy of antiseizure medications lacosamide and rufinamide, each administered in combination with ketamine (NMDAR antagonist) and midazolam (benzodiazepine), in combatting soman (GD)-induced seizure, epileptogenesis, and brain pathology over that provided by midazolam monotherapy, or dual therapy of midazolam and lacosamide (or rufinamide) in rats. Administration of lacosamide as adjunct to midazolam and ketamine was particularly effective against GD-induced toxicity. However, protection was incomplete, suggesting the need for further study.

Preventing Long-Term Brain Damage by Nerve Agent-Induced Status Epilepticus in Rat Models Applicable to Infants: Significant Neuroprotection by Tezampanel Combined with Caramiphen but Not by Midazolam Treatment.

Acute exposure to nerve agents induces a peripheral cholinergic crisis and prolonged status epilepticus (SE), causing death or long-term brain damage. To provide preclinical data pertinent to the protection of infants and newborns, we compared the antiseizure and neuroprotective effects of treating soman-induced SE with midazolam (MDZ) versus tezampanel (LY293558) in combination with caramiphen (CRM) in 12- and 7-day-old rats. The anticonvulsants were administered 1 hour after soman exposure; neuropathology data were collected up to 6 months postexposure. In both ages, the total duration of SE within 24 hours after soman exposure was significantly shorter in the LY293558 plus CRM groups compared with the MDZ groups. Neuronal degeneration was substantial in the MDZ-treated groups but absent or minimal in the groups treated with LY293558 plus CRM. Loss of neurons and interneurons in the basolateral amygdala and CA1 hippocampal area was significant in the MDZ-treated groups but virtually absent in the LY293558 plus CRM groups. Atrophy of the amygdala and hippocampus occurred only in MDZ-treated groups. Neuronal/interneuronal loss and atrophy of the amygdala and hippocampus deteriorated over time. Reduction of inhibitory activity in the basolateral amygdala and increased anxiety were found only in MDZ groups. Spontaneous recurrent seizures developed in the MDZ groups, deteriorating over time; a small percentage of rats from the LY293558 plus CRM groups also developed seizures. These results suggest that brain damage can be long lasting or permanent if nerve agent-induced SE in infant victims is treated with midazolam at a delayed timepoint after SE onset, whereas antiglutamatergic treatment with tezampanel and caramiphen provides significant neuroprotection. SIGNIFICANCE STATEMENT: To protect the brain and the lives of infants in a mass exposure to nerve agents, an anticonvulsant treatment must be administered that will effectively stop seizures and prevent neuropathology, even if offered with a relative delay after seizure onset. The present study shows that midazolam, which was recently approved by the Food and Drug Administration for the treatment of nerve agent-induced status epilepticus, is not an effective neuroprotectant, whereas brain damage can be prevented by targeting glutamate receptors.

Evaluation of Midazolam-Ketamine-Allopregnanolone Combination Therapy against Cholinergic-Induced Status Epilepticus in Rats.

Status epilepticus (SE) is a life-threatening development of self-sustaining seizures that becomes resistant to benzodiazepines when treatment is delayed. Benzodiazepine pharmacoresistance is thought in part to result from internalization of synaptic GABAA receptors, which are the main target of the drug. The naturally occurring neurosteroid allopregnanolone is a therapy of interest against SE for its ability to modulate all isoforms of GABAA receptors. Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has been partially effective in combination with benzodiazepines in mitigating SE-associated neurotoxicity. In this study, allopregnanolone as an adjunct to midazolam or midazolam-ketamine combination therapy was evaluated for efficacy against cholinergic-induced SE. Adult male rats implanted with electroencephalographic (EEG) telemetry devices were exposed to the organophosphorus chemical (OP) soman (GD) and treated with an admix of atropine sulfate and HI-6 at 1 minute after exposure followed by midazolam, midazolam-allopregnanolone, or midazolam-ketamine-allopregnanolone 40 minutes after seizure onset. Neurodegeneration, neuronal loss, and neuroinflammation were assessed 2 weeks after GD exposure. Seizure activity, EEG power integral, and epileptogenesis were also compared among groups. Overall, midazolam-ketamine-allopregnanolone combination therapy was effective in reducing cholinergic-induced toxic signs and neuropathology, particularly in the thalamus and hippocampus. Higher dosage of allopregnanolone administered in combination with midazolam and ketamine was also effective in reducing EEG power integral and epileptogenesis. The current study reports that there is a promising potential of neurosteroids in combination with benzodiazepine and ketamine treatments in a GD model of SE. SIGNIFICANCE STATEMENT: Allopregnanolone, a naturally occurring neurosteroid, reduced pathologies associated with soman (GD) exposure such as epileptogenesis, neurodegeneration, and neuroinflammation, and suppressed GD-induced toxic signs when used as an adjunct to midazolam and ketamine in a delayed treatment model of soman-induced status epilepticus (SE) in rats. However, protection was incomplete, suggesting that further studies are needed to identify optimal combinations of antiseizure medications and routes of administration for maximal efficacy against cholinergic-induced SE.

Neurosteroids as Novel Anticonvulsants for Refractory Status Epilepticus and Medical Countermeasures for Nerve Agents: A 15-Year Journey to Bring Ganaxolone from Bench to Clinic.

This article describes recent advances in the use of neurosteroids as novel anticonvulsants for refractory status epilepticus (RSE) and as medical countermeasures (MCs) for organophosphates and chemical nerve agents (OPNAs). We highlight a comprehensive 15-year journey to bring the synthetic neurosteroid ganaxolone (GX) from bench to clinic. RSE, including when caused by nerve agents, is associated with devastating morbidity and permanent long-term neurologic dysfunction. Although recent approval of benzodiazepines such as intranasal midazolam and intranasal midazolam offers improved control of acute seizures, novel anticonvulsants are needed to suppress RSE and improve neurologic function outcomes. Currently, few anticonvulsant MCs exist for victims of OPNA exposure and RSE. Standard-of-care MCs for postexposure treatment include benzodiazepines, which do not effectively prevent or mitigate seizures resulting from nerve agent intoxication, leaving an urgent unmet medical need for new anticonvulsants for RSE. Recently, we pioneered neurosteroids as next-generation anticonvulsants that are superior to benzodiazepines for treatment of OPNA intoxication and RSE. Because GX and related neurosteroids that activate extrasynaptic GABA-A receptors rapidly control seizures and offer robust neuroprotection by reducing neuronal damage and neuroinflammation, they effectively improve neurologic outcomes after acute OPNA exposure and RSE. GX has been selected for advanced, Biomedical Advanced Research and Development Authority-supported phase 3 trials of RSE and nerve agent seizures. In addition, in mechanistic studies of neurosteroids at extrasynaptic receptors, we identified novel synthetic analogs with features that are superior to GX for current medical needs. Development of new MCs for RSE is complex, tedious, and uncertain due to scientific and regulatory challenges. Thus, further research will be critical to fill key gaps in evaluating RSE and anticonvulsants in vulnerable (pediatric and geriatric) populations and military persons. SIGNIFICANCE STATEMENT: Following organophosphate and nerve agent intoxication, refractory status epilepticus (RSE) occurs despite benzodiazepine treatment. RSE occurs in 40% of status epilepticus patients, with a 35% mortality rate and significant neurological morbidity in survivors. To treat RSE, neurosteroids are better anticonvulsants than benzodiazepines. Our pioneering use of neurosteroids for RSE and nerve agents led us to develop ganaxolone as a novel anticonvulsant and neuroprotectant with significantly improved neurological outcomes. This article describes the bench-to-bedside journey of bringing neurosteroid therapy to patients, with ganaxolone leading the way.

Sex Differences in Organophosphate Model of Benzodiazepine-Refractory Status Epilepticus and Neuronal Damage.

Sex differences are common in human epilepsy. Although men are more susceptible to seizure than women, the mechanisms underlying sex-specific vulnerabilities to seizure are unclear. The organophosphate (OP) diisopropylfluorophosphate (DFP) is known to cause neurotoxicity and status epilepticus (SE), a serious neurologic condition that causes prolonged seizures and brain damage. Current therapies for OP poisoning and SE do not consider neuronal variations between male and female brains. Therefore, we investigated sex-dependent differences in electrographic seizure activity and neuronal injury using the DFP model of refractory SE in rats. Electroencephalogram recordings were used to monitor DFP-induced SE, and the extent of brain injury was determined using fluoro-jade-B staining to detect cellular necrosis. After DFP exposure, we observed striking sex-dependent differences in SE and seizure activity patterns as well as protective responses to midazolam treatment. Following acute DFP exposure, male animals displayed more severe SE with intense epileptiform spiking and greater mortality than females. In contrast, we observed significantly more injured cells and cellular necrosis in the hippocampus and other brain regions in females than in males. We also observed extensive neuronal injury in the somatosensory cortex of males. The anticonvulsant effect of midazolam against SE was limited in this model and found to be similar in males and females. However, unlike males, females exhibited substantially more protection against neuronal damage after midazolam treatment. Overall, these results demonstrate significant sex-dependent differences in DFP-induced refractory SE and neuronal damage patterns, suggesting that it may be possible to develop sex-specific neuroprotective strategies for OP intoxication and refractory SE. SIGNIFICANCE STATEMENT: Sex-dependent differences in neurotoxicity and status epilepticus (SE) are key biological variables after organophosphate (OP) exposure. Here, we investigated sex-dependent differences in SE and brain injury after acute diisopropylfluorophosphate exposure. Male rats had more severe SE and less survival than females, while females had more neuronal damage. Females had more neuroprotection to midazolam than males, while both sexes had similar but partial anticonvulsant effects. These findings suggest that a sex-specific therapeutic approach may prevent neurological complications of OP-induced SE.

Neuroprotectant Activity of Novel Water-Soluble Synthetic Neurosteroids on Organophosphate Intoxication and Status Epilepticus-Induced Long-Term Neurological Dysfunction, Neurodegeneration, and Neuroinflammation.

Organophosphates (OPs) and nerve agents are potent neurotoxic compounds that cause seizures, status epilepticus (SE), brain injury, or death. There are persistent long-term neurologic and neurodegenerative effects that manifest months to years after the initial exposure. Current antidotes are ineffective in preventing these long-term neurobehavioral and neuropathological changes. Additionally, there are few effective neuroprotectants for mitigating the long-term effects of acute OP intoxication. We have pioneered neurosteroids as novel anticonvulsants and neuroprotectants for OP intoxication and seizures. In this study, we evaluated the efficacy of two novel synthetic, water-soluble neurosteroids, valaxanolone (VX) and lysaxanolone (LX), in combating the long-term behavioral and neuropathological impairments caused by acute OP intoxication and SE. Animals were exposed to the OP nerve agent surrogate diisopropylfluorophosphate (DFP) and were treated with VX or LX in addition to midazolam at 40 minutes postexposure. The extent of neurodegeneration, along with various behavioral and memory deficits, were assessed at 3 months postexposure. VX significantly reduced deficits of aggressive behavior, anxiety, memory, and depressive-like traits in control (DFP-exposed, midazolam-treated) animals; VX also significantly prevented the DFP-induced chronic loss of NeuN(+) principal neurons and PV(+) inhibitory neurons in the hippocampus and other regions. Additionally, VX-treated animals exhibited a reduced inflammatory response with decreased GFAP(+) astrogliosis and IBA1(+) microgliosis in the hippocampus, amygdala, and other regions. Similarly, LX showed significant improvement in behavioral and memory deficits, and reduced neurodegeneration and cellular neuroinflammation. Together, these results demonstrate the neuroprotectant effects of the novel synthetic neurosteroids in mitigating the long-term neurologic dysfunction and neurodegeneration associated with OP exposure. SIGNIFICANCE STATEMENT: Survivors of nerve agents and organophosphate (OP) exposures suffer from long-term neurological deficits. Currently, there is no specific drug therapy for mitigating the impact of OP exposure. However, novel synthetic neurosteroids that activate tonic inhibition provide a viable option for treating OP intoxication. The data from this study indicates the neuroprotective effects of synthetic, water-soluble neurosteroids for attenuation of long-term neurological deficits after OP intoxication. These findings establish valaxanolone and lysaxanolone as potent and efficacious neuroprotectants suitable for injectable dosing.

Pulsed-field versus cryoballoon ablation for atrial fibrillation-Impact of energy source on sedation and analgesia requirement.

INTRODUCTION: Pulsed field ablation (PFA) represents a novel, nonthermal energy modality that can be applied for single-shot pulmonary vein isolation (PVI) in atrial fibrillation (AF). Comparative data with regard to deep sedation to established single-shot modalities such as cryoballoon (CB) ablation are scarce. The aim of this study was to compare a deep sedation protocol in patients receiving PVI with either PFA or CB. METHODS: Prospective, consecutive AF patients undergoing PVI with a pentaspline PFA catheter were compared to a retrospective CB-PVI cohort of the same timeframe. Study endpoints were the requirements of analgesics, cardiorespiratory stability, and sedation-associated complications. RESULTS: A total of 100 PVI patients were included (PFA n = 50, CB n = 50, mean age 66 ± 10.6, 61% male patients, 65% paroxysmal AF). Requirement of propofol, midazolam, and sufentanyl was significantly higher in the PFA group compared to CB [propofol 0.14 ± 0.04 mg/kg/min in PFA vs. 0.11 ± 0.04 mg/kg/min in CB (p = .001); midazolam 0.00086 ± 0.0004 mg/kg/min in PFA vs. 0.0006295 ± 0.0003 mg/kg/min in CB (p = .002) and sufentanyl 0.0013 ± 0.0007 µg/kg/min in PFA vs. 0.0008 ± 0.0004 µg/kg/min in CB (p < .0001)]. Sedation-associated complications did not differ between both groups (PFA n = 1/50 mild aspiration pneumonia, CB n = 0/50, p > .99). Nonsedation-associated complications (PFA: n = 2/50, 4%, CB: n = 1/50, 2%, p > .99) and procedure times (PFA 75 ± 31, CB 84 ± 32 min, p = .18) did not differ between groups. CONCLUSIONS: PFA is associated with higher sedation and especially analgesia requirements. However, the safety of deep sedation does not differ to CB ablation.

Midazolam Prevents the Adverse Outcome of Neonatal Asphyxia.

OBJECTIVE: Birth asphyxia (BA) is the most frequent cause of neonatal death as well as central nervous system (CNS) injury. BA is often associated with neonatal seizures, which only poorly respond to anti-seizure medications and may contribute to the adverse neurodevelopmental outcome. Using a non-invasive rat model of BA, we have recently reported that the potent benzodiazepine, midazolam, prevents neonatal seizures in ~50% of rat pups. In addition to its anti-seizure effect, midazolam exerts anti-inflammatory actions, which is highly relevant for therapeutic intervention following BA. The 2 major aims of the present study were to examine (1) whether midazolam reduces the adverse outcome of BA, and (2) whether this effect is different in rats that did or did not exhibit neonatal seizures after drug treatment. METHODS: Behavioral and cognitive tests were performed over 14 months after asphyxia, followed by immunohistochemical analyses. RESULTS: All vehicle-treated rats had seizures after asphyxia and developed behavioral and cognitive abnormalities, neuroinflammation in gray and white matter, neurodegeneration in the hippocampus and thalamus, and hippocampal mossy fiber sprouting in subsequent months. Administration of midazolam (1 mg/kg i.p.) directly after asphyxia prevented post-asphyctic seizures in ~50% of the rats and resulted in the prevention or decrease of neuroinflammation and the behavioral, cognitive, and neurodegenerative consequences of asphyxia. Except for neurodegeneration in the thalamus, seizures did not seem to contribute to the adverse outcome of asphyxia. INTERPRETATION: The disease-modifying effect of midazolam identified here strongly suggests that this drug provides a valuable option for improving the treatment and outcome of BA. ANN NEUROL 2023;93:226-243.

Physiologically Based Pharmacokinetic Modeling to Predict the Impact of Liver Cirrhosis on Glucuronidation via UGT1A4 and UGT2B7/2B4-A Case Study with Midazolam.

Hepatic impairment, due to liver cirrhosis, decreases the activity of cytochrome P450 enzymes (CYPs). The use of physiologically based pharmacokinetic (PBPK) modeling to predict this effect for CYP substrates has been well-established, but the effect of cirrhosis on uridine-glucuronosyltransferase (UGT) activities is less studied and few PBPK models have been reported. UGT enzymes are involved in primary N-glucuronidation of midazolam and glucuronidation of 1'-OH-midazolam following CYP3A hydroxylation. In this study, Simcyp was used to establish PBPK models for midazolam, its primary metabolites midazolam-N-glucuronide (UGT1A4) and 1'-OH midazolam (CYP3A4/3A5), and the secondary metabolite 1'-OH-midazolam-O-glucuronide (UGT2B7/2B4), allowing to simulate the impact of liver cirrhosis on the primary and secondary glucuronidation of midazolam. The model was verified in noncirrhotic subjects before extrapolation to cirrhotic patients of Child-Pugh (CP) classes A, B, and C. Our model successfully predicted the exposures of midazolam and its metabolites in noncirrhotic and cirrhotic patients, with 86% of observed plasma concentrations within 5th-95th percentiles of predictions and observed geometrical mean of area under the plasma concentration curve between 0 hours to infinity and maximal plasma concentration within 0.7- to 1.43-fold of predictions. The simulated metabolic ratio defined as the ratio of the glucuronide metabolite AUC over the parent compound AUC (AUCglucuronide/AUCparent, metabolic ratio [MR]), was calculated for midazolam-N-glucuronide to midazolam (indicative of UGT1A4 activity) and decreased by 40% (CP A), 48% (CP B), and 75% (CP C). For 1'-OH-midazolam-O-glucuronide to 1'-OH-midazolam, the MR (indicative of UGT2B7/2B4 activity) dropped by 35% (CP A), 51% (CP B), and 64% (CP C). These predicted MRs were corroborated by the observed data. This work thus increases confidence in Simcyp predictions of the effect of liver cirrhosis on the pharmacokinetics of UGT1A4 and UGT2B7/UGT2B4 substrates. SIGNIFICANCE STATEMENT: This article presents a physiologically based pharmacokinetic model for midazolam and its metabolites and verifies the accurate simulation of pharmacokinetic profiles when using the Simcyp hepatic impairment population models. Exposure changes of midazolam-N-glucuronide and 1'-OH-midazolam-O-glucuronide reflect the impact of decreases in UGT1A4 and UGT2B7/2B4 glucuronidation activity in cirrhotic patients. The approach used in this study may be extended to verify the modeling of other uridine glucuronosyltransferase enzymes affected by liver cirrhosis.

Assessing Pleiotropic Effects of a Mixed-Mode Perpetrator Drug, Rifampicin, by Multiple Endogenous Biomarkers in Dogs.

Rifampicin (RIF) is a mixed-mode perpetrator that produces pleiotropic effects on liver cytochrome P450 enzymes and drug transporters. To assess the complex drug-drug interaction liabilities of RIF in vivo, a known probe substrate, midazolam (MDZ), along with multiple endogenous biomarkers were simultaneously monitored in beagle dogs before and after a 7-day treatment period by RIF at 20 mg/kg per day. Confirmed by the reduced MDZ plasma exposure and elevated 4ß-hydroxycholesterol (4ß-HC, biomarker of CYP3A activities) level, CYP3A was significantly induced after repeated RIF doses, and such induction persisted for 3 days after cessation of the RIF administration. On the other hand, increased plasma levels of coproporphyrin (CP)-I and III [biomarkers of organic anion transporting polypeptides 1b (Oatp1b) activities] were observed after the first dose of RIF. Plasma CPs started to decline as RIF exposure decreased, and they returned to baseline 3 days after cessation of the RIF administration. The data suggested the acute (inhibitory) and chronic (inductive) effects of RIF on Oatp1b and CYP3A enzymes, respectively, and a 3-day washout period is deemed adequate to remove superimposed Oatp1b inhibition from CYP3A induction. In addition, apparent self-induction of RIF was observed as its terminal half-life was significantly altered after multiple doses. Overall, our investigation illustrated the need for appropriate timing of modulator dosing to differentiate between transporter inhibition and enzyme induction. As further indicated by the CP data, induction of Oatp1b activities was not likely after repeated RIF administration. SIGNIFICANCE STATEMENT: This investigation demonstrated the utility of endogenous biomarkers towards complex drug-drug interactions by rifampicin (RIF) and successfully determined the optimal timing to differentiate between transporter inhibition and enzyme induction. Based on experimental evidence, Oatp1b induction following repeated RIF administration was unlikely, and apparent self-induction of RIF elimination was observed.

In Vitro to In Vivo Scalars for Drug Clearance in Nonalcoholic Fatty Liver and Steatohepatitis.

In vitro-in vivo extrapolation (IVIVE) allows prediction of clinical outcomes across populations from in vitro data using specific scalars tailored to the biologic characteristics of each population. This study experimentally determined scalars for patients with varying degrees of nonalcoholic fatty liver disease (NAFLD), ranging from fatty liver to nonalcoholic steatohepatitis (NASH) and cirrhosis. Microsomal, S9, and cytosol fractions were extracted from 36 histologically normal and 66 NAFLD livers (27 nonalcoholic fatty liver [NAFL], 13 NASH, and 26 NASH with cirrhosis). Corrected microsomal protein per gram liver (MPPGL) progressively decreased with disease severity (26.8, 27.4, and 24.3 mg/g in NAFL, NASH, and NASH/cirrhosis, respectively, compared with 35.6 mg/g in normal livers; ANOVA, P < 0.001). Homogenate, S9, and cytosolic protein showed a consistent trend of decline in NASH/cirrhosis relative to normal control (post-hoc t test, P < 0.05). No differences across the groups were observed in homogenate, S9, cytosolic, and microsomal protein content in matched kidney samples. MPPGL-based scalars that combine protein content with liver size revealed that the reduction in MPPGL in NAFL and NASH was compensated by the reported increase in liver size (relative scalar ratios of 0.96 and 0.99, respectively), which was not the case with NASH/cirrhosis (ratio of 0.63), compared with healthy control. Physiologically based pharmacokinetics-informed global sensitivity analysis of the relative contribution of IVIVE scalars (hepatic CYP3A4 abundance, MPPGL, and liver size) to variability in exposure (area under the curve) to three CYP3A substrates (alprazolam, midazolam, and ibrutinib) revealed enzyme abundance as the most significant parameter, followed by MPPGL, whereas liver volume was the least impactful factor. SIGNIFICANCE STATEMENT: Nonalcoholic fatty liver disease-specific scalars necessary for extrapolation from in vitro systems to liver tissue are lacking. These are required in clearance prediction and dose selection in nonalcoholic fatty liver and steatohepatitis populations. Previously reported disease-driven changes have focused on cirrhosis, with no data on the initial stages of liver disease. The authors obtained experimental values for microsomal, cytosolic, and S9 fractions and assessed the relative impact of microsomal scalars on predicted exposure to substrate drugs using physiologically based pharmacokinetics.

Drug Disposition in Neonatal Göttingen Minipigs: Exploring Effects of Perinatal Asphyxia and Therapeutic Hypothermia.

Asphyxiated neonates often undergo therapeutic hypothermia (TH) to reduce morbidity and mortality. Since both perinatal asphyxia (PA) and TH influence physiology, altered pharmacokinetics (PK) and pharmacodynamics (PD) are expected. Given that TH is the standard of care for PA with moderate to severe hypoxic-ischemic encephalopathy, disentangling the effect of PA versus TH on PK/PD is not possible in clinical settings. However, animal models can provide insights into this matter. The (neonatal) Göttingen Minipig, the recommended strain for nonclinical drug development, was selected as translational model. Four drugs-midazolam (MDZ), fentanyl (FNT), phenobarbital (PHB), and topiramate (TPM)-were intravenously administered under four conditions: control (C), therapeutic hypothermia (TH), hypoxia (H), and hypoxia plus TH (H+TH). Each group included six healthy male neonatal Göttingen Minipigs anesthetized for 24 hours. Blood samples were drawn at 0 (predose) and 0.5, 2, 2.5, 3, 4, 4.5, 6, 8, 12, and 24 hours post drug administration. Drug plasma concentrations were determined using validated bioanalytical assays. The PK parameters were estimated through compartmental and noncompartmental PK analysis. The study showed a statistically significant decrease in FNT clearance (CL; 66% decrease), with an approximately threefold longer half-life (t1/2) in the TH group. The H+TH group showed a 17% reduction in FNT CL, with a 62% longer t1/2 compared with the C group; however, it was not statistically significant. Although not statistically significant, trends toward lower CL and longer t1/2 were observed in the TH and H+TH groups for MDZ and PHB. Additionally, TPM demonstrated a 28% decrease in CL in the H group compared with controls. SIGNIFICANCE STATEMENT: The overarching goal of this study using the neonatal Göttingen Minipig model was to disentangle the effects of systemic hypoxia and TH on PK using four model drugs. Such insights can subsequently be used to inform and develop a physiologically based pharmacokinetic model, which is useful for drug exposure prediction in human neonates.

Human Pharmacokinetic and CYP3A Drug-Drug Interaction Prediction of GDC-2394 Using Physiologically Based Pharmacokinetic Modeling and Biomarker Assessment.

Physiologically based pharmacokinetic (PBPK) modeling was used to predict the human pharmacokinetics and drug-drug interaction (DDI) of GDC-2394. PBPK models were developed using in vitro and in vivo data to reflect the oral and intravenous PK profiles of mouse, rat, dog, and monkey. The learnings from preclinical PBPK models were applied to a human PBPK model for prospective human PK predictions. The prospective human PK predictions were within 3-fold of the clinical data from the first-in-human study, which was used to optimize and validate the PBPK model and subsequently used for DDI prediction. Based on the majority of PBPK modeling scenarios using the in vitro CYP3A induction data (mRNA and activity), GDC-2394 was predicted to have no-to-weak induction potential at 900 mg twice daily (BID). Calibration of the induction mRNA and activity data allowed for the convergence of DDI predictions to a narrower range. The plasma concentrations of the 4ß-hydroxycholesterol (4ß-HC) were measured in the multiple ascending dose study to assess the hepatic CYP3A induction risk. There was no change in plasma 4ß-HC concentrations after 7 days of GDC-2394 at 900 mg BID. A dedicated DDI study found that GDC-2394 has no induction effect on midazolam in humans, which was reflected by the totality of predicted DDI scenarios. This work demonstrates the prospective utilization of PBPK for human PK and DDI prediction in early drug development of GDC-2394. PBPK modeling accompanied with CYP3A biomarkers can serve as a strategy to support clinical pharmacology development plans. SIGNIFICANCE STATEMENT: This work presents the application of physiologically based pharmacokinetic modeling for prospective human pharmacokinetic (PK) and drug-drug interaction (DDI) prediction in early drug development. The strategy taken in this report represents a framework to incorporate various approaches including calibration of in vitro induction data and consideration of CYP3A biomarkers to inform on the overall CYP3A-related DDI risk of GDC-2394.

Comparison of 1Beta- and 5Beta-hydroxylation of Deoxycholate and Glycodeoxycholate as In Vitro Index Reactions for Cytochrome P450 3A Activities.

Cytochrome P450 3A (CYP3A) participates in the metabolism of more than 30% of clinical drugs. The vast intra- and inter-individual variations in CYP3A activity pose great challenges to drug development and personalized medicine. It has been disclosed that human CYP3A4 and CYP3A7 are exclusively responsible for the tertiary oxidations of deoxycholic acid (DCA) and glycodeoxycholic acid (GDCA) regioselectivity at C-1ß and C-5ß This work aimed to compare the 1ß- and 5ß-hydroxylation of DCA and GDCA as potential in vitro CYP3A index reactions in both human liver microsomes and recombinant P450 enzymes. The results demonstrated that the metabolic activity of DCA 1ß- and 5ß-hydroxylation was 5-10 times higher than that of GDCA, suggesting that 1ß-hydroxyglycodeoxycholic acid and 5ß-hydroxyglycodeoxycholic acid may originate from DCA oxidation followed by conjugation in humans. Metabolic phenotyping data revealed that DCA 1ß-hydroxylation, DCA 5ß-hydroxylation, and GDCA 5ß-hydroxylation were predominantly catalyzed by CYP3A4 (>80%), while GDCA 1ß-hydroxylation had approximately equal contributions from CYP3A4 (41%) and 3A7 (58%). Robust Pearson correlation was established for the intrinsic clearance of DCA 1ß- and 5ß-hydroxylation with midazolam (MDZ) 1'- and 4-hydroxylation in fourteen single donor microsomes. Although DCA 5ß-hydroxylation exhibited a stronger correlation with MDZ oxidation, DCA 1ß-hydroxylation exhibited higher reactivity than DCA 5ß-hydroxylation. It is therefore suggested that DCA 1ß- and 5ß-hydroxylations may serve as alternatives to T 6ß-hydroxylation as in vitro CYP3A index reactions. SIGNIFICANCE STATEMENT: The oxidation of DCA and GDCA is primarily catalyzed by CYP3A4 and CYP3A7. This work compared the 1ß- and 5ß-hydroxylation of DCA and GDCA as in vitro index reactions to assess CYP3A activities. It was disclosed that the metabolic activity of DCA 1ß- and 5ß-hydroxylation was 5-10 times higher than that of GDCA. Although DCA 1ß-hydroxylation exhibited higher metabolic activity than DCA 5ß-hydroxylation, DCA 5ß-hydroxylation demonstrated stronger correlation with MDZ oxidation than DCA 1ß-hydroxylation in individual liver microsomes.

Arsenite-Induced Drug-Drug Interactions in Rats.

Arsenite is an important heavy metal. Some Chinese traditional medicines contain significant amounts of arsenite. The aim of this study was to investigate subacute exposure of arsenite on activities of cytochrome P450 enzymes and pharmacokinetic behaviors of drugs in rats. Midazolam, tolbutamide, metoprolol, omeprazole, caffeine, and chlorzoxazone, the probe substrates for cytochrome P450 (CYP) s3A, 2C6, 2D, 2C11, 1A, and 2E, were selected as probe drugs for the pharmacokinetic study. Significant decreases in areas under the curves of probe substrates were observed in rats after consecutive 30-day exposure to As at 12 mg/kg. Microsomal incubation study showed that the subacute exposure to arsenite resulted in little change in effects on the activities of P450 enzymes examined. However, everted gut sac study demonstrated that such exposure induced significant decreases in intestinal absorption of these drugs by both passive diffusion and carrier-mediated transport. In addition, in vivo study showed that the arsenite exposure decreased the rate of peristaltic propulsion. The decreases in intestinal permeability of the probe drugs and peristaltic propulsion rate most likely resulted in the observed decreases in the internal exposure of the probe drugs. Exposure to arsenite may lead to the reduction of the efficiencies of pharmaceutical agents coadministered resulting from the observed drug-drug interactions. SIGNIFICANCE STATEMENT: Exposure to arsenite may lead to the reduction of the efficiencies of pharmaceutical agents coadministered resulting from the observed drug-drug interactions. The present study, we found that P450 enzyme probe drug exposure was reduced in arsenic-exposed animals (areas under the curve) and the intestinal absorption of the drug was reduced in the animals. Subacute arsenic exposure tends to cause damage to intestinal function, which leads to reduced drug absorption.

Efficacy of a combined anti-seizure treatment against cholinergic established status epilepticus following a sarin nerve agent insult in rats.

The development of refractory status epilepticus (SE) following sarin intoxication presents a therapeutic challenge. Here, we evaluated the efficacy of delayed combined double or triple treatment in reducing abnormal epileptiform seizure activity (ESA) and the ensuing long-term neuronal insult. SE was induced in rats by exposure to 1.2 LD50 sarin followed by treatment with atropine and TMB4 (TA) 1 min later. Double treatment with ketamine and midazolam or triple treatment with ketamine, midazolam and levetiracetam was administered 30 min post-exposure, and the results were compared to those of single treatment with midazolam alone or triple treatment with ketamine, midazolam, and valproate, which was previously shown to ameliorate this neurological insult. Toxicity and electrocorticogram activity were monitored during the first week, and behavioral evaluations were performed 2 weeks post-exposure, followed by biochemical and immunohistopathological analyses. Both double and triple treatment reduced mortality and enhanced weight recovery compared to TA-only treatment. Triple treatment and, to a lesser extent, double treatment significantly ameliorated the ESA duration. Compared to the TA-only or the TA+ midazolam treatment, both double and triple treatment reduced the sarin-induced increase in the neuroinflammatory marker PGE2 and the brain damage marker TSPO and decreased gliosis, astrocytosis and neuronal damage. Finally, both double and triple treatment prevented a change in behavior, as measured in the open field test. No significant difference was observed between the efficacies of the two triple treatments, and both triple combinations completely prevented brain injury (no differences from the naïve rats). Delayed double and, to a greater extent, triple treatment may serve as an efficacious delayed therapy, preventing brain insult propagation following sarin-induced refractory SE.

Seizure suppression and neuroprotection in soman-exposed rats following delayed intramuscular treatment of adenosine A1 receptor agonist as an adjunct to standard medical treatment.

Soman produces excitotoxic effects by inhibiting acetylcholinesterase in the cholinergic synapses and neuromuscular junctions, resulting in soman-induced sustained status epilepticus (SSE). Our previous work showed delayed intramuscular (i.m.) treatment with A1 adenosine receptor agonist N-bicyclo-[2.2.1]-hept-2-yl-5'-chloro-5'-deoxyadenosine (ENBA) alone suppressed soman-induced SSE and prevented neuropathology. Using this same rat soman seizure model, we tested if delayed therapy with ENBA (60 mg/kg, i.m.) would terminate seizure, protect neuropathology, and aid in survival when given in conjunction with current standard medical countermeasures (MCMs): atropine sulfate, 2-PAM, and midazolam (MDZ). Either 15- or 30-min following soman-induced SSE onset, male rats received atropine and 2-PAM plus either MDZ or MDZ + ENBA. Electroencephalographic (EEG) activity, physiologic parameters, and motor function were recorded. Either 2- or 14-days following exposure surviving rats were euthanized and perfused for histology. All animals treated with MDZ + ENBA at both time points had 100% EEG seizure termination and reduced total neuropathology compared to animals treated with MDZ (2-day, p = 0.015 for 15-min, p = 0.002 for 30-min; 14-day, p < 0.001 for 15-min, p = 0.006 for 30-min), showing ENBA enhanced MDZ's anticonvulsant and neuroprotectant efficacy. However, combined MDZ + ENBA treatment, when compared to MDZ treatment groups, had a reduction in the 14-day survival rate regardless of treatment time, indicating possible enhancement of MDZ's neuronal inhibitory effects by ENBA. Based on our findings, ENBA shows promise as an anticonvulsant and neuroprotectant in a combined treatment regimen following soman exposure; when given as an adjunct to standard MCMs, the dose of ENBA needs to be adjusted.

Acute Dissociation and Ketamine's Antidepressant and Anti-Suicidal Ideation Effects in a Midazolam-Controlled Trial.

OBJECTIVE: We sought to explore relationships of acute dissociative effects of intravenous ketamine with change in depression and suicidal ideation and with plasma metabolite levels in a randomized, midazolam-controlled trial. METHODS: Data from a completed trial in suicidal, depressed participants (n = 40) randomly assigned to ketamine was used to examine relationships between ketamine treatment-emergent dissociative and psychotomimetic symptoms with pre/post-infusion changes in suicidal ideation and depression severity. Nonparametric correlational statistics were used. These methods were also used to explore associations between dissociative or psychotomimetic symptoms and blood levels of ketamine and metabolites in a subset of participants (n = 28) who provided blood samples immediately post-infusion. RESULTS: Neither acute dissociative nor psychotomimetic effects of ketamine were associated with changes in suicidal ideation or depressive symptoms from pre- to post-infusion. Norketamine had a trend-level, moderate inverse correlation with dissociative symptoms on Day 1 post-injection (P = .064; P =.013 removing 1 outlier). Dehydronorketamine correlated with Clinician-Administered Dissociative States Scale scores at 40 minutes (P = .034), 230 minutes (P = .014), and Day 1 (P = .012). CONCLUSION: We did not find evidence that ketamine's acute, transient dissociative, or psychotomimetic effects are associated with its antidepressant or anti-suicidal ideation actions. The correlation of higher plasma norketamine with lower dissociative symptoms on Day 1 post-treatment suggests dissociation may be more an effect of the parent drug.

Association of midazolam route of administration and need for recurrent dosing among children with seizures cared for by emergency medical services.

OBJECTIVE: National guidelines in the United States recommend the intramuscular and intranasal routes for midazolam for the management of seizures in the prehospital setting. We evaluated the association of route of midazolam administration with the use of additional benzodiazepine doses for children with seizures cared for by emergency medical services (EMS). METHODS: We conducted a retrospective cohort study from a US multiagency EMS dataset for the years 2018-2022, including children transported to the hospital with a clinician impression of seizures, convulsions, or status epilepticus, and who received an initial correct weight-based dose of midazolam (.2 mg/kg intramuscular, .1 mg/kg intravenous, .2 mg/kg intranasal). We evaluated the association of route of initial midazolam administration with provision of additional benzodiazepine dose in logistic regression models adjusted for age, vital signs, pulse oximetry, level of consciousness, and time spent with the patient. RESULTS: We included 2923 encounters with patients who received an appropriate weight-based dose of midazolam for seizures (46.3% intramuscular, 21.8% intranasal, 31.9% intravenous). The median time to the first dose of midazolam from EMS arrival was similar between children who received intramuscular (7.3 min, interquartile range [IQR] = 4.6-12.5) and intranasal midazolam (7.8 min, IQR = 4.5-13.4) and longer for intravenous midazolam (13.1 min, IQR = 8.2-19.4). At least one additional dose of midazolam was given to 21.4%. In multivariable models, intranasal midazolam was associated with higher odds (odds ratio [OR] = 1.39, 95% confidence interval [CI] = 1.10-1.76) and intravenous midazolam was associated with similar odds (OR = 1.00, 95% CI = .80-1.26) of requiring additional doses of benzodiazepines relative to intramuscular midazolam. SIGNIFICANCE: Intranasal midazolam was associated with greater odds of repeated benzodiazepine dosing relative to initial intramuscular administration, but confounding factors could have affected this finding. Further study of the dosing and/or the prioritization of the intranasal route for pediatric seizures by EMS clinicians is warranted.