Mechanisms of respiratory depression induced by the combination of buprenorphine and diazepam in rats.

BACKGROUND: The safety profile of buprenorphine has encouraged its widespread use. However, fatalities have been attributed to benzodiazepine/buprenorphine combinations, by poorly understood mechanisms of toxicity. Mechanistic hypotheses include (i) benzodiazepine-mediated increase in brain buprenorphine (pharmacokinetic hypothesis); (ii) benzodiazepine-mediated potentiation of buprenorphine interaction with opioid receptors (receptor hypothesis); and (iii) combined effects of buprenorphine and benzodiazepine on respiratory parameters (pharmacodynamic hypothesis). METHODS: We studied the neuro-respiratory effects of buprenorphine (30 mg kg-1, i.p.), diazepam (20 mg kg-1, s.c.), and diazepam/buprenorphine combination in rats using arterial blood gas analysis, plethysmography, and diaphragm electromyography. Pretreatments with various opioid and gamma-aminobutyric acid receptor antagonists were tested. Diazepam impact on brain 11C-buprenorphine kinetics and binding to opioid receptors was studied using positron emission tomography imaging. RESULTS: In contrast to diazepam and buprenorphine alone, diazepam/buprenorphine induced early-onset sedation (P<0.05) and respiratory depression (P<0.001). Diazepam did not alter 11C-buprenorphine brain kinetics or binding to opioid receptors. Diazepam/buprenorphine-induced effects on inspiratory time were additive, driven by buprenorphine (P<0.0001) and were blocked by naloxonazine (P<0.01). Diazepam/buprenorphine-induced effects on expiratory time were non-additive (P<0.001), different from buprenorphine-induced effects (P<0.05) and were blocked by flumazenil (P<0.01). Diazepam/buprenorphine-induced effects on tidal volume were non-additive (P<0.01), different from diazepam- (P<0.05) and buprenorphine-induced effects (P<0.0001) and were blocked by naloxonazine (P<0.05) and flumazenil (P<0.05). Compared with buprenorphine, diazepam/buprenorphine decreased diaphragm contraction amplitude (P<0.01). CONCLUSIONS: Pharmacodynamic parameters and antagonist pretreatments indicate that diazepam/buprenorphine-induced respiratory depression results from a pharmacodynamic interaction between both drugs on ventilatory parameters.

Complex Cytochrome P450 Kinetics Due to Multisubstrate Binding and Sequential Metabolism. Part 2. Modeling of Experimental Data.

Three CYP3A4 substrates, midazolam, ticlopidine, and diazepam, display non-Michaelis-Menten kinetics, form multiple primary metabolites, and are sequentially metabolized to secondary metabolites. We generated saturation curves for these compounds and analyzed the resulting datasets using a number of single-substrate and multisubstrate binding models. These models were parameterized using rate equations and numerical solutions of the ordinary differential equations. Multisubstrate binding models provided results superior to single-substrate models, and simultaneous modeling of multiple metabolites provided better results than fitting the individual datasets independently. Although midazolam datasets could be represented using standard two-substrate models, more complex models that include explicit enzyme-product complexes were needed to model the datasets for ticlopidine and diazepam. In vivo clearance predictions improved markedly with the use of in vitro parameters from the complex models versus the Michaelis-Menten equation. The results highlight the need to use sufficiently complex kinetic schemes instead of the Michaelis-Menten equation to generate accurate kinetic parameters. SIGNIFICANCE STATEMENT: The metabolism of midazolam, ticlopidine, and diazepam by CYP3A4 results in multiple metabolites and sequential metabolism. This study evaluates the use of rate equations and numerical methods to characterize the in vitro enzyme kinetics. Use of complex cytochrome P450 kinetic models is necessary to obtain accurate parameter estimates for predicting in vivo disposition.

High-throughput and trace analysis of diazepam in plasma using DART-MS/MS and its pharmacokinetic application.

A high-throughput quantitative analytical method based on Direct Analysis in Real Time tandem mass spectrometry (DART-MS/MS) has been developed and validated for the determination of diazepam in rat plasma, whereby analyzing of each sample needs merely 25 µL plasma, simple solid phase extraction sample preparation and 15 s acquisition time. The multiple reaction monitoring (MRM) transitions at m/z 285.2 â†’ 193.1 and 316.0 â†’ 270.0 were selected for the monitoring of diazepam and its internal standard clonazepam respectively. A good linearity within the range of 10-2000 ng/mL, an intra- and inter-day precisions within <7.78% as to an accuracy ranging from 1.04% to 7.92% have been achieved. The method has been successfully applied to the pharmacokinetic study of diazepam in rats' plasma after a single intragastric administration at a dose of 10 mg/kg. The results indicate that this method fulfills the requirements of the bioanalysis in sensitivity and accuracy. It shows considerable promise for application of DART-MS to the quantitative investigation of other drugs.

Bioavailability and safety of diazepam intranasal solution compared to oral and rectal diazepam in healthy volunteers.

OBJECTIVE: The study assesses the bioavailability of diazepam after intranasal administration (diazepam nasal spray) in healthy volunteers. Comparative agents were diazepam rectal gel, which served as the regulatory reference product; and oral diazepam, a product with decades of clinical use. Tolerability of diazepam nasal spray was also assessed. METHODS: This was a phase 1, open-label, randomized, single-dose, three-treatment, three-period, six-sequence crossover study in 48 healthy adult subjects that consisted of a screening period, a baseline period, and an open-label treatment period. Interperiod intervals were at least 28 days. RESULTS: Forty-eight healthy volunteer subjects were enrolled, two of whom discontinued before receiving study medication. For all routes of administration, the onset of diazepam absorption was rapid, with measurable concentrations of drug present by the first sample time point. The tmax (time to reach maximum plasma concentration) was similar for diazepam nasal spray and diazepam rectal gel, both of which were slower than oral diazepam in fasted individuals. Variability (as defined by % coefficient of variation of geometric mean) in peak plasma concentration and area under the curve0-∞ was lowest with oral diazepam, followed by diazepam nasal spray, with diazepam rectal gel showing the greatest variability. Overall, 131 treatment-emergent adverse events (TEAEs) were considered mild (42 subjects, 91.3%), four TEAEs were considered moderate (four subjects, 8.3%), and no TEAEs were considered severe. The most commonly reported TEAE was somnolence at 56.5% (26/46) during diazepam nasal spray treatment, 89.1% (41/46) with the rectal diazepam gel treatment, and 82.6% (38/46) with oral diazepam treatment. No nasal irritation was observed for the majority of the subjects at any time point after administration, with no score higher than 2 ("minor bleeding that stops within 1 minute"). SIGNIFICANCE: Diazepam nasal spray shows predicable pharmacokinetics and represents a potential novel therapeutic approach to control bouts of increased seizure activity (cluster seizures, acute repetitive seizures).

Pharmacokinetics and safety of VALTOCO (NRL-1; diazepam nasal spray) in patients with epilepsy during seizure (ictal/peri-ictal) and nonseizure (interictal) conditions: A phase 1, open-label study.

OBJECTIVE: To assess pharmacokinetics and safety of diazepam nasal spray (NRL-1; VALTOCO®) in pediatric and adult patients with epilepsy in seizure and nonseizure states. METHODS: A single dose of diazepam nasal spray (5, 10, 15, or 20 mg based on weight) was administered during each of two conditions (ictal/peri-ictal and interictal condition) to patients 6-65 years old with partial or generalized epilepsy with motor seizures or seizures with clear alteration of awareness; a second dose was permitted if needed for persistent seizures. Dosing could be interictal or ictal/peri-ictal first, with a washout of ≥14 days. Blood samples for pharmacokinetic analysis were taken at prespecified time points. Treatment-emergent adverse events (TEAEs), sedation, nasal irritation, nasal mucosal pain, and olfactory changes were assessed. RESULTS: Of 57 patients in the study (mean age = 28.1 years [range = 6-59], 54.4% female, 80.7% white), 49 were included in the primary pharmacokinetic analyses. Diazepam pharmacokinetic profiles were similar under both conditions, with approximately 2-hour median time to mean (SD) maximum plasma concentrations of 164 (88) and 189 (110) ng/mL for ictal/peri-ictal and interictal conditions, respectively; drug exposure during the first 6 hours postdosing was 532 (313) and 615 (368) h•ng/mL, respectively. Seventeen patients (29.8%) reported TEAEs, of whom eight (14%) had treatment-related TEAEs, with those reported in ≥2 patients being dysgeusia (n = 3, 5.3%) and nasal discomfort (n = 2, 3.5%). One patient had serious TEAEs (recurrent seizures, metabolic encephalopathy), which were deemed unrelated to study treatment. No changes in respiratory rate were observed, nor were there clinically relevant changes in sedation, olfaction, nasal irritation, or acute nasal mucosal pain. SIGNIFICANCE: The epileptic conditions (ictal/peri-ictal, interictal) had minimal impact on diazepam nasal spray pharmacokinetics in patients with epilepsy. Therefore, diazepam nasal spray can be administered ictally and interictally. Diazepam nasal spray safety was consistent with the profile of diazepam.

Intestinal Permeability and Oral Absorption of Selected Drugs Are Reduced in a Mouse Model of Familial Alzheimer's Disease.

Compared with the significant number of studies reporting altered abundance and function of drug transporters at the blood-brain barrier (BBB) in Alzheimer's disease (AD), the impact of AD on the abundance of intestinal drug transporters and the subsequent effects on oral drug absorption have received little attention. We have reported the altered abundance of some small intestinal drug transporters in a familial mouse model of AD; however, whether this leads to altered oral drug absorption is unknown. The current study examined plasma concentrations of caffeine and diazepam (markers for transcellular passive transport), digoxin (P-glycoprotein substrate), and valsartan (multidrug resistance-associated protein 2 substrate) following oral administration to 8-10 month old female wild-type (WT) and APPswe/PSEN1dE9 (APP/PS1) transgenic mice, a commonly used mouse model of familial AD. The plasma exposure of valsartan and digoxin was significantly (p < 0.05) lower in APP/PS1 animals compared with WT mice, whereas the plasma concentrations of the passive transcellular markers caffeine and diazepam did not significantly differ between the two genotypes. To assess whether the reduced oral absorption of valsartan and digoxin was due to decreased intestinal transport, the ex vivo transport of the previously mentioned drugs and mannitol (a marker of paracellular transport) across the jejunum of WT and APP/PS1 mice was assessed over 120 min. In line with the in vivo absorption studies, the permeability of caffeine and diazepam did not significantly differ between WT and APP/PS1 mice. The permeability of 3H-digoxin through the APP/PS1 mouse jejunum was lower than that measured through the WT jejunum; the average amount (relative to dose applied) permeating the tissue over 120 min was 0.22 ± 0.11% (mean ± SD) for the APP/PS1 jejunum and 0.85 ± 0.3% for the WT jejunum. A 1.9-fold reduction in the average amount of valsartan permeating the jejunum of APP/PS1 mice relative to that of WT mice was also detected. Although no apparent morphological alterations were observed in the jejunal tissue of APP/PS1 mice, the permeability of 14C-mannitol across the jejunum from APP/PS1 mice was lower than that across the WT jejunum (Papp= 10.7 ± 3.7 × 10-6 and 6.0 ± 3.4 × 10-6 cm/s, respectively), suggesting tightened paracellular junctions in APP/PS1 mice. These studies are the first to demonstrate, in APP/PS1 mice, reduced intestinal permeability and the absorption of drugs commonly prescribed to people with AD for their comorbidities. If these findings translate to people with AD, then modified dosing regimens may be necessary for selected drugs to ensure that their plasma concentrations remain in the effective range.

Intranasal Coadministration of a Diazepam Prodrug with a Converting Enzyme Results in Rapid Absorption of Diazepam in Rats.

Intranasal administration is an attractive route for systemic delivery of small, lipophilic drugs because they are rapidly absorbed through the nasal mucosa into systemic circulation. However, the low solubility of lipophilic drugs often precludes aqueous nasal spray formulations. A unique approach to circumvent solubility issues involves coadministration of a hydrophilic prodrug with an exogenous converting enzyme. This strategy not only addresses poor solubility but also leads to an increase in the chemical activity gradient driving drug absorption. Herein, we report plasma and brain concentrations in rats following coadministration of a hydrophilic diazepam prodrug, avizafone, with the converting enzyme human aminopeptidase B Single doses of avizafone equivalent to diazepam at 0.500, 1.00, and 1.50 mg/kg were administered intranasally, resulting in 77.8% ± 6.0%, 112% ± 10%, and 114% ± 7% bioavailability; maximum plasma concentrations 71.5 ± 9.3, 388 ± 31, and 355 ± 187 ng/ml; and times to peak plasma concentration 5, 8, and 5 minutes for each dose level, respectively. Both diazepam and a transient intermediate were absorbed. Enzyme kinetics incorporated into a physiologically based pharmacokinetic model enabled estimation of the first-order absorption rate constants: 0.0689 ± 0.0080 minutes-1 for diazepam and 0.122 ± 0.022 minutes-1 for the intermediate. Our results demonstrate that diazepam, which is practically insoluble, can be delivered intranasally with rapid and complete absorption by coadministering avizafone with aminopeptidase B. Furthermore, even faster rates of absorption might be attained simply by increasing the enzyme concentration, potentially supplanting intravenous diazepam or lorazepam or intramuscular midazolam in the treatment of seizure emergencies.

Vaginal Diazepam for Nonrelaxing Pelvic Floor Dysfunction: The Pharmacokinetic Profile.

BACKGROUND: Vaginal diazepam is frequently used to treat pelvic floor tension myalgia and pelvic pain despite limited knowledge of systemic absorption. AIM: To determine the pharmacokinetic and adverse event profile of diazepam vaginal suppositories. METHODS: We used a prospective pharmacokinetic design with repeated assessments of diazepam levels. Eight healthy volunteers were administered a 10-mg compounded vaginal diazepam suppository in the outpatient gynecologic clinic. Serum samples were collected at 0, 45, 90, 120, and 180 minutes; 8, 24, and 72 hours; and 1 week following administration of a 10-mg vaginal suppository. The occurrence of adverse events was assessed using the alternate step and tandem walk tests, the Brief Confusion Assessment Method, and numerical ratings. Plasma concentrations of diazepam and active long-acting metabolites were measured. Pharmacokinetic parameters were calculated by standard noncompartmental methods. RESULTS: The mean peak diazepam concentration (Cmax) of 31.0 ng/mL was detected at a mean time (Tmax) of 3.1 hours after suppository placement. The bioavailability was found to be 70.5%, and the mean terminal elimination half-life was 82 hours. The plasma levels of temazepam and nordiazepam peaked at 0.8 ng/mL at 29 hours and 6.4 ng/mL at 132 hours, respectively. Fatigue was reported by 3 of 8 participants. CLINICAL IMPLICATIONS: Serum plasma concentrations of vaginally administered diazepam are low; however the half-life is prolonged. STRENGTHS & LIMITATIONS: Strengths include use of inclusion and exclusion criteria aimed at mitigating clinical factors that could adversely impact diazepam absorption and metabolism, and the use of an ultrasensitive LC-MS/MS assay. Limitations included the lack of addressing the efficacy of vaginal diazepam in lieu of performing a pure pharmacokinetic study with healthy participants. CONCLUSION: Vaginal administration of diazepam results in lower peak serum plasma concentration, longer time to peak concentration, and lower bioavailability than standard oral use. Providers should be aware that with diazepam's long half-life, accumulating levels would occur with chronic daily doses, and steady-state levels would not be reached for up to 1 week. This profile would favor intermittent use to allow participation in physical therapy and intimacy. Larish AM, Dickson RR, Kudgus RA, et al. Vaginal Diazepam for Nonrelaxing Pelvic Floor Dysfunction: The Pharmacokinetic Profile. J Sex Med 2019;16;763-766.

Population pharmacokinetic reanalysis of a Diazepam PBPK model: a comparison of Stan and GNU MCSim.

The aim of this study is to benchmark two Bayesian software tools, namely Stan and GNU MCSim, that use different Markov chain Monte Carlo (MCMC) methods for the estimation of physiologically based pharmacokinetic (PBPK) model parameters. The software tools were applied and compared on the problem of updating the parameters of a Diazepam PBPK model, using time-concentration human data. Both tools produced very good fits at the individual and population levels, despite the fact that GNU MCSim is not able to consider multivariate distributions. Stan outperformed GNU MCSim in sampling efficiency, due to its almost uncorrelated sampling. However, GNU MCSim exhibited much faster convergence and performed better in terms of effective samples produced per unit of time.

Brain targeting of chitosan-based diazepam mucoadhesive microemulsions via nasal route: formulation optimization, characterization, pharmacokinetic and pharmacodynamic evaluation.

OBJECTIVE: The aim of present investigation was to develop microemulsions (MEs) and mucoadhesive microemulsions (MME) of diazepam for brain uptake through nasal administration for the treatment of seizure emergency. SIGNIFICANCE: Status epilepticus (SE) is a medical emergency, requires intravenous administration of diazepam which requires hospitalization of patient. Initiation of therapy at home via nasal administration of diazepam could prevent the damage of brain due to delay of therapy initiation. METHODS: Diazepam MEs were prepared by phase titration method, optimized by using Box-Behnken design. The influence of independent variables oleic acid, surfactant mixture (tween 80:propylene glycol), and water on dependent variables size, flux, and zeta potential was investigated. Optimized MEs, MMEs, and Calmpose (i.v route) were evaluated for pharmacokinetic and pharmacodynamic studies on rats. RESULTS: MME2 composed of oleic acid (5), surfactant mixture (50), water (45), and chitosan (0.5) showed size of 96.45 nm, PDI 0.21 and zeta potential 13.52 mV. MME2 showed significantly high flux of 846.96 ± 34 µg/cm2/h and AUCbrain 1206.49 ± 145.8. The drug targeting efficiency (314%) and direct nose-to-brain transport (68.1%) of MME2 were significantly high compared to Calmpose (i.v) and ME. The latency periods of minimal clonal seizures and generalized tonic-clonic seizures of MME2 was significantly increased (p < 0.0001) compared to drug solution and Calmpose (i.v). CONCLUSION: The brain uptake of diazepam from chitosan-based MMEs via nasal route is significantly high compared to i.v route.

Determination of minimal steady-state plasma level of diazepam causing seizure threshold elevation in rats.

OBJECTIVE: Diazepam, administered by the intravenous, oral, or rectal routes, is widely used for the management of acute seizures. Dosage forms for delivery of diazepam by other routes of administration, including intranasal, intramuscular, and transbuccal, are under investigation. In predicting what dosages are necessary to terminate seizures, the minimal exposure required to confer seizure protection must be known. Here we administered diazepam by continuous intravenous infusion to obtain near-steady-state levels, which allowed an assessment of the minimal levels that elevate seizure threshold. METHODS: The thresholds for various behavioral seizure signs (myoclonic jerk, clonus, and tonus) were determined with the timed intravenous pentylenetetrazol seizure threshold test in rats. Diazepam was administered to freely moving animals by continuous intravenous infusion via an indwelling jugular vein cannula. Blood samples for assay of plasma levels of diazepam and metabolites were recovered via an indwelling cannula in the contralateral jugular vein. RESULTS: The pharmacokinetic parameters of diazepam following a single 80-µg/kg intravenous bolus injection were determined using a noncompartmental pharmacokinetic approach. The derived parameters Vd , CL, t1/2α (distribution half-life) and t1/2ß (terminal half-life) for diazepam were, respectively, 608 mL, 22.1 mL/min, 13.7 minutes, and 76.8 minutes, respectively. Various doses of diazepam were continuously infused without or with an initial loading dose. At the end of the infusions, the thresholds for various behavioral seizure signs were determined. The minimal plasma diazepam concentration associated with threshold elevations was estimated at approximately 70 ng/mL. The active metabolites nordiazepam, oxazepam, and temazepam achieved levels that are expected to make only minor contributions to the threshold elevations. SIGNIFICANCE: Diazepam elevates seizure threshold at steady-state plasma concentrations lower than previously recognized. The minimally effective plasma concentration provides a reference that may be considered when estimating the diazepam exposure required for acute seizure treatment.

Evaluation of IV to Enteral Benzodiazepine Conversion Calculations in a Pediatric Intensive Care Setting.

OBJECTIVES: To evaluate if institutionally established calculations for transitioning continuous IV midazolam to enteral benzodiazepines maintain Withdrawal Assessment Tool-Version 1 scores equal to or less than preconversion values. DESIGN: Retrospective cohort study evaluating the effectiveness and safety of benzodiazepine conversion calculations embedded within an institution-specific clinical pathway for sedation and weaning of mechanically ventilated pediatric patients. SETTING: A 55-bed, mixed-medical, noncardiac surgical PICU in a tertiary care children's hospital. PATIENTS: All patients age 6 months to 18 years who received continuous midazolam for 5 days or longer while mechanically ventilated for 5-21 days and were then converted to either enteral diazepam or lorazepam following extubation (or return to baseline ventilator settings in tracheostomy-dependent patients) between January 1, 2015, and June 30, 2016. INTERVENTIONS: Benzodiazepine conversion calculations were applied according to institutional clinical pathway guidance. MEASUREMENTS AND MAIN RESULTS: Withdrawal Assessment Tool-Version 1 scores were compared pre and post benzodiazepine conversion. Patient demographics, benzodiazepine dose escalations, as needed benzodiazepine requirements, and severe adverse events within 48 hours of conversion were assessed. Seventy-one patient encounters were analyzed (median age, 2.5 yr; interquartile range, 1.2-5.3). The median Withdrawal Assessment Tool-Version 1 scores pre conversion and post conversion were not significantly different (1 [interquartile range, 0.75-2] and 1 [interquartile range, 0.25-2], respectively, p = 0.1). As needed benzodiazepine doses were administered in 38% of encounters post conversion, but escalation of a scheduled enteral benzodiazepine regimen was only required in 2.8% of encounters. Post conversion, one patient (1.4%) had increased seizure activity, and four patients (5.6%) required fluid boluses secondary to tachycardia or dehydration, but not hypotension. CONCLUSIONS: These findings suggest that standardized benzodiazepine conversions successfully achieved consistent Withdrawal Assessment Tool-Version 1 scores compared with preconversion values. Severe adverse events associated with oversedation and/or withdrawal were minimal and confounded by underlying disease states.

A phase I dose-escalation study of apatorsen (OGX-427), an antisense inhibitor targeting heat shock protein 27 (Hsp27), in patients with castration-resistant prostate cancer and other advanced cancers.

BACKGROUND: Heat shock protein 27 (Hsp27) is a chaperone protein that regulates cell survival via androgen receptor and other signaling pathways, thereby mediating cancer progression. Apatorsen (OGX-427) is a 2'-methoxyethyl-modified antisense oligonucleotide that inhibits Hsp27 expression. This study evaluated the safety profile and recommended phase II dosing of apatorsen in patients with advanced cancer. PATIENTS AND METHODS: Patients with castration-resistant prostate (CRPC), breast, ovary, lung, or bladder cancer were enrolled to this phase I dose-escalation study. Apatorsen was administered i.v. weekly in 21-day cycles following 3 loading doses and over 5 dose levels (200-1000 mg). Apatorsen plasma concentrations, circulating tumor cells (CTCs) and CTC Hsp27 expression, and serum Hsp27 levels were evaluated. RESULTS: Forty-two patients were accrued, of which 52% had CRPC. Patients were heavily pretreated, with 57% having had ≥3 prior chemotherapy regimens. During the loading dose/cycle 1 and overall study period, 93% and 100% of patients (N = 42) experienced treatment-related adverse events, respectively; most were grade 1-2 and included chills, pruritus, flushing, prolonged aPTT, lymphopenia, and anemia. One patient experienced a dose-limiting toxicity at the 600 mg dose level (intracranial hemorrhage in a previously undiagnosed brain metastasis). A maximum tolerated dose was not defined. Apatorsen Cmax increased proportionally with dose. Decreases in tumor markers and declines in CTCs were observed, with a prostate-specific antigen decline >%50% occurring in 10% of patients with CRPC; 29/39 assessable patients (74%) had reductions from ≥5 CTC/7.5 ml at baseline to <5 CTC/7.5 ml post-treatment. Twelve patients had stable measurable disease as best response. CONCLUSIONS: Apatorsen was tolerated at the highest dose evaluated (1000 mg). Single-agent activity was suggested by changes in tumor markers, CTC, and stable measurable disease. Phase II studies evaluating apatorsen are underway. CLINICALTRIALSGOV ID: NCT00487786.

Ontogeny of plasma proteins, albumin and binding of diazepam, cyclosporine, and deltamethrin.

BACKGROUND: To characterize the ontogeny of plasma albumin and total proteins, due to the lack of a comprehensive pediatric database. Secondly, to establish the magnitude and duration of maturational changes in binding of highly-bound drugs/chemicals. METHODS: Anonymized plasma samples from 296 donors were pooled in 6 age brackets from birth to adolescence. Total protein and albumin levels were measured in each age group, as was the age-dependency of plasma binding of diazepam (DZP), cyclosporine (CYC), and deltamethrin (DLM), a pyrethroid insecticide. RESULTS: Plasma levels of albumin and total proteins steadily increased for the first 1-3 y of life. Unbound DZP and CYC fractions were elevated three- to fourfold in neonates, but decreased to adult levels after 1 and 3 y, respectively. Unbound DLM levels exceeded those in adults for just 1 mo. CONCLUSION: Neonates and infants under 1-3 y may be at risk from increased amounts of free drug, when given standard doses of some highly-bound drugs. Pyrethroid insecticides might be anticipated to pose increased risk for 1 mo.

Characterization of equine cytochrome P450: role of CYP3A in the metabolism of diazepam.

Research on drug metabolism and pharmacokinetics in large animal species including the horse is scarce because of the challenges in conducting in vivo studies. The metabolic reactions catalyzed by cytochrome P450s (CYPs) are central to drug pharmacokinetics. This study elucidated the characteristics of equine CYPs using diazepam (DZP) as a model compound as this drug is widely used as an anesthetic and sedative in horses, and is principally metabolized by CYPs. Diazepam metabolic activities were measured in vitro using horse and rat liver microsomes to clarify the species differences in enzyme kinetic parameters of each metabolite (temazepam [TMZ], nordiazepam [NDZ], p-hydroxydiazepam [p-OH-DZP], and oxazepam [OXZ]). In both species microsomes, TMZ was the major metabolite, but the formation rate of p-OH-DZP was significantly less in the horse. Inhibition assays with a CYP-specific inhibitors and antibody suggested that CYP3A was the main enzyme responsible for DZP metabolism in horse. Four recombinant equine CYP3A isoforms expressed in Cos-7 cells showed that CYP3A96, CYP3A94, and CYP3A89 were important for TMZ formation, whereas CYP3A97 exhibited more limited activity. Phylogenetic analysis suggested diversification of CYP3As in each mammalian order. Further study is needed to elucidate functional characteristics of each equine CYP3A isoform for effective use of diazepam in horses.

Synthesis, biological activities and pharmacokinetic properties of new fluorinated derivatives of selective PDE4D inhibitors.

A new series of selective PDE4D inhibitors has been designed and synthesized by replacing 3-methoxy group with 3-difluoromethoxy isoster moiety in our previously reported cathecolic structures. All compounds showed a good PDE4D3 inhibitory activity, most of them being inactive toward other PDE4 isoforms (PDE4A4, PDE4B2 and PDE4C2). Compound 3b, chosen among the synthesized compounds as the most promising in terms of inhibitory activity, selectivity and safety, showed an improved pharmacokinetic profile compared to its non fluorinated analogue. Spontaneous locomotor activity, assessed in an open field apparatus, showed that, differently from rolipram and diazepam, selective PDE4D inhibitors, such as compounds 3b, 5b and 7b, did not affect locomotion, whereas compound 1b showed a tendency to reduce the distance traveled and to prolong the immobility period, possibly due to a poor selectivity.

Simultaneous Quantification of Diazepam and Dexamethasone in Plasma by High-Performance Liquid Chromatography with Tandem Mass Spectrometry and Its Application to a Pharmacokinetic Comparison between Normoxic and Hypoxic Rats.

In order to investigate the pharmacokinetics of a combination of diazepam and dexamethasone under hypoxic conditions, a novel, sensitive and specific liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of diazepam and dexamethasone in rat plasma was developed and validated. The chromatographic separation of analytes was successfully achieved on an XTerra® MS C18 column using a gradient elution of methanol and water containing 0.1% formic acid at a flow rate of 0.5 mL/min. This method demonstrated good linearity and no endogenous material interferences. The linear ranges were 1.0-100 ng/mL for diazepam and 2.0-200 ng/mL for dexamethasone. The intra- and inter-day precision for the two compounds in plasma were lower than 10.0%, and the accuracy was between -7.9% and 11.5%. Our method was then successfully applied in a pharmacokinetic comparison between normoxic and hypoxic rats. The results indicated that there were significant differences in the main pharmacokinetics parameters of diazepam and dexamethasone between normoxic and hypoxic rats. The results provide the important and valuable information for discovering and developing novel anti-hypoxia drug combinations, as well as a better understanding of the safety and efficacy of these drugs.

Dosing feasibility and tolerability of intranasal diazepam in adults with epilepsy.

OBJECTIVE: To determine the feasibility of administering a diazepam nasal spray formulation (diazepam-NS) to adults with epilepsy during a generalized tonic-clonic seizure or in the postictal period following a tonic-clonic or other seizure type, to assess pharmacokinetics and to assess tolerability. METHODS: An open-label study was conducted in patients admitted to the epilepsy monitoring unit. Eligible patients received a single dose of diazepam-NS approximating 0.2 mg/kg. Plasma diazepam concentrations were measured serially up to 12 h postdose, and maximum observed plasma concentration (Cmax ); time to maximum concentration (Tmax ); and the area under the plasma concentration-time curve for time zero to last sampling time (AUC0-12 ) were estimated and dose-normalized. Pharmacodynamic assessments included Kaplan-Meier analysis to determine the time-to-next seizure. Safety and tolerability were assessed. RESULTS: Of the 78 patients who consented, 30 had treatment and pharmacokinetic data. Ten patients were treated during a convulsive tonic-clonic seizure, seven within 5 min following the last clonic jerk, and 13 in the postictal period ≥ 5 min after a tonic-clonic or following other seizure-types. Diazepam median Tmax was 45 min. Dose-normalized mean Cmax and AUC0-12 values of diazepam were comparable among patients regardless of the timing of diazepam-NS administration in relation to seizure. Of those treated, 65% were seizure-free during the 12-h observation period and 35% had post-dose seizures. Treatment was well tolerated, with no unexpected safety findings: 74% had mild and 25% had moderate adverse events. Nasopharyngeal signs were resolved by 12 h postdose. SIGNIFICANCE: Diazepam can be delivered in effective therapeutic concentrations by a nasal spray device during the convulsive phase of tonic-clonic seizures or in the postictal periods following tonic-clonic or other seizure types.

Age-related pharmacokinetic changes of acetaminophen, antipyrine, diazepam, diphenhydramine, and ofloxacin in male cynomolgus monkeys and beagle dogs.

1. The pharmacokinetics of acetaminophen (marker of gastric emptying), antipyrine (marker of hepatic metabolic activity and total body water), diazepam (lipophilic and highly distributed), diphenhydramine (hepatic blood flow-limited and alpha-1 acid glycoprotein bound) and ofloxacin (renally eliminated) were evaluated in cynomolgus monkeys (3-18 years old) and beagle dogs (2-11 years old) as models in elderly persons. 2. Gastric pH fluctuated with aging in monkeys and dogs. The concentration of alpha-1 acid glycoprotein appeared to be increased by aging. There were no age-related differences in the absorption rates of the drugs under the conditions used in the study. Total body fat increased and water decreased in monkeys, but these parameters did not change in dogs. 3. Hepatic blood flow decreased in both species, but a significant decrease of hepatic clearance was only seen in monkeys. Renal clearance decreased significantly with age in monkeys and showed a tendency to decrease in dogs. 4. Age-related alterations of physiological parameters in monkeys are in agreement with clinical observations in humans, except for the lack of a change in the plasma albumin concentration. Therefore, this study suggests that monkey might be a suitable animal model for prediction of age-related changes in pharmacokinetics in humans.