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Propofol, one of the most commonly used intravenous general anesthetics, modulates neuronal function by interacting with ion channels. The mechanisms that link propofol binding to the modulation of distinct ion channel states, however, are not understood. To tackle this problem, we investigated prokaryotic ancestors of eukaryotic voltage-gated Na+ channels (Navs) using unbiased photoaffinity labeling with a photoacitivatable propofol analog (AziPm), electrophysiological methods and mutagenesis. The results directly demonstrate conserved propofol binding sites involving the S4 voltage sensors and the S4-S5 linkers in NaChBac and NavMs, and also suggest state-dependent changes at these sites. Then, using molecular dynamics simulations to elucidate the structural basis of propofol modulation, we show that the S4 voltage sensors and the S4-S5 linkers shape two distinct propofol binding sites in a conformation-dependent manner. These interactions help explain how propofol binding promotes activation-coupled inactivation to inhibit Nav channel function.

Despite extensive use in clinical practice, the mechanisms of propofol action on sodium channels are not fully understood. Yang et al. incorporate complementary biophysical approaches (electrophysiology and molecular dynamics simulations) to demonstrate that propofol inhibits two prokaryotic voltage-gated sodium channels, NaChBac and NavMs, by modulating both activation and inactivation gating., Propofol is widely used in the clinic for the induction and maintenance of general anesthesia. As with most general anesthetics, however, our understanding of its mechanism of action remains incomplete. Local and general anesthetics largely inhibit voltage-gated Na+ channels (Navs) by inducing an apparent stabilization of the inactivated state, associated in some instances with pore block. To determine the biophysical and molecular basis of propofol action in Navs, we investigated NaChBac and NavMs, two prokaryotic Navs with distinct voltage dependencies and gating kinetics, by whole-cell patch clamp electrophysiology in the absence and presence of propofol at clinically relevant concentrations (2–10 µM). In both Navs, propofol induced a hyperpolarizing shift of the pre-pulse inactivation curve without any significant effects on recovery from inactivation at strongly hyperpolarized voltages, demonstrating that propofol does not stabilize the inactivated state. Moreover, there was no evidence of fast or slow pore block by propofol in a non-inactivating NaChBac mutant (T220A). Propofol also induced hyperpolarizing shifts of the conductance-voltage relationships with negligible effects on the time constants of deactivation at hyperpolarized voltages, indicating that propofol does not stabilize the open state. Instead, propofol decreases the time constants of macroscopic activation and inactivation. Adopting a kinetic scheme of Nav gating that assumes preferential closed-state recovery from inactivation, a 1.7-fold acceleration of the rate constant of activation and a 1.4-fold acceleration of the rate constant of inactivation were sufficient to reproduce experimental observations with computer simulations. In addition, molecular dynamics simulations and molecular docking suggest that propofol binding involves interactions with gating machinery in the S4–S5 linker and external pore regions. Our findings show that propofol is primarily a positive gating modulator of prokaryotic Navs, which ultimately inhibits the channels by promoting activation-coupled inactivation.

General anesthetics inhibit voltage-gated sodium channels by unknown molecular mechanisms. Using computation-guided NMR and electrophysiology analyses, Wang et al. show that propofol binds to the prokaryotic sodium channel NaChBac at multiple distinct sites., Voltage-gated sodium (NaV) channels are important targets of general anesthetics, including the intravenous anesthetic propofol. Electrophysiology studies on the prokaryotic NaV channel NaChBac have demonstrated that propofol promotes channel activation and accelerates activation-coupled inactivation, but the molecular mechanisms of these effects are unclear. Here, guided by computational docking and molecular dynamics simulations, we predict several propofol-binding sites in NaChBac. We then strategically place small fluorinated probes at these putative binding sites and experimentally quantify the interaction strengths with a fluorinated propofol analogue, 4-fluoropropofol. In vitro and in vivo measurements show that 4-fluoropropofol and propofol have similar effects on NaChBac function and nearly identical anesthetizing effects on tadpole mobility. Using quantitative analysis by 19F-NMR saturation transfer difference spectroscopy, we reveal strong intermolecular cross-relaxation rate constants between 4-fluoropropofol and four different regions of NaChBac, including the activation gate and selectivity filter in the pore, the voltage sensing domain, and the S4–S5 linker. Unlike volatile anesthetics, 4-fluoropropofol does not bind to the extracellular interface of the pore domain. Collectively, our results show that propofol inhibits NaChBac at multiple sites, likely with distinct modes of action. This study provides a molecular basis for understanding the net inhibitory action of propofol on NaV channels.

Voltage-gated ion channels (VGICs) of excitable tissues are emerging as targets likely involved in both the therapeutic and toxic effects of inhaled and intravenous general anesthetics. Whereas sevoflurane and propofol inhibit voltage-gated Na+ channels (Navs), sevoflurane potentiates certain voltage-gated K+ channels (Kvs). The combination of these effects would dampen neural excitability and, therefore, might contribute to the clinical endpoints of general anesthesia. As the body of work regarding the interaction of general anesthetics with VGICs continues to grow, a multidisciplinary approach involving functional, biochemical, structural, and computational techniques, many of which are detailed in other chapters, has increasingly become necessary to solve the molecular mechanism of general anesthetic action on VGICs. Here, we focus on electrophysiological and modeling approaches and methodologies to describe how our work has elucidated the biophysical basis of the inhibition Navs by propofol and the potentiation of Kvs by sevoflurane.

Voltage-gated sodium channels (Navs), critical for action potential generation and propagation in the central nervous system, are inhibited by clinically relevant concentrations of many general anesthetics. The molecular mechanism for this inhibition, however, is still largely unclear. Here, we investigated the electrophysiological response of the bacterial voltage-gated sodium channel NaChBac to the widely used injectable anesthetic propofol at concentrations near the clinically relevant EC50. At 2 μM, propofol induces a −7.0 ± 1.1 mV leftward shift of the steady-state inactivation curve (V½ control = −49 ± 2.2 mV, V½ 2μM = −56 ± 2.3 mV, n=3) and a ∼24% decrease in the time constant of inactivation at −10 mV (τ control = 188 ± 26 ms, τ 2μM = 142 ± 11 ms, n=3). 5 μM propofol induces a −7.0 ± 1.3 mV leftward shift of the steady-state inactivation curve (V½ control = −55 ± 0.6 mV, V½ 5μM = −62 ± 1.2 mV, n=8) and a ∼37% decrease in the time constant of inactivation at −10 mV (τ control = 187 ± 8.7ms, τ 5μM = 117 ± 5.5 ms, n=8). Consistent with behavior in response to isoflurane (Ouyang et al., 2007) and sevoflurane (Barber et al., 2014), propofol inhibits channel function by promoting and stabilizing the inactivated state. Structurally diverse general anesthetics might inhibit NaChBac by acting on slow inactivation, a mechanism also present in mammalian Navs that possibly involves the pore domain. Therefore, to assess the role of slow inactivation in the mechanism of general anesthetic action, we are evaluating sites in the S6 helix that alter inactivation kinetics (G219, T220, and F227). Furthermore, aided by molecular dynamics simulations, we are validating putative general anesthetic binding sites in the pore domain of NaChBac.

Compare treatment persistence, healthcare utilisation and costs for patients treated with escitalopram versus other SSRI/SNRIs in a real-world setting.Patients with a diagnosis for major depressive disorder (MDD) and at least one prescription for an SSRI or SNRI were identified from the Ingenix Impact Database (2002-2005). The baseline and study observation periods were defined as the 6 months before and after the index date (first date for an SSRI /SNRI pharmacy claim). Comparisons were made between patients initiated on escitalopram versus other SSRI/SNRIs using descriptive statistics and multivariate regressions.Escitalopram patients (n=10,465) had better treatment persistence compared to patients initiated on other SSRI/SNRIs (n=28,310): the hazard ratio of all discontinuation was 0.96 (95% confidence interval [CI]=0.94-0.99) for the escitalopram therapy (p=0.003), and the hazard ratio of switching to another second-generation antidepressant was 0.91 (95% CI=0.87-0.94) for the escitalopram therapy (p0.001). Escitalopram patients also had fewer inpatient service and emergency room visits. Adjusted average total all-cause healthcare costs and inpatient services costs were $839 and $405 lower in the escitalopram group (both p0.05).Escitalopram may be associated with lower healthcare utilisation and costs among adult MDD patients compared to other SSRI/SNRIs.

To assess treatment persistence, hospitalization outcomes and mean healthcare costs of geriatric major depressive disorder (MDD) patients treated with escitalopram compared to other selective serotonin reuptake inhibitors (SSRIs) and serotonin and norepinephrine reuptake inhibitors (SNRIs).Patients agedor = 65 years with at least one inpatient claim or two independent claims associated with MDD diagnosis were identified in the IHCIS National Managed Care Database (2003-2005). Patients were continuously enrolled for at leastor = 12 months, filled at least one prescription for an SSRI/SNRI and did not use any second-generation antidepressant during the 6 months pre-index date. Unadjusted and multivariate analyses adjusting for baseline characteristics were conducted.Treatment persistence, hospitalization utilization, and average prescription drug, medical, and total healthcare costs were compared between patients initiated on escitalopram versus other SSRI/SNRIs.Escitalopram-treated patients (N = 459) were less likely to discontinue treatment (HR = 0.85, p = 0.012) or switch to another second-generation antidepressant (HR = 0.76, p = 0.006) compared to patients treated with other SSRI/SNRIs (N = 1517). Escitalopram-treated patients had 39% fewer hospitalization days (p = 0.004). Both groups had similar mean prescription drug costs ($1659 vs. $1630, p = 0.687). After controlling for baseline characteristics, escitalopram-treated patients had lower mean total medical service costs ($9425 vs. $12,703, p0.001) and mean total healthcare costs ($11,043 vs. $14,163, p0.001).This study's limitations include its small sample size, short observational periods and exclusivity of indirect costs.Geriatric patients treated with escitalopram had higher treatment persistence, fewer hospitalization days and lower total healthcare costs than patients on other SSRI/SNRIs after controlling for baseline characteristics. Most of the cost savings were due to reductions in hospitalizations.

To compare escitalopram versus citalopram for the treatment of major depressive disorder (MDD) in geriatric patients.Administrative claims data (2003-2005) were analyzed for patients agedor =65 years with at least one inpatient claim or two independent medical claims associated with MDD diagnosis. Patients were continuously enrolled for at least 12 months, filled at least one prescription for citalopram or escitalopram and had no second generation antidepressant use during the 6-month pre-index date. Contingency table analysis and survival analysis were used to compare outcomes between the two treatment groups.Treatment persistence, hospitalization utilization, and prescription drug, medical, and total healthcare costs were analyzed. Outcomes were compared between patients initiated on escitalopram and those initiated on citalopram both descriptively and using multivariate analysis adjusting for baseline characteristics.Among 691 geriatric patients, escitalopram-treated patients (n=459) were less likely to discontinue treatment (hazard ratio [HR]=0.83, p=0.049) or switch to another second generation antidepressant (HR=0.62, p=0.001) compared to patients treated with citalopram (n=232). Patients treated with escitalopram had a significantly lower hospitalization rate (31.2% vs. 38.8%, p=0.045) and 66% fewer hospitalization days based on negative binomial regression (p0.001). While escitalopram patients had comparable prescription drug costs, they had lower total medical service costs (regression: $9748 vs. $19,208, p0.001) and lower total healthcare costs (regression: $11,434 vs. $20,601, p0.001).This study's limitations include its small sample size, short observational periods and exclusivity of indirect costs.Geriatric patients treated with escitalopram had better treatment persistence, fewer hospitalizations, and lower medical and total healthcare costs than patients treated with citalopram. Most of the cost reduction was attributable to significantly lower hospitalizations and total medical costs.

To describe dosing patterns for tumor necrosis factor (TNF) antagonists in patients with rheumatoid arthritis from health care provider and payer point of interest.Using privately insured US claims data from 31 large employers covering 31 companies across the US, rheumatoid arthritis (RA) patients were identified and three cohorts were defined based on first TNF-antagonist treatment (adalimumab, etanercept, or infliximab) administered after January 1, 2003. Dosage-adjustment patterns were assessed during the following 12-month period. Changes in dosage (both increases and decreases) and maintenance of a stable dosage were evaluated. For the health care provider point of interest, a new algorithm was developed to assess treatment patterns with chronic injectable therapies that incorporated the potential inconsistency between days of supply and prescription-gap data, thus providing the actual use of TNF-antagonist treatment. For the payer, usage data addressed whether the TNF antagonist was used at a greater dosage than recommended. Differences in baseline characteristics and dosage change rates between cohorts were tested using Chi-Square tests for categorical variables and Wilcoxon tests for continuous variables.From the health care provider point of interest, 83.4% of adalimumab-treated patients (n = 205) initially received the recommended dosage, 10.2% received less, and 6.3% received more; 87.7% of etanercept-treated patients (n = 455) initially received the recommended dosage, 11.2% received less, and 1.1% received more; and 83.8% of infliximab-treated patients (n = 148) started with 2-4 vials (the recommended dosage is based on the weight of the patient, not total milligrams). All treatments had similar dosage decrease and discontinuation rates. Maintenance of stable dosage was lower for infliximab (20.9%) than adalimumab (37.1%) and etanercept (39.1%); both p0.01. The infliximab dosage-increase rate (35.1%) was greater than adalimumab (3.9%) and etanercept (0); both p0.01. From the payer point of interest, dosage-increase rate was greater for infliximab (28.3%) than adalimumab (8.7%) and etanercept (6.9%), both p0.01.Infliximab had greater dosage-increase rates than adalimumab and etanercept. Adalimumab and etanercept had similar dosage-increase rates. All treatments had similar dosage-decrease and discontinuation rates. Maintenance of stable dosage was lower for infliximab than for adalimumab and etanercept. The study has the usual limitation of claims data analysis in that clinical details might be insufficient to draw causal inference.

To compare the cost of care for rheumatoid arthritis (RA) patients treated with adalimumab, infliximab, and etanercept.RA patients were identified from a privately insured database. Three mutually exclusive treatment cohorts were formed based on the date of first tumor necrosis factor (TNF) antagonist treatment (index date) after January 1, 2003. Baseline characteristics were assessed in the 3-month pretreatment period. Healthcare (i.e., medical service and prescription medications) utilization and cost were assessed for the following 12 months. RA-related medical cost included the total cost for medical service associated with RA diagnosis. RA-related healthcare cost included RA-related medical and drug cost. Uneven distribution of baseline characteristics were adjusted with the propensity score method. Cost was compared between treatment cohorts.Twelve-month TNF-antagonist therapy cost ($12 853 vs. 17 299, p = 0.002), total RA-related drug cost ($13 794 vs. 17 647, p = 0.006), total RA-related medical cost ($971 vs. 2920, p0.001), total RA-related healthcare cost ($14 764 vs. 20 566, p = 0.002), and total drug cost ($16 210 vs. 19 769, p = 0.028) were significantly less for adalimumab (n = 217) than infliximab (n = 234). Twelve-month healthcare cost for adalimumab was comparable to etanercept (n = 546).Annual healthcare cost for adalimumab patients was significantly less than for infliximab patients and was comparable to etanercept patients. This analysis is subject to the usual limitation of claims data analyses in that few clinical details are available and causal inference conclusions are limited.

Many therapies exist for treating adult attention-deficit/hyperactivity disorder (ADHD), also referred to as attention-deficit disorder (ADD), but there is no research regarding cost differences associated with initiating alternative ADD/ADHD drug therapies in adults.To compare from the perspective of a large self-insured employer the risk-adjusted direct health care costs associated with 3 alternative drug therapies for ADD in newly treated patients: extended-release methylphenidate (osmotic release oral system-MPH), mixed amphetamine salts extended release (MAS-XR), or atomoxetine.We analyzed data from a US claims database of 5 million beneficiaries from 31 large self-insured employers (1999-2004). Analysis was restricted to adults aged 18 to 64 years with at least 1 diagnosis of ADD/ADHD (International Classification of Diseases, Ninth Revision, Clinical Modification [ICD-9-CM] codes 314.0x--attention deficit disorder; 314.00--attention deficit disorder without hyperactivity; or 314.01--attention-deficit disorder with hyperactivity) and at least 1 pharmacy claim for OROS-MPH, MAS-XR, or atomoxetine identified using National Drug Codes. In preliminary analysis, we calculated the duration of index ADHD drug therapy as time from index therapy initiation to a minimum 60-day gap. Because the median duration of index ADHD drug therapy was found to be approximately 90 days, the primary measures were total direct medical plus drug costs and medical-only costs computed over 6 months following therapy initiation. Adults were required to have continuous eligibility 6 months before and 6 months after their latest drug therapy initiation and no ADHD therapy during the previous 6 months. Cost was measured as the payment amount made by the health plan to the provider rather than billed charges, and it excluded patient copayments and deductibles. Medical costs included costs incurred for all-cause inpatient and outpatient/other services. Costs were adjusted for inflation to 2004 U.S. dollars using the consumer price index for medical care. T tests were used for descriptive cost comparisons. Generalized linear models (GLMs) were used to compare costs of adults receiving alternative therapies, adjusting for demographic characteristics, substance abuse, depression, and the Charlson Comorbidity Index.Of the 4,569 patients who received 1 of these 3 drug therapies for ADHD, 31.8% received OROS-MPH for a median duration of 99 days of therapy, 34.0% received MAS-XR for a median 128 days, and 34.2% received atomoxetine for a median 86 days. In the 6-month follow-up period, the mean (standard deviation) total medical and drug costs were $2,008 ($3,231) for OROS-MPH, $2,169 ($4,828) for MAS-XR, and $2,540 ($4,269) for atomoxetine-treated adults. The GLM for patient characteristics suggested that 6-month, risk-adjusted mean medical costs, excluding drug costs, for adults treated with OROS-MPH were $142 less (10.4%, $1,220 vs. $1,362) compared with MAS-XR (P =0.022) and $132 less (9.8%, $1,220 vs. $1,352) compared with atomoxetine (P =0.033); risk-adjusted mean medical costs were not significantly different between MAS-XR and atomoxetine. The GLM comparison of risk-adjusted total direct costs, including drug cost, was on average $156 less (8.0%, $1,782 vs. $1,938) for OROS-MPH compared with MAS-XR (P = 0.017) and $226 less (11.3%, $1,782 vs. $2,008) compared with atomoxetine (P0.001); the risk-adjusted total direct costs were not significantly different between MAS-XR and atomoxetine. Two high-cost outliers (greater than 99.96th percentile, 1 each for OROS-MPH and atomoxetine) accounted for $47 (30%) of the $156 cost difference between OROS-MPH and MAS-XR and $11 (5%) of the $226 cost difference between OROS-MPH and atomoxetine, and the medical diagnoses for the highest-cost claims for these 2 outlier patients were unrelated to ADHD.After adjusting for patient characteristics including substance abuse, depression, and the Charlson Comorbidity Index, adults treated with OROS-MPH had, on average, slightly lower medical and total medical and drug costs than those treated with MAS-XR or atomoxetine over the 6-month period after drug therapy initiation. Approximately 30% of the cost difference compared with MAS-XR was attributable to 1 high-cost outlier with medical diagnoses for the highest-cost claim that were unrelated to ADHD.

Forty patients (A.S.A. class I or II), 18 to 75 years of age, who were undergoing elective surgery were studied to determine the clinical and subclinical neuromuscular blocking effects of two antibiotics, tobramycin and gentamicin and to compare these effects with those produced by cefazolin, an aminoglycoside not known to produce paralysis. Patients were prospectively and randomly assigned in approximately equal numbers to one of four groups: group A received 1 mg/kg of tobramycin; group B, 1 mg/kg of gentamicin; group C, 500 mg of cefazolin; or group D, control (no antibiotic). Antibiotics were administered intravenously 45 minutes immediately preceding the study. The ulnar nerve was stimulated supramaximally and neuromuscular function was measured electromyographically. Anesthesia was induced with thiopental, 4 mg/kg IV, and maintained with endotracheal enflurane 1.0% to 1.5% (inspired) and N2O-O2 (2 L:1 L) after intubation. Succinylcholine (1 mg/kg) was administered after induction of anesthesia and the magnitude and duration of neuromuscular block monitored. d-Tubocurarine (0.1 mg/kg) was given 5 to 10 minutes after full recovery from succinylcholine and repeated as required. At the end of the operation, atropine, 0.02 mg/kg, and neostigmine, 0.4 mg/kg, were used to reverse the block. Base line neuromuscular data, duration of block of succinylcholine, and potency, duration of block, recovery rate, train-of-four fade, tetanic trend, response to double stimuli, post-tetanic effect, and reversibility of the subsequent d--tubocurarine-induced neuromuscular block were not significantly different (p less than 0.01) between any two groups. Tobramycin, gentamicin, and cefazolin, in recommended single doses, lack clinical neuromuscular blocking and subclinical relaxant-potentiating effects.

Interactions of neuromuscular effects of edrophonium, alpha-bungarotoxin, and beta-bungarotoxin were studied in 12 chickens using the sciatic-gastrocnemius nerve-muscle preparation to elucidate the mechanism of action of each drug. Modification by the toxins of neuromuscular effects of edrophonium depended on the level of block pre-established by the toxins. Edrophonium-induced augmentation of muscle twitch ("facilitation") was decreased by both toxins. As the block reached 50 per cent, the facilitation was nearly abolished. Edrophonium-induced contracture of the muscle was blocked by alpha-bungarotoxin only. At 25 per cent block, it was no longer observable in five of six preparations. Beta-bungarotoxin enhanced the contracture. At complete block, the contracture reached 156 (SE 11, n = 6) per cent of control. The authors conclude that edrophonium facilitates neuromuscular transmission by a prejunctional mechanism and causes contracture of the chicken muscle by a post-junctional activation. The beta-bungarotoxin-blocked nerve-muscle preparation of the chicken is a model of acute denervation potentially useful for the study of drug effects on the postjunctional membrane.