Development and application of a multiple reaction monitoring method for the simultaneous quantification of sodium channels Nav 1.1, Nav 1.2, and Nav 1.6 in solubilized membrane proteins from stable HEK293 cell lines, rodents, and human brain tissues.

RATIONALE: Nav 1.1, 1.2, and 1.6 are transmembrane proteins acting as voltage-gated sodium channels implicated in various forms of epilepsy. There is a need for knowing their actual concentration in target tissues during drug development. METHODS: Unique peptides for Nav 1.1, Nav 1.2, and Nav 1.6 were selected as quantotropic peptides for each protein and used for their quantification in membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. RESULTS: Nav 1.1, 1.2, and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1 ± 0.2, 6.4 ± 1.2, and 4.0 ± 0.6 fmol/µg membrane protein, respectively. In brains, Nav 1.2 showed the highest expression, with approximately three times higher (P < 0.003) in rodents than in humans at 3.05 ± 0.57, with 3.35 ± 0.56 in mouse and rat brains and 1.09 ± 0.27 fmol/µg in human brain. Both Nav 1.1 and 1.6 expressions were much lower in the brains, with approximately 40% less expression in human Nav 1.1 than rodent Nav 1.1 at 0.49 ± 0.1 (mouse), 0.43 ± 0.3 (rat), and 0.28 ± 0.04 (humans); whereas Nav 1.6 had approximately 60% less expression in humans than rodents at 0.27 ± 0.09 (mouse), 0.26 ± 0.06 (rat), and 0.11 ± 0.02 (humans) fmol/µg membrane proteins. CONCLUSIONS: Multiple reaction monitoring was used to quantify sodium channels Nav 1.1, 1.2, and 1.6 expressed in stably transfected HEK293 cells and brain tissues from mice, rats, and humans. We found significant differences in the expression of these channels in mouse, rat, and human brains. Nav expression ranking among the three species was Nav 1.2 ≫ Nav 1.1 > Nav 1.6, with the human brain expressing much lower concentrations overall compared to rodent brain.

Predictive validity of a pressure injury risk assessment tool at different time-points in patients admitted to the intensive care unit.

BACKGROUND: Multiple risk assessment scales are available for predicting the development of pressure injuries (PIs) in patients in the intensive care unit (ICU). Most PI risk assessment tools have been validated at the time of admission; however, another time point during treatment could better reflect clinical changes and therefore, the risk of PIs. AIMS: The study aimed to examine the predictive validity of PI risk assessment scale designed for ICU patients, the conscious level, mobility, haemodynamic, oxygenation and nutrition (COMHON) index, at several time points or intervals during ICU stay. STUDY DESIGN: This was an observational prospective study undertaken over a period of 1 year (July 2021-June 2022). Patients admitted to ICU for >3 days were included. The number, location and degree of the PIs were recorded. The level of risk for developing PIs during the stay was determined by calculating the COMHON scores at admission, and 72 h, as well as the highest and mean score. Predictive validity was studied using accuracy parameters and areas under the receiver operating characteristic curve (AUC). The best cutoff point was also determined and used to compare risk between categories. RESULTS: Of the 286 patients included in the study, 160 (59%) were male. The level of severity evaluated using the APACHE II scale was 18.4 ± 5.8 points. Invasive mechanical ventilation was used in 32.1% (n = 92) of the patients and 20.6% (n = 59) received high flow oxygen therapy. The incidence of PI was 15.4% (n = 44), with sacral location in 47.7% (n = 21) and grade II in 75% (n = 33) of the patients. The AUC was 0.907 (0.872-0.942); 0.881 (0.842-0.920); 0.877 (0.835-0.920) and 0.749 (0.667-0.831) at the mean, the highest, 72 h and ICU admission scores, respectively. The best cutoff point was 13 in all patients. The risk of developing a PI was 6.4 times higher in the high-risk group (>13 points). CONCLUSIONS: The best predictive capacity for the COMHON index risk assessment was the mean and highest scores. The predictive accuracy was higher on the third day of the patient's stay than on admission, and this was attributed to the clinical changes observed in some patients over the course of their critical illness. RELEVANCE FOR CLINICAL PRACTICE: Patients in ICU are at high risk of developing PIs, therefore, preventive measures should be maximized. Risk assessment should be carried out sequentially owing to the changes that patients present throughout their ICU stay and preventive measures should be used according to the risk level.

Potent, Gut-Restricted Inhibitors of Divalent Metal Transporter 1: Preclinical Efficacy against Iron Overload and Safety Evaluation.

Divalent metal transporter 1 (DMT1) cotransports ferrous iron and protons and is the primary mechanism for uptake of nonheme iron by enterocytes. Inhibitors are potentially useful as therapeutic agents to treat iron overload disorders such as hereditary hemochromatosis or ß-thalassemia intermedia, provided that inhibition can be restricted to the duodenum. We used a calcein quench assay to identify human DMT1 inhibitors. Dimeric compounds were made to generate more potent compounds with low systemic exposure. Direct block of DMT1 was confirmed by voltage clamp measurements. The lead compound, XEN602, strongly inhibits dietary nonheme iron uptake in both rats and pigs yet has negligible systemic exposure. Efficacy is maintained for >2 weeks in a rat subchronic dosing assay. Doses that lowered iron content in the spleen and liver by >50% had no effect on the tissue content of other divalent cations except for cobalt. XEN602 represents a powerful pharmacological tool for understanding the physiologic function of DMT1 in the gut. SIGNIFICANCE STATEMENT: This report introduces methodology to develop potent, gut-restricted inhibitors of divalent metal transporter 1 (DMT1) and identifies XEN602 as a suitable compound for in vivo studies. We also report novel animal models to quantify the inhibition of dietary uptake of iron in both rodents and pigs. This research shows that inhibition of DMT1 is a promising means to treat iron overload disorders.

Identification of aryl sulfonamides as novel and potent inhibitors of NaV1.5.

We describe the synthesis and biological evaluation of a series of novel aryl sulfonamides that exhibit potent inhibition of NaV1.5. Unlike local anesthetics that are currently used for treatment of Long QT Syndrome 3 (LQT-3), the most potent compound (-)-6 in this series shows high selectivity over hERG and other cardiac ion channels and has a low brain to plasma ratio to minimize CNS side effects. Compound (-)-6 is also effective inshortening prolonged action potential durations (APDs) in a pharmacological model of LQT-3 syndrome in pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Unlike most aryl sulfonamide NaV inhibitors that bind to the channel voltage sensors, these NaV1.5 inhibitors bind to the local anesthetic binding site in the central pore of the channel.

On the feasibility of quantifying sodium channel Nav 1.6 protein in mouse brain using targeted ultra-high-performance/electrospray ionization multiple reaction monitoring mass spectrometry.

RATIONALE: Nav 1.6 is a transmembrane voltage gated sodium channel implicated in various forms of epilepsy. Modulation of its activity in epilepsy animal models can be accomplished using inhibitors which may result in changes in its expression. There is a need to generate reliable quantitative measurements of Nav 1.6 expression in animal models. This research explores the feasibility of quantifying Nav 1.6 expression in mouse brains using targeted multiple reaction monitoring (MRM) mass spectrometry. METHODS: A combination of in silico tryptic Nav 1.6 peptides and MRM transitions were used to select target peptides. This was followed by a simple proteomic work-up including plasma membrane isolation, trypsin-based proteolysis and ultra-high-performance/electrospray ionization tandem mass spectrometry (UHPLC/ESI-MS/MS) to detect the presence of Nav 1.6 in induced HEK293 cells. The unique Nav 1.6 peptide, DSLFIPR, was selected as probe for quantifying Nav 1.6 levels in brains from C57BL/6J wild-type mice as well as two kinds of mutants including Scn8aN1768D/+ and heterozygous null Scn8a+/- mice using isotope dilution targeted mass spectrometry. RESULTS: The feasibility of using targeted MRM for quantifying Nav 1.6 expression in mice brains was demonstrated. Expression of Nav 1.6 in brains (hippocampi) from wild-type and mutant Scn8aN1768D/+ mice were found to be around 0.40 fmol/µg. Mutant null Scn8a+/- heterozygous mice, on the other hand, showed levels of 0.22 fmol/µg as expected based on this particular mutation which only generates 50% of the expression in wild-type mice. Nav 1.6-overexpressed HEK293 cells showed 3.7 fmol/µg of Nav 1.6 expression, suitable for screening new compounds for Nav 1.6 blocking activity. CONCLUSIONS: The results of the present feasibility study support the use of DSLFIPIR for quantification of Nav1.6 in brain tissues using UHPL/ESI-MS/MS.

Oxidation of catechols during positive ion electrospray mass spectrometric analysis: evidence for in-source oxidative dimerization.

RATIONALE: Catechols are an important class of analytes occurring in many natural and synthetic products. Electrospray ionization in negative mode is the preferred way of ion generation for these compounds; however, studies in positive ion mode can reveal their potential for in-source oxidation and further structural changes, some of which may also occur in the solution phase. Therefore in-source oxidation can provide a forward look into the potential for solution oxidation. METHODS: 1:1 Acetonitrile/water solutions of catechol (Cat), 4,5-dichlorocatechol (4,5-DCC), 3,4-dichlorocatechol (3,4-DCC) and tetrachlorocatechol (TCC) were analyzed by positive ion ultrahigh-performance liquid chromatography (UHPLC/ESI-MS) and UHPLC/ESI-MS/MS under various emitter voltages to assess their liability towards in-source oxidation. Structural information for in-source generated compounds was obtained through the use of product ion scans. RESULTS: Using catechols as probe compounds, we have demonstrated that under the conditions used in many analytical laboratories in-source oxidation can severely affect the sensitivity and response functions of an analyte. Under standard UHPLC conditions (300 µL/min flow rate), Cat, 3,4-DCC, 4,5-DCC and TCC can undergo in-source oxidation. The extent of oxidation is dependent either on the instrument or on the characteristics of the emitter. This is evident by a change in the isotopic pattern of these compounds and the generation of ions at lower m/z values due to a loss of 1 and/or 2 hydrogens and electrons. In the case of catechol, the formation of a dimer resulting from in-source oxidation reactions was observed. This dimer has the same fragmentation pattern as the dimer generated by oxidation in the solution phase. CONCLUSIONS: The present work demonstrates the potential of positive ion ESI for oxidizing electroactive compounds during regular analytical operation using commercially available mass spectrometers. Using Cat and some of its chlorinated analogues as probe compounds, we have demonstrated that under the conditions used in many analytical laboratories in-source oxidation and dimerization can and does take place.

Opioid-Sparing Nonsteroid Anti-inflammatory Drugs Protocol in Patients Undergoing Intramedullary Nailing of Tibial Shaft Fractures: A Randomized Control Trial.

INTRODUCTION: Nonsteroidal anti-inflammatory drugs (NSAIDs) are effective analgesics commonly used in fracture management. Although previously associated with delayed fracture healing, multiple studies have demonstrated their safety, with minimal risks of fracture healing. Given the current opioid crisis in the United States, alternate pain control modalities are essential to reduce opioid consumption. This study aims to determine whether the combination of oral acetaminophen and intravenous ketorolac is a viable alternative to opioid-based pain management in closed tibial shaft fractures treated with intramedullary nailing. METHODS: We conducted a randomized controlled trial evaluating postoperative pain control and opioid consumption in patients with closed tibial shaft fractures who underwent intramedullary nailing. Patients were randomized into an NSAID-based pain control group (52 patients) and an opioid-based pain control group (44 patients). Visual analog scale (VAS) scores and morphine milligram equivalents (MMEs) were evaluated at 12-hour postoperative intervals during the first 48 hours after surgery. Nonunion and delayed healing rates were recorded for both groups. RESULTS: A statistically significant decrease in MMEs was noted at every measured interval (12, 24, 36, and 48 hours) in the NSAID group compared with the opioid group ( P -value 0.001, 0.001, 0.040, 0.024, respectively). No significant change in visual analog scale scores was observed at 12, 36, and 48 hours between both groups ( P -value 0.215, 0.12, and 0.083, respectively). A significant decrease in VAS scores was observed at the 24-hour interval in the NSAID group compared with the opioid group ( P -value 0.041). No significant differences in union rates were observed between groups ( P -value 0.820). DISCUSSION: Using an NSAID-based postoperative pain protocol led to a decrease in opioid consumption without affecting pain scores or union rates. Owing to the minimal risk of short-term NSAID use, their role in the perioperative management of tibia shaft fractures is justified, especially when they reduce opioid consumption markedly. LEVEL OF EVIDENCE: Therapeutic Level I.

The contribution of NaV1.6 to the efficacy of voltage-gated sodium channel inhibitors in wild type and NaV1.6 gain-of-function (GOF) mouse seizure control.

BACKGROUND AND PURPOSE: Inhibitors of voltage-gated sodium channels (NaVs) are important anti-epileptic drugs, but the contribution of specific channel isoforms is unknown since available inhibitors are non-selective. We aimed to create novel, isoform selective inhibitors of Nav channels as a means of informing the development of improved antiseizure drugs. EXPERIMENTAL APPROACH: We created a series of compounds with diverse selectivity profiles enabling block of NaV1.6 alone or together with NaV1.2. These novel NaV inhibitors were evaluated for their ability to inhibit electrically evoked seizures in mice with a heterozygous gain-of-function mutation (N1768D/+) in Scn8a (encoding NaV1.6) and in wild-type mice. KEY RESULTS: Pharmacologic inhibition of NaV1.6 in Scn8aN1768D/+ mice prevented seizures evoked by a 6-Hz shock. Inhibitors were also effective in a direct current maximal electroshock seizure assay in wild-type mice. NaV1.6 inhibition correlated with efficacy in both models, even without inhibition of other CNS NaV isoforms. CONCLUSIONS AND IMPLICATIONS: Our data suggest NaV1.6 inhibition is a driver of efficacy for NaV inhibitor anti-seizure medicines. Sparing the NaV1.1 channels of inhibitory interneurons did not compromise efficacy. Selective NaV1.6 inhibitors may provide targeted therapies for human Scn8a developmental and epileptic encephalopathies and improved treatments for idiopathic epilepsies.

Discovery of XEN445: a potent and selective endothelial lipase inhibitor raises plasma HDL-cholesterol concentration in mice.

Endothelial lipase (EL) activity has been implicated in HDL metabolism and in atherosclerotic plaque development; inhibitors are proposed to be efficacious in the treatment of dyslipidemia related cardiovascular disease. We describe here the discovery of a novel class of anthranilic acids EL inhibitors. XEN445 (compound 13) was identified as a potent and selective EL inhibitor, that showed good ADME and PK properties, and demonstrated in vivo efficacy in raising plasma HDLc concentrations in mice.

Patient Experience and Perspective on Medical Cannabis as an Alternative for Musculoskeletal Pain Management.

INTRODUCTION: The current rate of opioid prescription is disquieting because of their high abuse potential, adverse effects, and thousands of overdose deaths. This situation imposes urgency in seeking alternatives for adequate pain management. From this perspective, this study aimed to evaluate the experience and the perceived analgesic efficacy of medical cannabis in managing the pain associated with musculoskeletal conditions. METHODS: A 28-question survey was distributed to patients at a major medical cannabis center in Puerto Rico for 2 months. Demographics, medical history, cannabis usage, cannabis use perspective, and analgesic efficacy were assessed in the questionnaire. RESULTS: One hundred eighty-four patients completed our survey. The majority (67%) were males, and the participants' average age was 38 years. This study showed an average pain reduction score of 4.02 points on the Numeric Rating Scale among all the participants. Those with musculoskeletal conditions reported a notable average pain reduction score of 4.47 points. In addition, 89% of the participants considered medical cannabis to be more effective than narcotics for adequate pain management. CONCLUSIONS: This study demonstrated that the use of medical cannabis among patients with musculoskeletal conditions effectively reduced pain levels based on their Numeric Rating Scale reported scores.

NBI-921352, a first-in-class, NaV1.6 selective, sodium channel inhibitor that prevents seizures in Scn8a gain-of-function mice, and wild-type mice and rats.

NBI-921352 (formerly XEN901) is a novel sodium channel inhibitor designed to specifically target NaV1.6 channels. Such a molecule provides a precision-medicine approach to target SCN8A-related epilepsy syndromes (SCN8A-RES), where gain-of-function (GoF) mutations lead to excess NaV1.6 sodium current, or other indications where NaV1.6 mediated hyper-excitability contributes to disease (Gardella and Møller, 2019; Johannesen et al., 2019; Veeramah et al., 2012). NBI-921352 is a potent inhibitor of NaV1.6 (IC500.051 µM), with exquisite selectivity over other sodium channel isoforms (selectivity ratios of 756 X for NaV1.1, 134 X for NaV1.2, 276 X for NaV1.7, and >583 Xfor NaV1.3, NaV1.4, and NaV1.5). NBI-921352is a state-dependent inhibitor, preferentially inhibiting inactivatedchannels. The state dependence leads to potent stabilization of inactivation, inhibiting NaV1.6 currents, including resurgent and persistent NaV1.6 currents, while sparing the closed/rested channels. The isoform-selective profile of NBI-921352 led to a robust inhibition of action-potential firing in glutamatergic excitatory pyramidal neurons, while sparing fast-spiking inhibitory interneurons, where NaV1.1 predominates. Oral administration of NBI-921352 prevented electrically induced seizures in a Scn8a GoF mouse,as well as in wild-type mouse and ratseizure models. NBI-921352 was effective in preventing seizures at lower brain and plasma concentrations than commonly prescribed sodium channel inhibitor anti-seizure medicines (ASMs) carbamazepine, phenytoin, and lacosamide. NBI-921352 waswell tolerated at higher multiples of the effective plasma and brain concentrations than those ASMs. NBI-921352 is entering phase II proof-of-concept trials for the treatment of SCN8A-developmental epileptic encephalopathy (SCN8A-DEE) and adult focal-onset seizures.

Blunt Abdominopelvic Trauma Complicated by Traumatic Testicular Dislocation in a 19-Year-Old Male Patient: A Case Report.

CASE: A 19 year-old male patient presented with testicular dislocation after abdominopelvic trauma. During open reduction and internal fixation, consult to urology was placed after discovering the presence of the intra-abdominal testicle. The testicle was repositioned into the scrotum with orchiopexy, and pelvic fixation was completed with 1 sacroiliac percutaneous screw and pubic symphysis fixation. Postoperative recovery was uneventful, and the patient was discharged home on postoperative day 3. CONCLUSION: Testicular dislocation is an uncommon finding after blunt abdominopelvic trauma; hence, it may be overlooked. Prompt diagnosis of testicular dislocation given the need for operative management to preserve testicle viability is crucial.

Level of Supervision and Radiation Exposure of Senior Orthopedic Residents During Surgical Treatment of Proximal Femur Fracture.

The sustained use of intraoperative fluoroscopy has led to increased use of minimally invasive surgical techniques, enhanced surgeon proficiency, improved anatomic corrections, reduced patient morbidity, earlier functional recovery, and decreased length of hospital stay. As a result, orthopedic attending surgeons and residents are exposed to more radiation, increasing the risk of cancer and radiation-induced cataracts compared with the general population and those who work in other surgical specialties. The magnitude of radiation exposure depends on the susceptibility of the tissues affected, medical specialty, the position of the C-arm, distance from the radiation beam, level of difficulty of the surgical procedure, surgeon experience, level of resident training, and level of supervision by the attending surgeon. However, little information is available on the effect of supervision level on radiation exposure for orthopedic senior residents. The goal of this study was to investigate whether level of supervision by the attending surgeon affects the radiation exposure of orthopedic senior residents during surgical treatment of proximal femur fracture with cephalomedullary nail fixation. This retrospective cohort study was performed from January 2019 to March 2019. No significant relationship between supervision level and radiation exposure of senior residents was observed. Supervision level does not significantly affect radiation exposure for senior residents; therefore, the implementation of standardized training in radiation safety may be a more essential measure to decrease radiation exposure. [Orthopedics. 2021;44(3):e402-e406.].

Discovery of Acyl-sulfonamide Nav1.7 Inhibitors GDC-0276 and GDC-0310.

Nav1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 (1) and GDC-0310 (2), selective Nav1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3. This resulted in the discovery of GDC-0276 (1), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Nav1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 (2), which possesses improved Nav selectivity and pharmacokinetic profile over 1.

Anterior tibialis artery pseudoaneurysm after minimally invasive plate osteosynthesis in the proximal tibia: a case report.

Tibial plateau fractures are common fractures associated with high-energy trauma. The treatment of these fractures remains a challenge today. Recent management of fractures has moved from open surgery toward minimally invasive surgery to reduce complications and improve functional outcomes. Nevertheless, such a minimalistic approach makes visualization of neurovascular structures difficult, placing them at risk. We report the case of a 39-year-old male who developed a pseudoaneurysm of the anterior tibial artery following minimally invasive plate osteosynthesis of the right proximal tibia. Diagnosis was made through noninvasive duplex ultrasound and was referred to endovascular service. Understanding of the anatomy of the surgical site is vital to minimize complications. In addition, proper postsurgical patient evaluation is important to monitor the insurgence of such complications.

Socioeconomic Factors Influencing Self-reported Outcomes After Posterior Wall Fractures of the Acetabulum: Lessons Learned From a Hispanic Population.

INTRODUCTION: Demographic and socioeconomic factors are important determinants that may affect patient self-reported outcomes after acetabular fracture surgery. Hispanics, as a minority group, have an increased tendency to suffer demographic and socioeconomic disparities. At the present time, there is scant information regarding their role among Hispanic patients with acetabular fractures. The aim of this study was to investigate whether demographic or socioeconomic factors would affect satisfaction and self-reported functional outcomes in Hispanic patients who endured open reduction and internal fixation (ORIF) of posterior wall fractures of the acetabulum. METHODS: A cross-sectional study of 78 patients with posterior wall fracture of the acetabulum treated with ORIF from 2011 to 2017 was performed. Data from demographics (age, sex, and body mass index [BMI]) and socioeconomic factors (household income, highest educational level achieved, employment status, type of medical insurance, workers' compensation involvement, and injury-related litigation process) were obtained and compared with the Short Musculoskeletal Form Assessment-46 (SMFA-46) questionnaire using a multivariable logistic regression analysis. RESULTS: Unemployment was the most significant variable associated with dissatisfaction among 15 of the 46 responses of the SMFA-46 (P < 0.048). Patients who had an overweight or obese BMI and had an age of 41 years or older exhibited significantly worse outcomes in 7 of the 46 questions (P < 0.049). Finally, women were less likely to be satisfied in 1 of the 46 questions (P = 0.028). No notable difference was observed in any of the SMFA-46 responses regarding insurance plan, educational level, workers' compensation involvement, and injury-related litigation process. DISCUSSION: A strong association exists between dissatisfaction after ORIF of posterior wall fractures of the acetabulum and employment status, BMI, and age among the Hispanic population. Addressing socioeconomic factors can be critical to overcome dissatisfaction and improve functional status among Hispanic patients with acetabular fractures.

Identification of CNS-Penetrant Aryl Sulfonamides as Isoform-Selective NaV1.6 Inhibitors with Efficacy in Mouse Models of Epilepsy.

Nonselective antagonists of voltage-gated sodium (NaV) channels have been long used for the treatment of epilepsies. The efficacy of these drugs is thought to be due to the block of sodium channels on excitatory neurons, primarily NaV1.6 and NaV1.2. However, these currently marketed drugs require high drug exposure and suffer from narrow therapeutic indices. Selective inhibition of NaV1.6, while sparing NaV1.1, is anticipated to provide a more effective and better tolerated treatment for epilepsies. In addition, block of NaV1.2 may complement the anticonvulsant activity of NaV1.6 inhibition. We discovered a novel series of aryl sulfonamides as CNS-penetrant, isoform-selective NaV1.6 inhibitors, which also displayed potent block of NaV1.2. Optimization focused on increasing selectivity over NaV1.1, improving metabolic stability, reducing active efflux, and addressing a pregnane X-receptor liability. We obtained compounds 30-32, which produced potent anticonvulsant activity in mouse seizure models, including a direct current maximal electroshock seizure assay.

Identification of Selective Acyl Sulfonamide-Cycloalkylether Inhibitors of the Voltage-Gated Sodium Channel (NaV) 1.7 with Potent Analgesic Activity.

Herein, we report the discovery and optimization of a series of orally bioavailable acyl sulfonamide NaV1.7 inhibitors that are selective for NaV1.7 over NaV1.5 and highly efficacious in in vivo models of pain and hNaV1.7 target engagement. An analysis of the physicochemical properties of literature NaV1.7 inhibitors suggested that acyl sulfonamides with high fsp3 could overcome some of the pharmacokinetic (PK) and efficacy challenges seen with existing series. Parallel library syntheses lead to the identification of analogue 7, which exhibited moderate potency against NaV1.7 and an acceptable PK profile in rodents, but relatively poor stability in human liver microsomes. Further, design strategy then focused on the optimization of potency against hNaV1.7 and improvement of human metabolic stability, utilizing induced fit docking in our previously disclosed X-ray cocrystal of the NaV1.7 voltage sensing domain. These investigations culminated in the discovery of tool compound 33, one of the most potent and efficacious NaV1.7 inhibitors reported to date.

Selective NaV1.7 Antagonists with Long Residence Time Show Improved Efficacy against Inflammatory and Neuropathic Pain.

Selective block of NaV1.7 promises to produce non-narcotic analgesic activity without motor or cognitive impairment. Several NaV1.7-selective blockers have been reported, but efficacy in animal pain models required high multiples of the IC50 for channel block. Here, we report a target engagement assay using transgenic mice that has enabled the development of a second generation of selective Nav1.7 inhibitors that show robust analgesic activity in inflammatory and neuropathic pain models at low multiples of the IC50. Like earlier arylsulfonamides, these newer acylsulfonamides target a binding site on the surface of voltage sensor domain 4 to achieve high selectivity among sodium channel isoforms and steeply state-dependent block. The improved efficacy correlates with very slow dissociation from the target channel. Chronic dosing increases compound potency about 10-fold, possibly due to reversal of sensitization arising during chronic injury, and provides efficacy that persists long after the compound has cleared from plasma.

Design of Conformationally Constrained Acyl Sulfonamide Isosteres: Identification of N-([1,2,4]Triazolo[4,3- a]pyridin-3-yl)methane-sulfonamides as Potent and Selective hNaV1.7 Inhibitors for the Treatment of Pain.

The sodium channel NaV1.7 has emerged as a promising target for the treatment of pain based on strong genetic validation of its role in nociception. In recent years, a number of aryl and acyl sulfonamides have been reported as potent inhibitors of NaV1.7, with high selectivity over the cardiac isoform NaV1.5. Herein, we report on the discovery of a novel series of N-([1,2,4]triazolo[4,3- a]pyridin-3-yl)methanesulfonamides as selective NaV1.7 inhibitors. Starting with the crystal structure of an acyl sulfonamide, we rationalized that cyclization to form a fused heterocycle would improve physicochemical properties, in particular lipophilicity. Our design strategy focused on optimization of potency for block of NaV1.7 and human metabolic stability. Lead compounds 10, 13 (GNE-131), and 25 showed excellent potency, good in vitro metabolic stability, and low in vivo clearance in mouse, rat, and dog. Compound 13 also displayed excellent efficacy in a transgenic mouse model of induced pain.