Evaluation of different blood tests in dogs with extrahepatic portosystemic shunts to assess shunt closure after surgical treatment.

OBJECTIVE: To determine the sensitivity and specificity of different individual and combined blood tests to assess extrahepatic portosystemic shunt (EHPSS) closure after gradual attenuation of EHPSS in dogs. STUDY DESIGN: Clinical prospective study. ANIMALS: Twenty client-owned dogs with EHPSS. METHODS: Fasting ammonia (FA), preprandial, postprandial, and paired serum bile acids (SBA), the lidocaine/monoethylglycylxylidide (L/MEGX) test, and serum hyaluronic acid (SHA) were performed at diagnosis, and 1, 3, and 6 months postoperatively. Transsplenic portal scintigraphy was performed to determine EHPSS closure 3 months postoperatively. Their sensitivity and specificity in determining shunt closure postoperatively were calculated. RESULTS: When assessing a single blood parameter, FA had the highest specificity (100%), whereas SHA and MEGX measured 15 min after lidocaine administration (T15) had the highest sensitivity (96.9% and 96.2%, respectively) for determining shunt closure postoperatively. The most promising blood test combinations were SHA (sensitivity 96.9%, specificity 81.8%), combined with the L/MEGX test (MEGX at T15: sensitivity 100%, specificity 72.4%) or the L/MEGX test (MEGX at T15) combined with either FA (sensitivity 100%, specificity 82.8%) or postprandial SBA (sensitivity 100%, specificity 81.5%). CONCLUSION: Both SHA and the L/MEGX test were sensitive tests for determining shunt closure after gradual attenuation of EHPSS. Test performances could even be improved by combining these tests with each other or with traditional tests such as FA or postprandial SBA. CLINICAL SIGNIFICANCE: Although SHA and the L/MEGX test are sensitive blood tests for determining EHPSS closure, especially when combined with traditional blood tests, imaging is still needed to confirm EHPSS closure.

The 14-day repeated-dose toxicity study of a fixed-dose combination, QXOH/levobupivacaine, via subcutaneous injection in beagle dogs.

QXOH-Levobupivacaine (LB) is a fixed-dose combination of 35-mM QXOH and 10-mM LB. It was developed for perioperative analgesia because of its long-acting analgesic effect. The purpose of this study was to evaluate the potential toxicity of QXOH-LB in beagle dogs in accordance with the Guidance on the repeated-dose toxicity published by the China Food and Drug Administration. Groups of five male and five female beagle dogs received normal saline, QXOH-LB (2, 4, and 8 mg/kg, calculated as QXOH), QXOH (2, 4, and 8 mg/kg), or LB (2 mg/kg, equals the concentration of LB in 8-mg/kg QXOH-LB group) at the volume of 1 mL/kg once per day for 14 days through subcutaneous injection. No mortality was observed. Dogs in the control group as well as animals treated with 2-mg/kg QXOH or QXOH-LB exhibited normal behaviors. Clinical signs of toxicity in dogs treated with 4 and 8 mg/kg of QXOH or QXOH-LB included decreased activity, unsteady gait, jerks, tremors, vocalization, emesis, ataxia, lateral/sternal recumbency, deep/rapid respiration, and gasping. Additionally, neurological function was found to be affected by QXOH and QXOH-LB at the doses of 4 and 8 mg/kg. All clinical signs were recovered within 24 h. The no-observed-adverse-effect level of QXOH and QXOH-LB was considered to be 2 mg/kg. Toxicokinetic data showed that exposure to QXOH and LB increased as QXOH-LB doses were increased from 4 to 8 mg/kg. There was no evidence of drug accumulation or any effect of gender.

Atractylodin Produces Antinociceptive Effect through a Long-Lasting TRPA1 Channel Activation.

Atractylodin (ATR) is a bioactive component found in dried rhizomes of Atractylodes lancea (AL) De Candolle. Although AL has accumulated empirical evidence for the treatment of pain, the molecular mechanism underlying the anti-pain effect of ATR remains unclear. In this study, we found that ATR increases transient receptor potential ankyrin-1 (TRPA1) single-channel activity in hTRPA1 expressing HEK293 cells. A bath application of ATR produced a long-lasting calcium response, and the response was completely diminished in the dorsal root ganglion neurons of TRPA1 knockout mice. Intraplantar injection of ATR evoked moderate and prolonged nociceptive behavior compared to the injection of allyl isothiocyanate (AITC). Systemic application of ATR inhibited AITC-induced nociceptive responses in a dose-dependent manner. Co-application of ATR and QX-314 increased the noxious heat threshold compared with AITC in vivo. Collectively, we concluded that ATR is a unique agonist of TRPA1 channels, which produces long-lasting channel activation. Our results indicated ATR-mediated anti-nociceptive effect through the desensitization of TRPA1-expressing nociceptors.

Efficacy of a Subcutaneous Implantable Cardioverter Defibrillator in a Child with Early Repolarization Syndrome.

An early repolarization (ER) pattern or J waves are considered to be a benign finding observed in the healthy population, however, it has been pointed out that the ER pattern seen in the inferolateral leads could be an independent risk factor for fatal arrhythmias. We present a pediatric case in which early repolarization syndrome (ERS) was suspected due to the presence of ER or J waves in the inferior leads, which eventually disappeared after the administration of pilsicainide. During the follow-up period, several fatal ventricular arrhythmias were recorded after implantation of a subcutaneous implantable cardiac defibrillator (S-ICD). This report describes the efficacy of S-ICDs in a child with an ER pattern after aborted sudden cardiac death.

Concurrent increases in post-pacing action potential duration and contractility predict occurrence of ventricular arrhythmia.

Excitation-contraction coupling from the integration of action potential duration (APD) and muscle contractility plays an important role in arrhythmogenesis. We aimed to determine whether distinctive excitation-contraction coupling contributes to the genesis of ventricular tachycardias (VTs). Action potential (AP) and mechanical activity were simultaneously recorded under electrical pacing (cycle lengths from 1000 to 100 ms) in the tissue model created from isolated rabbit right ventricular outflow tracts treated with NS 5806 (10 µM, transient outward potassium current enhancer), pinacidil (2 µM, ATP-sensitive potassium channel opener), and pilsicainide (5 µM, sodium channel blocker). There were 15 (9.9%) inducible VT episodes (group 1) and 136 (90.1%) non-inducible VT episodes (group 2) in our tissue model. Group 1 had greater post-pacing increases of the first occurrence of AP at 90% repolarization (ΔAPD90, p < 0.001) and contractility (ΔContractility, p = 0.003) compared with group 2. Triggered VT episodes were common (72.7%) in cases with a ΔAPD90 > 15% and a ΔContractility > 270%, but were undetectable in those with a ΔAPD90 < 15% and a ΔContractility < 270%. In those with pacing-induced VTs, KB-R7943 (10 µM, a Na+-Ca2+ exchanger inhibitor, NCX inhibitor) significantly reduced the occurrence of VTs from 100.0 to 20.0% (15/15 to 3/15 episodes, p < 0.001). Concurrent increases in both post-pacing APD and contractility resulted in the occurrence of ventricular arrhythmias. NCX inhibition may be a potential therapeutic strategy for ventricular arrhythmias.

Clinical and Electrocardiographic Differences in Brugada Syndrome With Spontaneous or Drug-Induced Type 1 Electrocardiogram.

BACKGROUND: Spontaneous type 1 electrocardiogram (ECG) in the right precordial lead is a dominant predictor of ventricular fibrillation (VF) in Brugada syndrome (BrS). In some BrS patients with VF, however, spontaneous type 1 ECG is undetectable, even in repeated ECG and immediately after VF. This study investigated differences between BrS patients with spontaneous or drug-induced type 1 ECG. Methods and Results: We evaluated 15 BrS patients with drug-induced (D-BrS) and 29 with spontaneous type 1 ECG (SP-BrS). All patients had had a previous VF episode. In each D-BrS patient, ECG was recorded more than 15 times (mean, 46±34) during 7.2±5.1 years of follow-up. Age and family history were comparable between groups. Inferolateral early repolarization (ER) was observed in 13 D-BrS (87%) at least once but in only 3 SP-BrS (10%, P<0.01). Immediately after VF, inferolateral ER was accentuated in 9 of 10 D-BrS, while type 1 ECG was accentuated in 12 of 16 SP-BrS. Fragmented QRS in the right precordial lead and aVR sign were absent in D-BrS but present in 20 (69%, P<0.01) and 11 (38%, P<0.01) SP-BrS, respectively. There was no prognostic difference between groups. CONCLUSIONS: Although having similar clinical profiles, there are obvious ECG differences between VF-positive BrS patients with spontaneous or drug-induced type 1 ECG. The inferolateral lead rather than the right precordial lead on ECG may be particularly crucial in some BrS patients.

Involvement of the persistent Na+ current in the diastolic depolarization and automaticity of the guinea pig pulmonary vein myocardium.

The role of the Na+ current in the automaticity of the pulmonary vein myocardium was examined in isolated guinea pig pulmonary vein cardiomyocytes and tissue preparations. Tetrodotoxin inhibited the automaticity of pulmonary vein tissue preparations by suppressing the diastolic depolarization of the action potential. ATX-II, which increased the density of persistent component of the Na+ current (late INa), induced a depolarization of the resting membrane potential followed by spontaneous firing of action potentials. GS-458967, which inhibited the late INa, suppressed the diastolic depolarization and the firing of action potentials. Pilsicainide, which inhibited only the transient component of Na+ current (peak INa), had no effect on the firing frequency. GS-458967 had no effect on the contractile force of the working myocardium. In conclusion, late INa is involved in the diastolic depolarization and automaticity of the pulmonary vein myocardium. Late INa inhibitors appear to be effective therapeutic agents for atrial fibrillation with minimum adverse effects on the working myocardium.

Addition of dexmedetomidine to QX-314 enhances the onset and duration of sciatic nerve block in rats.

An experimental set-up was designed to observe whether adding dexmedetomidine to QX-314 would enhance the onset and duration of sensory and motor function in a rat sciatic nerve block model. Fifty-six Sprague-Dawley rats received unilateral sciatic nerve blocks with 0.2 mL of 35 mmol/L QX-314 alone, dexmedetomidine (5.3 µmol/L (1 µg/kg), 26.4 µmol/L (5 µg/kg), 52.8 µmol/L (10 µg/kg)) alone, or a combination of the two. Thermal nociception and motor function were assessed by an investigator blinded to the drug treatment, and sciatic nerves and perineural tissues were harvested at 14 days after injection. In addition, we examined the effects of these solutions on compound action potentials in isolated frog sciatic nerves. Dexmedetomidine added to QX-314 enhanced the onset and duration of thermal nociception block and motor block (P < 0.05) without aggravating histopathological injuries. Furthermore, 52.8 µmol/L dexmedetomidine added to 35 mmol/L QX-314 showed less inflammation than QX-314 alone at 14 days (P = 0.003). Dexmedetomidine plus QX-314 was shown to dose-dependently reduce the compound action potentials relative to QX-314 alone (P < 0.05). It was concluded that co-administration of QX-314 with a clinical dose of dexmedetomidine produced a synergistic anesthetic effect to enhance the effect of sciatic nerve block.

Pilsicainide Poisoning: An Autopsy Case and Review of Literature.

We describe an autopsy case of fatal poisoning due to accidental overdose of pilsicainide, which is a Vaughan Williams class IC antiarrhythmic drug (a pure sodium channel blocker). A man in his 50s was found dead in his home at approximately noon. He had ischemic heart disease and insomnia, and had previously demonstrated improper prescription drug adherence. The autopsy revealed old coronary artery bypass grafting and mild fibrosis of myocardium, but no acute myocardial infarction was found in microscopic examination. Toxicological analysis also identified a high blood concentration of pilsicainide (femoral vein blood, 14.9 µg/mL), more than 15 times higher than reported therapeutic levels. The blood concentrations of other drugs were at therapeutic levels, and no alcohol was detected. We concluded that the cause of death was pilsicainide poisoning, based on the results of the autopsy and the toxicological examination. This is the first autopsy report of fatal poisoning due to pilsicainide as a single agent.

Pharmacokinetics of intravenous, subcutaneous, and topical administration of lidocaine hydrochloride and metabolites 3-hydroxylidocaine, monoethylglycinexylidide, and 4-hydroxylidocaine in horse.

Intravenous (iv), subcutaneous (sq), and topical (tp) lidocaine was administered to six horses in a cross-over, randomized design study. Samples were collected for up to 72 hr. Compartmental models were used to investigate the pharmacokinetics of (LD) and its metabolites 3-hydroxylidocaine (3-OH), 4-hydroxylidocaine (4-OH), and monoethylglycinexylidide (MEGX). Metabolites 3-OH and 4-OH were present in conjugated forms, whereas LD and metabolite MEXG were present primarily in the un-conjugated form. Plasma concentrations of LD after iv administration (100 mg) were described by three-compartment model with an additional three compartments to describe the elimination of metabolites. Median (range) elimination micro-constants (Ke ) for LD, 3-OH, 4-OH, and MEXG were 4.12 (2.62-6.23), 1.25 (1.10-2.15), 1.79 (1.22-2.39), and 1.69 (1.03-1.99)/hr, respectively. Median (range) values of alpha (t½α ), beta (t½ß ), and gamma (t½Î³ ) half-lives were 0.08 (0.07-0.13), 0.57 (0.15-1.25), and 4.11 (0.52-7.36) hr. Plasma concentrations of LD after sq (200 mg) administration were described by absorption and two-compartment elimination model. The median (range) of the LD absorption half-life (t½ab ) was 0.47 (0.29-0.61) hr. The Ke for LD, 3-OH, 4-OH, and MEXG was 3.91 (1.48-9.25), 1.00 (0.78-1.08), 1.76 (0.96-2.11), and 1.13 (0.69-1.33)/hr. The median (range) of t½α and t½ß was 0.15 (0.06-0.27) and 3.04 (2.53-6.39) hr. Plasma concentrations of LD after tp (400 mg) application were described by one-compartment model with a t½ab of 8.49 (5.16-11.80) hr. The Ke for LD, 3-OH, and MEXG was 0.24 (0.10-0.81), 0.41 (0.08-0.93), and 0.38 (0.26-1.14)/hr.

Functional Human α7 Nicotinic Acetylcholine Receptor (nAChR) Generated from Escherichia coli.

Human Cys-loop receptors are important therapeutic targets. High-resolution structures are essential for rational drug design, but only a few are available due to difficulties in obtaining sufficient quantities of protein suitable for structural studies. Although expression of proteins in E. coli offers advantages of high yield, low cost, and fast turnover, this approach has not been thoroughly explored for full-length human Cys-loop receptors because of the conventional wisdom that E. coli lacks the specific chaperones and post-translational modifications potentially required for expression of human Cys-loop receptors. Here we report the successful production of full-length wild type human α7nAChR from E. coli Chemically induced chaperones promote high expression levels of well-folded proteins. The choice of detergents, lipids, and ligands during purification determines the final protein quality. The purified α7nAChR not only forms pentamers as imaged by negative-stain electron microscopy, but also retains pharmacological characteristics of native α7nAChR, including binding to bungarotoxin and positive allosteric modulators specific to α7nAChR. Moreover, the purified α7nAChR injected into Xenopus oocytes can be activated by acetylcholine, choline, and nicotine, inhibited by the channel blockers QX-222 and phencyclidine, and potentiated by the α7nAChR specific modulators PNU-120596 and TQS. The successful generation of functional human α7nAChR from E. coli opens a new avenue for producing mammalian Cys-loop receptors to facilitate structure-based rational drug design.

Characterization of microminipigs as an in vivo experimental model for cardiac safety pharmacology.

We pharmacologically characterized microminipigs as an in vivo experimental model by assessing cardiovascular effects of pilsicainide, verapamil and E-4031, which can preferentially inhibit cardiac Na+, Ca2+ and K+ channels, respectively. Intravenous infusion of 1 mg/kg of pilsicainide (n = 4), 0.1 mg/kg of verapamil (n = 4) and 0.01 followed by 0.1 mg/kg of E-4031 (n = 5) over 10 min decreased the heart rate, mean blood pressure and ventricular contractility. Moreover, pilsicainide prolonged the PR interval, QRS width and QTc; verapamil prolonged the PR interval, but shortened the QRS width and QTc; and E-4031 prolonged the QTc, whereas no substantial change was detected in the PR interval or QRS width. Peak plasma concentrations of pilsicainide, verapamil and E-4031 in microminipigs were 1.7-4.8 times higher than those expected in humans and dogs, possibly due to smaller effective volume of drug distribution. The extent of the drug-induced cardiovascular responses was generally greater in microminipigs than in humans and dogs, which could be explained by the following possibilities; namely unique pharmacokinetic profile, less great reflex-mediated increase of sympathetic tone and/or smaller repolarization reserve in microminipigs. These information may make it feasible to apply this new-type animal to a tool for assessing cardiac safety profiles of new chemical entities.

Utility of 12-lead and signal-averaged Holter electrocardiograms after pilsicainide provocation for risk stratification in Brugada syndrome.

Non-invasive risk stratification for ventricular fibrillation (VF) in Brugada syndrome (BrS) has not been fully evaluated. The aim of this study was to assess the utility of signal-averaged Holter electrocardiogram (Holter SAECG) and 12-lead Holter electrocardiogram (Holter ECG) after a pilsicainide provocation test for non-invasive risk stratification in BrS. We enrolled 30 consecutive patients with BrS [divided into 2 groups: the VF group, those with a previous history of VF (n = 10); and the non-VF group, those without a history of VF (n = 20)] and 10 control subjects without type 1 ECG. We evaluated late potentials [LP: filtered QRS (f-QRS), RMS40, and LAS40] on the Holter SAECG for 4 h after the pilsicainide provocation and in the same patients on another day without performing the pilsicainide provocation. Furthermore, we measured QRS duration and QTc interval in leads V2 and V5, and J amplitude in lead V2 on the Holter ECG after the pilsicainide provocation. On the Holter SAECG, the f-QRS at 1 h and LAS40 at 3 h after the pilsicainide provocation were significantly larger in the VF group than in the non-VF group (f-QRS at 1 h: 113.9 ± 8.9 vs. 104.9 ± 8 ms; p = 0.01, LAS40 at 3 h: 45.4 ± 5.9 vs. 35.5 ± 7.4 ms; p < 0.001). The receiver-operating characteristic curve analysis for a single parameter of VF occurrence was determined [f-QRS at 1 h: area under the curve (AUC) 0.8, with sensitivity 80% and specificity 80%; and LAS40 at 3 h: AUC 0.87, with sensitivity 90% and specificity 75%]. On the Holter ECG, there were no significant differences in these parameters between the VF and non-VF groups. In conclusion, the LP after the pilsicainide provocation using Holter SAECG may be useful for risk stratification of VF episodes in patients with BrS.

Significance of electrocardiogram recording in high intercostal spaces in patients with early repolarization syndrome.

AIMS: Published reports regarding inferolateral early repolarization (ER) syndrome (ERS) before 2013 possibly included patients with Brugada-pattern electrocardiogram (BrP-ECG) recorded only in the high intercostal spaces (HICS). We investigated the significance of HICS ECG recording in ERS patients. METHODS AND RESULTS: Fifty-six patients showing inferolateral ER in the standard ECG and spontaneous ventricular fibrillation (VF) not linked to structural heart disease underwent drug provocation tests by sodium channel blockade with right precordial ECG (V1-V3) recording in the 2nd-4th intercostal spaces. The prevalence and long-term outcome of ERS patients with and without BrP-ECG in HICS were investigated. After 18 patients showing type 1 BrP-ECG in the standard ECG were excluded, 38 patients (34 males, mean age; 40.4 ± 13.6 years) were classified into four groups [group A (n = 6;16%):patients with ER and type 1 BrP-ECG only in HICS, group B (n = 5;13%):ERS with non-type 1 BrP-ECG only in HICS, group C (n = 8;21%):ERS with non-type 1 BrP-ECG in the standard ECG, and group D (n = 19;50%):ERS only, spontaneously or after drug provocation test]. During follow-up of 110.0 ± 55.4 months, the rate of VF recurrence including electrical storm was significantly higher in groups A (4/6:67%), B (4/5:80%), and C (4/8:50%) compared with D (2/19:11%) (A, B, and C vs. D, P < 0.05). CONCLUSIONS: Approximately 30% of the patients with ERS who had been diagnosed with the previous criteria showed BrP-ECG only in HICS. Ventricular fibrillation mostly recurred in patients showing BrP-ECG in any precordial lead including HICS; these comprised 50% of the ERS cohort.

Selective Gating of Neuronal Activity by Intrinsic Properties in Distinct Motor Rhythms.

Many neural circuits show fast reconfiguration following altered sensory or modulatory inputs to generate stereotyped outputs. In the motor circuit of Xenopus tadpoles, I study how certain voltage-dependent ionic currents affect firing thresholds and contribute to circuit reconfiguration to generate two distinct motor patterns, swimming and struggling. Firing thresholds of excitatory interneurons [i.e., descending interneurons (dINs)] in the swimming central pattern generator are raised by depolarization due to the inactivation of Na(+) currents. In contrast, the thresholds of other types of neurons active in swimming or struggling are raised by hyperpolarization from the activation of fast transient K(+) currents. The firing thresholds are then compared with the excitatory synaptic drives, which are revealed by blocking action potentials intracellularly using QX314 during swimming and struggling. During swimming, transient K(+) currents lower neuronal excitability and gate out neurons with weak excitation, whereas their inactivation by strong excitation in other neurons increases excitability and enables fast synaptic potentials to drive reliable firing. During struggling, continuous sensory inputs lead to high levels of network excitation. This allows the inactivation of Na(+) currents and suppression of dIN activity while inactivating transient K(+) currents, recruiting neurons that are not active in swimming. Therefore, differential expression of these currents between neuron types can explain why synaptic strength does not predict firing reliability/intensity during swimming and struggling. These data show that intrinsic properties can override fast synaptic potentials, mediate circuit reconfiguration, and contribute to motor-pattern switching.

Auditory Golgi cells are interconnected predominantly by electrical synapses.

The mossy fiber-granule cell-parallel fiber system conveys proprioceptive and corollary discharge information to principal cells in cerebellum-like systems. In the dorsal cochlear nucleus (DCN), Golgi cells inhibit granule cells and thus regulate information transfer along the mossy fiber-granule cell-parallel fiber pathway. Whereas excitatory synaptic inputs to Golgi cells are well understood, inhibitory and electrical synaptic inputs to Golgi cells have not been examined. Using paired recordings in a mouse brain slice preparation, we find that Golgi cells of the cochlear nucleus reliably form electrical synapses onto one another. Golgi cells were only rarely electrically coupled to superficial stellate cells, which form a separate network of electrically coupled interneurons in the DCN. Spikelets had a biphasic effect on the excitability of postjunctional Golgi cells, with a brief excitatory phase and a prolonged inhibitory phase due to the propagation of the prejunctional afterhyperpolarization through gap junctions. Golgi cells and stellate cells made weak inhibitory chemical synapses onto Golgi cells with low probability. Electrical synapses are therefore the predominant form of synaptic communication between auditory Golgi cells. We propose that electrical synapses between Golgi cells may function to regulate the synchrony of Golgi cell firing when electrically coupled Golgi cells receive temporally correlated excitatory synaptic input.

JM25-1, a Lidocaine Analog Combining Airway Relaxant and Antiinflammatory Properties: Implications for New Bronchospasm Therapy.

BACKGROUND: Inhaled lidocaine antagonized bronchospasm in animal models and patients, but adverse effects limited its efficacy. This study evaluated the antibronchospasm potential of the analog JM25-1, exploring in vitro mechanisms and translation to an animal model. METHODS: The effectiveness of JM25-1 was assessed in GH3 cells, rat tracheal rings, mouse lymphocytes, and human eosinophil systems in vitro, assessing changes in Na current, contraction, proliferation, and survival, respectively. Lung function and inflammatory changes were studied in ovalbumin-sensitized mice. RESULTS: The efficacy of JM25-1 was higher than lidocaine in inhibiting carbachol-induced and calcium-induced tracheal contractions (maximum effect inhibition at 1 mM [%]: 67 ± 10 [JM25-1] vs. 41 ± 11 [lidocaine] [P < 0.001] for carbachol; 100 ± 3 [JM25-1] vs. 36 ± 26 [lidocaine] [P < 0.001] for Ca; mean ± SD; n = 9 each) but lower in Na current (50% inhibitory concentration = 151.5, n = 8 vs. 0.2 mM; n = 5; P < 0.001). JM25-1 also inhibited eosinophil survival (dead cells [%]: 65 ± 6; n = 4; P < 0.001 at 1 mM) and lymphocyte proliferation (cells in phase S + G2 [%]: 94 ± 10; n = 6; P < 0.001) at 0.6 mM. Aerosolized JM25-1 (1%) decreased lung eosinophil numbers from 13.2 ± 2.4 to 1.7 ± 0.7 × 10/µm (n = 6; P < 0.001) and neutrophils from 1.9 ± 0.4 to 0.2 ± 0.1 × 10/µm (n = 7; P < 0.001). Other parameters, including airway hyperreactivity, cytokines, mucus, and extracellular matrix deposition, were also sensitive to aerosolized JM25-1. CONCLUSION: These findings highlight the potential of JM25-1, emphasizing its putative value in drug development for clinical conditions where there is bronchospasm.

Quaternary Lidocaine Derivative QX-314 Activates and Permeates Human TRPV1 and TRPA1 to Produce Inhibition of Sodium Channels and Cytotoxicity.

BACKGROUND: The relatively membrane-impermeable lidocaine derivative QX-314 has been reported to permeate the ion channels transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential cation channel, subfamily A, member 1 (TRPA1) to induce a selective inhibition of sensory neurons. This approach is effective in rodents, but it also seems to be associated with neurotoxicity. The authors examined whether the human isoforms of TRPV1 and TRPA1 allow intracellular entry of QX-314 to mediate sodium channel inhibition and cytotoxicity. METHODS: Human embryonic kidney 293 (HEK-293) cells expressing wild-type or mutant human (h) TRPV1 or TRPA1 constructs as well as the sodium channel Nav1.7 were investigated by means of patch clamp and ratiometric calcium imaging. Cytotoxicity was examined by flow cytometry. RESULTS: Activation of hTRPA1 by carvacrol and hTRPV1 by capsaicin produced a QX-314-independent reduction of sodium current amplitudes. However, permeation of QX-314 through hTRPV1 or hTRPA1 was evident by a concentration-dependent, use-dependent inhibition of Nav1.7 activated at 10 Hz. Five and 30 mM QX-314 activated hTRPV1 via mechanisms involving the intracellular vanilloid-binding domain and hTRPA1 via unknown mechanisms independent of intracellular cysteins. Expression of hTRPV1, but not hTRPA1, was associated with a QX-314-induced cytotoxicity (viable cells 48 ± 5% after 30 mM QX-314) that was ameliorated by the TRPV1 antagonist 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide (viable cells 81 ± 5%). CONCLUSIONS: The study data demonstrate that QX-314 directly activates and permeates the human isoforms of TRPV1 and TRPA1 to induce inhibition of sodium channels, but also a TRPV1-dependent cytotoxicity. These results warrant further validation of this approach in more intact preparations and may be valuable for the development of this concept into clinical practice.

Systemic QX-314 Reduces Bone Cancer Pain through Selective Inhibition of Transient Receptor Potential Vanilloid Subfamily 1-expressing Primary Afferents in Mice.

BACKGROUND: The aim of this study was to determine whether systemic administration of QX-314 reduces bone cancer pain through selective inhibition of transient receptor potential vanilloid subfamily 1 (TRPV1)-expressing afferents. METHODS: A mouse model of bone cancer pain was used. The authors examined the effects of bolus (0.01 to 3 mg/kg, n = 6 to 10) and continuous (5 mg kg h, n = 5) administration of QX-314 on both bone cancer pain-related behaviors and phosphorylated cyclic adenosine monophosphate response element-binding protein expression in dorsal root ganglion neurons (n = 3 or 6) and the effects of ablation of TRPV1-expressing afferents on bone cancer pain-related behaviors (n = 10). RESULTS: The numbers of flinches indicative of ongoing pain in QX-314-treated mice were smaller than those in vehicle-treated mice at 10 min (3 mg/kg, 4 ± 3; 1 mg/kg, 5 ± 3 vs. 12 ± 3; P < 0.001; n = 8 to 9), 24 h (3 ± 2 vs. 13 ± 3, P < 0.001), and 48 h (4 ± 1 vs. 12 ± 2, P < 0.001; n = 5 in each group) after QX-314 administration, but impaired limb use, weight-bearing including that examined by the CatWalk system, and rotarod performance indicative of movement-evoked pain were comparable. QX-314 selectively inhibited the increase in phosphorylated cyclic adenosine monophosphate response element-binding protein expression in TRPV1-positive, but not in TRPV1-negative, dorsal root ganglion neurons compared to that in the case of vehicle administration (32.2 ± 3.0% vs. 52.6 ± 5.9%, P < 0.001; n = 6 in each group). Ablation of TRPV1-expressing afferents mimicked the effects of QX-314. CONCLUSION: This study showed that systemic administration of QX-314 in mice inhibits some behavioral aspects of bone cancer pain through selective inhibition of TRPV1-expressing afferents without coadministration of TRPV1 agonists.

Antitussive effect of carcainium chloride in patients with chronic cough and idiopathic interstitial pneumonias: A pilot study.

BACKGROUND: Cough is a common presenting symptom in patients with idiopathic interstitial pneumonia (IIP); it is often disabling, and lacks effective treatment. Studies in animals suggest that carcainium chloride, a quaternary derivative of the local anesthetic lidocaine, is able to inhibit experimentally induced cough by a mechanism of action distinct from that of lidocaine. OBJECTIVE: To determine the effectiveness of aerosolised carcainium chloride (VRP700) in controlling cough in patients with IIP. METHODS: Eight female patients (mean age 71 years) with IIP were investigated in a double blind, randomised, placebo controlled crossover, adaptive contingency study design (EudraCT Number 2010-021350-19). The study consisted of a screening visit to assess the eligibility of patients, and two separated (48-72 h) study days. On the two study days, patients were randomised to receive either nebulized VRP700 (1.0 mg/kg) on the first study visit followed by nebulised placebo (sodium chloride 0.9%) on the second visit, or placebo on the first visit followed by VRP700 on the second visit. The primary endpoint was cough frequency over a 4-h assessment period; secondary endpoints were subjective cough-related level of discomfort as assessed by a visual analogue scale (VAS) and the subjective response to treatment as assessed by a quality of life question. Safety (ECG, spirometry, urine and blood tests) and adverse events occurring during the trial were also investigated. RESULTS: In all patients both VRP700 and placebo decreased cough frequency; however, mean decreases in cough frequency after treatment with VRP700 were significantly (P < 0.001) higher than with placebo. Similarly, mean reductions in VAS score were significantly (P < 0.001) higher after treatment with VRP 700 compared with placebo. All but one patient indicated that they felt better after receiving VRP700. No adverse events were reported during the study, nor were any changes in ECG variables, spirometry, urine and blood tests noted. CONCLUSION: The results of this exploratory study indicate that nebulised VRP700 improved cough and quality of life in hospitalised IIP patients with no significant side effects. A larger trial is warranted to assess these promising results.