Highly sensitive analysis of tetrodotoxin based on free-label fluorescence aptamer sensing system.

Tetrodotoxin (TTX) specifically can bind to its nucleic acid aptamer (TTX-aptamer) and cause the conformation of TTX-aptamer to be switched from the single-strand random coiling form to the compact neck ring structure. Based on the microenvironment difference of the fluorescence reporter, berberine in between the single-stranded coil oligonucleotides and the structure of the neck ring, a simple, rapid and sensitive label-free fluorescence aptamer sensing system for detection of TTX was developed. Various factors affecting the analysis of TTX were optimized, including the concentration of berberine, ion strength, pH, reaction time, the concentration of TTX-aptamer. Under the optimal experimental conditions, the fluorescence intensity of the sensing system and the concentration of TTX showed a good linear relationship in the range of 0.1 nM to 500 nM, with the detection limit of 0.074 nM. The standard recovery test result exhibited that the recoveries of TTX in serum samples were 96.54%-106.40%. The established method has the advantages of high specificity, good sensitivity, quickness and convenience, low cost, and can be used for the detection of TTX in serum samples.

Optimal sampling time and clinical implication of the SCN5A promoter haplotype in propafenone therapeutic drug monitoring.

PURPOSE: The clinical usefulness of therapeutic drug monitoring (TDM) of propafenone, a sodium channel blocker, has been unclear due to the lack of information regarding optimal blood sampling time and therapeutic concentration range. Antiarrhythmic effects of sodium channel blockers are affected by the activity of the cardiac sodium channel (SCN5A). We investigated the optimal sampling time and the clinical implication of the SCN5A promoter haplotype in propafenone TDM. METHODS: We evaluated serum concentrations of propafenone, the SCN5A promoter haplotype, and antiarrhythmic efficacy in 55 patients with supraventricular tachy-arrhythmias. Blood samples obtained 1.5-6 and 10-24 h after the last dose were categorized as peak and trough samples, respectively. RESULTS: The peak propafenone concentration was significantly higher in effectively treated patients than that in patients showing insufficient response (337 ± 213 vs. 177 ± 93 ng/mL, P = 0.005), but the trough propafenone concentration was not significantly different between the two groups (68 ± 48 vs. 42 ± 36 ng/mL). Clinically relevant propafenone efficacy was achieved significantly more often in SCN5A haplotype B carriers than in wild-type haplotype A homozygotes (90 vs. 60%, P < 0.05). Among the haplotype A homozygotes, peak propafenone concentration was higher in effectively treated patients than that in patients showing insufficient response (299 ± 177 vs. 177 ± 93 ng/mL, P = 0.061). CONCLUSION: The present study found that antiarrhythmic efficacy of propafenone was associated with peak propafenone concentration rather than trough concentration and was affected by the SCN5A promoter haplotype.

Carbamazepine, a beta-cell protecting drug, reduces type 1 diabetes incidence in NOD mice.

Pancreatic beta-cells are selectively destroyed by the host immune system in type 1 diabetes. Thus, drugs that preserve beta-cell mass and/or function have the potential to prevent or slow the progression of this disease. We recently reported that the use-dependent sodium channel blocker, carbamazepine, protects beta-cells from inflammatory cytokines in vitro. Here, we tested the effects of carbamazepine treatment in female non-obese diabetic (NOD) mice by supplementing LabDiet 5053 with 0.5% w/w carbamazepine to achieve serum carbamazepine levels of 14.98 ± 3.19 µM. Remarkably, diabetes incidence over 25 weeks, as determined by fasting blood glucose, was ~50% lower in carbamazepine treated animals. Partial protection from diabetes in carbamazepine-fed NOD mice was also associated with improved glucose tolerance at 6 weeks of age, prior to the onset of diabetes in our colony. Less insulitis was detected in carbamazepine treated NOD mice at 6 weeks of age, but we did not observe differences in CD4+ and CD8+ T cell composition in the pancreatic lymph node, as well as circulating markers of inflammation. Taken together, our results demonstrate that carbamazepine reduces the development of type 1 diabetes in NOD mice by maintaining functional beta-cell mass.

Safety and Efficacy of a Topical Sodium Channel Inhibitor (TV-45070) in Patients With Postherpetic Neuralgia (PHN): A Randomized, Controlled, Proof-of-Concept, Crossover Study, With a Subgroup Analysis of the Nav1.7 R1150W Genotype.

OBJECTIVE: The objective was to evaluate the safety and efficacy of TV-45070 ointment, as a treatment for postherpetic neuralgia, and to explore the response in patients with the Nav1.7 R1150W gain-of-function polymorphism. MATERIALS AND METHODS: This was a randomized, placebo-controlled, 2-period, 2-treatment crossover trial. Patients with postherpetic neuralgia with moderate or greater pain received TV-45070 and placebo ointments, each applied twice daily for 3 weeks. The primary efficacy measure was the difference in change in mean daily pain score from baseline compared with the last week of placebo and active treatment. Secondary endpoints included responder rate analyses and a further exploratory analysis of response in carriers of the Nav1.7 R1150W polymorphism was conducted. RESULTS: Seventy patients were enrolled and 54 completed the study. TV-45070 was safe and well tolerated. No statistical difference was observed between treatments for the primary endpoint. However, the proportion of patients with ≥50% reduction in mean pain scores at week 3 was greater on TV-45070 than on placebo (26.8% vs. 10.7%, P=0.0039). Similarly, a greater proportion of patients on TV-45070 had a ≥30% reduction in mean pain scores at week 3 (39.3% on TV-45070 vs. 23.2% on placebo, P=0.0784). Of note, 63% of patients with the R1150W polymorphism versus 35% of wild-type carriers had a ≥30% reduction in mean pain score on TV-45070 at week 3 (no inferential analysis performed). CONCLUSIONS: The 50% responder analysis suggests a subpopulation may exist with a more marked analgesic response to TV-45070.The trend toward a larger proportion of responders within Nav1.7 R1150W carriers warrants further investigation.

A novel substituted aminoquinoline selectively targets voltage-sensitive sodium channel isoforms and NMDA receptor subtypes and alleviates chronic inflammatory and neuropathic pain.

Recent understanding of the systems that mediate complex disease states, has generated a search for molecules that simultaneously modulate more than one component of a pathologic pathway. Chronic pain syndromes are etiologically connected to functional changes (sensitization) in both peripheral sensory neurons and in the central nervous system (CNS). These functional changes involve modifications of a significant number of components of signal generating, signal transducing and signal propagating pathways. Our analysis of disease-related changes which take place in sensory neurons during sensitization led to the design of a molecule that would simultaneously inhibit peripheral NMDA receptors and voltage sensitive sodium channels. In the current report, we detail the selectivity of N,N-(diphenyl)-4-ureido-5,7-dichloro-2-carboxy-quinoline (DCUKA) for action at NMDA receptors composed of different subunit combinations and voltage sensitive sodium channels having different α subunits. We show that DCUKA is restricted to the periphery after oral administration, and that circulating blood levels are compatible with its necessary concentrations for effects at the peripheral cognate receptors/channels that were assayed in vitro. Our results demonstrate that DCUKA, at concentrations circulating in the blood after oral administration, can modulate systems which are upregulated during peripheral sensitization, and are important for generating and conducting pain information to the CNS. Furthermore, we demonstrate that DCUKA ameliorates the hyperalgesia of chronic pain without affecting normal pain responses in neuropathic and inflammation-induced chronic pain models.

Investigation of Metabolite Profile of YM758, a Novel If Channel Inhibitor.

BACKGROUND: YM758 monophosphate is a novel If channel inhibitor that has an inhibitory action for If current and shows a strong and specific activity, selectively lowering the heart rate and decreasing oxygen consumption by heart muscle. OBJECTIVES: The objectives of the current study were to investigate the in vivo metabolic profiles of YM758 in mice, rats, rabbits, dogs, and monkeys and to elucidate the structures of YM758 metabolites. METHODS: Biological samples were analyzed by liquid chromatography hyphenated with a radiometric detection system and liquid chromatography coupled with a mass spectrometer to clarify their metabolic patterns. To elucidate their structures, metabolites were isolated and analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy. RESULTS: Our results from in vivo metabolic profiling in humans and animals indicated there is no significant species difference in the metabolism of YM758, and the metabolic pathways of YM758 are considered to be oxidation, hydration, and demethylation followed by sulfate or glucuronide conjugation.

Effect of ranolazine on glycaemic control in patients with type 2 diabetes treated with either glimepiride or metformin.

AIM: To report the results of two phase III trials assessing the efficacy of ranolazine for glycaemic control in patients with type 2 diabetes on metformin or glimepiride background therapy. METHODS: In two double-blind trials we randomized 431 and 442 patients with type 2 diabetes to ranolazine 1000 mg twice daily versus placebo added to either glimepiride (glimepiride add-on study) or metformin background therapy (metformin add-on study). Patients receiving ranolazine added to metformin had their metformin dose halved (with the addition of a metformin-matched placebo) relative to the placebo group to correct for a metformin-ranolazine pharmacokinetic interaction. The primary endpoint of the trials was the change from baseline in glycated haemoglobin (HbA1c) at week 24. RESULTS: When added to glimepiride, ranolazine caused a 0.51% least squares mean [95% confidence interval (CI) 0.71, 0.32] decrease from baseline in HbA1c at 24 weeks relative to placebo and roughly doubled the proportion of patients achieving an HbA1c of <7% (27.1 vs 14.1%; p = 0.001). When added to metformin background therapy, there was no significant difference in the 24-week HbA1c change from baseline [placebo-corrected LS mean difference -0.11% (95% CI -0.31, 0.1)]. CONCLUSIONS: Compared with placebo, addition of ranolazine in patients with type 2 diabetes treated with glimepiride, but not metformin, significantly reduced HbA1c over 24 weeks. The decreased dose of metformin used in the metformin add-on study complicates the interpretation of this trial. Whether an effective regimen of ranolazine added to metformin for glycaemic control can be identified remains unclear.

Chronic toxicity study of neosaxitoxin in rats.

Neosaxitoxin (NeoSTX) is a specific reversible blocker of voltage gated sodium channels on excitable cells. In the last decade, it has been tested in a number of interesting clinical trials, however there is still little information available on mammalian toxicity. Rats were treated for 12 weeks with doses of 1, 3 or 6 µg/kg of subcutaneous NeoSTX. At weeks 12 and 17, animals were sacrificed and blood samples collected for hematological and biochemical analysis. Organs were harvested for weight determination and histopathological assessments. The lowest acute toxicity via the intraperitoneal (ip) route was (30.35 µg/kg) and there was no significant difference between intramuscular and subcutaneous routes (11.4 and 12.41 µg/kg). The NeoSTX adiministration did not produce lethality at week 12 and after five weeks of suspension. NeoSTX 6 µg/kg ip produced reductions (p < 0.05) in body weight and food intake, and increased blood level of total and direct bilirubin, GGT and SGOT at week 12; all of these were reversed in the recovery period. NeoSTX 1 and 3 µg/kg ip did not show significant changes with the control group. Histopathological presentations were normal in all groups. This study revealed that NeoSTX is safe in vivo, giving a reliable security margin for its use like a local anesthetic.

In vivo and ex vivo inhibition of spinal nerve ligation-induced ectopic activity by sodium channel blockers correlate to in vitro inhibition of NaV1.7 and clinical efficacy: a pharmacokinetic-pharmacodynamic translational approach.

PURPOSE: In vivo and ex vivo inhibition of ectopic activity of clinically used and newly developed sodium channel (NaV) blockers were quantified in the rat spinal nerve ligation (SNL) model using a pharmacokinetic-pharmacodynamic (PKPD) approach and correlated to in vitro NaV1.7 channel inhibition and clinical effective concentrations. METHODS: In vivo, drug exposure and inhibition of ectopic activity were assessed in anaesthetized SNL rats at two dose levels. Ex vivo, compounds were applied at increasing concentrations to dorsal root ganglias isolated from SNL rats. The inhibitory potency (IC 50 ) was estimated using PKPD analysis. In vitro IC 50 was estimated using an electrophysiology-based assay using recombinant rat and human NaV1.7 expressing HEK293 cells. RESULTS: In vivo and ex vivo inhibition of ectopic activity correlated well with the in vitro inhibition on the rat NaV1.7 channel. The estimated IC 50s for inhibition of ectopic activity in the SNL model occurred at similar unbound concentrations as clinical effective concentrations in humans. CONCLUSIONS: Inhibition of ectopic activity in the SNL model could be useful in predicting clinical effective concentrations for novel sodium channel blockers. In addition, in vitro potency could be used for screening, characterization and selection of compounds, thereby reducing the need for in vivo testing.

Influence of quercetin on the pharmacokinetics of ranolazine in rats and in vitro models.

The aim of our study was to enhance the bioavailability of ranolazine by using herbal-bioenhancer quercetin in rats and to study the role of P-glycoprotein (P-gp) in vitro models. In single dose study (SDS), rats were divided into four groups, Group I was treated with 0.5% sodium carboxy methyl cellulose (SCMC), Group II was treated with ranolazine (14 mg/kg), Group III was treated with quercetin (20 mg/kg) and Group IV was treated with both ranolazine and quercetin. The blood samples were collected at 0.5, 1, 2, 3, 4, 6, 8 and 12 h, and the concentration of ranolazine in the plasma was estimated by reverse phase high performance liquid chromatography (RP-HPLC) method. In multiple dose study (MDS), rats were treated with same drugs for 7 days. On 8th day, the concentration of ranolazine in plasma was estimated. In vitro study performed on the rat and chick intestinal sacs to study the intestinal transport of ranolazine in the presence and absence of quercetin and verapamil (P-gp-inhibitor). Quercetin increased the peak concentration (Cmax) of ranolazine from 254 ± 8.45 to 324 ± 10.21 and 331 ± 9.65 ng/mL in SDS and MDS, respectively. Quercetin also increased area under the curve (AUC) of ranolazine from 1565.12 ± 52.24 to 2016.98 ± 142.65 and 2070.85 ± 271.60 ng/mL/h in SDS and MDS, respectively. The transport of ranolazine from mucosal side to serosal side was increased in presence of quercetin. Quercetin is an inhibitor of CYP3A4 and P-gp. So it increased the AUC and Cmax of ranolazine.

Analytical challenges: determination of tetrodotoxin in human urine and plasma by LC-MS/MS.

Tetrodotoxin (TTX) is a powerful sodium channel blocker found in puffer fish and some marine animals. Cases of TTX poisoning most often result from puffer fish ingestion. Diagnosis is mainly from patient's signs and symptoms or the detection of TTX in the leftover food. If leftover food is unavailable, the determination of TTX in the patient's urine and/or plasma is essential to confirm the diagnosis. Although various methods for the determination of TTX have been published, most of them are for food tissue samples. Dealing with human urine and blood samples is much more challenging. Unlike in food, the amount of toxin in the urine and blood of a patient is generally extremely low; therefore a very sensitive method is required to detect it. In this regard, mass spectrometry (MS) methods are the best choice. Since TTX is a very polar compound, there will be lack of retention on conventional reverse-phase columns; use of ion pair reagent or hydrophilic interaction liquid chromatography (HILIC) can help solve this problem. The problem of ion suppression is another challenge in analyzing polar compound in biological samples. This review will discuss different MS methods and their pros and cons.

Translation of flecainide- and mexiletine-induced cardiac sodium channel inhibition and ventricular conduction slowing from nonclinical models to clinical.

INTRODUCTION: Nonclinical in vivo models used for cardiovascular safety testing have not previously been studied for their sensitivity for detection of conduction slowing resulting from cardiac sodium channel block. The goal of this study was to examine the sensitivity of in vivo models to cardiac sodium channel block, and translation of the effect from in vitro to in vivo models using sodium channel inhibitors flecainide and mexiletine; flecainide, but not mexiletine is commonly associated with QRS complex prolongation in humans. METHODS: Inhibition of cloned cardiac sodium channels (hNav1.5) was studied using the IonWorks platform. Conduction slowing was measured in vitro in the rabbit isolated ventricular wedge (RVW) and in vivo in the conscious telemetered rat and dog, and anaesthetised dog. RESULTS: Flecainide and mexiletine inhibited hNav1.5 channels with IC50 values of 10.7 and 67.2 µM respectively. In the RVW, QRS was increased by flecainide at 60 bpm, and at 120bpm, there was an increased effect of both drugs. In conscious rats, flecainide significantly increased QRS complex duration; mexiletine had no significant effect, but there was an increase at the highest dose in 4/6 animals. QRS complex was increased by flecainide and mexiletine in anaesthetised dogs but this was not statistically significant; in conscious dog, only flecainide produced a significant increase in QRS complex. DISCUSSION: When compared to clinical data, effects of flecainide and mexiletine in RVW and conscious dog compared well with effects in patients and healthy volunteers in terms of sensitivity. The anaesthetised dog was least sensitive for detection of changes in QRS. All assays showed some differentiation between the expected conduction slowing activity of flecainide and mexiletine. Based on these data, RVW and conscious dog were most predictive for effects of compounds on QRS complex and cardiac conduction.

Development and validation of a high-throughput double solid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination of tetrodotoxin in human urine and plasma.

A sensitive analytical method for the determination of tetrodotoxin (TTX) in urine and plasma matrices was developed using double solid phase extraction (C18 and hydrophilic interaction liquid chromatography) and subsequent analysis by HPLC coupled with tandem mass spectrometry. The double SPE sample cleanup efficiently reduced matrix and ion suppression effects. Together with the use of ion pair reagent in the mobile phase, isocratic elution became possible which enabled a shorter analysis time of 5.5 min per sample. The assay results were linear up to 500 ng mL(-1) for urine and 20 ng mL(-1) for plasma. The limit of detection and limit of quantification were 0.13 ng mL(-1) and 2.5 ng mL(-1), respectively, for both biological matrices. Recoveries were in the range of 75-81%. To eliminate the effect of dehydration and variations in urinary output, urinary creatinine-adjustment was made. TTX was quantified in eight urine samples and seven plasma samples from eight patients suspected of having TTX poisoning. TTX was detected in all urine samples, with concentrations ranging from 17.6 to 460.5 ng mL(-1), but was not detected in any of the plasma samples. The creatinine-adjusted TTX concentration in urine (ranging from 7.4 to 41.1 ng µmol(-1) creatinine) correlated well with the degree of poisoning as observed from clinical symptoms.

[Flecainide poisoning]. / Intoxicación por flecainida.

Flecainide is an antiarrhythmic drug that blocks sodium channels during phase 0 of cardiac action potential, delaying conduction and reducing contractility. Intoxication by this drug is rare. Onset of effect, which is rapid, takes the form of hypotension and cardiac arrhythmias; mortality is high. No antidote is available and management is based on the few cases that have been reported. The metabolism of flecainide is affected by both kidney and liver failure, which lead to accumulation of the drug. Flecainide should not be used in patients with such failure unless the potential benefits clearly outweigh the risks. If flecainide is prescribed, diligent clinical, electrocardiographic, and hemodynamic vigilance is imperative and plasma levels of the drug should be monitored. We report a case of flecainide poisoning in which the drug was prescribed to treat atrial fibrillation in a woman with resolving sepsis with renal and hepatic complications.

Lidocaine-magnesium blood cardioplegia was equivalent to potassium blood cardioplegia in left ventricular function of canine heart.

This study evaluated the effects of lidocaine-magnesium blood cardioplegia on left ventricular function compared with potassium blood cardioplegia. Crystalloid cardioplegia which contains lidocaine has been reported but blood cardioplegia is rare. Thirteen dogs received 60 min of global ischemia under hypothermic cardioplumonary bypass (30 degrees C). Potassium blood cardioplegia was administered every 20 min in group A (n=6), and lidocaine-magnesium blood cardioplegia in group B (n=7). We compared the ratio of Emax obtained during IVC occlusion at pre- and post-global ischemia (%Emax) and LVSW (%LVSV). Cardiac function was evaluated prior to CPB and 60 min after reperfusion. There was no difference in time required for cardiac arrest between the two groups (group A: 78+/-3 s, group B: 89+/-9 s). Percentage maximal elastance was significantly better in group B (group A: 63+/-3%, group B: 76+/-4%, P<0.05). Percentage tissue water content of the myocardium after CPB was significantly lower in group B (group A: 82.3+/-4%, group B: 75.5+/-2%, P<0.05). Lidocaine-magnesium blood cardioplegia was equivalent to potassium blood cardioplegia in systolic left ventricular function and reduced myocardial edema in canine heart.

Phase 1A safety assessment of intravenous amitriptyline.

UNLABELLED: The antidepressant amitriptyline is used as an adjuvant in the treatment of chronic pain. Among its many actions, amitriptyline blocks Na+ channels and nerves in several animal and human models. As perioperative intravenous lidocaine has been suggested to decrease postoperative pain, amitriptyline, because of its longer half-life time, might be more useful than lidocaine. However, the use of intravenous amitriptyline is not approved by the US Food and Drug Administration. We therefore investigated the adverse effects of preoperative intravenous amitriptyline in a typical phase 1A trial. After obtaining written Food and Drug Administration and institutional review board approval, we obtained written consent for preoperative infusion of amitriptyline in an open-label, dose-escalating design (25, 50, and 100 mg, n=5 per group). Plasma levels of amitriptyline/nortriptyline were determined, and adverse effects were recorded in a predetermined symptom list. Infusion of 25 and 50 mg amitriptyline appears to be well tolerated; however, the study was terminated when 1 subject in the 100-mg group developed severe bradycardia. Intravenous infusion of amitriptyline (25 to 50 mg over 1 hour) did not create side effects beyond dry mouth and drowsiness, or dizziness, in 2 of our 10 otherwise healthy participants receiving the 25- to 50-mg dose. An appropriately powered future trial is necessary to determine a potential role of amitriptyline in decreasing postoperative pain. PERSPECTIVE: Amitriptyline potently blocks the persistently open Na+ channels, which are known to be instrumental in various pain states. As this occurs at very low plasma concentrations, a single preoperative intravenous infusion of amitriptyline could provide long-lasting pain relief and decrease the incidence of chronic pain.

Effect of gender on the pharmacokinetics of eslicarbazepine acetate (BIA 2-093), a new voltage-gated sodium channel blocker.

PURPOSE: To determine the effect of gender on the pharmacokinetics of eslicarbazepine acetate, a novel voltage-gated sodium channel blocker in the development for the treatment of epilepsy and bipolar disorder. METHODS: Single-centre, open-label, parallel-group study in 12 female and 12 male healthy subjects. The study consisted of a single-dose (600 mg) period and a multiple-dose (600 mg, once-daily, for 8 days) period, separated by 4 days. RESULTS: Eslicarbazepine acetate was rapidly and extensively metabolized to eslicarbazepine (S-licarbazepine), the main active metabolite. Following a single-dose, arithmetic mean eslicarbazepine maximum plasma concentrations (C(max)) and area under the plasma concentration-time curve over 24 h (AUC(0-24)) and from 0 to infinity (AUC(0-infinity)) were, respectively, 9.3 microg/ml, 128.5 microg h/ml and 171.9 microg h/ml in male subjects and 10.1 microg/ml, 150.1 microg h/ml and 205.0 microg h/ml in female subjects. At steady-state, C(max), AUC(0-24) and AUC(0-infinity) were 15.5 microg/ml, 207.8 microg h/ml and 295.8 microg h/ml in male subjects, and 16.8 microg/ml, 214.5 microg h/ml and 295.2 microg h/ml in female subjects. Steady-state plasma concentrations were attained at 4 to 5 days of administration in both groups. Eslicarbazepine C(max), AUC(0-24) and AUC(0-infinity) female:male geometric mean ratios (90%CI) were, respectively, 1.09 (0.94; 1.24), 1.16 (1.00; 1.33) and 1.17 (0.99; 1.38) following single-dose, and 1.10 (0.97; 1.25), 1.04 (0.92; 1.17) and 1.01 (0.88; 1.16) at steady-state. CONCLUSION: At steady-state, the pharmacokinetic profile of eslicarbazepine acetate was not affected by gender.

Sample preparation and RPHPLC determination of diuretics in human body fluids.

This article describes reverse phase high-performance liquid chromatography (RPHPLC) methods for determination of diuretics in different human body fluids (whole blood, plasma, serum or urine). Sample preparation procedures, including solid-phase extraction, liquid-liquid extraction, dilution, precipitation as well as automated RPHPLC procedures, are discussed in order to present the advantages and disadvantages of each type of sample preparation. Also, values of analytical recovery of each procedure used for sample preparation are summarized. The most important RPHPLC parameters (detection mode, stationary phase, mobile phase, sensitivity, etc.) are also summarized and discussed.