Quantitative determination of talatisamine and its pharmacokinetics and bioavailability in mouse plasma by UPLC-MS/MS.

Talatisamine, as the efficacy ingredient of Aconitum, was known as a novel specific blocker for the delayed rectifier K+ channels in rat hippocampal neurons. In this study, a rapid, selective and reproducible UPLC-MS/MS separation method was established and fully validated for the quantitative determination of talatisamine levels in ICR (Institute of Cancer Research) mouse blood. A total of 24 healthy male ICR mice were divided into four groups that was administered talatisamine via intravenous at a dose of 1 mg/kg and oral administration of three doses (2, 4, 8 mg/kg). All blood samples were protein precipitate by using acetonitrile with an internal standard (IS) deltaline. The effective chromatographic separation was carried out through an UPLC BEH C18 analytical column (2.1 mm × 50 mm, 1.7 µm) with an initial mobile phase that consisted of acetonitrile and 10 mmol/L ammonium acetate aqueous solution (containing 0.1% formic acid) with a gradient elution pumped at a flow rate of 0.4 mL/min. Also, an electrospray ionization (ESI) was applied to quantify the talatisamine in the positive ions mode. The method validation demonstrated good linearity over the range of 1-1000 ng/mL (r2 ≥ 0.9993) for talatisamine in mouse blood with a lower limit of quantification (LLOQ) at 1 ng/mL. The accuracy values of the method were within 89.4% to 113.3%, and the matrix effects were between 103.2% and 106.3%. The mean extraction recoveries for talatisamine obtained from four concentrations of QC blood samples were exceeded 71.7%, and the relative standard deviation (RSD) both of intra- and inter-day precision values for replicate quality control samples did not exceed 15% respectively for all analytes during the assay validation. This method was successfully applied to the evaluation of the pharmacokinetic of talatisamine, regardless of intragastric or intravenous administration in mice. Based on the pharmacokinetics data, the bioavailability of talatisamine in mice was >65.0% after oral administration, exhibiting an excellent oral absorption.

Population Pharmacokinetics/Pharmacodynamics of 3,4-Diaminopyridine Free Base in Patients With Lambert-Eaton Myasthenia.

Lambert-Eaton myasthenia (LEM) is a rare autoimmune disorder associated with debilitating muscle weakness. There are limited treatment options and 3,4-diaminopyridine (3,4-DAP) free base is an investigational orphan drug used to treat LEM-related weakness. We performed a population pharmacokinetic/pharmacodynamic (PK/PD) analysis using 3,4-DAP and metabolite concentrations collected from a phase II study in patients with LEM. The Triple Timed Up & Go (3TUG) assessment, which measures lower extremity weakness, was the primary outcome measure. A total of 1,270 PK samples (49 patients) and 1,091 3TUG data points (32 randomized patients) were included in the PK/PD analysis. A two-compartment and one-compartment model for parent and metabolite, respectively, described the PK data well. Body weight and serum creatinine partially explained the variability in clearance for the final PK model. A fractional inhibitory maximum effect (Emax ) model characterized the exposure-response relationship well. The PK/PD model was applied to identify a suggested dosing approach for 3,4-DAP free base.

Efavirenz Inhibits the Human Ether-A-Go-Go Related Current (hERG) and Induces QT Interval Prolongation in CYP2B6*6*6 Allele Carriers.

BACKGROUND: Efavirenz (EFV) has been associated with torsade de pointes despite marginal QT interval lengthening. Since EFV is metabolized by the cytochrome P450 (CYP) 2B6 enzyme, we hypothesized that EFV would lengthen the rate-corrected QT (QTcF) interval in carriers of the CYP2B6*6 decreased functional allele. OBJECTIVE: The primary objective of this study was to evaluate EFV-associated QT interval changes with regard to CYP2B6 genotype and to explore mechanisms of QT interval lengthening. METHODS: EFV was administered to healthy volunteers (n = 57) as a single 600 mg dose followed by multiple doses to steady-state. Subjects were genotyped for known CYP2B6 alleles and ECGs and EFV plasma concentrations were obtained serially. Whole-cell, voltage-clamp experiments were performed on cells stably expressing hERG and exposed to EFV in the presence and absence of CYP2B6 expression. RESULTS: EFV demonstrated a gene-dose effect and exceeded the FDA criteria for QTcF interval prolongation in CYP2B6*6/*6 carriers. The largest mean time-matched differences ∆∆QTcF were observed at 6 hours (14 milliseconds; 95% CI [1; 27]), 12 hours (18 milliseconds; 95% CI [-4; 40]), and 18 hours (6 milliseconds; 95% CI [-1; 14]) in the CYP2B6*6/*6 genotype. EFV concentrations exceeding 0.4 µg/mL significantly inhibited outward hERG tail currents (P < 0.05). CONCLUSIONS: This study demonstrates that homozygous carriers of CYP2B6*6 allele may be at increased risk for EFV-induced QTcF interval prolongation via inhibition of hERG.

Non-cardiovascular drugs that inhibit hERG-encoded potassium channels and risk of sudden cardiac death.

BACKGROUND: Virtually all QTc-prolonging drugs act by blocking the human ether a go-go-related gene (hERG)-encoded potassium channels (hERG channels), whereas not all QTc-prolonging drugs are associated with an increased risk of serious cardiac arrhythmias. This study assessed whether non-cardiovascular hERG channel blockers are associated with an increased risk of sudden cardiac death (SCD) and whether hERG-channel-inhibiting capacity is an indicator of the risk of SCD. METHODS AND RESULTS: The risk of SCD was studied in the Integrated Primary Care Information database, a longitudinal general practice research database. A case-control study was performed, matched for age, gender and calendar time. Odds ratios were calculated with conditional logistic regression, multivariably adjusted. In addition, the hERG-channel-inhibiting capacity of the different drugs was compared, defined as the effective free therapeutic plasma concentration (ETCP(unbound)) divided by the concentration that inhibits 50% of the potassium channels (IC50), with the risk of SCD. 1424 cases of SCD and 14 443 controls were identified. Current use of hERG channel blockers was associated with an increased risk of SCD. The risk of SCD was significantly increased in users of antipsychotic drugs. Patients using hERG channel blockers with a high ETCP(unbound)/IC50 ratio (≥ 0.033) had a higher risk of SCD than patients using drugs with a low ETCP(unbound)/IC50 ratio (<0.033). CONCLUSIONS: The current use of hERG channel blockers was associated with an increased risk of SCD in the general population. In addition, drugs with a high hERG-channel-inhibiting capacity had a higher risk of SCD than drugs with a low hERG-channel-inhibiting capacity.

Preclinical electrophysiology assays of mitemcinal (GM-611), a novel prokinetic agent derived from erythromycin.

Mitemcinal (GM-611) is a novel erythromycin-derived prokinetic agent that acts as an agonist at the motilin receptor. Erythromycin has shown QT prolongation and torsades de pointes (TdP) in humans and cisapride, a second class of prokinetic agents typified by the 5-HT(4) receptor agonist, has been terminated due to TdP. In this study an extended series of safety pharmacology protocols and evaluations have been undertaken to assess the potential risk of mitemcinal on QT prolongation or proarrhythmic effects. Mitemcinal and its metabolites, GM-577 and GM-625, inhibited the human ether-a-go-go-related gene (HERG) tail current in a concentration-dependent manner with IC(50) values of 20.2, 41.7, and 55.0 microM, respectively. Administration of 10 mg/kg mitemcinal in anesthetized guinea pigs resulted in a slight prolongation of the monophasic action potential (MAP) duration during atrial pacing at the plasma concentration of mitemcinal 1.1 microM, with low maximum increases in MAPD(70) (6.6%) and MAPD(90) (4.6%) relative to vehicle. A 10-min infusion of 20 mg/kg of mitemcinal in a proarrhythmic rabbit model did not evoke TdP even when QT and corrected QT (QTc) intervals were significantly prolonged. In this study, the Cmax plasma-free concentration of mitemcinal indicates that the prolongation was more than 400-fold that of the therapeutic dose. Our findings of a wide safety margin and the absence of TdP within this margin suggest that mitemcinal may provide sufficient safety in clinical use.

Targeting effector memory T cells with the small molecule Kv1.3 blocker PAP-1 suppresses allergic contact dermatitis.

The voltage-gated potassium channel Kv1.3 has been recently identified as a molecular target that allows for selective pharmacological suppression of effector memory T (T(EM)) cells without affecting the function of naïve and central memory T cells. We here investigated whether PAP-1, a small molecule Kv1.3 blocker (EC50=2 nM), could suppress allergic contact dermatitis (ACD). In a rat model of ACD, we first confirmed that the infiltrating cells in the elicitation phase are indeed CD8+ CD45RC- memory T cells with high Kv1.3 expression. In accordance with its selective effect on T(EM) cells, PAP-1 did not impair sensitization, but potently suppressed oxazolone-induced inflammation by inhibiting the infiltration of CD8+ T cells and reducing the production of the inflammatory cytokines IFN-gamma, IL-2, and IL-17 when administered intraperitoneally or orally during the elicitation phase. PAP-1 was equally effective when applied topically, demonstrating that it effectively penetrates skin. We further show that PAP-1 is not a sensitizer or an irritant and exhibits no toxicity in a 28-day toxicity study. Based on these results we propose that PAP-1 could potentially be developed into a drug for the topical treatment of inflammatory skin diseases such as psoriasis.

Effect of the oral hypoglycaemic sulphonylurea glibenclamide, a blocker of ATP-sensitive potassium channels, on walking distance in patients with intermittent claudication.

AIMS: The oral hypoglycaemic sulphonylurea glibenclamide stimulates endogenous insulin secretion through blockade of ATP-sensitive potassium (KATP) channels on pancreatic beta cells, but also blocks cardiovascular KATP channels, leading to increased peripheral vascular resistance and reduced peripheral blood flow in non-diabetic subjects. Therefore, this study examined whether a single oral dose of glibenclamide adversely affected the pain-free or maximal walking distance in patients with intermittent claudication. METHODS: In a double-blind, randomized crossover study, 12 non-diabetic patients with intermittent claudication were given a single oral dose of glibenclamide (5.25 mg) or placebo separated by a washout period of 1 week. A treadmill test was carried out 180 min after glibenclamide/placebo intake for determination of pain-free and maximal walking distance. Plasma glucose concentrations were kept constant by an euglycemic clamp. Changes in ankle/brachial blood pressure index (ABI), serum insulin, and serum glibenclamide were also assessed. RESULTS: The pain-free walking distance was 62.8 +/- 9.8 metres (mean +/- sem) after glibenclamide and 52.6 +/- 5.9 metres after placebo (P = 0.52). The maximal walking distance was 142.7 +/- 18.7 metres after glibenclamide and 132.6 +/- 16.6 metres after placebo (P = 0.23). The ABI was not significantly changed by glibenclamide compared with placebo. Serum glibenclamide was 0.51 +/- 0.08 microm 180 min after administration of the drug. Glibenclamide produced an 8-fold increase in circulating insulin compared with placebo (P < 0.001). CONCLUSIONS: Glibenclamide given as a single oral dose commonly used in glucose-lowering drug therapy does not reduce pain-free or maximal walking distance in non-diabetic patients with intermittent claudication.

Anti-HERG activity and the risk of drug-induced arrhythmias and sudden death.

AIMS: Drug-induced QTc-prolongation, resulting from inhibition of HERG potassium channels may lead to serious ventricular arrhythmias and sudden death. We studied the quantitative anti-HERG activity of pro-arrhythmic drugs as a risk factor for this outcome in day-to-day practice. METHODS AND RESULTS: All 284,426 case reports of suspected adverse drug reactions of drugs with known anti-HERG activity received by the International Drug Monitoring Program of the World Health Organization (WHO-UMC) up to the first quarter of 2003, were used to calculate reporting odds ratios (RORs). Cases were defined as reports of cardiac arrest, sudden death, torsade de pointes, ventricular fibrillation, and ventricular tachycardia (n = 5591), and compared with non-cases regarding the anti-HERG activity, defined as the effective therapeutic plasma concentration (ETCPunbound) divided by the HERG IC50 value, of suspected drugs. We identified a significant association of 1.93 (95% CI: 1.89-1.98) between the anti-HERG activity of drugs, measured as log10 (ETCPunbound/IC50), and reporting of serious ventricular arrhythmias and sudden death to the WHO-UMC database. CONCLUSION: Anti-HERG activity is associated with the risk of reports of serious ventricular arrhythmias and sudden death in the WHO-UMC database. These findings are in support of the value of pre-clinical HERG testing to predict pro-arrhythmic effects of medicines.

A comparison of the receptor binding and HERG channel affinities for a series of antipsychotic drugs.

Many antipsychotic drugs produce QT interval prolongation on the electrocardiogram (ECG). Blockade of the human cardiac K(+) channel known as human ether-a-go-go-related gene (HERG) often underlies such clinical findings. In fact, HERG channel inhibition is now commonly used as a screen to predict the ability of a drug to prolong QT interval. However, the exact relationship between HERG channel blockade, target receptor binding affinity and clinical QT prolongation is not known. Using patch-clamp electrophysiology, we examined a series of seven antipsychotic drugs for their ability to block HERG, and determined their IC(50) values. We then compared these results to their binding affinities (K(i) values) for the dopamine D(2) receptor, the 5-HT(2A) receptor and, where available, to clinical QT prolongation data. We found that sertindole, pimozide and thioridazine displayed little (<10-fold) or no selectivity for dopamine D(2) or 5-HT(2A) receptors relative to their HERG channel affinities. This lack of selectivity likely underlies the significant QT interval prolongation observed with administration of these drugs. Of the other drugs tested (ziprasidone, quetiapine, risperidone and olanzapine), olanzapine displayed the greatest selectivity for dopamine D(2) and 5-HT(2A) receptor binding (100-1000-fold) compared to its HERG channel IC(50). We also compared these HERG channel IC(50) values to QT interval prolongation and plasma drug levels obtained in a recent clinical study. We found that the ratio of total plasma drug concentration to HERG IC(50) value was indicative of the degree of QT prolongation observed. Target receptor affinity and expected clinical plasma levels are important parameters to consider for the interpretation of HERG channel data.