Na+ channel modulation and force-frequency relationship in human myocardium

The enhancement of force of contraction (FOC) following increasing frequencies of stimulation is an important mechanism of positive inotropy in human myocardium. The present study aimed to investigate the influence of alterations in Na⁺ influx on FFR in human myocardium. Isometric FOC of electrically stimulated right auricular trabeculae (AUT, n=12) from human nonfailing hearts (n=8) was measured at different stimulation rates (0.5-3 Hz) under control conditions, after increasing Na⁺ influx by the addition of (±)BDF 9148 (BDF, 3 μmol l^-1) and after decreasing Na⁺ influx by the addition of lidocaine (LIDO, 10 μmol l^-1). Additionally, the rate dependent changes in diastolic tension (DT) were measured in all experiments. Under control conditions FOC increased with increasing frequencies of stimulation. The rate at which maximal FOC was observed (SFmax) was 2.0±0.2 Hz and maximal increase in FOC (PIEmax) by increasing frequency of stimulation was +1.5±0.5 mN. After increase of Na⁺ influx by BDF (3 μmol l^-1) SFmax was decreased to 0.8±0.1 Hz (p<0.05 versus control) and PIEmax was +0.1±0.3 mN (p<0.05). When Na⁺ influx was diminished by LIDO (10 μmol l^-1) SFmax and PIEmax were increased compared to control (2.4±0.1 Hz and +4.1±0.9 mN, p<0.05 versus control). The diastolic tension (DT) of AUT at 3 Hz was not changed at higher rates in the control group and after application of LIDO (10 μmol l^-1), whereas after enhancement of Na⁺ influx by BDF there was an increase in DT of +0.7±0.2 at 3Hz (p<0.05 versus control and LIDO). An enhanced Na⁺ influx leads to a decrease in the optimal frequency and to a smaller force potentiation by higher stimulation rates which could be at least partly due to incomplete relaxation at higher frequencies, whereas a reduced Na⁺ influx is followed by opposite alterations. It is concluded that besides Ca²⁺ handling also Na⁺ influx and Na⁺ homeostasis might determine the frequency-induced force potentiation in human myocardium. Thus, the negative FFR in diseased human myocardium might result from changes in cellular Ca²⁺ or Na⁺ regulatory sites. Received: 20 November 1996 / Accepted: 21 February 1997     

The effect of omeprazole on the pharmacokinetics of metronidazole and hydroxymetronidazole in human plasma, saliva and gastric juice

Aims To evaluate the effect of omeprazole on the pharmacokinetics of metronidazole and hydroxymetronidazole in plasma, gastric juice and saliva following intravenous infusion or oral dosing of metronidazole. Methods Eight volunteers received single doses of metronidazole (400 mg) intravenously and orally, whilst taking placebo or omeprazole (40 mg, twice daily for 5 days) in a randomized 4-way crossover study. Metronidazole and hydroxymetronidazole concentrations in plasma, saliva and gastric juice samples were determined by h.p.l.c. Pharmacokinetic parameters for metronidazole and hydroxymetronidazole were calculated, and the significance of the mean differences in parameters between omeprazole and placebo co-administration was assessed using a two-tailed, paired t-test. Results There were no significant differences (P<0.05) in any of the plasma or saliva pharmacokinetic parameter values for metronidazole between volunteers receiving omeprazole or placebo when metronidazole was administered either as an intravenous infusion or orally. Following intravenous administration of metronidazole to the placebo group and omeprazole treated group respectively, the gastric transfer of metronidazole was significantly reduced from 15.5±10.4% to 2.6±1.0% of the dose (P=0.007; 95% CI of difference 4.8 to 21.0) with concomitant changes in the metronidazole AUC (from 77.5±18.0 μmol l⁻¹ h to 352.6±182.1 μmol l⁻¹ h; P=0.0003; 95% CI of difference 127.6 to 422.7), C_max (from 61.4±26.5 μmol l⁻¹ to 271.8±104.3 μmol l⁻¹; P=0.0001; 95% CI of difference 118.6 to 302.1). Similarly, the gastric juice AUC of hydroxymetronidazole was significantly reduced from 3.2±1.9 μmol l⁻¹ h to 1.5±0.8 μmol l⁻¹ h of the dose (P=0.0043; 95% CI of difference 0.4 to 3.0) with a concomitant change in C_max (from 5.0±2.5 μmol l⁻¹ to 3.0±1.2 μmol l⁻¹; P=0.0007; 95% CI of difference 0.7 to 3.4). Conclusions Omeprazole had little effect on the plasma and salivary pharmacokinetics of metronidazole (or its hydroxymetabolite) after intravenous or oral administration, but it did have a substantial effect on the pharmacokinetics of metronidazole and hydroxymetronidazole in gastric juice.     

Na+-channel modulating effect of the inotropic compound S(-)BDF 9196 in human myocardium

S(-)BDF 9196, the active enantiomer of racemic (±)BDF 9148, has been shown to increase force of contraction in myocardium from different species including humans. The present study aimed to investigate the mechanism of the positive inotropic action of the active enantiomer S(-)BDF 9196 in human myocardium. In electrically driven human left ventricular papillary muscle strips (dilated cardiomyopathy, NYHA IV, cardiac transplantation, n=9), S(-)BDF 9196 increased force of contraction concentration-dependently. The maximal positive inotropic effect remained unchanged after the addition of carbachol (1 mmol/l), indicating a cAMP-independent mode of action of S(-)BDF 9196. While [³H]ouabain binding in human myocardial membranes was not influenced by S(-)BDF 9196 up to 10 μmol/l, the inward Na⁺-current in isolated human left ventricular myocytes was increased significantly by S(-)BDF 9196 (1 μmol/l, n=5). These results provide evidence that S(-)BDF 9196 increases force of contraction in human myocardium primarily by enhancing Na⁺-influx, while cAMP-dependent or Na⁺,K⁺-ATPase blocking effects do not seem to play a role. Received: 12 May 1998 / Accepted: 5 October 1998     

In vitro adrenaline and collagen-induced mobilization of platelet calcium and its inhibition by naftopidil, doxazosin and nifedipine

Aims The aim of the study was to obtain further information regarding the modes of action of doxazosin, naftopidil and nifedipine on platelet function.Methods We conducted an in vitro study of drug influences on adrenaline and collagen-induced mobilization of platelet calcium.‘fn2\Results In the presence of fibrinogen (300 μg ml⁻¹ ) both collagen (5 μg ml⁻¹ ) and adrenaline (16 μm ) stimulated the aggregation of washed platelets. Collagen induced a transient rise (+4.97±0.63 μm ) in platelet Ca²⁺ concentration, [Ca²⁺]_i, as measured using the photoprotein aequorin, which coincided with the onset of aggregation. Adrenaline induced a smaller rise (+3.6±0.96 μm ) which, however, occurred after the onset of aggregation. Naftopidil, an α₁-adrenoreceptor antagonist produced a concentration-dependent inhibition of collagen-induced Ca²⁺ mobilization, maximum inhibition (22.9±4%, P<0.05) occurring with 40 μm naftopidil. The inhibition of Ca²⁺ mobilization was not reflected by a concentration-dependent inhibition of platelet aggregation, although 40 μm naftopidil produced statistically significant inhibition (23.3±11.7%, P<0.05). The adrenaline-induced rise in [Ca²⁺]_i was inhibited dose dependently by naftopidil (e.g. 40 μm naftopidil, 100±0%, P<0.05), as was aggregation (40 μm naftopidil, 100±0%, P<0.05). Doxazosin, another α₁-adrenoreceptor blocker, inhibited Ca²⁺ mobilization induced by collagen to similar extents as for naftopidil (30 μm doxazosin, 17.4±2.5%, P<0.05), but did not inhibit platelet aggregation. It also inhibited the adrenaline-induced rise in [Ca²⁺]_i in a concentration-dependent manner (30 μm doxazosin, 37.6±13.7%, P<0.05), significant inhibitions of platelet aggregation also being produced (30 μm, 49.6±17.2%, P<0.05). As expected, the calcium channel blocker nifedipine produced concentration-dependent inhibitions of both collagen-induced Ca²⁺ mobilization (e.g. 28 μm nifedipine, 47.8±2.7%, P<0.05) and aggregation (28 μm, 55.1±9.2%, P<0.05).Conclusions These data indicate that the α₁-adrenoreceptor blockers, naftopidil and doxazosin, inhibit Ca²⁺ mobilization, this mechanism being possibly the means whereby these drugs inhibit platelet aggregation.     

Inhibition of vasoconstriction by potassium channel opener aprikalim in human conduit arteries used as bypass grafts

Aims Potassium channel openers (KCOs) are of potential therapeutic value. Little is known about the effect of these drugs on human conduit arteries used as coronary bypass grafts. The purpose of this study was to determine the effect of the KCO aprikalim (RP52891) on human arteries used as coronary bypass grafts with emphasis on the possible difference in the inhibitory effect on depolarizing agent-mediated rather than receptor-mediated contraction. Methods Human internal mammary artery segments (IMA, n=88) taken from 28 patients were studied. Concentration-relaxation curves for aprikalim were established in IMA precontracted with three vasoconstrictors (K⁺, U46619, and phenylephrine). In IMA rings incubated with aprikalim (1 or 30 μm ) for 10 min concentration-contraction curves for the three vasoconstrictors were constructed. Results Aprikalim-induced relaxation was less in K⁺ (37.3±6.4%) than in U46619 (80.2±7.7%, P=0.002), or phenylephrine (67.5±7.0%, P=0.038) -precontracted IMA. The EC₅₀ for K⁺-(−5.40±0.12 log m ) was significantly higher than that for phenylephrine (−6.43±0.30 log m, P=0.007) but not significant compared with that for U46619 (−5.81±0.11, P >0.05). Pretreatment with aprikalim depressed the contraction by phenylephrine from 140.6±27.6% to 49.3±14.1% (P=0.002) and shifted the EC₅₀ 11.0-fold higher in rings treated with 1 μm aprikalim (P=0.007). Treatment of aprikalim did not significantly reduce the K⁺ and U46619-induced contraction (P >0.05) but shifted the concentration-contraction curves rightward (2.8-fold higher for K⁺, P<0.05 and 2.2-fold higher for U46619, P<0.05). Conclusions This study demonstrates that aprikalim has vasorelaxant effects in human conduit arteries used as coronary artery bypass grafts contracted by a variety of vasoconstrictors and this effect is vasoconstrictor-selective with greater potency for α₁-adrenoceptor agonists than for depolarizing agent K⁺. These findings provide information on the possible use of this KCO in various clinical settings.     

Effect of the Na+-channel modulator BDF 9148 on Ca2+-sensitivity and force of contraction of hypertrophic myocardium from transgene rats harboring the mouse Renin gene (TG(mREN2)27)

The present study aimed to investigate the inotropic effect of the Na⁺-channel modulator BDF 9148 in hypertrophic myocardium compared to control tissue. Thus, TG(mREN2)27 rats (TGR), a model with hypertension induced cardiac hypertrophy, was compared with age matched Sprague-Dawley rats (SPDR). The effect of BDF 9148 (0.01–10 μM) on force of contraction (1 Hz, 37°C), the force-frequency relationship (0.5–7 Hz) and the frequency-dependent diastolic tension (0.5–7 Hz) was studied on leftventricular papillary muscles from SPDR and TGR. Chemically skinned muscle fibers of the same hearts were used to examine the influence of BDF 9148 on the Ca²⁺-sensitivity of the contractile proteins. For control the Ca²⁺-sensitizer EMD 57033 was examined. In addition the Na⁺/K⁺-ATPase activity was measured in both, SPDR and TGR. BDF 9148 showed a concentration dependent positive inotropic effect in SPDR and TGR cardiac preparations. Comparing SPDR and TGR, a higher effectiveness of BDF 9148 on TGR was found, while the potency was unchanged. With increasing stimulation rates a significant higher decrease in force of contraction in TGR compared to SPDR was observed. In addition, a significant higher increase in diastolic tension was found in TGR. After exposure to 1 μM BDF 9148 the decrease in force of contraction was significantly reduced in both SPDR and TGR, while only in TGR the increase in diastolic tension was reduced. BDF 9148 had no effect on the Ca²⁺-sensitivity or maximal developed tension of skinned fiber preparations from SPDR or TGR. In contrast, the Ca²⁺-sensitizer EMD 57033 increased the Ca²⁺-sensitivity. The activity of the Na⁺/K⁺-ATPase was significantly reduced in TGR compared to controls. Conclusions: The Na⁺-channel modulator BDF 9148 was more effective in hypertrophic compared to control myocardium in increasing force of contraction, enhancing frequency-dependent force generation and reducing diastolic tension. These effects were not mediated via interaction with the contractile apparatus. The enhanced effectiveness of Na⁺-channel modulation in hypertrophic myocardium could result from alterations of the Na⁺ homeostasis, i. e. a reduced Na⁺/K⁺-ATPase activity. Received: 13 August 1997 / Accepted: 4 February 1998     

The involvement of CYP1A2 and CYP3A4 in the metabolism of clozapine

Aims Clozapine (CLZ), an atypical neuroleptic with a high risk of causing agranulocytosis, is metabolized in the liver to desmethylclozapine (DCLZ) and clozapine N-oxide (CLZ-NO). This study investigated the involvement of different CYP isoforms in the formation of these two metabolites. Methods Human liver microsomal incubations, chemical inhibitors, specific antibodies, and different cytochrome P450 expression systems were used. Results K_m and V_max values determined in human liver microsomes were lower for the demethylation (61±21 μm, 159±42 pmol min⁻¹ mg protein⁻¹ mean±s.d.; n=4), than for the N-oxidation of CLZ (308±1.5 μm, 456±167pmol min⁻¹ mg protein⁻¹; n=3). Formation of DCLZ was inhibited by fluvoxamine (53±28% at 10 μm ), triacetyloleandomycin (33±15% at 10 μm ), and ketoconazole (51±28% at 2 μm ) and by antibodies against CYP1A2 and CYP3A4. CLZ-NO formation was inhibited by triacetyloleandomycin (34±16% at 10 μm ) and ketoconazole (51±13% at 2 μm ), and by antibodies against CYP3A4. There was a significant correlation between CYP3A content and DCLZ formation in microsomes from 15 human livers (r=0.67; P=0.04). A high but not significant correlation coefficient was found for CYP3A content and CLZ-NO formation (r=0.59; P=0.09). Using expression systems it was shown that CYP1A2 and CYP3A4 formed DCLZ and CLZ-NO. K_m and V_max values were lower in the CYP1A2 expression system compared to CYP3A4 for both metabolic reactions. Conclusions It is concluded that CYP1A2 and CYP3A4 are involved in the demethylation of CLZ and CYP3A4 in the N-oxidation of CLZ. Close monitoring of CLZ plasma levels is recommended in patients treated at the same time with other drugs affecting these two enzymes.     

The influence of the sparteine/debrisoquine genetic polymorphism on the disposition of dexfenfluramine

1 To determine whether dexfenfluramine is a substrate of cytochrome P450 2D6 (CYP2D6), its disposition has been studied in nine extensive (EM) and eight poor metabolizers (PM) of debrisoquine. 2 Following a 30 mg dose of dexfenfluramine hydrochloride, urine was collected in all subjects for 96 h post-dose and plasma samples were collected in 11 subjects (six EMs and five PMs). Dexfenfluramine and nordexfenfluramine were measured in urine by h.p.l.c. and in plasma by g.c. 3 Urinary recovery of dexfenfluramine was greater in PMs than EMs (4136±1509 μg vs 1986±792 μg; 95% CI of difference 926–3374; P<0.05) whereas that of nordexfenfluramine was similar in both phenotypes (PM: 1753±411 μg vs 1626±444 μg). 4 Dexfenfluramine AUC was higher in PMs (677±348 μg l⁻¹ h) than EMs (359±250 μg l⁻¹ h). The apparent oral clearance of dexfenfluramine was greater in EMs than PMs (93.6±42.4 l h⁻¹vs 45.6±19.5 l h⁻¹; 95% CI of difference 1.2–94.7; P<0.05). The renal clearance was similar in both phenotypes (EMs: 5.88±2.83 l h⁻¹; PMs 6.60±2.01 l h⁻¹), indicating that the higher urinary recovery of dexfenfluramine in PMs reflects higher plasma concentrations, rather than phenotype differences in the renal handling, of dexfenfluramine. 5 The apparent nonrenal clearance of dexfenfluramine was substantially lower (P<0.05; 95% CI of difference 3.0–94.1) in PMs (39.0±19.5 l h⁻¹) than EMs (87.6±41.2 l h⁻¹). 6 There was a significant inverse correlation (r_s=−0.776 95% CI −0.31–−0.94; n=11; P=0.005) between the debrisoquine metabolic ratio and the apparent nonrenal clearance of dexfenfluramine. 7 PMs had a higher incidence of adverse effects (nausea and vomiting) than EMs. 8 In conclusion, the metabolism of dexfenfluramine is impaired in PMs. Thus CYP2D6, the isoenzyme deficient in poor metabolizers of debrisoquine, must catalyse at least one pathway of dexfenfluramine biotransformation.     

Comparison of nitroprusside and nitroglycerin in inhibition of angiotensin II and other vasoconstrictor-mediated contraction in human coronary bypass conduits

Aims To compare the effect of nitroprusside (SNP) and nitroglycerin (NTG) on angiotensin II (ANGII), endothelin-1 (ET-1), and α₁-adrenoceptor (phenylephrine, PE)-mediated contraction in internal mammary artery (IMA). Methods Human IMA segments (n=120) taken from 37 patients were studied. Concentration-relaxation curves for SNP and NTG were established in IMA precontracted with these vasoconstrictors. Concentration-contraction curves were also constructed in IMA rings incubated with SNP and NTG (0.1 and 1 μm ) for 10 min. Results Both SNP and NTG caused full relaxation with similar EC₅₀ s except NTG was four-fold more potent than SNP in PE-induced contraction (−7.92±0.06 vs−7.32±0.2 log m, mean±s.e. mean, P<0.01; 95% confidence interval for the difference of the means: 0.19, 1.01 log m ). Pretreatment with SNP (0.1 and 1 μm ) significantly depressed the contraction by ANGII from 56.6±7.7% (of 100 mm K⁺-contraction) to 18.3±8.6% and 3.9±2.1% (P=0.0001). In four rings treated with SNP, the contraction to ANGII was abolished whereas NTG did not depress ANGII-mediated contraction. Pretreatment with SNP (1 μm ), but not NTG, significantly depressed the magnitude of the PE-induced contraction from 4.7±1.2 to 1.7±0.4 g (P<0.05). Treatment with both SNP and NTG significantly increased the EC₅₀ (−5.09±0.17 log m, P=0.0007 for SNP and −5.40±0.06 log M, P=0.02 for NTG). Pretreatment with SNP did not significantly change either the magnitude or the EC₅₀ of the ET-1-induced contraction. Conclusions SNP may be advantageous compared with NTG in preventing coronary arterial graft contraction. However, once grafts have constricted to ANGII, α₁-adrenoceptor agonists, and ET-1, NTG may be only marginally advantageous.     

Metergoline inhibits the neuronal Navl.2 voltage- dependent Na^＋ channels expressed in Xenopus oocytes

Aim： Metergoline is an ergot-derived psychoactive drug that acts as a ligand for serotonin and dopamine receptors. The aim of this study was to investigate the regulatory effects of metergoline on the neuronal Nav1.2 voltage-dependent Na^＋ channels in vitro. Methods： Xenopus oocytes were injected with cRNAs encoding rat brain Nav1.2 α and β1 subunits. Voltage-activated Na^＋ currents were recorded using two-electrode voltage clamp technique. Drugs were applied though perfusion. Results： Both metergoline and lidocaine reversibly and concentration-dependently inhibited the peak of Na^＋ currents with IC50 values of 3.6±4.2 and 916.9±98.8 μmol/L, respectively. Metergoline （3 pmol/L） caused a 6.8±1.2 mV depolarizing shift of the steady-state activation curve of the Na^＋ currents, and did not alter the inactivation curve. In contrast, lidocaine （3 μmol/L） caused a 12.7±1.2 mV hyperpolarizing shift of the inactivation curve of the Na^＋ currents without changing the steady-state activation curve. Both metergoline and lidocaine produced tonic and use-dependent inhibition on the peak of Na^＋ currents. Conclusion： Metergoline exerts potent inhibition on the activity of neuronal Nav1.2 channels, which may contribute to its actions on the central nervous system.     

Role of Na+-K+ ATPase enzyme in vascular response of goat ruminal artery

Objective:

To study the role of Na⁺, K⁺- ATPase enzyme in the vascular response of goat ruminal artery.

Materials and Methods:

Ruminal artery was obtained in chilled aerated modified Krebs-Henseleit solution (KHS) from a local slaughterhouse and transported in ice for further processing. The endothelium intact arterial ring was mounted in a thermostatically controlled (37 ± 0.5°C) organ bath containing 20 ml of modified KHS (pH 7.4) bubbled with oxygen (95%) and CO₂ (5%) under 2g tension. An equilibration of 90 min was allowed before addition of drugs into the bath. The responses were recorded isometrically in an automatic organ bath connected to PowerLab data acquisition system. In order to examine intact functional endothelium, ACh (10 μM) was added on the 5-HT (1.0 μM) - induced sustained contractile response. Similarly, functional characterization of Na⁺, K⁺-ATPase activity was done by K⁺-induced relaxation (10 μM-10 mM) in the absence and presence of ouabain (0.1 μM/ 0.1 mM), digoxin (0.1 μM) and barium (30 μM).

Results:

ACh (10⁻⁵ M) did not produce any relaxing effect on 5-HT-induced sustained contractile response suggesting that vascular endothelium has no significant influence on the activation of sodium pump by extracellular K⁺ in ruminal artery. Low concentration of Ba²⁺ (30 μM) (IC₅₀: 0.479 mM) inhibited K⁺-induced relaxation suggesting K_ir (inward rectifier) channel in part had role in K⁺-induced vasodilatation in ruminal artery. Vasorelaxant effect of KCl (10 μM-10 mM) in K⁺-free medium is also blocked by ouabain (0.1 μM and 0.1 mM) (IC₅₀:0.398 mM and IC₃₅: 1.36 mM), but not by digoxin (0.1 μM) (IC₅₀ 0.234 mM) suggesting that ouabain sensitive Na⁺, K⁺-ATPase isoform is present in the ruminal artery.

Conclusion:

In the goat ruminal artery functional regulation of sodium pump is partly mediated by K⁺ channel and ouabain sensitive Na⁺, K⁺ ATPase.     

The effects of combined treatment with β1-selective receptor antagonists and lipid-lowering drugs on fat metabolism and measures of fatigue during moderate intensity exercise: a placebo-controlled study in healthy subjects

Aims We examined the effects of different combinations of β₁-selective adrenoceptor blockers and lipid-lowering drugs, on fat metabolism and fatigue during moderate intensity exercise in 14 healthy young volunteers. Methods The study was a randomized crossover design, each subject completing 5, 90 min walks at 50% of predetermined maximal oxygen uptake (VO₂ max), one following each 3 day treatment period with either: atenolol 100 mg and bezafibrate 400 mg, atenolol 100 mg and fluvastatin 40 mg, metoprolol CR 100 mg and bezafibrate 400 mg, metoprolol CR 100 mg and fluvastatin 40 mg, or placebo. Results Plasma free fatty acid (FFA) concentration during exercise was significantly reduced on all treatments, in comparison with placebo, P=0.0001. Following 90 min of exercise FFA levels were as follows: placebo 573 μmol l⁻¹ (105–1041), metoprolol CR+fluvastatin 277 μmol l⁻¹ (0–647), metoprolol CR+bezafibrate 182 μmol l⁻¹ (0–396), atenolol+fluvastatin 211 μmol l⁻¹ (0–511), and atenolol+bezafibrate 123 μmol l⁻¹ (0–352). Total fat oxidation during exercise was also reduced on all treatments in comparison with placebo: 38.1% (2–74), compared with 29.1% (0–61) on metoprolol CR+fluvastatin, P=0.02, 26.2% (2–51) on metoprolol CR+bezafibrate, P=0.002, 25.5% (3–48) on atenolol+fluvastatin, P=0.009, and 22.8% (0–47) on atenolol+bezafibrate treatment, P=0.0002. Plasma ammonia concentration was elevated on all treatments during exercise in comparison with placebo. After 90 min of exercise, plasma ammonia levels were as follows: placebo 37 μmol l⁻¹ (0–84), metoprolol CR+fluvastatin 56 μmol l⁻¹ (2–110), metoprolol CR+bezafibrate 79 μmol l⁻¹ (0–167), atenolol+fluvastatin 90 μmol l⁻¹ (10–170), and atenolol+bezafibrate 100 μmol l⁻¹ 26–174). In comparison with placebo, metoprolol CR+fluvastatin had the least adverse impact on measures of perceived exertion and the ‘feeling scale’ during exercise. Metoprolol CR+bezafibrate, atenolol+fluvastatin, and atenolol+bezafibrate treatments had greater adverse effects, particularly on perceived ‘cardiorespiratory effort’ and ‘feeling scale’ scores. Conclusions In healthy volunteers, combinations of β₁-selective blockers and lipid-lowering drugs were associated with significant reductions in fat metabolism, increased plasma ammonia levels, and raised the perception of effort during exercise, in comparison with placebo. Metoprolol CR+fluvastatin had the least effect, combinations metoprolol CR+bezafibrate and atenolol+fluvastatin had intermediate effects, and atenolol+bezafibrate had the most adverse effect.     

Dopamine natriuresis in salt-repleted, water-loaded humans: a dose-response study

Aims The purpose of the present study was to define the dose-response relationship between exogenous dopamine and systemic haemodynamics, renal haemodynamics, and renal excretory function at infusion rates in the range 0 to 12.5 μg kg⁻¹ min⁻¹ in normal volunteers. Methods While undergoing water diuresis, eight subjects were infused with 0, 1, 2, 3, 5, 7.5, 10 or 12.5 μg of dopamine kg⁻¹ min⁻¹ over 2 h in a randomized and double-blind fashion. On each study day, renal clearance studies were performed during a 1 h baseline period and subsequently during the second 1 h infusion period. Lithium clearance (CL_Li ) was used to estimate proximal tubular outflow. Results Cardiac output increased with the four highest doses. Mean arterial pressure followed a biphasic pattern with a decrease during the two lowest doses and a dose-dependent increase from the 7.5 μg kg⁻¹ min⁻¹ dose onwards. Effective renal plasma flow increased with all doses of dopamine, but peaked with the 3 μg kg⁻¹ min⁻¹ infusion rate [ from 617 (585–649) ml min⁻¹ with placebo to 915 (824–1006) ml min⁻¹ (means with 95% CI, P<0.001)]. None of the doses changed glomerular filtration rate (GFR). Sodium clearance (CL_Na ) and CL_Li were elevated with the four lowest doses but increased further from 7.5 μg kg⁻¹ min⁻¹ onwards. Compared with placebo, the percentage increase in CL_Na with increasing dose was 77 (5–159), 93 (13–172), 107 (24–190), 121 (60–181), 253 (65–441), 284 (74–494), and 212 (111–312) %, respectively. There were only small, inconsistent decreases in absolute proximal reabsorption rate (APR=GFR-CL_Li ). Fractional distal reabsorption of sodium (FDR_Na=(CL_Li-CL_Na )/CL_Li ) decreased with all doses, reaching its nadir with 7.5 μg kg⁻¹ min⁻¹ [from 95.9 (94.6–97.2) % with placebo to 91.5 (90.0–93.0) % (P<0.01)] whereafter a flat dose-response curve was observed. Conclusions In conclusion, the renal vasodilating effect of dopamine was maximal with 3 μg kg⁻¹ min⁻¹. The dose-dependent attenuation seen with higher doses is consistent with an increased α-adrenergic stimulation opposing the effect on dopaminergic receptors. The present CL_Li studies confirm that dopamine increases proximal tubular outflow. The results suggest that the natriuretic effect of depressor doses of dopamine was primarily caused by attenuation of the increase in distal sodium reabsorption normally seen after an increase in proximal tubular outflow. Pressor doses further increased sodium excretion, indicating the presence of pressure natriuresis at these high doses.     

Single-dose pharmacokinetics of ampicillin and tobramycin administered by hypodermoclysis in young and older healthy volunteers

1To test the feasibility of administering antibiotics by subcutaneous infusion to the elderly, we compared the pharmacokinetics of tobramycin (single dose of 80 mg) given by hypodermoclysis (HDC) with the kinetics of the antibiotic injected intravenously (i.v.) in 10 young (<50 years old) and 10 elderly (>65 years old) healthy volunteers. Similar studies were performed with ampicillin (single dose of 1 g) in 12 young and 10 older healthy volunteers. 2Compared with the i.v. route, HDC delayed the time to reach the maximal plasma concentration (t_max) of tobramycin in young volunteers: 32±6 (s.d.) min vs 88±46, P<0.005, and older volunteers: 27±4 min vs 89±15, P<0.005. Administration of the antibiotics by HDC was well tolerated. The plasma concentration of tobramycin 30 min after the end of infusion (C₆₀) was lower (P<0.05) following HDC than after the i.v. route in both young, 2.2±0.7 vs 3.5±0.8 μg ml⁻¹, and elderly subjects, 2.2±0.8 vs 3.8±0.9. μg ml⁻¹. 3The area under the curve (AUC) of tobramycin given by HDC was slightly smaller than when given i.v., i.e. in young subjects: 740±225 (s.d.) vs 893±223 μg ml⁻¹ min, NS, and in the elderly: 980±228 vs 1056±315 μg ml⁻¹ min, NS. 4When ampicillin was administered by HDC, the t_max was also delayed in young volunteers: 45±18 vs 23±6 min, and in the elderly: 49±18 vs 27±4 min, P<0.005, the AUC was greater by HDC than i.v. in the young volunteers: 4527±1658 μg ml⁻¹ min vs 3810±1033 μg ml⁻¹ min and in the elderly: 6795±2094 μg ml⁻¹ min vs 4217±1518 μg ml⁻¹ min, and the C₆₀ was higher by HDC in the young: 27±7 vs 24±9 μg ml⁻¹, and in the elderly: 32±9 vs 23±11 μg ml⁻¹, P<0.05. 5In conclusion, HDC delays the entry of the antibiotic into the systemic circulation, but did not affect the amount available. HDC was well tolerated and could become an adequate method for antibiotic administration to the elderly.     

Pharmacokinetics and response of obese patients with chronic hepatitis C treated with different doses of PEG-IFN α-2a (40KD) (PEGASYS®)

AIMS

To evaluate whether higher doses of peginterferon α-2a (40KD) [PEG-IFN α-2a (40KD)] can compensate for lower exposure observed among obese patients with chronic hepatitis C (CHC) treated with the standard dose of PEG-IFN α-2a (40KD).

METHODS

Noncirrhotic, obese (body mass index ≥30 kg m⁻²) patients with CHC participated in a single-centre, open-label study. Patients were randomized to 180 or 270 µg week⁻¹ PEG-IFN α-2a (40KD) + ribavirin (1000/1200 mg day⁻¹) for 48 weeks. Blood samples were collected predose and up to 168 h after the first dose and at week 12 for pharmacokinetic analysis. Trough serum concentrations (C_trough) were determined up to week 24.

RESULTS

In the 180 µg week⁻¹ group mean ± SD steady-state (week 12) estimates of AUC_0–168 (ng h⁻¹ ml⁻¹), C_max (ng ml⁻¹) and CL/F (l h⁻¹) were 2154 ± 919, 13.8 ± 6.7 and 0.102 ± 0.051, respectively. In the 270 µg week⁻¹ group, estimates were 3374 ± 1844, 23.4 ± 10.7 and 0.090 ± 0.042, respectively. The mean (range) C_trough (ng ml⁻¹) was 11.2 (4.4–18.5) in the 180 µg week⁻¹ group and 16.1 (0.4–44.2) in the 270 µg week⁻¹ group. Overall, 14 of 20 (70%) and 16 of 20 (80%) patients in the 180 µg week⁻¹ and 270 µg week⁻¹ groups were infected with hepatitis C virus genotype 1 or 4. In the 180 µg week⁻¹ and 270 µg week⁻¹ groups 14 of 20 (70%) and 15 of 19 (79%) patients, respectively, achieved a sustained viral response. Safety was similar between groups.

CONCLUSIONS

Mean PEG-IFN α-2a (40KD) exposure was dose proportional from 180 to 270 µg week⁻¹. Increasing PEG-IFN α-2a (40KD) from 180 to 270 µg week⁻¹ achieves higher serum drug exposure in obese patients.     

Effects of oral and inhaled corticosteroid on lymphocyte β2-adrenoceptor function in asthmatic patients

Aims We have previously demonstrated that a single dose of oral prednisolone but not single doses of inhaled fluticasone had facilitatory effects on lymphocyte β₂-adrenoceptor (AR) function. To address possible differences in steady-state time-course, the aim of this study was to determine if repeated dosing with inhaled fluticasone would have facilitatory effects on lymphocyte β₂-AR. Plasma cortisol was also evaluated as a measure of systemic bioactivity. Methods Ten asthmatic subjects, mean (s.e.mean) age 29 (3) years, FEV₁ 89 (5) % predicted, were randomised in a double-blind crossover study to receive inhaled placebo (PL), inhaled fluticasone 1000 μg day⁻¹ (F1000) and inhaled fluticasone 2000 μg day⁻¹, each for 4 days and also a single dose of oral prednisolone 50 mg (PRED). Prednisolone was given as open medication. The last dose of study drug was taken at 22.00 h and subjects attended the laboratory at 08.00 h the following day. Results β₂-AR density (B_max; fmol/10⁶ cells) was significantly increased after PRED compared with PL and inhaled fluticasone. B_max (geometric mean) after each treatment were: PL 1.51, F1000 1.20, F2000 1.20 and PRED 2.14 (a 1.4 fold difference PRED vs PL; 95% CI 1.05 to 1.95; P<0.001). There was significant (P<0.001) suppression of plasma cortisol (nmol l⁻¹ ) following F2000 and PRED compared with PL: 393.8, F1000 302.1, F2000 205.0 (95% CI F2000 vs PL 58.1 to 319.4) and PRED 87.0 (95% CI PRED vs PL 176.2 to 437.5). The estimated milligram equivalence ratio for adrenal suppression was calculated at 1:11 for fluticasone vs prednisolone. Conclusions Repeated dosing with high-dose inhaled fluticasone did not up-regulate lymphocyte β₂-AR as compared with a single dose of oral prednisolone, despite having significantly suppressed early morning plasma cortisol. This study confirms our previous finding of a dissociation in sensitivity between effects of inhaled corticosteroid on adrenal suppression and lymphocyte β₂-AR regulation, at least for doses up to 2 mg day⁻¹ of fluticasone.     

State-dependent blockade of human ether-a-go-go- related gene （hERG） K＋ channels by changrolin in stably transfected HEK293 cells

Aim:

To study the effect of changrolin on the K⁺ channels encoded by the human ether-a-go-go-related gene (hERG).

Methods:

hERG channels were heterologously stably expressed in human embryonic kidney 293 cells, and the hERG K⁺ currents were recorded using a standard whole-cell patch-clamp technique.

Results:

Changrolin inhibited hERG channels in a concentration-dependent and reversible manner (IC₅₀=18.23 μmol/L, 95% CI: 9.27–35.9 μmol/L; Hill coefficient=−0.9446). In addition, changrolin shifted the activation curve of hERG channels by 14.3±1.5 mV to more negative potentials (P<0.01, n=9) but did not significantly affect the steady-state inactivation of hERG (n=5, P>0.05). The relative block of hERG channels by changrolin was close to zero at the time point of channel opening by the depolarizing voltage step and quickly increased afterwards. The maximal block was achieved in the inactivated state, with no further development of the open channel block. In the “envelope of tails” experiments, the time constants of activation were found to be 287.8±46.2 ms and 174.2±18.4 ms, respectively, for the absence and presence of 30 μmol/L changrolin (P<0.05, n=7). The onset of inactivation was accelerated significantly by changrolin between −40 mV and +60 mV (P<0.05, n=7).

Conclusion:

The results demonstrate that changrolin is a potent hERG blocker that preferentially binds to hERG channels in the open and inactivated states.     

Inhibition of the INa/K and the activation of peak INa contribute to the arrhythmogenic effects of aconitine and mesaconitine in guinea pigs

Aconitine(ACO),a main active ingredient of Aconitum,is well-known for its cardiotoxicity.However,the mechanisms of toxic action of ACO remain unclear.In the current study,we investigated the cardiac effects of ACO and mesaconitine(MACO),a structurally related analog of ACO identified in Aconitum with undocumented cardiotoxicity in guinea pigs.We showed that intravenous administration of ACO or MACO(25μg/kg)to guinea pigs caused various types of arrhythmias in electrocardiogram(ECG)recording,including ventricular premature beats(VPB),atrioventricular blockade(AVB),ventricular tachycardia(VT),and ventricular fibrillation(VF).MACO displayed more potent arrhythmogenic effect than ACO.We conducted whole-cell patch-clamp recording in isolated guinea pig ventricular myocytes,and observed that treatment with ACO(0.3,3μM)or MACO(0.1,0.3μM)depolarized the resting membrane potential(RMP)and reduced the action potential amplitude(APA)and durations(APDs)in a concentration-dependent manner.The ACO-and MACO-induced AP remodeling was largely abolished by an INa blocker tetrodotoxin(2μM)and partly abolished by a specific Na+/K+pump(NKP)blocker ouabain(0.1μM).Furthermore,we observed that treatment with ACO or MACO attenuated NKP current(INa/K)and increased peak INa by accelerating the sodium channel activation with the EC50 of 8.36±1.89 and 1.33±0.16μM,respectively.Incubation of ventricular myocytes with ACO or MACO concentration-dependently increased intracellular Na+and Ca2+concentrations.In conclusion,the current study demonstrates strong arrhythmogenic effects of ACO and MACO resulted from increasing the peak INa via accelerating sodium channel activation and inhibiting the INa/K.These results may help to improve our understanding of cardiotoxic mechanisms of ACO and MACO,and identify potential novel therapeutic targets for Aconitum poisoning.     

Sodium/hydrogen exchange inhibition with cariporide reduces leukocyte adhesion via P-selectin suppression during inflammation

Background and purpose:

The Na⁺/H⁺ exchange (NHE) inhibitor cariporide is known to ameliorate ischaemia/reperfusion (I/R) injury by reduction of cytosolic Ca²⁺ overload. Leukocyte activation and infiltration also mediates I/R injury but whether cariporide reduces I/R injury by affecting leukocyte activation is unknown. We studied the effect of cariporide on thrombin and I/R induced leukocyte activation and infiltration models and examined P-selectin expression as a potential mechanism for any identified effects.

Experimental approach:

An in vivo rat mesenteric microcirculation microscopy model was used with stimulation by thrombin (0.5 μ ml⁻¹) superfusion or ischaemia (by haemorrhagic shock for 60 min) and reperfusion (90 min).

Key results:

Treatment with cariporide (10 mg kg⁻¹ i.v.) significantly reduced leukocyte rolling, adhesion and extravasation after thrombin exposure. Similarly, cariporide reduced leukocyte rolling (54±6.2 to 2.4±1.0 cells min⁻¹, P<0.01), adherence (6.3±1.9 to 1.2±0.4 cells 100 μm⁻¹, P<0.01) and extravasation (9.1±2.1 to 2.4±1.1 cells per 20 × 100 μm perivascular space, P<0.05), following haemorrhagic shock induced systemic ischaemia and reperfusion. The cell adhesion molecule P-selectin showed a profound decrease in endothelial expression following cariporide administration in both thrombin and I/R stimulated groups (35.4±3.2 vs 14.2±4.1% P-selectin positive cells per tissue section, P<0.01).

Conclusions and implications:

The NHE inhibitor cariporide is known to limit reperfusion injury by controlling Ca²⁺ overload but these data are novel evidence for a vasculoprotective effect of NHE inhibition at all levels of leukocyte activation, an effect which is likely to be mediated at least in part by a reduction of P-selectin expression.     

Application of HPLC for the simultaneous determination of aceclofenac, paracetamol and tramadol hydrochloride in pharmaceutical dosage form

A simple, precise and accurate reversed-phase liquid chromatographic method has been developed for the simultaneous estimation of aceclofenac (ACF), paracetamol (PCM) and tramadol hydrochloride (TRM) in pharmaceutical dosage form. The chromatographic separation was achieved on a HiQ-Sil™ HS C18 column (250×4.6 mm i.d., 5 μm particle size), kromatek analytical column at ambient temperature. The mobile phase consisted of 40: 60 (v/v); phosphate buffer (pH 6.0): methanol. The flow rate was set to 1.0 mL min⁻¹ and UV detection was carried out at 270 nm. The retention time (t_R) for ACF, PCM and TRM were found to be 14.567 ± 0.02, 3.133 ± 0.01 and 7.858 ± 0.02 min, respectively. The validation of the proposed method was carried out for linearity, precision, robustness, limit of detection, limit of quantitation, speci city, accuracy and system suitability. The linear dynamic ranges were from 40–160 μg mL⁻¹ for ACF, 130–520 μg mL⁻¹ for PCM and 15–60 μg mL⁻¹ for TRM. The developed method can be used for routine quality control analysis of titled drugs in pharmaceutical dosage form.