Pharmacokinetics of 619C89, a novel neuronal sodium channel inhibitor, in acute stroke patients after loading and discrete maintenance infusions

1This was a multi-centre, placebo controlled, randomized, dose-escalating design study in which five dosing regimens of 619C89/placebo were evaluated in 48 stroke patients. Loading infusions of 0.5, 1, 1.5, 2 and 2.5 mg kg⁻¹ over 1 h were followed by respective maintenance infusions of 0.25, 0.5, 0.75, 1 and 1.25 mg kg⁻¹ over 30 min at 8 hourly intervals for 3 days. 2Plasma concentrations of 619C89 and its N-oxide, 341C90, and N-demethylated, 78C90, metabolites were assayed using an LC–MS–MS method. Plasma concentration-time profiles after the final maintenance infusion were subjected to conventional noncompartmental pharmacokinetic analysis. 3For 619C89, geometric CL means ranged between 0.71 and 0.99 l h⁻¹ kg⁻¹ for maintenance infusions up to 1.25 mg kg⁻¹ over 30 min, with an overall mean of 0.85 l h⁻¹ kg⁻¹ (95% CI: 0.70–1.04 l h⁻¹ kg⁻¹). Geometric V_ss means ranged between 13.2 and 27.9 l kg⁻¹ for the same doses, with an overall mean of 22.5 l kg⁻¹ (95% CI: 16.4–30.9 l kg⁻¹). The ANOVA results revealed that neither CL, V_ss nor t_1/2 were significantly different across the five dosing regimens (P values: 0.82, 0.54 and 0.61, respectively). 4Average AUC for 341C90 was 270% and that for 78C90 was 62% of the AUC for 619C89. The AUC_m/AUC_p-ratios were similar at all dose levels for each metabolite. Values of t_1/2 for 341C90 were similar to those of 619C89 whereas t_1/2 for 78C90 was about three-fold longer than that of parent drug. 5In conclusion, the pharmacokinetics of 619C89 are independent of dose in acute stroke patients. The pharmacokinetics of 341C90 are probably formation rate-limited and those of 78C90 are elimination rate-limited and are also dose-independent.     

Enzyme kinetic modelling as a tool to analyse the behaviour of cytochrome P450 catalysed reactions: application to amitriptyline N-demethylation

1To determine kinetic parameters (V_max, K_m) for cytochrome P450 (CYP) mediated metabolic pathways, nonlinear least squares regression is commonly used to fit a model equation (e.g., Michaelis Menten [MM]) to sets of data points (reaction velocity vs substrate concentration). This method can also be utilized to determine the parameters for more complex mechanisms involving allosteric or multi-enzyme systems. Akaike''s Information Criterion (AIC), or an estimation of improvement of fit as successive parameters are introduced in the model ( F-test), can be used to determine whether application of more complex models is helpful. To evaluate these approaches, we have examined the complex enzyme kinetics of amitriptyline (AMI) N-demethylation in vitro by human liver microsomes. 2For a 15-point nortriptyline (NT) formation rate vs substrate (AMI) concentration curve, a two enzyme model, consisting of one enzyme with MM kinetics (V_max=1.2 nmol min⁻¹ mg⁻¹, K_m=24 μm) together with a sigmoidal component (described by an equation equivalent to the Hill equation for cooperative substrate binding; V_max=2.1 nmol min⁻¹ mg⁻¹, K′=70 μm; Hill exponent n=2.34), was favoured according to AIC and the F-test. 3Data generated by incubating AMI under the same conditions but in the presence of 10 μm ketoconazole (KET), a CYP3A3/4 inhibitor, were consistent with a single enzyme model with substrate inhibition (V_max=0.74 nmol min⁻¹ mg⁻¹, K_m=186 μm, K₁=0.0028 μm⁻¹). 4Sulphaphenazole (SPA), a CYP2C9 inhibitor, decreased the rate of NT formation in a concentration dependent manner, whereas a polyclonal rat liver CYP2C11 antibody, inhibitory for S-mephenytoin 4′-hydroxylation in humans, had no important effect on this reaction. 5Incubation of AMI with 50 μm SPA resulted in a curve consistent with a two enzyme model, one with MM kinetics (V_max=0.72 nmol min⁻¹ mg⁻¹, K_m=54 μm) the other with ‘Hill-kinetics’ (V_max=2.1 nmol min⁻¹ mg⁻¹, K′=195 μm; n=2.38). 6A fourth data-set was generated by incubating AMI with 10 μm KET and 50 μm SPA. The proposed model of best fit describes two activities, one obeying MM-kinetics (V_max=0.048 nmol min⁻¹ mg⁻¹, K_m=7 μm) and the other obeying MM kinetics but with substrate inhibition (V_max=0.8 nmol min⁻¹ mg⁻¹, K_m=443 μm, K₁=0.0041 μm⁻¹). 7The combination of kinetic modelling tools and biological data has permitted the discrimination of at least three CYP enzymes involved in AMI N-demethylation. Two are identified as CYP3A3/4 and CYP2C9, although further work in several more livers is required to confirm the participation of the latter.     

Slow delayed rectifier K+ current block by HMR 1556 increases dispersion of repolarization and promotes Torsades de Pointes in rabbit ventricles

Background and purpose:

The slow delayed rectifier K⁺ current (I_Ks) contributes to ventricular repolarization when the action potential (AP) is prolonged. I_Ks block during drug-induced AP prolongation may promote Torsades de Pointes (TdP), but whether this is due to additional AP prolongation is uncertain.

Experimental approach:

In bradycardic perfused rabbit ventricles, the incidence of spontaneous TdP, monophasic AP duration at 90% repolarization (MAPD₉₀) and ECG interval between the peak and the end of T wave (T_peak−end) (index of dispersion of repolarization) were measured after the administration of veratridine (125 nM, slows Na⁺ channel inactivation), dofetilide (7.5 or 10 nM, a rapid delayed rectifier blocker) and HMR 1556 (HMR, 100 nM, an I_Ks blocker), alone or in combinations (n=6 each).

Key results:

HMR did not prolong MAPD₉₀, whereas veratridine or 7.5 nM dofetilide prolonged MAPD₉₀ (P<0.01) without inducing TdP. Veratridine+7.5 nM dofetilide additively prolonged MAPD₉₀ (P<0.05), induced 4±6 TdP per heart and prolonged T_peak−end by 12±10 ms. Subsequent addition of HMR did not further prolonged MAPD₉₀, but increased the number of TdP to 22±18 per heart and increased T_peak−end by 39±21 ms (P<0.05). Increasing dofetilide concentration from 7.5 to 10 nM (added to veratridine) produced a longer MAPD₉₀, but fewer TdP (5±5 per heart) and less T_peak−end prolongation (17±8 ms) compared to the veratridine+7.5 nM dofetilide+HMR group (P<0.05).

Conclusions and implications:

Adding I_Ks block markedly increases TdP incidence in hearts predisposed to TdP development by increasing the dispersion of repolarization, but without additional AP prolongation.     

Effects of excitatory and inhibitory neurotransmission on motor patterns of human sigmoid colon in vitro

Background and purpose:

To characterize the in vitro motor patterns and the neurotransmitters released by enteric motor neurons (EMNs) in the human sigmoid colon.

Experimental approach:

Sigmoid circular strips were studied in organ baths. EMNs were stimulated by electrical field stimulation (EFS) and through nicotinic ACh receptors.

Key results:

Strips developed weak spontaneous rhythmic contractions (3.67±0.49 g, 2.54±0.15 min) unaffected by the neurotoxin tetrodotoxin (TTX; 1 μM). EFS induced strong contractions during (on, 56%) or after electrical stimulus (off, 44%), both abolished by TTX. Nicotine (1–100 μM) inhibited spontaneous contractions. Latency of off-contractions and nicotine responses were reduced by N^G-nitro-L-arginine (1 mM) and blocked after further addition of apamin (1 μM) or the P2Y₁ receptor antagonist MRS 2179 (10 μM) and were unaffected by the P2X antagonist NF279 (10 μM) or α-chymotrypsin (10 U mL⁻¹). Amplitude of on- and off-contractions was reduced by atropine (1 μM) and the selective NK₂ receptor antagonist Bz-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH₂ (1 μM). MRS 2179 reduced the amplitude of EFS on- and off-contractions without altering direct muscular contractions induced by ACh (1 nM–1 mM) or substance P (1 nM–10 μM).

Conclusions and implications:

Latency of EFS-induced off-contractions and inhibition of spontaneous motility by nicotine are caused by stimulation of inhibitory EMNs coreleasing NO and a purine acting at muscular P2Y₁ receptors through apamin-sensitive K⁺ channels. EFS-induced on- and off-contractions are caused by stimulation of excitatory EMNs coreleasing ACh and tachykinins acting on muscular muscarinic and NK₂ receptors. Prejunctional P2Y₁ receptors might modulate the activity of excitatory EMNs. P2Y₁ and NK₂ receptors might be therapeutic targets for colonic motor disorders.     

Pharmacokinetic and pharmacodynamic interaction trial after repeated oral doses of imdapril and digoxin in healthy volunteers

Aims To investigate the potential pharmacokinetic and pharmacodynamic interaction between imidapril and digoxin. Methods AUC, C_max and t_max of imidapril, imidaprilat and digoxin were calculated and evaluated in a randomized, doubleblind three-period cross-over design in 12 healthy volunteers after 8 days treatment with the following combinations: digoxin 0.25 mg day⁻¹+placebo (D+P); imidapril 10 mg day⁻¹+placebo (I+P); imidapril 10 mg day⁻¹+digoxin 0.25 mg day⁻¹ (I+D). Results Mean AUC (0, 24 h) of digoxin was 10.4 (±4.9 s.d.) ng ml⁻¹ h (D+P) and 10.7 (±3.9 s.d.) ng ml⁻¹ h (I+D), respectively (90%-confidence intervals [ CI] for the ratio of (D+P) and (I+D): 0.91–1.27, point estimator [PE]: 1.06). Mean AUC (0, 24 h) of imidapril was 133 (±86 s.d.) ng ml⁻¹ h (I+P) and 108 (±52 s.d.) ng ml⁻¹ h (I+D), respectively (90%-CI: 0.76–0.94, PE 0.85). AUC (0, 24 h) of imidaprilat was 215 (±91 s.d.) ng ml⁻¹ h (I+P) and 194 (±54 s.d.) ng ml⁻¹ h (I+D), respectively (90%-CI: 0.80–1.08, PE 0.93). C_max was 19.9 (±8.7 s.d.) ng ml⁻¹ (I+P) and 15.9 (±5.3 s.d.) ng ml⁻¹ (I+D) (90%-CI: 0.67–1.00, PE 0.82). The results indicate a slight reduction of imidapril and imidaprilat plasma levels when coadministered with digoxin without any effect on digoxin plasma levels. Maximal ACE-inhibition was 79% (I+P) and 67% (I+D). Conclusions Grouped data analysis of imidaprilat plasma levels vs ACE-activity showed that for maximal inhibition of plasma ACE activity, imidaprilat plasma levels should exceed 10 ng ml⁻¹. Under digoxin and imidapril, more plasma concentrations of imidaprilat were seen under this level as after imidapril alone, this reduces the integral of the ACE-inhibition/time curves by about 20 to 30%.     

H2S-donating sildenafil (ACS6) inhibits superoxide formation and gp91phox expression in arterial endothelial cells: role of protein kinases A and G

Background and purpose:

Superoxide (O₂^•−), derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, is associated with acute respiratory distress syndrome (ARDS). NADPH oxidase activity and expression are blocked by nitric oxide (NO) and sildenafil. As another gas, hydrogen sulphide (H₂S) is formed by blood vessels, the effect of sodium hydrosulphide (NaHS) and the H₂S-donating derivative of sildenafil, ACS6, on O₂^•− formation and the expression of gp91^phox (a catalytic subunit of NADPH oxidase) in porcine pulmonary arterial endothelial cells (PAECs) was investigated.

Experimental approach:

PAECs were incubated with 10 ng mL⁻¹ tumour necrosis factor-α (TNFα) (±NaHS or ACS6), both of which released H₂S, for 2 h or 16 h. O₂^•− was measured. Expression of gp91^phox was measured by western blotting and the role of cyclic AMP (cAMP) and/or cyclic GMP was assessed using protein kinase inhibitors.

Key results:

After either 2- or 16-h incubations, O₂^•− formation by PAECs was inhibited by NaHS or ACS6, with IC₅₀ values of about 10 nM and less than 1 nM, respectively. Both 100 nM NaHS and 1 nM ACS6 completely inhibited gp91^phox expression induced by TNFα. The effects of NaHS were blocked by the inhibition of protein kinase A (PKA), but not PKG, and not by the inhibition of guanylyl cyclase. Effects of ACS6 were blocked by inhibition of both PKA and PKG. Both NaHS and ACS6 augmented cAMP formation.

Conclusion and implications:

H₂S inhibited O₂^•− formation and upregulation of NADPH oxidase in PAECs through the adenylyl cyclase-PKA pathway. ACS6 may be effective in treating ARDS through both elevation of cAMP and inhibition of phosphodiesterase type 5 activity.     

A pharmacokinetic and safety study of single dose intravenous combretastatin A4 phosphate in Chinese patients with refractory solid tumours

AIMS

This trial was conducted to evaluate the safety and pharmacokinetics of combretastatin A4 phosphate (CA4P) given intravenously as a single dose to Chinese patients with refractory solid tumours.

METHODS

Twenty-five patients were treated with single doses of CA4P according to a dose escalation scheme: 5, 10, 20, 33, 50, 65 and 85 mg m⁻² infused intravenously over 30 min.

RESULTS

CA4P was generally well tolerated at ≤65 mg m⁻². Transient, moderate increases in the heart rate-corrected QT interval occurred at all doses. CA4P produced a transient dose-dependent increase in neural and gastrointestinal toxicities. Acute renal failure occurred in one dehydrated patient who had also taken paracetamol. There were seven episodes of dose-limiting toxicity at doses ≥65 mg m⁻², including two episodes of reversible ataxia at 85 mg m⁻². For CA4P, at 50 mg m⁻², mean (SD) peak plasma concentration (C_max) was 0.99 (0.33) µm, area under the curve from time zero to time of last quantifiable concentration (AUC(0,t)) was 1.42 (0.30) µm h and terminal elimination half-life (t_1/2) was 1.81 (0.61) h. At 65 mg m⁻², C_max was 1.73 (0.62) µm, AUC(0,t) was 3.19 (1.47) µm h and t_1/2 was 1.90 (0.61) h. One patient with nasopharyngeal carcinoma had an obvious clinical response with central necrosis in the metastatic lung mass.

CONCLUSION

Doses ≤65 mg m⁻² given as 30 min infusions define the maximum tolerated dose in East Asian patients, and doses in the range of 50–65 mg m⁻² have been selected for further studies.     

Natriuretic efficiency of frusemide as a consequence of drug input rate

Aims The aim of the present study was to investigate the influence of the rate of delivery of frusemide to its site of action on the effect and efficiency of the drug. Methods Frusemide 30 mg was administered as a bolus dose, a slow-rate infusion and a bolus dose in combination with 2 g of probenecid in a three way cross-over design to seven healthy volunteers. Urinary volume and contents of frusemide and sodium were measured in samples collected over 10 h. Results Total natriuretic response was 40% higher (P<0.001) after the infusion and 20% higher (P<0.05) after the combined treatment with probenecid, as compared with the bolus dose. Total natriuretic efficiency did not differ between the infusion (0.013 mmol μg⁻¹ ) and the combined treatment with probenecid (0.015 mmol μg⁻¹ ), but was significantly higher as compared with the bolus dose (0.009 mmol μg⁻¹ ). Natriuretic effect data were modeled according to the sigmoid E_max model and the frusemide excretion rate with maximum efficiency (ER_effmax) was calculated from the estimated parameters. For both the frusemide infusion and the combined treatment with probenecid, the time course of delivery of frusemide into the urine consistently approached ER_effmax more closely than was the case for the bolus dose. The natriuretic effect vs frusemide excretion rate curves were shifted to the right, and the estimated values of the sigmoid E_max model were higher for EC₅₀ and lower for the slope factor after the bolus dose, which may indicate tolerance development for this treatment. Conclusions Slowing the delivery of frusemide to the site of action increased the efficiency of the drug, leading to an increased natriuretic response.     

Bradykinin regulates human colonic ion transport in vitro

Background and purpose:

Kinins are acknowledged as important regulators of intestinal function during inflammation; however, their effects on human intestinal ion transport have not been reported. Here, we used muscle-stripped human colonic tissue and cultured T₈₄-cell monolayers to study bradykinin (BK) actions on human intestinal ion transport.

Experimental approach:

Ion transport was measured as changes in short-circuit current (I_sc) across colonic epithelia mounted in Ussing chambers.

Key results:

In intact tissue, there was a distinct polarity to BK-elicited I_sc responses. Whereas basolateral BK stimulated sustained responses (EC₅₀=0.5±0.1 μM), those to apical BK were more rapid and transient (EC₅₀=4.1±1.2 nM). In T₈₄ cells, responses to both apical and basolateral BK were similar to those seen upon apical addition to intact tissues. Cross-desensitization between apical and basolateral domains was not observed. BK-induced responses were largely due to Cl⁻ secretion as shown by their sensitivity to bumetanide and removal of Cl⁻ from the bathing solution. Studies using selective agonists and antagonists indicate responses to BK are mediated by B₂ receptors. Finally, responses to basolateral BK in intact tissues were inhibited by tetrodotoxin (1 μM), atropine (1 μM), capsaicin (100 μM) and piroxicam (10 μM). BK-stimulated prostaglandin (PG)E₂ release from colonic tissue.

Conclusions:

BK stimulates human colonic Cl⁻ secretion by activation of apical and basolateral B₂ receptors. Responses to apical BK reflect a direct action on epithelial cells, whereas those to basolateral BK are amplified by stimulation of enteric nerves and PG synthesis.     

Alteration of flow-induced dilatation in mesenteric resistance arteries of L-NAME treated rats and its partial association with induction of cyclo-oxygenase-2

1. We investigated the response to pressure (myogenic tone) and flow of rat mesenteric resistance arteries cannulated in an arteriograph which allowed the measurement of intraluminal diameter for controlled pressures and flows. Rats were treated for 3 weeks with N^G-nitro-L-arginine methyl ester (L-NAME, 50 mg kg⁻¹ day⁻¹) or L-NAME plus the angiotensin I converting enzyme inhibitor (ACEI) quinapril (10 mg kg⁻¹ day⁻¹).
2. Mean blood pressure increased significantly in chronic L-NAME-treated rats (155±4 mmHg, n=8, vs control 121±6 mmHg, n=10; P<0.05). L-NAME-treated rats excreted significantly more dinor-6-keto prostaglandin F_1α (dinor-6-keto PGF_1α), the stable urinary metabolite of prostacyclin, than control rats. The ACEI prevented the rise in blood pressure and the rise in urinary dinor-6-keto PGF_1α due to L-NAME.
3. Isolated mesenteric resistance arteries, developed myogenic tone in response to stepwise increases in pressure (42±6 to 847±10 mN mm⁻¹, from 25 to 150 mmHg, n=9). Myogenic tone was not significantly affected by the chronic treatment with L-NAME or L-NAME+ACEI.
4. Flow (100 μl min⁻¹) significantly attenuated myogenic tone by 50±6% at 150 mmHg (n=10). Flow-induced dilatation was significantly attenuated by chronic L-NAME to 22±6% at 150 mmHg (n=10, P=0.0001) and was not affected in the L-NAME+ACEI group.
5. Acute in vitro N^G-nitro-L-arginine (L-NOARG, 10 μM) significantly decreased flow-induced dilatation in control but not in L-NAME or L-NAME+ACEI rats. Both acute indomethacin (10 μM) and acute NS 398 (cyclo-oxygenase-2 (COX-2) inhibitor, 1 μM) did not change significantly flow-induced dilatation in controls but they both decreased flow-induced dilatation in the L-NAME and L-NAME+ACEI groups. Acute Hoe 140 (bradykinin receptor inhibitor, 1 μM) induced a significant contraction of the isolated mesenteric arteries which was the same in the 3 groups.
6. Immunofluorescence analysis of COX-2 showed that the enzyme was expressed in resistance mesenteric arteries in L-NAME and L-NAME+ACEI groups but not in control. COX-1 expression was identical in all 3 groups.
7. We conclude that chronic inhibition of nitric oxide synthesis is associated with a decreased flow-induced dilatation in resistance mesenteric arteries which was compensated by an overproduction of vasodilator prostaglandins resulting in part from COX-2 expression. The decrease in flow-induced dilatation was prevented by the ACEI, quinapril.

     

A pharmacokinetic and pharmacodynamic interaction study between nebivolol and the H2-receptor antagonists cimetidine and ranitidine

Aims The study was designed to investigate the effects of the H₂-receptor antagonists, cimetidine and ranitidine on the pharmacokinetics and pharmacodynamics of nebivolol in healthy volunteers. Methods Twelve healthy volunteers took part in a randomized placebo-controlled cross-over study. Each subject received on three separate occasions placebo, cimetidine (400 mg twice daily) or ranitidine (150 mg twice daily) for 24 h before and 48 h after a single oral dose of nebivolol (5 mg). Nebivolol and its individual (+) and (−) enantiomers were determined tby h.p.l.c. Results Ranitidine had no significant effect on nebivolol pharmacokinetics. Cimetidine, however, resulted in a 21–23% increase in C_max of unchanged nebivolol and of each enantiomer plus its hydroxylated metabolites. Cimetidine significantly (P<0.05) increased the AUC [mean±s.d. (95% C.I. of differences in mean)] for unchanged (±)-nebivolol [7.76±3.07 ng ml⁻¹h with placebo; 11.50±5.40 (1.75, 8.76) ng ml⁻¹h with cimetidine], (+)-nebivolol plus its hydroxylated metabolites [73.0±18.0 ng ml⁻¹h with placebo; 91.5±25.7 (1.0, 23.1) ng ml⁻¹h with cimetidine] and (−)-nebivolol plus its hydroxylated metabolites [101±32 ng ml⁻¹h with placebo; 123±38 (3.3, 27.0) ng ml⁻¹h with cimetidine]. Statistical analysis of the resting blood pressure and heart rate and exercise data did not suggest any consistent effects of ranitidine or cimetidine upon the pharmacodynamic effects of nebivolol. Conclusions There was no interaction between ranitidine and nebivolol. Although cimetidine inhibited nebivolol metabolism, it did not have a significant influence on the pharmacodynamics of the drug.     

Pharmacological characterization of CGRP receptors mediating relaxation of the rat pulmonary artery and inhibition of twitch responses of the rat vas deferens

1. CGRP receptors mediating vasorelaxation of the rat isolated pulmonary artery and inhibition of contractions of the rat isolated prostatic vas deferens were investigated using CGRP agonists, homologues and the antagonist CGRP_8-37.
2. In the pulmonary artery, human (h)α-CGRP-induced relaxation of phenylephrine-evoked tone was abolished either by removal of the endothelium or by N^G-nitro-L-arginine (10⁻⁵ M). The inhibitory effect of N^G-nitro-L-arginine was stereoselectively reversed by L- but not by D-arginine (10⁻⁴ M). Thus, CGRP acts via nitric oxide released from the endothelium.
3. In the endothelium-intact artery, hα-CGRP, hβ-CGRP and human adrenomedullin (10⁻¹⁰–3×10⁻⁷ M), dose-dependently relaxed the phenylephrine-induced tone with similar potency. Compared with hα-CGRP, rat amylin was around 50 fold less potent, while [Cys(ACM^2,7)] hα-CGRP (10⁻⁷–10⁻⁴ M) was at least 3000 fold less potent. Salmon calcitonin was inactive (up to 10⁻⁴ M).
4. Human α-CGRP_8-37 (3×10⁻⁷–3×10⁻⁶ M) antagonized hα-CGRP (pA₂ 6.9, Schild plot slope 1.2±0.1) and hβ-CGRP (apparent pK_B of 7.1±0.1 for hα-CGRP_8-37 10⁻⁶ M) in the pulmonary artery. Human β-CGRP_8-37 (10⁻⁶ M) antagonized hα-CGRP responses with a similar affinity (apparent pK_B 7.1±0.1). Human adrenomedullin responses were not inhibited by hα-CGRP_8-37 (10⁻⁶ M).
5. In the prostatic vas deferens, hα-CGRP, hβ-CGRP and rat β-CGRP (10⁻¹⁰–3×10⁻⁷ M) concentration-dependently inhibited twitch responses with about equal potency, while rat amylin (10⁻⁸–10⁻⁵ M) was around 10 fold less potent and the linear analogue [Cys(ACM^2,7)] hα-CGRP was at least 3000 fold weaker. Salmon calcitonin was inactive (up to 10⁻⁴ M).
6. The antagonist effect of hα-CGRP_8-37 (10⁻⁵–3×10⁻⁵) in the vas deferens was independent of the agonist, with pA₂ values against hα-CGRP of 6.0 (slope 0.9±0.1), against hβ-CGRP of 5.8 (slope 1.1±0.1), and an apparent pK_B value of 5.8±0.1 against both rat β-CGRP and rat amylin. Human β-CGRP_8-37 (3×10⁻⁵–10⁻⁴ M) competitively antagonized hα-CGRP responses (pA₂ 5.6, slope 1.1±0.2). The inhibitory effect of hα-CGRP on noradrenaline-induced contractions in both the prostatic and epididymal vas deferens was antagonized by hα-CGRP_8-37 (pA₂ 5.8 and 5.8, slope 1.0±0.2 and 1.0±0.3, respectively).
7. The effects of hα-CGRP and hα-CGRP_8-37 in both rat pulmonary artery and vas deferens were not significantly altered by pretreatment with peptidase inhibitors (amastatin, bestatin, captopril, phosphoramidon and thiorphan, all at 10⁻⁶ M). The weak agonist activity of [Cys(ACM^2,7)] hα-CGRP in the vas deferens was not increased by peptidase inhibitors.
8. These data demonstrate that two different CGRP receptors may exist in the rat pulmonary artery and vas deferens, a CGRP₁ receptor subtype in the rat pulmonary artery (CGRP_8-37 pA₂ 6.9), while the lower affinity for CGRP_8-37 (pA₂ 6.0) in the vas deferens is consistent with a CGRP₂ receptor.

     

ATP and vasoactive intestinal polypeptide relaxant responses in hamster isolated proximal urethra

1. Nitric oxide (NO) is known from previous studies to be the principle transmitter in NANC inhibitory nerves supplying the hamster urethra. However, the identity of the cotransmitter(s) responsible for the responses remaining following block with L-N^G-nitroarginine methyl ester (L-NAME) is not known.
2. Electrical field stimulation (EFS) of circular strips of hamster proximal urethra precontracted with arginine vasopressin (AVP 10⁻⁸ M), and in the presence of phentolamine (10⁻⁶ M), propranolol (10⁻⁶ M) and atropine (10⁻⁶ M), caused frequency-dependent relaxation, which was attenuated by suramin (10⁻⁴ M) and reactive blue 2 (RB2; 2×10⁻⁴ M), but not by pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 10⁻⁴ M), α-chymotrypsin (10–50 u ml⁻¹) or by the vasoactive intestinal polypeptide (VIP) antagonist, [Lys¹, Pro^2,5, Arg^3,4, Tyr⁶]-VIP, (5×10⁻⁷–10⁻⁶ M). In the presence of indomethacin (10⁻⁶ M) frequency-dependent relaxations to EFS were enhanced, particularly at the lower frequencies of stimulation. EFS-induced relaxation was blocked by tetrodotoxin (10⁻⁶ M), indicating its neurogenic origin.
3. Exogenous ATP (10⁻⁷–10⁻³ M) produced concentration-related relaxations which were attenuated by the P2-purinoceptor antagonists suramin (10⁻⁴ M) and RB2 (2×10⁻⁴ M) but not by PPADS (10⁻⁴ M). ATP-induced relaxations were also reduced significantly by indomethacin (10⁻⁶ M). The inhibitory responses to ATP were urothelium- and NO-independent, since they were not affected by either removal of urothelium or by L-NAME (10⁻⁴ M).
4. Exogenous VIP (10⁻⁹–10⁻⁷ M) induced concentration-related relaxations which were not affected by urothelium removal, L-NAME (10⁻⁴ M), α-chymotrypsin (10–50 u ml⁻¹) or by [Lys¹, Pro^2,5, Arg^3,4, Tyr⁶]-VIP (3×10⁻⁷–10⁻⁶ M). Nevertheless, suramin (10⁻⁴ M) and RB2 (2×10⁻⁴ M) but not PPADS (10⁻⁴ M) antagonized the VIP-induced relaxant responses. Calcitonin gene-related peptide (CGRP: 10⁻⁹–10⁻⁷ M) was devoid of any effect or only elicited a small relaxant response in AVP-precontracted strips.
5. Exogenous prostaglandin E₂ (PGE₂; 10⁻⁹–3×10⁻⁶ M) and the NO donor, sodium nitroprusside (SNP; 10⁻⁸–3×10⁻⁵ M) elicited concentration-related relaxations on the hamster proximal urethra which were not attenuated by suramin (10⁻⁴ M), RB2 (2×10⁻⁴ M), or by PPADS (10⁻⁴ M), indicating a specific inhibitory effect of the antagonists used.
6. In summary, these results are consistent with the view that ATP is an inhibitory transmitter released from inhibitory nerves supplying the NANC relaxation of hamster proximal urethra. The relaxant effect of ATP is NO- and urothelium-independent. The present study did not demonstrate whether VIP is released from parasympathetic nerves during EFS, since both α-chymotrypsin and [Lys¹, Pro^2,5, Arg^3,4, Tyr⁶]-VIP were ineffective on neurogenic responses.

     

Influence of a selective guanylate cyclase inhibitor,and of the contraction level,on nitrergic relaxations in the gastric fundus

1. The influence of the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) on non-adrenergic non-cholinergic (NANC) relaxations and the possible role of a nerve-derived hyperpolarizing factor in NANC relaxation were investigated in the rat gastric fundus.
2. ODQ (10⁻⁶ and 10⁻⁵ M) concentration-dependently inhibited the short-lasting relaxations by NO (2×10⁻⁶ M–10⁻⁴ M) administered as a bolus without influencing the relaxation by 3×10⁻⁸ M isoprenaline. The relaxation by an infusion of NO was reduced to the same extent by 10⁻⁶ and 10⁻⁵ M ODQ.
3. The electrically induced short-lasting and sustained relaxations (40 V, 1 ms, 0.5–16 Hz, 10 s trains at 2 min interval or cumulative increase in the frequency every 2 min) in NANC conditions were inhibited to a similar extent by 10⁻⁶ and 10⁻⁵ M ODQ, and by the NO synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 3×10⁻⁴ M).
4. ODQ (10⁻⁶ M) and L-NAME (3×10⁻⁴ M), administered after 5, 10 or 20 min of long-term stimulation, reversed the relaxation to a similar extent (approximately 50% at 2 Hz and 20% at 8 Hz).
5. When the tissues were contracted to 40% of maximum by adapting the concentration of prostaglandin F_2α (PGF_2α), the inhibitory effect of 3×10⁻⁴ M L-NAME on relaxations induced by train and cumulative stimulation was the same as when tissues were contracted with 3×10⁻⁷ M PGF_2α.
6. The findings of this study illustrate that the relaxation by exogenous and endogenous NO in the rat gastric fundus is due to activation of soluble guanylate cyclase. During long-term electrical stimulation, the partial contribution of NO to NANC relaxation is maintained but it is small at higher frequencies of stimulation. Evidence for the contribution of a nerve-derived hyperpolarizing factor to NANC relaxation was not obtained.

     

Response of normoxic pulmonary arteries of the rat in the resting and contracted state to NO synthase blockade

1. The pulmonary vasculature is normally in a low resting state of tone. It has been hypothesized that this basal tone is actively maintained by the continuous release of a vasodilator in the resting state. However, evidence for basal release of nitric oxide (NO) is inconclusive.
2. We studied the release of NO in arteries from the pulmonary circulation of male Wistar-Kyoto rats by examining the effects of the L-arginine analogue N^G-nitro-L-arginine methyl ester (L-NAME) on resting pulmonary arteries and on vessels pre-contracted with prostaglandin F_2α (PGF_2α).
3. Rats (n=21) were killed by an overdose with pentobarbitone. Pulmonary arteries were dissected (mean internal diameter 459±11 μm) and mounted in a small vessel wire myograph. Resting tensions were set to simulate transmural pressures of 17.5 mmHg.
4. L-NAME (100 μM) was found to produce a contraction of 0.64±0.09 mN mm⁻¹ in resting pulmonary arteries when added alone to the myograph bath. This contraction was not produced following removal of the endothelium. Vessel contraction to PGF_2α (100 μM) was found to be significantly greater when carried out in the presence of L-NAME (100 μM)–1.37±0.15 mN mm⁻¹ compared with 1.96±0.17 mN mm⁻¹. Dilatation following acetylcholine (ACh) (1 μM) was abolished in the presence of L-NAME (100 μM).
5. Rat pulmonary artery contraction in response to the addition of L-NAME and the absence of contraction upon removal of the endothelium provides supportive evidence of the active release of nitric oxide for the maintenance of resting tone.

     

Evaluation of sulfonamide detoxification pathways in haematologic malignancy patients prior to intermittent trimethoprim-sulfamethoxazole prophylaxis

AIMS

Patients with haematologic malignancies have a reportedly high incidence of sulfamethoxazole (SMX) hypersensitivity. The objective of this study was to determine whether deficiencies in sulfonamide detoxification pathways, to include glutathione (GSH) and ascorbate (AA), and cytochrome b₅ (b5) and cytochrome b₅ reductase (b5R), were prevalent in these patients. A secondary pilot objective was to determine whether the incidence of drug hypersensitivity following intermittent trimethoprim-SMX (TMP-SMX) prophylaxis approached that reported for high dose daily regimens.

METHODS

Forty adult patients with haematologic malignancies (HM) and 35 healthy adults were studied; an additional 13 HM patients taking ascorbate supplements (HM-AA) were also evaluated. Twenty-two of 40 HM patients were prescribed and were compliant with TMP-SMX 960 mg three to four times weekly.

RESULTS

There were no significant differences between HM and healthy groups in plasma AA (median 37.2 µmvs. 33.9 µm) or red blood cell GSH (1.9 mmvs. 1.8 mm). However, plasma AA was correlated significantly with leucocyte b5/b5R reduction (r = 0.39, P = 0.002). Deficient b5/b5R activities were not found in HM patients. In fact, patients with chronic lymphocytic leukaemia or myeloma had significantly higher median activities (80.7 µmol mg^–1 min⁻¹) than controls (18.9 µmol mg^–1 min⁻¹, P = 0.008). After 3–4 weeks of treatment, no patients developed SMX-specific T cells and only one patient developed rash.

CONCLUSIONS

Deficiencies of blood antioxidants and b5/b5R reduction were not found in this population with haematologic malignancies, and the development of skin rash and drug-specific T cells appeared to be uncommon with intermittent TMP-SMX prophylaxis.     

Evidence for a dilator function of 8-iso prostaglandin F2α in rat pulmonary artery

1. 8-Iso prostaglandin F_2α (8-iso PGF_2α) is one of a series of prostanoids formed independently of the cyclo-oxygenase pathway. It has been shown to be upregulated in many conditions of oxidant stress where its formation is induced by free radical-catalysed actions on arachidonic acid. As 8-iso PGF_2α is formed in vivo in diseases in which oxidant stress is high such as septic shock, we have assessed the relative potency and efficacy of this compound in pulmonary arteries from control and lipopolysaccharide (LPS)-treated rats.
2. Several studies have characterized the contractile actions of 8-iso PGF_2α on various smooth muscle preparations, but its potential dilator actions have not been addressed. Thus these studies examined both the contractile and dilator actions of 8-iso PGF_2α in rat pulmonary artery rings. The thromboxane mimetic U46619, PGE₂ sodium nitroprusside (SNP) and acetyl choline (ACh) were used for comparison. Each prostanoid had to be dissolved in ethanol to a maximum concentration of 1×10⁻² M. At high concentrations, ethanol directly contracted pulmonary vessels. We were therefore limited by the actions of the vehicle such that we were unable to add prostanoids at concentrations higher than 1×10⁻⁴ M. In some cases this meant that maximum responses were not achieved and in these cases the E_max and pD₂ values are apparent estimates.
3. The following rank order of potency was obtained from contractile studies; U46619>8-iso PGF_2α>PGE₂, each prostanoid producing concentration-dependent contractions (10⁻¹⁰3×10⁻⁴ M, 10⁻⁹10⁻⁴ M, 10⁻⁸10⁻⁴ M, respectively). As has been shown previously for other smooth muscle preparations, the thromboxane receptor (TP) antagonist ICI 192605, (1×10⁻⁶, 1×10⁻⁵ and 1×10⁻⁴ M), inhibited the contractions of 8-iso PGF_2α in a concentration-dependent fashion.
4. The nitric oxide synthase inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME; 1×10⁻⁴ M), enhanced the contractile function of both 8-iso PGF_2α and PGE₂, but had no effect on that caused by U46619. Similarly, L-NAME inhibited the dilator function of all agents tested except the exogenous nitric oxide (NO) donor SNP, indicating that PGE₂ and 8-iso PGF_2α like ACh, act through the release of NO. The specificity of the effects of L-NAME were confirmed in studies with the inactive enantiomer D-NAME (1×10⁻⁴ M), which did not affect the contractile or the dilator actions of 8-iso PGF_2α. Furthermore, ICI 192605 enhanced the dilator actions of 8-iso PGF_2α, suggesting that the dilator component of 8-iso PGF_2α was achieved via activation of a non-TP receptor.
5. Isoprostanes may modulate vascular tone by a direct action on TP receptors to cause contraction and via a distinct receptor leading to the release of NO to cause dilatation.

     

Clozapine, but not haloperidol, enhances glial D-serine and L-glutamate release in rat frontal cortex and primary cultured astrocytes

BACKGROUND AND PURPOSE

Deficient transmission at the glutamate NMDA receptor is considered a key component of the pathophysiology of schizophrenia. However, the effects of antipsychotic drugs on the release of the endogenous NMDA receptor partial agonist, d-serine, remain to be clarified.

EXPERIMENTAL APPROACH

We determined the interaction between antipsychotic drugs (clozapine and haloperidol) and transmission-modulating toxins (tetanus toxin, fluorocitrate, tetrodotoxin) on the release of L-glutamate and d-serine in the medial prefrontal cortex (mPFC) of freely moving rats, using microdialysis, and primary cultures of astrocytes using extreme high-pressure liquid chromatography.

KEY RESULTS

Release of L-glutamate and d-serine in the mPFC and in cultured astrocytes was inhibited by tetanus toxin (a synaptobrevin inhibitor) and fluorocitrate (a glial toxin), whereas tetrodotoxin (a voltage-sensitive Na⁺ blocker) inhibited depolarization-induced L-glutamate release in the mPFC without affecting that of d-serine. Clozapine (1 and 5 mg·kg⁻¹), but not haloperidol (0.5 and 1 mg·kg⁻¹), dose-dependently increased L-glutamate and d-serine release from both astrocytes and mPFC. Clozapine-induced release of L-glutamate and d-serine was also reduced by tetanus toxin and fluorocitrate. Tetrodotoxin reduced clozapine-induced mPFC L-glutamate release but not that of d-serine. Clozapine-induced L-glutamate release preceded clozapine-induced d-serine release. MK-801 (a NMDA receptor antagonist) inhibited the delayed clozapine-induced L-glutamate release without affecting that of d-serine.

CONCLUSIONS AND IMPLICATIONS

Clozapine predominantly activated glial exocytosis of d-serine, and this clozapine-induced d-serine release subsequently enhances neuronal L-glutamate release via NMDA receptor activation. The enhanced d-serine associated glial transmission seems a novel mechanism of action of clozapine but not haloperidol.     

Potentiation by vasopressin of adrenergic vasoconstriction in the rat isolated mesenteric artery

1. The aim of the present study was to investigate in rat mesenteric artery rings whether low concentrations of vasopressin could modify the contractile responses to noradrenaline and electrical stimulation of perivascular nerves.
2. Vasopressin (10⁻¹⁰–10⁻⁷ M) caused concentration-dependent contractions (pD₂=8.36±0.09). The V₁-receptor antagonist d(CH₂)₅Tyr(Me)AVP (10⁻⁹–10⁻⁸ M) produced parallel rightward shifts of the control curve for vasopressin. Schild analysis yielded a pA₂ value of 9.83 with a slope of 1.10±0.14.
3. Vasopressin (3×10 ⁻¹⁰ and 10⁻⁹ M) caused concentration-dependent potentiation of the contractions elicited by electrical stimulation (2–8 Hz; 0.2 ms duration for 30 s) and produced leftward shifts of the concentration-response curve for noradrenaline. The V₁-receptor antagonist induced concentration-dependent inhibitions of potentiation induced by vasopressin. The selective V₁-receptor agonist [Phe^*, Orn⁸]-vasotocin (3×10 ⁻¹⁰ and 10⁻⁹ M) induced potentiation of electrical stimulation-evoked responses which was also inhibited in the presence of the V₁ antagonist (10⁻⁸ M). In contrast, the V₂-receptor agonist deamino-8-D-arginine vasopressin (desmopressin 10⁻⁸–10⁻⁷ M) did not modify the electrical stimulation-induced responses and the V₂-receptor antagonist [d(CH₂)₅, D-Ile^*, Ile⁴, Arg⁸]-vasopressin (10⁻⁸–10⁻⁷ M) did not affect the potentiation evoked by vasopressin.
4. In artery rings contracted by 10⁻⁶ M noradrenaline in the presence of 10⁻⁶ M guanethidine and 10⁻⁶ M atropine, electrical stimulation (2, 4 and 8 Hz) produced frequency-dependent relaxations which were unaffected by 10⁻⁹ M vasopressin but abolished by 10⁻⁶ M tetrodotoxin.
5. Vasopressin also potentiated contractions elicited by KCl and contractions induced by addition of CaCl₂ to KCl depolarized vessels. The augmenting effects were inhibited by the V₁ antagonist.
6. In the presence of the calcium antagonist nifedipine (10⁻⁶ M), vasopressin failed to enhance the contractile responses to electrical stimulation, noradrenaline and KCl.
7. The results demonstrate that low concentrations of vasopressin strongly potentiate the contractions to adrenergic stimulation and KCl depolarization. This effect appears to be mediated by V₁ receptor stimulation which brings about an increase in calcium entry through dihydropyridine-sensitive calcium channels.