Rationale and design of the iCORONARY trial: improving the cost-effectiveness of coronary artery disease diagnosis.

BACKGROUND: In patients with stable coronary artery disease (CAD), revascularisation decisions are based mainly on the visual grading of the severity of coronary stenosis on invasive coronary angiography (ICA). However, invasive fractional flow reserve (FFR) is the current standard to determine the haemodynamic significance of coronary stenosis. Non-invasive and less-invasive imaging techniques such as computed-tomography-derived FFR (FFR-CT) and angiography-derived FFR (QFR) combine both anatomical and functional information in complex algorithms to calculate FFR. TRIAL DESIGN: The iCORONARY trial is a prospective, multicentre, non-inferiority randomised controlled trial (RCT) with a blinded endpoint evaluation. It investigates the costs, effects and outcomes of different diagnostic strategies to evaluate the presence of CAD and the need for revascularisation in patients with stable angina pectoris who undergo coronary computed tomography angiography. Those with a Coronary Artery Disease-Reporting and Data System (CAD-RADS) score between 0-2 and 5 will be included in a prospective registry, whereas patients with CAD-RADS 3 or 4A will be enrolled in the RCT. The RCT consists of three randomised groups: (1) FFR-CT-guided strategy, (2) QFR-guided strategy or (3) standard of care including ICA and invasive pressure measurements for all intermediate stenoses. The primary endpoint will be the occurrence of major adverse cardiac events (death, myocardial infarction and repeat revascularisation) at 1 year. CLINICALTRIALS: gov-identifier: NCT04939207. CONCLUSION: The iCORONARY trial will assess whether a strategy of FFR-CT or QFR is non-inferior to invasive angiography to guide the need for revascularisation in patients with stable CAD. Non-inferiority to the standard of care implies that these techniques are attractive, less-invasive alternatives to current diagnostic pathways.

Calculation of plasma concentrations of intra-arterially infused compounds in forearm plethysmography.

OBJECTIVE: Forearm blood flow plethysmography is a widely accepted in vivo technique for pharmacologic and functional studies in peripheral resistance vessels and veins. Pharmacological effects on forearm blood flow (FBF) are usually expressed by means of dose-response relationships. This approach does not consider the influence of variations in FBF on the actual plasma concentrations of compounds infused, and is less suitable for quantitative comparison of the pharmacologic characteristics of different compounds. The aim of this study was to validate an equation to estimate the plasma concentrations of intra-arterially infused compounds. This was done at different levels of FBF, using an indicator dilution technique with constant rate infusions of indocyanine green (ICG) and inulin. METHODS: ICG (0.5 mg/min) and insulin (5 mg/min) were infused into the brachial artery in the presence of sodium nitroprusside (10 ng/kg/min; to obtain high FBF), vehicle (0.9% saline; for intermediate FBF), and methoxamine (1 microgram/kg/min; for low FBF), FBF was measured using venous occlusion plethysmography in six healthy male volunteers. Plasma concentrations of the indicators, measured in venous blood samples, were compared with the calculated values. RESULTS: Excellent correspondence was observed between calculated and measured plasma concentrations for both ICG and inulin. Venous plasma concentrations of ICG (> or = 95% protein binding) reached steady-state within four min independent of FBF. Alternatively, the time required for venous plasma concentrations of inulin (not bound to protein) to reach steady-state appeared dependent on FBF. CONCLUSION: Total plasma concentrations of intra-arterially infused drugs can be appropriately estimated at the level of the arterioles by the proposed equation.

The interaction between methylene blue and the cholinergic system.

1. The inhibitory effects of methylene blue (MB) on different types of cholinesterases and [3H]-N-methylscopolamine ([3H]-NMS) binding to muscarinic receptors were studied. 2. Human plasma from young healthy male volunteers, purified human pseudocholinesterase and purified bovine true acetylcholinesterase were incubated with acetylcholine and increasing concentrations of MB (0.1-100 mumol l-1) in the presence of the pH-indicator m-nitrophenol for 30 min at 25 degrees C. The amount of acetic acid produced by the enzymatic hydrolysis of acetylcholine was determined photometrically. 3. Rat cardiac left ventricle homogenate was incubated with [3H]-NMS and with increasing concentrations of MB (0.1 mmol l-1 mumol l-1) at 37 degrees C for 20 min. THe binding of [3H]-NMS to the homogenate was quantified by a standard liquid scintillation technique. 4. MB inhibited the esterase activity of human plasma, human pseudocholinesterase and bovine acetylcholinesterase concentration-dependently with IC50 values of 1.05 +/- 0.05 mumol l-1, 5.32 +/- 0.36 mumol l-1 and 0.42 +/- 0.09 mumol l-1, respectively. MB induced complete inhibition of the esterase activity of human plasma and human pseudocholinesterase, whereas bovine acetylcholinesterase was maximally inhibited by 73 +/- 3.3%. 5. MB was able to inhibit specific [3H]-NMS binding to rat cardiac left ventricle homogenate completely with an IC50 value of 0.77 +/- 0.03 mumol l-1, which resulted in a Ki value for MB of 0.58 +/- 0.02 mumol l-1. 6. In conclusion, MB may be considered as a cholinesterase inhibitor with additional, relevant affinity for muscarinic binding sites at concentrations at which MB is used for investigations into the endothelial system. In our opinion these interactions between MB and the cholinergic system invalidate the use of MB as a tool for the investigation of the L-arginine-NO-pathway, in particular when muscarinic receptor stimulation is involved.

Comparison of cholinergic vasodilator responses to acetylcholine and methacholine in the human forearm.

In order to study the contribution of the nitric oxide (NO)-pathway to cholinergic vasodilatation in the resistance vessels of the human forearm, we infused acetylcholine (ACh; 0.1 1000 ng/kg/min) or methacholine (MCh; 0.1 A 100 ng/kg/min) in the presence of saline, the NO-scavenger and guanylate cyclase inhibitor methylene blue (MB; 1000 ng/kg/min), or the NO-synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 30 micrograms/kg/min) into the brachial artery of normotensive volunteers (n = 32), using venous occlusion plethysmography. We calculated the plasma concentrations of the infused compounds to obtain EC50-values (-log mol/l). ACh and MCh both caused concentration-dependent vasodilatation (EC50-values of 6.43 +/- 0.05 and 7.24 +/- 0.08, respectively). MB (13 mumol/l) did not change basal forearm blood flow (FBF) when administered alone, but it markedly potentiated the vasodilator response to ACh, shifting the concentration-response curve (CRC) leftwards by 1.5 log-step (p < 0.001). MB did not affect MCh-induced vasodilatation. L-NMMA (1 mmol/l) alone caused dose-dependent vasoconstriction that was subject to tachyphylaxis. In addition, L-NMMA caused a steepening of the slopes of the CRCs of ACh, and MCh L-NMMA attenuated the ACh-/MCh-induced vasodilator responses in the lowest concentration ranges (p < 0.05) only, but did not alter the response at higher concentrations. The 10-fold higher potency of MCh compared to ACh can be explained by the more rapid degradation of ACh by cholinesterases. The observation that high concentrations of L-NMMA only affect vasodilation mediated by low concentrations of ACh or MCh, suggests a second mechanism in cholinergic vasodilatation, such as a direct effect on smooth muscle cells or the release of a relaxing factor other than NO.

Cholinergic receptor-mediated responses in the arteriolar and venous vascular beds of the human forearm.

In arterioles, acetylcholine (ACh) is a well known vasodilator. However, in veins a wide variation in responses to ACh has been reported. In the present study the effects of the cholinergic agonists acetylcholine and methacholine (MCh) were determined simultaneously both in arterial and venous vasculature in the forearm vascular bed of healthy volunteers by means of venous occlusion plethysmography. The vasodilator sodium nitroprusside (SNP) served as an endothelium-independent control agent. The vascular beds were preconstricted by the selective alpha 1-adrenoceptor agonist methoxamine. Atropine, a non-selective muscarinic receptor antagonist, was used to antagonize the dilator effect of MCh. Overall we observed a weaker relaxant effect of ACh, MCh and SNP in the veins compared with their dilator responses in the arteries. ACh, which is highly sensitive to the hydrolytic inactivation by choline esterases, failed to induce a significant vasodilation in the venous vascular bed. Atropine blocked the dilator effects of MCh, indicating the involvement of muscarinic receptors. In arteries, MCh did not induce a significantly stronger vasodilatation than SNP on a molar basis. However, in veins, MCh had a weaker relaxant effect (p < 0.05).

In vivo characterization of muscarinic receptor subtypes that mediate vasodilatation in patients with essential hypertension.

Attenuated cholinergic vasodilatation has been suggested as an endothelium-related mechanism involved in essential hypertension. We investigated the role of muscarinic (M) receptor subtypes in the forearm resistance vasculature. In eight white men with essential hypertension and eight matched normotensive control subjects (age of both groups, 47 +/- 4 years; mean +/- SEM), we infused the nonselective agonist methacholine in the presence of saline and the antagonists atropine (nonselective), pirenzepine (M1-selective), and AF-DX 116 (M2-selective) into the brachial artery and measured forearm blood flow and forearm vascular resistance using venous occlusion plethysmography. Affinity constants (pKb values) were determined from calculated plasma concentrations of the infused compounds and EC50 values. Sodium nitroprusside was given as an endothelium-independent control, and minimal forearm vascular resistance after 10 minutes of ischemia was used as a marker of structural vascular changes. Hypertensive patients showed higher minimal forearm vascular resistance, indicating structural vascular changes. However, sodium nitro-prusside- and methacholine-induced vasodilatation was similar in both groups, with apparent EC50 values (log moles per liter; mean +/- SEM) of -7.32 +/- 0.13 and -7.51 +/- 0.21 in hypertensive patients and -7.37 +/- 0.13 and -7.45 +/- 0.02 in control subjects, respectively. Atropine, pirenzepine, and AF-DX 116 caused a shift to the right of the concentration-response curve of methacholine, with apparent pKb values of 8.63 +/- 0.08, 6.81 +/- 0.13, and 5.51 +/- 0.29 in hypertensive individuals and 8.62 +/- 0.10, 6.98 +/- 0.08, and 5.49 +/- 0.09 in control subjects, respectively. Again, there were no statistically significant differences in these pharmacological parameters between hypertensive patients and normotensive subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of peripheral vascular effects of antimigraine drugs in humans.

The vascular beds of the forearm and finger can be used to study the peripheral effects of antimigraine drugs under normal and pathologic circumstances. We have investigated the novel antimigraine drug sumatriptan, a selective agonist for 5HT1 receptors. Its antimigraine effect may be attributed, at least in part, to constriction of cranial arteriovenous anastomoses (AVAs). In assessing the peripheral vascular effects of sumatriptan we used a forearm and finger blood flow model. Forearm blood flow (FBF) is mainly determined by resistance vessels, whereas finger blood flow (FiBF) mainly involves skin vessels, which contain many AVAs. Changes in FBF and FiBF can be assessed using venous occlusion plethysmography. Changes in AVA flow are determined by measuring the patency of the vascular beds of the forearm and hand to well-defined radiolabelled microspheres, which are injected into the brachial artery. We report the effects of sumatriptan on FBF, FiBF and AVA flow when administered into the brachial artery of healthy volunteers, and discuss the peripheral vascular effects of therapeutic doses of sumatriptan when given subcutaneously in migraine patients during and between attacks.

No functional involvement of 5-hydroxytryptamine1A receptors in nitric oxide-dependent dilatation caused by serotonin in the human forearm vascular bed.

The vascular effects of serotonin (5-hydroxytryptamine, 5-HT) are complex and heterogeneous. In human forearm, we showed that low doses of 5-HT cause marked but transient vasodilatation followed by a persistent vasodilator response. In in vitro and in animal experiments, 5-HT induced release of nitric oxide (NO) through stimulation of endothelial 5-HT1-like receptors. In the present study, we investigated involvement of the "NO pathway" and possible involvement of the 5-HT1A receptor subtype in 5-HT-induced persistent vasodilator response. In 8 healthy volunteers, we infused 5-HT (0.1, 0.3, and 1 ng/kg/min) and the selective 5-HT1A receptor agonist flesinoxan (15, 45, and 150 ng/kg/min) intraarterially (i.a.) with NG-monomethyl-L-arginine (L-NMMA 30 micrograms/kg/min) or saline. Forearm blood flow (FBF) was measured by automated R-wave-triggered venous occlusion plethysmography. Forearm vascular resistance (FVR) was derived from simultaneously recorded i.a. blood pressure (BP) and FBF. 5-HT dose-dependently decreased FVR (p < 0.001). The persistent vasodilator response to 5-HT appears to be mediated by NO release, as suggested by its complete abolition by L-NMMA (p < 0.001). Flesinoxan decreased FVR slightly, but only at high doses (p < 0.05). The present findings indicate that 5-HT1A receptors are not functionally involved in 5-HT-mediated vasodilatation in human forearm.

Interactions between serotonin and endogenous and exogenous noradrenaline in the human forearm.

We investigated the interactions between serotonin (5-hydroxytryptamine, 5-HT) and exogenous or endogenous noradrenaline (NA) in the forearm of normotensive volunteers (n = 24), using venous occlusion plethysmography. Endogenous release of NA was stimulated by lower body negative pressure (LBNP; -10, -20 and -40 mmHg) and by intra-arterial (i.a.) infusions of tyramine (0.1 to 1 microgram/kg/min). Exogenous NA was infused in cumulative doses (0.3 to 10 ng/kg/min). All experiments were performed in the presence of either vehicle (0.4 mL/min), sodium nitroprusside (SNP; 3 or 5 ng/kg/min), or 5-HT (0.1 or 1 ng/kg/min). NA or 5-HT (1 ng/kg/min) were also infused with the selective 5-HT2 receptor antagonist ritanserin (500 ng/kg/min). Angiotensin II (Ang II; 0.03 to 0.3 ng/kg/min) was given as a vasoconstrictor control (n = 6). Vasoconstriction to exogenous NA was significantly enhanced by 5-HT (1 ng/kg/min; p < 0.05) in one group of subjects, but this could not be reproduced in a second group. In contrast, the vasoconstrictor responses to endogenous NA remained unaffected by 5-HT. SNP caused the same degree of vasodilation as 5-HT, and was followed by a similar response to NA. Ritanserin did not alter the vasoconstriction to infused NA, nor vasoconstriction to infused NA when combined with 5-HT. The vasoconstrictor response to Ang II was not influenced by 5-HT (0.1 ng/kg/min). Apparently, a synergistic effect between 5-HT and NA in the periphery involves a nonspecific mechanism. The latter seems to be confined to infused NA. The precise role of the 5-HT2 receptor in this interaction could not be elucidated.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo characterization of vasodilating muscarinic-receptor subtypes in humans.

The role of muscarinic (M)-receptor subtypes in the regulation of vascular tone has not yet been defined in humans. To analyze the role of M-receptor subtypes in the forearm resistance vasculature of normotensive volunteers (n = 20), we infused acetylcholine (ACh) and methacholine (MCh) in the presence of saline and the antagonists atropine (nonselective), pirenzepine (M1 selective), and AF-DX 116 (M2 selective), using automated R-wave-triggered venous occlusion plethysmography. Schild analysis was applied by calculating plasma concentrations of the infused compounds and determining EC50 values. ACh and MCh both caused dose-dependent vasodilation, with EC50 values of 537 and 52 nmol/L, respectively. The apparent 10-fold higher potency of MCh compared with ACh may be explained by rapid degradation of ACh in plasma. The concentration-response curve of MCh was shifted to the right by atropine, pirenzepine, and AF-DX 116, with apparent pA2 values of 8.03 +/- 0.03, 6.71 +/- 0.08, and 5.32 +/- 0.05, respectively, and slopes not different from unity. The present technique enabled us to perform M-receptor characterization by Schild analysis in humans. The affinity constants and rank order of potency--atropine > pirenzepine > AF-DX 116-suggest that cholinergic vasodilation in this vascular bed is predominantly mediated by the M3-receptor subtype. The EC50 value of MCh and the pA2 values of pirenzepine and AF-DX 116 are comparable to values reported for in vitro experiments.

Comparison of the hemodynamic effects of urapidil and flesinoxan in healthy volunteers.

The possible role of peripheral 5HT1A-receptors in the vasodilation caused by urapidil was studied by means of venous occlusion plethysmography in the forearm vascular bed of healthy volunteers. Urapidil is known to be an alpha 1-adrenoceptor antagonist and an agonist of 5HT1A-receptors. The hemodynamic effects of urapidil were compared with those of flesinoxan, a selective 5HT1A-receptor agonist virtually devoid of alpha 1-adrenoceptor antagonistic activity, and with the selective alpha 1-adrenoceptor antagonist doxazosin, which has no affinity for 5HT1A-receptors. Urapidil, as well as doxazosin, caused a dose-dependent decrease in forearm vascular resistance (FVR), thus reflecting vasodilation. Both urapidil and doxazosin were competitive antagonists of the vasoconstrictor effect of the selective alpha 1-adrenoceptor agonist methoxamine. On a molar base doxazosin proved more potent than urapidil (more than 10-fold). Flesinoxan slightly decreased FVR only at high doses. The nitric oxide (NO)-synthase inhibitor NG-monomethyl-L-arginine acetate (L-NMMA) depressed the vasodilatation caused by serotonin and also that by high-dose flesinoxan. The serotonin-induced vasodilatation is known to be NO-dependent. From the experiments it is concluded that peripheral 5HT1A-receptors cannot play an important role in the vasodilator response caused by urapidil, which is predominantly the result of postsynaptic alpha 1-adrenoceptor blockade. 5HT1A-receptors are clearly not involved in the NO-dependent dilatation caused by serotonin. During chronic treatment of hypertension with urapidil central but not peripheral 5HT1A-receptors may be assumed to play a role.

Serotonin-induced vasodilatation in the human forearm is mediated by the "nitric oxide-pathway": no evidence for involvement of the 5-HT3-receptor.

The "nitric oxide (NO)-pathway" is presumed to be involved in acetylcholine (ACh)- and serotonin (5-hydroxytryptamine, 5-HT)-mediated vasodilatation. In addition, both the 5-HT-induced transient and persistent vasodilator responses in the forearm vascular bed are abolished by the 5-HT3/5-HT4-receptor antagonist ICS 205-930 ([1H]-indol-3-carbonic-acid-tropine-ester HCl, tropisetron). We studied 5-HT-mediated vasodilatation in the forearm vascular bed of normotensive volunteers, using venous occlusion plethysmography. Intraarterial (i.a.) infusions of 5-HT, ACh, and sodium nitroprusside (SNP) all caused an increase in forearm blood flow (FBF). Single infusions of ondansetron and granisetron also caused an increase in FBF. Infusion of the NO scavenger and guanylate-cyclase antagonist methylene blue (MB) did not change FBF, whereas the arginine analogue NG-monomethyl-L-arginine (L-NMMA) caused a decrease in FBF, which became less pronounced when infusions were repeated. Unlike ICS 205-930, concomitant infusions of the selective 5-HT3-receptor antagonists ondansetron (OND) and granisetron (GRAN) did not antagonize the transient or persistent vasodilator responses to 5-HT. These findings suggest the involvement of a 5-HT4-receptor. L-NMMA and MB both reduced the persistent vasodilator response to 5-HT, indicating involvement of the NO pathway. Neither MB nor L-NMMA influenced the endothelium-independent vasodilator response to SNP. ACh-induced vasodilatation was markedly potentiated by MB but was not affected by L-NMMA. The mechanism by which MB enhances the vasodilator response to ACh remains unclear.