Effects of P2Y(1) receptor antagonism on the reactivity of platelets from patients with stable coronary artery disease using aspirin and clopidogrel

BACKGROUND AND PURPOSE

P2Y₁ is a purine receptor that triggers platelet aggregation. Its inhibition was studied in patients with stable coronary artery disease (CAD) receiving standard anti-platelet therapy.

EXPERIMENTAL APPROACH

Blood samples from 10 patients on aspirin therapy (ASA, 80 mg·day⁻¹) were withdrawn before and 24 h after the administration of 450 mg clopidogrel (ASA/C) and were anti-coagulated with citrate or hirudin/PPACK in the presence or absence of the P2Y₁ inhibitor MRS2179 (M, 100 µM). Platelet responses to ADP (2.5 µM) and TRAP (2.5 µM), and collagen-induced thrombosis under flow conditions were analysed.

KEY RESULTS

Compared with ASA, ASA + M strongly inhibited ADP-induced peak platelet aggregation (88%), late aggregation (84%), P-selectin expression (85%) and α_IIbβ₃ activation (62%) (28%, 65%, 70% and 51% inhibition, respectively, for ASA/C vs. ASA). ASA + M also inhibited platelet/monocyte and platelet/neutrophil conjugate formation by 69% and 71% (57% and 59% for ASA/C vs. ASA). In TRAP-activated blood, ASA + M unexpectedly inhibited α_IIbb₃ activation by 30%. In blood perfused in collagen-coated glass capillaries (shear rate of 1500 s⁻¹), ASA/C prevented thrombus growth beyond 5 min in relation to thrombus fragments embolization. ASA + M with or without clopidogrel completely prevented thrombus formation. Finally, ex vivo addition of MRS2179 and ASA to the blood of healthy donors markedly blocked thrombus formation on collagen in flow conditions, in contrast to ASA plus the P2Y₁₂ inhibitor 2-MeSAMP.

CONCLUSIONS AND IMPLICATIONS

Through particularly efficient complementarities with ASA to inhibit platelet activation and thrombus formation, the inhibition of P2Y₁ in the blood of patients with CAD appears to play a more important role than previously anticipated.     

Effects of drug interactions on biotransformation and antiplatelet effect of clopidogrel in vitro

BACKGROUND AND PURPOSE

The conversion of clopidogrel to its active metabolite, R-130964, is a two-step cytochrome P450 (CYP)-dependent process. The current investigations were performed to characterize in vitro the effects of different CYP inhibitors on the biotransformation and on the antiplatelet effect of clopidogrel.

EXPERIMENTAL APPROACH

Clopidogrel biotransformation was studied using human liver microsomes (HLM) or specific CYPs and platelet aggregation using human platelets activated with ADP.

KEY RESULTS

Experiments using HLM or specific CYPs (3A4, 2C19) revealed that at clopidogrel concentrations >10 µM, CYP3A4 was primarily responsible for clopidogrel biotransformation. At a clopidogrel concentration of 40 µM, ketoconazole showed the strongest inhibitory effect on clopidogrel biotransformation and clopidogrel-associated inhibition of platelet aggregation with IC₅₀ values of 0.03 ± 0.07 µM and 0.55 ± 0.06 µM respectively. Clarithromycin, another CYP3A4 inhibitor, impaired clopidogrel biotransformation and antiplatelet activity almost as effectively as ketoconazole. The CYP3A4 substrates atorvastatin and simvastatin both inhibited clopidogrel biotransformation and antiplatelet activity, less potently than ketoconazole. In contrast, pravastatin showed no inhibitory effect. As clopidogrel itself inhibited CYP2C19 at concentrations >10 µM, the CYP2C19 inhibitor lansozprazole affected clopidogrel biotransformation only at clopidogrel concentrations ≤10 µM. The carboxylate metabolite of clopidogrel was not a CYP substrate and did not affect platelet aggregation.

CONCLUSIONS AND IMPLICATIONS

At clopidogrel concentrations >10 µM, CYP3A4 is mainly responsible for clopidogrel biotransformation, whereas CYP2C19 contributes only at clopidogrel concentrations ≤10 µM. CYP2C19 inhibition by clopidogrel at concentrations >10 µM may explain the conflicting results between in vitro and in vivo investigations regarding drug interactions with clopidogrel.     

Pharmacokinetics, pharmacodynamics, safety and tolerability of multiple ascending doses of ticagrelor in healthy volunteers

AIM

To determine the pharmacokinetics, pharmacodynamics, safety and tolerability of multiple oral doses of ticagrelor, a P2Y₁₂ receptor antagonist, in healthy volunteers.

METHODS

This was a randomized, single-blind, placebo-controlled, ascending dose study. Thirty-two subjects received ticagrelor 50–600 mg once daily or 50–300 mg twice daily or placebo for 5 days at three dose levels in two parallel groups. Another group of 16 subjects received a clopidogrel 300 mg loading dose then 75 mg day⁻¹, or placebo for 14 days.

RESULTS

Ticagrelor was absorbed with median t_max 1.5–3 h, exhibiting predictable pharmacokinetics over the 50–600 mg dose range. Mean C_max and AUC for ticagrelor and its main metabolite, AR-C124910XX, increased approximately dose-proportionately (approximately 2.2- to 2.4-fold with a twofold dose increase) over the dose range. Inhibition of platelet aggregation (IPA) with ticagrelor was greater and better sustained at high levels with ticagrelor twice daily vs. once daily regimens. Throughout dosing, more consistent IPA was observed at doses ≥300 mg once daily and ≥100 mg twice daily compared with clopidogrel. Mean IPA with ticagrelor ≥100 mg twice daily was greater and less variable (93–100%, range 65–100%) than with clopidogrel (77%, range 11–100%) at trough concentrations. No safety or tolerability issues were identified.

CONCLUSIONS

Multiple dosing provided predictable pharmacokinetics of ticagrelor and its metabolite over the dose range of 50–600 mg once daily and 50–300 mg twice daily with C_max and AUC(0,t) increasing approximately dose-proportionally. Greater and more consistent IPA with ticagrelor at doses ≥100 mg twice daily and ≥300 mg once daily were observed than with clopidogrel. Ticagrelor at doses up to 600 mg day⁻¹ was well tolerated.     

Platelet P2Y12 receptors are involved in the haemostatic effect of notoginsenoside Ft1, a saponin isolated from Panax notoginseng

BACKGROUND AND PURPOSE

Saponins isolated from Panax notoginseng (Burk.) F.H. Chen have been shown to relieve thrombogenesis and facilitate haemostasis. However, it is not known which saponin accounts for this haemostatic effect. Hence, in the present study we aimed to identify which saponins contribute to its haemostatic activity and to elucidate the possible underlying mechanisms.

EXPERIMENTAL APPROACH

Platelet aggregation was analysed using a platelet aggregometer. Prothrombin time, activated partial thromboplastin time and thrombin time were measured using a blood coagulation analyser, which was further corroborated with bleeding time and thrombotic assays. The interaction of notoginsenoside Ft1 with the platelet P2Y₁₂ receptor was determined by molecular docking analysis, cytosolic Ca²⁺ and cAMP measurements, and phosphorylation of PI3K and Akt assays.

KEY RESULTS

Among the saponins examined, Ft1 was the most potent procoagulant and induced dose-dependent platelet aggregation. Ft1 reduced plasma coagulation indexes, decreased tail bleeding time and increased thrombogenesis. Moreover, it potentiated ADP-induced platelet aggregation and increased cytosolic Ca²⁺ accumulation, effects that were attenuated by clopidogrel. Molecular docking analysis suggested that Ft1 binds to platelet P2Y₁₂ receptors. The increase in intracellular Ca²⁺ evoked by Ft1 in HEK293 cells overexpressing P2Y₁₂ receptors could be blocked by ticagrelor. Ft1 also affected the production of cAMP and increased phosphorylation of PI3K and Akt downstream of P2Y₁₂ signalling pathways.

CONCLUSION AND IMPLICATIONS

Ft1 enhanced platelet aggregation by activating a signalling network mediated through P2Y₁₂ receptors. These novel findings may contribute to the effective utilization of this compound in the therapy of haematological disorders.     

The roles and mechanisms of PAR4 and P2Y12/phosphatidylinositol 3-kinase pathway in maintaining thrombin-induced platelet aggregation

BACKGROUND AND PURPOSE

Activation of human platelets by thrombin is mediated predominately through two proteinase-activated receptors (PARs), PAR1 and PAR4. Phosphatidylinositol 3-kinase (PI3K) inhibition leads to reversible PAR1-mediated platelet aggregation, but has no effect on the stability of platelet aggregation induced by thrombin. In the present study, the molecular mechanisms underlying this difference were investigated.

EXPERIMENTAL APPROACH

The functions of PI3K and PAR4 were assessed using specific inhibitors and aggregometry. The duration of platelet glycoprotein (GP) IIb/IIIa exposure was determined by flow cytometry with the antibody PAC-1. Western blotting and fluo-3 was used to evaluate the activation of Akt and protein kinase C (PKC) and intracellular Ca²⁺ mobilization respectively.

KEY RESULTS

When PAR4 function was inhibited either by the PAR4 antagonist YD-3 [1-benzyl-3-(ethoxycarbonylphenyl)-indazole] or by receptor desensitization, the PI3K inhibitor wortmannin turned thrombin-elicited platelet aggregation from an irreversible event to a reversible event. Moreover, wortmannin plus YD-3 markedly accelerated the inactivation of GPIIb/IIIa in thrombin-stimulated platelets. The aggregation-reversing activity mainly resulted from inhibition of both PI3K-dependent PKC activation and PAR4-mediated sustained intracellular Ca²⁺ rises. Blockade of ADP P2Y₁₂ receptor with 2-methylthioadenosine 5′-monophosphate triethylammonium salt mimicked the inhibitory effect of wortmannin on PI3K-dependent PKC activation and its ability to reverse PAR1-activating peptide-induced platelet aggregation. Co-administration of 2-methylthioadenosine 5′-monophosphate triethylammonium salt with YD-3 also decreased the stability of thrombin-induced platelet aggregation.

CONCLUSIONS AND IMPLICATIONS

These results suggest that PAR4 acts in parallel with the P2Y₁₂/PI3K pathway to stabilize platelet aggregates, and provide new insights into the mechanisms of thrombus stabilization and potential applications for antithrombotic therapy.     

P2Y12 polymorphisms and antiplatelet effects of aspirin in patients with coronary artery disease

Aims

Recently, two genetic polymorphisms of the platelet ADP receptor P2Y₁₂ (haplotypes H2 and 34T) have been implicated in increased platelet aggregation and atherothrombotic risk. It was suggested that these polymorphisms contribute to a diminished response to antiplatelet drugs. Therefore, we investigated the effects of these polymorphisms on platelet aggregation in aspirin-treated patients with coronary artery disease (CAD).

Methods

Platelet aggregation was studied in platelet-rich plasma from 124 patients with CAD treated with 100 mg aspirin day⁻¹. P2Y₁₂ ADP receptor polymorphisms were determined by PCR-RFLP. The 52G > T polymorphism was used as tag-SNP for the H2 haplotype. Aggregation was induced by 1 mg l⁻¹ collagen. In a subgroup (n = 72), a concentration-response curve to collagen (0.5–10 mg l⁻¹), aggregation at 2 μmol l⁻¹ ADP and 1 mmol l⁻¹ arachidonic acid were determined.

Results

Whereas arachidonic acid-induced aggregation was inhibited in all patients, collagen and ADP-induced aggregation were highly variable. However, aggregation did not differ significantly between carriers and noncarriers of the 52T-allele (1 mg l⁻¹ collagen: 32.7% (21.9–38.6%) vs. 32.5% (21.2–41.6%); P = 0.77; ADP: 33.1% (29.9–40.9%) vs. 39.1% (31.5–49.7%); P = 0.34), respectively. EC₅₀ values were 1.26 mg l⁻¹ (0.79–2.02) and 1.54 mg l⁻¹ (0.98–2.4) collagen in noncarriers and carriers of the H2 haplotype, respectively (P = 0.56). Moreover, the 34°C > T polymorphism did not significantly affect any of the aggregatory responses.

Conclusions

Low-dose aspirin inhibits platelet aggregation to the same extent in patients carrying or not carrying the P2Y₁₂ H2 haplotype and/or the 34T allele. Our data do not support the hypothesis that these polymorphisms contribute to an attenuated antiplatelet effect of aspirin.

What is already known about this subject

• Genetic polymorphisms of the P2Y₁₂ ADP receptor on platelets have been shown to contribute to variability in platelet aggregation in healthy humans.
• P2Y₁₂ ADP receptor polymorphisms are more frequently present in patients with vascular disease than in healthy people.
• The majority of patients with vascular disease receive acetylsalicylic acid as an anti-aggregatory agent, which has also been shown to induce a variable response; however, the role of P2Y₁₂ ADP receptor polymorphisms in the platelet response to acetylsalicylic acid in patients with vascular disease has not yet been studied.

What this study adds

• The present data show that the platelet response to acetylsalicylic acid is independent of the presence or absence of P2Y₁₂ ADP receptor polymorphisms in patients with stable coronary artery disease who have had their first myocardial infarction.
• This is important, as studies in healthy humans had suggested that carriers of P2Y₁₂ ADP receptor polymorphisms may be at increased risk of experiencing cardiovascular events.
• However, the observed variability of the platelet response to the cyclooxygenase inhibitor acetylsalicylic acid (in our study) and to the P2Y₁₂ ADP receptor blocker clopidogrel (in a study by Angiolillo et al.[18]) in patients with coronary artery disease is clearly not determined by common P2Y₁₂ ADP receptor polymorphisms.

     

Relative contribution of ecto-ATPase and ecto-ATPDase pathways to the biphasic effect of ATP on acetylcholine release from myenteric motoneurons

Background and purpose:

The relative contribution of distinct ecto-nucleotidases to the modulation of purinergic signalling may depend on differential tissue distribution and substrate preference.

Experimental approach:

Extracellular ATP catabolism (assessed by high-performance liquid chromatography) and its influence on [³H]acetylcholine ([³H]ACh) release were investigated in the myenteric plexus of rat ileum in vitro.

Key results:

ATP was primarily metabolized via ecto-ATPDase (adenosine 5′-triphosphate diphosphohydrolase) into AMP, which was then dephosphorylated into adenosine by ecto-5′-nucleotidase. Alternative conversion of ATP into ADP by ecto-ATPase (adenosine 5′-triphosphatase) was more relevant at high ATP concentrations. ATP transiently increased basal [³H]ACh outflow in a 2′,3′-O-(2,4,6-trinitrophenyl)adenosine-5′-triphosphate (TNP-ATP)-dependent, tetrodotoxin-independent manner. ATP and ATPγS (adenosine 5′-[γ-thio]triphosphate), but not α,β-methyleneATP, decreased [³H]ACh release induced by electrical stimulation. ADP and ADPβS (adenosine 5′[β-thio]diphosphate) only decreased evoked [³H]ACh release. Inhibition by ADPβS was prevented by MRS 2179 (2′-deoxy-N⁶-methyl adenosine 3′,5′-diphosphate diammonium salt, a selective P2Y₁ antagonist); blockade of ADP inhibition required co-application of MRS 2179 plus adenosine deaminase (which inactivates endogenous adenosine). Blockade of adenosine A₁ receptors with 1,3-dipropyl-8-cyclopentyl xanthine enhanced ADPβS inhibition, indicating that P2Y₁ stimulation is cut short by tonic adenosine A₁ receptor activation. MRS 2179 facilitated evoked [³H]ACh release, an effect reversed by the ecto-ATPase inhibitor, {"type":"entrez-protein","attrs":{"text":"ARL67156","term_id":"1186396857","term_text":"ARL67156"}}ARL67156, which delayed ATP conversion into ADP without affecting adenosine levels.

Conclusions and implications:

ATP transiently facilitated [³H]ACh release from non-stimulated nerve terminals via prejunctional P2X (probably P2X₂) receptors. Hydrolysis of ATP directly into AMP by ecto-ATPDase and subsequent formation of adenosine by ecto-5′-nucleotidase reduced [³H]ACh release via inhibitory adenosine A₁ receptors. Stimulation of inhibitory P2Y₁ receptors by ADP generated alternatively via ecto-ATPase might be relevant in restraining ACh exocytosis when ATP saturates ecto-ATPDase activity.     

A phase 1 study of prasugrel in patients with sickle cell disease: pharmacokinetics and effects on ex vivo platelet reactivity

Aims

Prasugrel is a novel thienopyridine P2Y₁₂ adenosine diphosphate (ADP) receptor antagonist that inhibits ADP-mediated platelet activation and aggregation. Accordingly, it may be useful in reducing platelet-related ischaemia in sickle cell disease (SCD). Exposure to prasugrel''s active metabolite (Pras-AM) and its antiplatelet activity in SCD have not been investigated.

Methods

Thirteen adult patients with SCD and an equal number of matched healthy control subjects were studied before and after 12 days of 5.0 or 7.5 mg day⁻¹ prasugrel treatment. Platelet reactivity was assessed by light transmission aggregometry (LTA), impedance aggregometry (MEA), VerifyNow® P2Y12, vasodilator-stimulated phosphoprotein (VASP) phosphorylation and Plateletworks. Exposure to Pras-AM was also assessed.

Results

At baseline, patients with SCD showed increased platelet reactivity vs. healthy control subjects with VerifyNow (408 vs. 323 P2Y12 reaction units (PRU), respectively, P = 0.003) and MEA (106 vs. 77 area under the aggregation curve (AU.min), P = 0.002); lower platelet reactivity index with VASP flow cytometry (59 vs. 79% platelet reactivity index (PRI), P = 0.018); and no significant differences with LTA, VASP enzyme-linked immunosorbent assay or Plateletworks. Relative to baseline, prasugrel significantly reduced platelet reactivity by all assays in both populations (all P < 0.05). Prasugrel was well tolerated, with no bleeding-related events in patients with SCD. The mean concentration–time profiles of Pras-AM were comparable between healthy subjects and patients with SCD following a single 10 mg prasugrel dose and following the 12th dose of 7.5 or 5 mg prasugrel.

Conclusions

Results demonstrate that in response to prasugrel, patients with SCD and healthy subjects have similar degrees of platelet inhibition and exposure to Pras-AM, and provide a basis for further study of prasugrel in patients with SCD.     

Self-augmentation of the lengthening of repolarization is related to the shape of the cardiac action potential: implications for reverse rate dependency

Background and purpose:

The aims of the present work were to study the mechanism of the reverse rate dependency of different interventions prolonging cardiac action potential duration (APD).

Experimental approach:

The reverse rate-dependent lengthening effect of APD-prolonging interventions and the possible involvement of I_Kr (rapid component of the delayed rectifier potassium current) and I_K1 (inward rectifier potassium current) were studied by using the standard microelectrode and the whole-cell patch-clamp techniques in dog multicellular ventricular preparations and in myocytes isolated from undiseased human and dog hearts.

Key results:

All applied drugs – dofetilide (1 µmol·L⁻¹), BaCl₂ (10 µmol·L⁻¹), BAY-K-8644 (1 µmol·L⁻¹), veratrine (1 µg·mL⁻¹) – lengthened APD in a reverse rate-dependent manner regardless of their mode of action, suggesting that reverse rate dependency may not represent a specific mechanism of APD prolongation. The E-4031-sensitive current (I_Kr) and the Ba²⁺-sensitive current (I_K1) were recorded during repolarizing voltage ramps having various steepness and also during action potential waveforms with progressively prolonged APD. Gradually delaying repolarization results in smaller magnitude of I_Kr and I_K1 currents at an isochronal phase of the pulses. This represents a positive feedback mechanism, which appears to contribute to the reverse rate-dependent prolongation of action potentials.

Conclusions and implications:

Action potential configuration may influence the reverse rate-dependent APD prolongation due to the intrinsic properties of I_Kr and I_K1 currents. Drugs lengthening repolarization by decreasing repolarizing outward, or increasing depolarizing inward, currents are expected to cause reverse rate-dependent APD lengthening with high probability, regardless of which current they modify.     

Influences of different proton pump inhibitors on the anti-platelet function of clopidogrel in relation to CYP2C19 genotypes

AIMS

The efficacy of clopidogrel is influenced by CYP2C19 genotypes and substrates of CYP2C19, such as proton pump inhibitors (PPIs). We assessed the influence of three different PPIs on the anti-platelet function of clopidogrel in relation to CYP2C19 genotype status.

METHODS

Thirty-nine healthy volunteers with different CYP2C19 genotypes took clopidogrel 75 mg with or without omeprazole 20 mg, lansoprazole 30 mg or rabeprazole 20 mg in the morning for 7 days. The influence of the three PPIs on the anti-platelet function of clopidogrel was determined. A less than 30% inhibition of platelet aggregation (IPA) during clopidogrel dosing was defined as a ‘low responder’. We also examined whether evening dosing of omeprazole could prevent the interaction with clopidogrel dosed in the morning.

RESULTS

In rapid metabolizers (RMs, *1/*1, n = 15) of CYP2C19, omeprazole and rabeprazole significantly attenuated the anti-platelet function of clopidogrel. In decreased metabolizers (DMs, carriers of *2 and/or *3, n = 24), there was a large variation in IPA and there was a trend but no significant decrease in IPA when placed on a concomitant PPI. Some DMs became ‘low-responders’ when placed on a concomitant PPI. Evening omeprazole dose in RMs did not seem to cause a significant decrease in IPA in contrast to morning dosing, but did so in DMs.

CONCLUSIONS

The three PPIs affected the efficacy of clopidogrel to different degrees. Both omeprazole and rabeprazole significantly decreased IPA in RMs but not DMs, although there was a trend towards lower IPA in DMs. Morning and evening dosing of omeprazole were both associated with lower IPA in DMs.     

NP-313, 2-acetylamino-3-chloro-1,4-naphthoquinone, a novel antithrombotic agent with dual inhibition of thromboxane A(2) synthesis and calcium entry

BACKGROUND AND PURPOSE

1,4-Naphthoquinones exhibit antiplatelet activity both in vivo and in vitro. In the present study, we investigated the antiplatelet effect of a novel naphthoquinone derivative NP-313, 2-acetylamino-3-chloro-1,4-naphthoquinone and its mechanism of action.

EXPERIMENTAL APPROACH

We measured platelet aggregation, Ca²⁺ mobilization, thromboxane B2 formation and P-selectin expression and examined several enzymatic activities. Furthermore, we used the irradiated mesenteric venules in fluorescein sodium–treated mice to monitor the antithrombotic effect of NP-313 in vivo.

KEY RESULTS

NP-313 concentration-dependently inhibited human platelet aggregation induced by collagen, arachidonic acid, thapsigargin, thrombin and A23187. NP-313 also inhibited P-selectin expression, thromboxane B₂ formation and [Ca²⁺]_i elevation in platelets stimulated by thrombin and collagen. NP-313 at 10 µM inhibited cyclooxygenase, thromboxane A₂ synthase, and protein kinase Cα, whereas it did not affect phospholipase A₂ or phospholipase C activity. In the presence of indomethacin and an adenosine 5-diphosphate scavenger, NP-313 concentration-dependently inhibited thrombin- and A23187-induced [Ca²⁺]_i increase through its inhibitory effects on Ca²⁺ influx, rather than blocking Ca²⁺ release from intracellular stores. NP-313 also inhibited thapsigargin-mediated Ca²⁺ influx through store-operated calcium channel but had no effect on Ca²⁺ influx through store-independent calcium channel evoked by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol. Nevertheless, it had little effect on cyclic AMP and cyclic GMP levels. Also, intravenously administered NP-313 dose-dependently inhibited the thrombus occlusion of the irradiated mesenteric vessels of fluorescein-pretreated mice.

CONCLUSIONS AND IMPLICATIONS

Taken together, these results indicate that NP-313 exerts its antithrombotic activity through dual inhibition of thromboxane A₂ synthesis and Ca²⁺ influx through SOCC.     

Reduced signal transduction by 5-HT4 receptors after long-term venlafaxine treatment in rats

BACKGROUND AND PURPOSE

The 5-HT₄ receptor may be a target for antidepressant drugs. Here we have examined the effects of the dual antidepressant, venlafaxine, on 5-HT₄ receptor-mediated signalling events.

EXPERIMENTAL APPROACH

The effects of 21 days treatment (p.o.) with high (40 mg·kg⁻¹) and low (10 mg·kg⁻¹) doses of venlafaxine, were evaluated at different levels of 5-HT₄ receptor-mediated neurotransmission by using in situ hybridization, receptor autoradiography, adenylate cyclase assays and electrophysiological recordings in rat brain. The selective noradrenaline reuptake inhibitor, reboxetine (10 mg·kg⁻¹, 21 days) was also evaluated on 5-HT₄ receptor density.

KEY RESULTS

Treatment with a high dose (40 mg·kg⁻¹) of venlafaxine did not alter 5-HT₄ mRNA expression, but decreased the density of 5-HT₄ receptors in caudate-putamen (% reduction = 26 ± 6), hippocampus (% reduction = 39 ± 7 and 39 ± 8 for CA1 and CA3 respectively) and substantia nigra (% reduction = 49 ± 5). Zacopride-stimulated adenylate cyclase activation was unaltered following low-dose treatment (10 mg·kg⁻¹) while it was attenuated in rats treated with 40 mg·kg⁻¹ of venlafaxine (% reduction = 51 ± 2). Furthermore, the amplitude of population spike in pyramidal cells of CA1 of hippocampus induced by zacopride was significantly attenuated in rats receiving either dose of venlafaxine. Chronic reboxetine did not modify 5-HT₄ receptor density.

CONCLUSIONS AND IMPLICATIONS

Our data indicate a functional desensitization of 5-HT₄ receptors after chronic venlafaxine, similar to that observed after treatment with the classical selective inhibitors of 5-HT reuptake.     

Peroxynitrite decreases arrhythmias induced by ischaemia reperfusion in anaesthetized dogs, without involving mitochondrial KATP channels

Background and purpose:

Exogenous peroxynitrite from nanomolar to micromolar concentrations exerts cardioprotection. Here, we have assessed its effects on ischaemia- and reperfusion-induced ventricular arrhythmias in vivo and a possible role for mitochondrial K_ATP channels in these effects, using the channel inhibitor 5-hydroxydecanoate (5-HD).

Experimental approach:

Chloralose-urethane-anaesthetized dogs were treated twice for 5 min with peroxynitrite (100 nM, by intracoronary infusions) in both the absence and presence of 5-HD (150 μg kg⁻¹ min⁻¹), and then subjected to 25 min occlusion of the left anterior descending coronary artery. The severity of ischaemia and of arrhythmias, as well as the levels of nitrotyrosine were assessed and compared with a group of control dogs, subjected only to a 25 min occlusion and reperfusion insult.

Key results:

Compared with controls, infusion of peroxynitrite markedly suppressed the number of ventricular premature beats (388±88 vs 133±44), the incidence of ventricular fibrillation both during occlusion (50% vs 10%) and reperfusion (100% vs 44%), and increased survival (0% vs 50%; all P<0.05). The severity of ischaemia (epicardial ST-segment changes, inhomogeneity of electrical activation) during occlusion and nitrotyrosine levels on reperfusion were significantly less in the peroxynitrite-treated dogs than in the controls. 5-HD did not modify the cardioprotective effects of peroxynitrite.

Conclusion and implications:

Exogenous peroxynitrite provided antiarrhythmic protection in vivo, which might have been on account of a reduction in endogenous peroxynitrite formation. This protection seemed not to be mediated through mitoK_ATP channels.     

Cannabinoid CB(2) receptor attenuates morphine-induced inflammatory responses in activated microglial cells

BACKGROUND AND PURPOSE

Among several pharmacological properties, analgesia is the most common feature shared by either opioid or cannabinoid systems. Cannabinoids and opioids are distinct drug classes that have been historically used separately or in combination to treat different pain states. In the present study, we characterized the signal transduction pathways mediated by cannabinoid CB₂ and µ-opioid receptors in quiescent and LPS-stimulated murine microglial cells.

EXPERIMENTAL APPROACH

We examined the effects of µ-opioid and CB₂ receptor stimulation on phosphorylation of MAPKs and Akt and on IL-1β, TNF-α, IL-6 and NO production in primary mouse microglial cells.

KEY RESULTS

Morphine enhanced release of the proinflammatory cytokines, IL-1β, TNF-α, IL-6, and of NO via µ-opioid receptor in activated microglial cells. In contrast, CB₂ receptor stimulation attenuated morphine-induced microglial proinflammatory mediator increases, interfering with morphine action by acting on the Akt-ERK1/2 signalling pathway.

CONCLUSIONS AND IMPLICATIONS

Because glial activation opposes opioid analgesia and enhances opioid tolerance and dependence, we suggest that CB₂ receptors, by inhibiting microglial activity, may be potential targets to increase clinical efficacy of opioids.     

A role for neuropeptide Y in the gender-specific gastrointestinal, corticosterone and feeding responses to stress

BACKGROUND AND PURPOSE

Exposure to an acute stress inhibits gastric emptying and stimulates colonic transit via central neuropeptide Y (NPY) pathways; however, peripheral involvement is uncertain. The anxiogenic phenotype of NPY^−/− mice is gender-dependent, raising the possibility that stress-induced gastrointestinal (GI) responses are female-dominant through NPY. The aim of this study was to determine GI transit rates, corticosterone levels and food intake after acute restraint (AR) or novel environment (NE) stress in male and female NPY^−/− and WT mice.

EXPERIMENTAL APPROACH

Upper gastrointestinal transit (UGIT) (established 30 min after oral gavage) and corticosterone levels were determined under basal or restrained conditions (30 min) and after treatment i.p. with Y₁ antagonist BIBO3304 or Y₂ antagonist BIIE0246. Faecal pellet output (FPO) was established after AR and treatment i.p. with NPY in the NE, as were colonic bead expulsion rates.

KEY RESULTS

UGIT and FPO were similar in unrestrained male and female mice. NPY^−/− females displayed significantly slower UGIT than NPY^−/− males after AR, but both genders displayed significantly higher FPO and reduced food intake relative to WT counterparts. Peripheral NPY treatment increased bead expulsion time in WT mice. AR male NPY^−/− mice had higher levels of corticosterone than male WT mice; whilst in AR WT mice, after peripheral Y₁ and Y₂ receptor antagonism in males, and Y₂ antagonism in females, corticosterone was significantly elevated.

CONCLUSIONS AND IMPLICATIONS

NPY possesses a role in the gender-dependent susceptibility to stress-induced GI responses. Furthermore, NPY inhibits GI motility through Y₂ receptors and corticosterone release via peripheral Y₁ and Y₂ receptors.     

Effects of pyrazole partial agonists on HCA(2) -mediated flushing and VLDL-triglyceride levels in mice

BACKGROUND AND PURPOSE

Niacin can effectively treat dyslipidaemic disorders. However, its clinical use is limited due to the cutaneous flushing mediated by the nicotinic acid receptor HCA₂. In the current study, we evaluated two partial agonists for HCA₂, LUF6281 and LUF6283, with respect to their anti-dyslipidaemic potential and cutaneous flushing effect.

EXPERIMENTAL APPROACH

In vitro potency and efficacy studies with niacin and the two HCA₂ partial agonists were performed using HEK293T cells stably expressing human HCA₂. Normolipidaemic C57BL/6 mice received either niacin or the HCA₂ partial agonists (400 mg·kg⁻¹·day⁻¹) once a day for 4 weeks for evaluation of their effects in vivo.

KEY RESULTS

Radioligand competitive binding assay showed K_i values for LUF6281 and LUF6283 of 3 and 0.55 µM. [³⁵S]-GTPγS binding revealed the rank order of their potency as niacin > LUF6283 > LUF6281. All three compounds reduced plasma VLDL-triglyceride concentrations similarly, while LUF6281 and LUF6283, in contrast to niacin, did not also exhibit the unwanted flushing side effect in C57BL/6 mice. Niacin reduced the expression of lipolytic genes HSL and ATGL in adipose tissue by 50%, whereas LUF6281 and LUF6283 unexpectedly did not. In contrast, the decrease in VLDL-triglyceride concentration induced by LUF6281 and LUF6283 was associated with a parallel >40% reduced expression of APOB within the liver.

CONCLUSIONS AND IMPLICATIONS

The current study identifies LUF6281 and LUF6283, two HCA₂ partial agonists of the pyrazole class, as promising drug candidates to achieve the beneficial lipid lowering effect of niacin without producing the unwanted flushing side effect.     

Antiplatelet and antithrombotic effect of F 16618, a new thrombin proteinase-activated receptor-1 (PAR1) antagonist

BACKGROUND AND PURPOSE

New antithrombotic agents with the potential to prevent atherothrombotic complications are being developed to target receptors on platelets and other cells involved in plaque growth. The aim of this study was to investigate the antiplatelet effects of F 16618, a new non-peptidic PAR1 (thrombin receptor) antagonist.

EXPERIMENTAL APPROACH

We investigated the inhibitory effect of F 16618 on human platelet aggregation ex vivo, in whole blood and washed platelets, by using a multiple-electrode platelet aggregometer based on impedance and an optical aggregometer, respectively. Its effects on whole-blood haemostasis (clot parameters) were analysed with the ROTEM thromboelastometry device and the platelet function analyser PFA-100. A guinea-pig model of arterial thrombosis was used to investigate its effects on thrombus formation in vivo.

KEY RESULTS

F 16618 inhibited PAR1 agonist peptide (SFLLR-peptide)-induced washed platelet aggregation ex vivo. This effect was concentration-dependent and exhibited a competitive inhibition profile. Washed platelet aggregation, as well as P-selectin expression induced by thrombin, were significantly inhibited by 10 µM F 16618. In whole-blood experiments, 20 µM F 16618 inhibited SFLLR-induced platelet aggregation by 49%. In contrast, it had no effect on whole-blood haemostasis. In the guinea-pig model of carotid thrombosis, 0.32 mg·kg⁻¹ F 16618 doubled the occlusion time.

CONCLUSIONS AND IMPLICATIONS

F 16618 was shown to have strong antithrombotic activity in vivo and moderate antiplatelet effects ex vivo. As these effects were not associated with major effects on physiological haemostasis, this molecule is a good antiplatelet drug candidate for use either alone or in combination with current treatments.     

Activation of ��-opioid receptors and block of KIR3 potassium channels and NMDA receptor conductance by l- and d-methadone in rat locus coeruleus

BACKGROUND AND PURPOSE

Methadone activates opioid receptors to increase a potassium conductance mediated by G-protein-coupled, inwardly rectifying, potassium (K_IR3) channels. Methadone also blocks K_IR3 channels and N-methyl-D-aspartic acid (NMDA) receptors. However, the concentration dependence and stereospecificity of receptor activation and channel blockade by methadone on single neurons has not been characterized.

EXPERIMENTAL APPROACH

Intracellular and whole-cell recording were made from locus coeruleus neurons in brain slices and the activation of µ-opioid receptors and blockade of K_IR3 and NMDA channels with l- and d-methadone was examined.

KEY RESULTS

The potency of l-methadone, measured by the amplitude of hyperpolarization was 16.5-fold higher than with d-methadone. A maximum hyperpolarization was caused by both enantiomers (∼30 mV); however, the maximum outward current measured with whole-cell voltage-clamp recording was smaller than the current induced by [Met]⁵enkephalin. The K_IR3 conductance induced by activation of α₂-adrenoceptors was decreased with high concentrations of l- and d-methadone (10–30 µM). In addition, methadone blocked the resting inward rectifying conductance (K_IR). Both l- and d-methadone blocked the NMDA receptor-dependent current. The block of NMDA receptor-dependent current was voltage-dependent suggesting that methadone acted as a channel blocker.

CONCLUSIONS AND IMPLICATIONS

Methadone activated µ-opioid receptors at low concentrations in a stereospecific manner. K_IR3 and NMDA receptor channel block was not stereospecific and required substantially higher concentrations. The separation in the concentration range suggests that the activation of µ-opioid receptors rather than the channel blocking properties mediate both the therapeutic and toxic actions of methadone.     

Rolofylline,an adenosine A1 receptor antagonist,inhibits osteoclast differentiation as an inverse agonist

BACKGROUND AND PURPOSE

Adenosine may be generated by hydrolysis of extracellular nucleotides by ectonucleotidases, including ectonucleoside triphosphate diphosphohydrolase 1 (CD39), ecto-5′-nucleotidase (CD73), nucleotide pyrophosphatase phosphodiesterase 1 (NPP-1) and tissue non-specific alkaline phosphatase (TNAP). Previous work from our laboratory has uncovered a critical role for adenosine A₁ receptors (A₁R) in osteoclastogenesis; blockade or deletion of these receptors diminishes osteoclast differentiation. Interestingly, selective A₁R agonists neither affect basal osteoclastogenesis nor do they reverse A₁R antagonist-mediated inhibition of osteoclastogenesis. In this study, we determined whether ectonucleotidase-mediated adenosine production was required for A₁R antagonist-mediated inhibition, and, when we saw no effect, determined whether A₁R was constitutively activated and the antagonist was acting as an inverse agonist to diminish osteoclast differentiation.

EXPERIMENTAL APPROACH

Osteoclast formation derived from wild-type, CD39 knockout (KO), CD73 KO, NPP-1 KO and TNAP KO mice was examined by tartrate-resistant acid phosphatase staining of receptor activator of NF-κB ligand–macrophage colony-stimulating factor-stimulated osteoclasts and osteoclast gene expression (Ctsk, Acp5, MMP-9 and NFATc1). Intracellular cAMP concentration was determined by elisa.

KEY RESULTS

Rolofylline inhibited osteoclast formation in a dose-dependent manner (IC₅₀ = 20–70 nM) in mice lacking all four of these phosphatases, although baseline osteoclast formation was significantly less in precursors from CD73 KO mice. Rolofylline (1 μM) stimulates cAMP production in bone marrow macrophages by 10.23 ± 0.89-fold.

CONCLUSIONS AND IMPLICATIONS

Based on these findings, we hypothesize that the A₁R is constitutively activated in osteoclast precursors, thereby diminishing basal AC activity, and that A₁R antagonists act as inverse agonists to release A₁R-mediated inhibition of basal AC activity and permit osteoclast differentiation. The constitutive activity of A₁R promotes osteoclast formation and down-regulation of this activity blocks osteoclast formation.     

Ezetimibe's effect on platelet aggregation and LDL tendency to peroxidation in hypercholesterolaemia as monotherapy or in addition to simvastatin

AIMS

To investigate the effect of lowering low-density lipoprotein-cholesterol (LDL-C) on platelet aggregation and LDL tendency to peroxidation by ezetimibe alone or with simvastatin in hypercholesterolaemia.

METHODS

Sixteen patients with LDL-C >3.4 mmol l⁻¹ received ezetimibe for 3 months (Part I). Twenty-two patients on fixed simvastatin dose with LDL-C >2.6 mmol l⁻¹ were enrolled (Part II). Part II patients continued simvastatin treatment 20 mg day⁻¹ for 6 weeks, then received 20 mg day⁻¹ simvastatin combined with ezetimibe 10 mg day⁻¹ for another 6 weeks. The tendency of LDL to peroxidation measured by lag time in minutes required for initiation of LDL oxidation and by LDL oxidation at maximal point (plateau) was measured before and after ezetimibe treatment.

RESULTS

Part I: Ezetimibe 10 mg daily for 3 months decreased plasma LDL-C level 16% (P = 0.002), prolonged lag time to LDL oxidation from 144 ± 18 min to 195 ± 16 min (P < 0.001), decreasing maximal aggregation from 83 ± 15% to 60 ± 36% (P = 0.04). Part II: Serum level LDL-C decreased 23% (P = 0.02) and lag time in minutes to LDL oxidation was prolonged from 55.9 ± 16.5 to 82.7 ± 11.6 (P < 0.0001) using combined simvastatin–ezetimibe therapy. There were no differences in platelet aggregation.

CONCLUSIONS

Ezetimibe was associated with decreased platelet aggregation and LDL tendency to peroxidation. Treatment with ezetimibe in addition to simvastatin has an additive antioxidative effect on LDL.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Statins demonstrate a pleiotropic effect which contributes beyond the hypocholesterolaemic effect to prevent atherosclerosis.

WHAT THIS STUDY ADDS

• Ezetimibe has an antioxidative effect when given as monotherapy or as an add-on to the statin, simvastatin.