Dalcetrapib: no off-target toxicity on blood pressure or on genes related to the renin-angiotensin-aldosterone system in rats

Background and purpose:

The association between torcetrapib and its off-target effects on blood pressure suggested a possible class-specific effect. The effects of dalcetrapib (RO4607381/JTT-705) and torcetrapib on haemodynamics and the renin-angiotensin-aldosterone system (RAAS) were therefore assessed in a rat model.

Experimental approach:

Arterial pressure (AP) and heart rate were measured by telemetry in normotensive and spontaneously hypertensive rats (SHR) receiving torcetrapib 10, 40 or 80 mg·kg⁻¹·day⁻¹; dalcetrapib 100, 300 or 500 mg⁻¹·kg·day⁻¹; or vehicle (placebo) for 5 days. Expression of RAAS genes in adrenal gland, kidney, aorta and lung from normotensive rats following 5 days'' treatment with torcetrapib 40 mg·kg⁻¹·day⁻¹, dalcetrapib 500 mg·kg⁻¹·day⁻¹ or vehicle was measured by quantitative polymerase chain reaction.

Key results:

Torcetrapib transiently increased mean AP in normotensive rats (+3.7 ± 0.1 mmHg), whereas treatment in SHR resulted in a dose-dependent and sustained increase [+6.5 ± 0.6 mmHg with 40 mg·kg⁻¹·day⁻¹ at day 1 (P < 0.05 versus placebo)], which lasted over the treatment period. No changes in AP or heart rate were observed with dalcetrapib. Torcetrapib, but not dalcetrapib, increased RAAS-related mRNAs in adrenal glands and aortas.

Conclusions and implications:

In contrast to torcetrapib, dalcetrapib did not increase blood pressure or RAAS-related gene expression in rats, suggesting that the off-target effects of torcetrapib are not a common feature of all compounds acting on cholesteryl ester transfer protein.     

Stimulation of angiotensin AT2 receptors by the non-peptide agonist,Compound 21, evokes vasodepressor effects in conscious spontaneously hypertensive rats

Background and purpose:

Angiotensin type 2 receptor (AT₂ receptor) stimulation evokes vasodilator effects in vitro and in vivo that oppose the vasoconstrictor effects of angiotensin type 1 receptors (AT₁ receptors). Recently, a novel non-peptide AT₂ receptor agonist, Compound 21, was described, which exhibited high AT₂ receptor selectivity.

Experimental approach:

Functional cardiovascular effects of the drug candidate Compound 21 were assessed, using mouse isolated aorta and rat mesenteric arteries in vitro and in conscious spontaneously hypertensive rats (SHR).

Key results:

Compound 21 evoked dose-dependent vasorelaxations in aortic and mesenteric vessels, abolished by the AT₂ receptor antagonist, PD123319. In vivo, Compound 21 administered alone, at doses ranging from 50 to 1000 ng·kg⁻¹·min⁻¹ over 4 h did not decrease blood pressure in conscious normotensive Wistar-Kyoto rats or SHR. However, when given in combination with the AT₁ receptor antagonist, candesartan, Compound 21 (300 ng·kg⁻¹·min⁻¹) lowered blood pressure in SHR only. Further analysis in separate groups of conscious SHR revealed that, at a sixfold lower dose, Compound 21 (50 ng·kg⁻¹·min⁻¹) still evoked a significant depressor response in adult SHR (∼30 mmHg) when combined with different doses of candesartan (0.01 or 0.1 mg·kg⁻¹). Moreover, the Compound 21-evoked depressor effect was abolished when co-infused (50 µg·kg⁻¹·min⁻¹ for 2 h) with the AT₂ receptor antagonist PD123319.

Conclusion and implications:

Collectively, our results indicate that acute administration of Compound 21 evoked blood pressure reductions via AT₂ receptor stimulation. Thus Compound 21 can be considered an excellent drug candidate for further study of AT₂ receptor function in cardiovascular disease.     

Relevance of conserved lysine and arginine residues in transmembrane helices for the transport activity of organic anion transporting polypeptide 1B3

Background and purpose:

Organic anion transporting polypeptide 1B3 (OATP1B3) (SLCO1B3) mediates the uptake of endogenous substrates (e.g. estrone-3-sulphate) and drugs (e.g. pravastatin) from blood into hepatocytes. Structure-based modelling of OATP1B3 suggested that a pore with a positive electrostatic potential contributes to the transport mechanism. Therefore, we investigated the role of conserved positively charged amino acids for OATP1B3-mediated uptake of sulphobromophthalein (BSP) and pravastatin.

Experimental approach:

Residues Lys28, Lys41 and Arg580 in OATP1B3 were substituted by alanine, arginine, glutamine, glycine or lysine. Using immunofluorescence, immunoblot analysis and cellular uptake assays, the effect of these mutations on protein expression and transport activity was investigated.

Key results:

Immunofluorescence revealed that all mutants were localized in the plasma membrane with partial intracellular retention of the Arg580>Ala and Arg580>Lys mutants. Lys41>Ala, Lys41>Gln, Lys41>Gly, Arg580>Gly and Arg580>Lys showed significantly reduced transport for BSP and pravastatin. Kinetic analyses of BSP transport revealed a significant reduction of V_max normalized to cell surface protein expression for Lys41>Ala (wild type: 190 ± 8, Lys41>Ala:16 ± 4 pmol (mg protein)⁻¹ min⁻¹, P < 0.001), whereas V_max of Lys41>Arg and Arg580>Lys (103 ± 8 and 123 ± 14 pmol (mg protein)⁻¹ min⁻¹, P > 0.05) did not change significantly. This suggests that the positive charges at positions 41 and 580 are important for transport activity of BSP. Structural modelling indicated that the positively charged side chain of Lys41 is flexible within the pore. The orientation of Arg580 is defined by adjacent residues Glu74 and Asn77, which was confirmed by kinetic analysis of Glu74>Ala.

Conclusions and implications:

We demonstrated that the conserved positively charged amino acids Lys41 and Arg580 are pivotal to the transport activity of OATP1B3.     

Cardiovascular changes in spontaneously hypertensive rats are improved by chronic treatment with zofenopril

Background and purpose:

The aim of this study was to investigate the effect of chronic treatment with antihypertensive and non-antihypertensive doses of zofenopril on cardiovascular changes in spontaneously hypertensive rats (SHR).

Experimental approach:

Male SHR were treated with 0.5 or 10 mg·kg⁻¹ per day of zofenopril (Z_0.5 and Z₁₀) for 3 months. SHR and Wistar-Kyoto rats (WKY) receiving vehicle were used as controls. Systolic blood pressure was measured using the tail cuff method. Left ventricular weight/body weight ratio was calculated as cardiac hypertrophy index. Angiotensin converting enzyme (ACE) activity was determined in plasma and tissues by a fluorimetric method. Vascular reactivity was evaluated on aortic rings by acetylcholine and sodium nitroprusside relaxations. Effects on vascular structure were assessed by lumen diameter, wall thickness and medial cross-sectional area determination. Superoxide anion generation was quantified using lucigenin-amplified chemiluminescence in aorta.

Results:

Long-term daily administration of zofenopril (10 mg·kg⁻¹) to SHR reduced blood pressure to WKY values, decreased cardiac hypertrophy, improved the acetylcholine-induced relaxant response and reversed the vascular remodelling. ACE inhibition and antioxidant activity were involved in these effects. 0.5 mg·kg⁻¹ per day of zofenopril slightly modified blood pressure and the other effects were weaker.

Conclusions and implications:

Antihypertensive effects of chronic treatment with zofenopril were accompanied by recovery of endothelial function and improvement of cardiovascular structure. Low-dose zofenopril had little effect on blood pressure, with some benefits on cardiovascular structure and function. Inhibition of ACE and antioxidant activity were involved in these effects.     

Synergism of irbesartan and amlodipine on hemodynamic amelioration and organ protection in spontaneously hypertensive rats

Aim:

To investigate the synergism of low-doses of amlodipine and irbesartan on reduction of blood pressure variability (BPV), amelioration of baroreflex sensitivity (BRS) and organ protection in spontaneously hypertensive rats (SHR).

Methods:

The rats were administered amlodipine (1 mg·kg⁻¹·d⁻¹) alone, irbesartan (10 mg·kg⁻¹·d⁻¹) alone, or the combination of the two drugs for 4 months. The drugs were mixed into the rat chow. Blood pressure (BP) was continuously monitored in conscious animals. After the determination of BRS, the rats were killed for morphological evaluation of organ damages.

Results:

The combination of low-dose irbesartan and amlodipine had statistically significant synergism on reduction of BP and BPV, amelioration of BRS and organ protection in SHR. Multiple regression analysis showed that the decrease in left ventricular hypertrophy was associated with the decrease in systolic BPV (r=0.665, P<0.01); the decrease in aortic hypertrophy was associated with the increase in BRS (r=0.656, P<0.01); and the amelioration in renal lesion was associated with the increase in BRS (r=0.763, P<0.01) and the decrease in systolic BPV (r=0.706, P<0.01).

Conclusion:

Long-term treatment with a combination of low-doses of amlodipine and irbesartan showed significant synergism on reduction of BP and BPV, restoration of BRS and organ protection in SHR. Besides BP reduction, the enhancement of BRS and reduction of BPV might contribute to the organ protection.     

Influence of rosuvastatin on the NAD(P)H oxidase activity in the retina and electroretinographic response of spontaneously hypertensive rats

Background and purpose:

Retinal complications may be encountered during the development of hypertension as a response to oxidative stress. Statins may reduce the risk of developing hypertension and ocular diseases. We evaluate the effects of rosuvastatin (ROSU) on retinal functionality and oxidative stress levels in normotensive and spontaneously hypertensive rats (SHR).

Experimental approach:

Wistar Kyoto (WKY) and SHR were treated for 3 weeks with rosuvastatin (10 mg kg⁻¹ day⁻¹). Electroretinograms (ERG) were recorded before and after rosuvastatin treatment. Reactive oxygen species (ROS) were determined in the retina with dihydroethidium staining and NAD(P)H oxidase activity was evaluated.

Key results:

Retinal ganglion cell ROS and retinal NAD(P)H oxidase activity were higher in SHR than in WKY rats, respectively (17.1±1.1 vs 10.2±1.2 AU, P<0.01; 38095±8900 vs 14081±5820 RLU mg⁻¹; P<0.05). The ERG b-wave amplitude in SHR was significantly lower than that in WKY rats. Rosuvastatin reduced SBP in SHR but did not change plasma lipid levels. Rosuvastatin treatment in SHR significantly decreased ROS levels (11.2±1.3, P<0.01), NAD(P)H activity in retinal ganglion cells (9889±4290; P<0.05), and increased retinal plasmalogen content in SHR, but did not modify the ERG response.

Conclusions and implications:

Rosuvastatin, beyond lowering cholesterol levels, was able to lower ROS in the retina induced by hypertension, but without improving retinal function in SHR. These findings point to a complex relationship between ROS in the pathogenesis of retinal disease and hypertension.     

The ecto-nucleotidase NTPDase1 differentially regulates P2Y1 and P2Y2 receptor-dependent vasorelaxation

Background and purpose:

Extracellular nucleotides produce vasodilatation through endothelial P2 receptor activation. As these autacoids are actively metabolized by the ecto-nucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), we studied the effects of this cell surface enzyme on nucleotide-dependent vasodilatation.

Experimental approach:

Vascular NTPDase expression and activity were evaluated by immunohistochemistry and histochemistry. The vascular effects of nucleotides were tested in vivo by monitoring mean arterial pressure, and in vitro comparing reactivity of aortic rings using wild-type and Entpd1^−/− (lacking NTPDase1) mice.

Key results:

The absence of NTPDase1 in Entpd1^−/− mice led to a dramatic drop in endothelial nucleotidase activity. This deficit was associated with an exacerbated decrease in blood pressure after nucleotide injection. Following ATP injection, mean arterial pressure was decreased in Entpd1^+/+ and Entpd1^−/− mice by 5.0 and 17%, respectively, and by 0.1 and 19% after UTP injection (10 nmole·kg⁻¹ both). In vitro, the concentration-response curves of relaxation to ADP and ATP were shifted to the left, revealing a facilitation of endothelial P2Y1 and P2Y2 receptor activation in Entpd1^−/− mice. EC₅₀ values in Entpd1^+/+ versus Entpd1^−/− aortic rings were 14 µM versus 0.35 µM for ADP, and 29 µM versus 1 µM for ATP. In Entpd1^−/− aortas, P2Y1 receptors were more extensively desensitized than P2Y2 receptors. Relaxations to the non-hydrolysable analogues ADPβS (P2Y1) and ATPγS (P2Y2) were equivalent in both genotypes confirming the normal functionality of these P2Y receptors in mutant mice.

Conclusions and implications:

NTPDase1 controls endothelial P2Y receptor-dependent relaxation, regulating both agonist level and P2 receptor reactivity.     

Modulation of gap junctions by nitric oxide contributes to the anti-arrhythmic effect of sodium nitroprusside?

Background and purpose:

Nitric oxide (NO) donors provide a preconditioning-like anti-arrhythmic protection in the anaesthetized dog. As NO may modulate gap junction (GJ) function, the present study investigated whether this anti-arrhythmic effect is due to a modification of GJs by NO, derived from the NO donor sodium nitroprusside (SNP).

Experimental approach:

In chloralose-urethane-anaesthetized, open-chest dogs, either saline (controls; n= 11) or SNP (0.2 µg·kg⁻¹·min⁻¹; n= 10) was infused at a rate of 0.5 mL·min⁻¹ by the intracoronary route. The infusions were started 20 min prior to and maintained throughout the entire 60 min occlusion period of the left anterior descending coronary artery. The severity of ischaemia and of arrhythmias, tissue electrical impedance and permeability, as well as the phosphorylation of connexin43, were assessed.

Key results:

Compared with the controls, SNP infusion markedly suppressed the total number of ventricular premature beats (666 ± 202 vs. 49 ± 18; P < 0.05), and the number of ventricular tachycardiac episodes (8.1 ± 2.3 vs. 0.2 ± 0.1; P < 0.05) without significantly modifying the incidence of ventricular tachycardia or ventricular fibrillation. The severity of ischaemia (epicardial ST-segment changes, inhomogeneity of electrical activation) and tissue electrical impedance changes were significantly less in the SNP-treated dogs. SNP improved GJ permeability and preserved the phosphorylated form of connexin43.

Conclusion and implications:

The anti-arrhythmic protection resulting from SNP infusion in the anaesthethized dog may, in part, be associated with the modulation of gap junctional function by NO.     

Inhibition of colonic motility and defecation by RS-127445 suggests an involvement of the 5-HT2B receptor in rodent large bowel physiology

Background:

5-HT_2B receptors are localized within the myenteric nervous system, but their functions on motor/sensory neurons are unclear. To explore the role of these receptors, we further characterized the 5-HT_2B receptor antagonist RS-127445 and studied its effects on peristalsis and defecation.

Experimental approach:

Although reported as a selective 5-HT_2B receptor antagonist, any interactions of RS-127445 with 5-HT₄ receptors are unknown; this was examined using the recombinant receptor and Biomolecular Interaction Detection technology. Mouse isolated colon was mounted in tissue baths for isometric recording of neuronal contractions evoked by electrical field stimulation (EFS), or under an intraluminal pressure gradient to induce peristalsis; the effects of RS-127445 on EFS-induced and on peristaltic contractions were measured. Faecal output of rats in grid-bottom cages was measured over 3 h following i.p. RS-127445 and separately, validation of the effective doses was achieved by determining the free, unbound fraction of RS-127445 in blood and brain.

Key results:

RS-127445 (up to 1 µmol·L⁻¹) did not interact with the 5-HT₄ receptor. RS-127445 (0.001–1 µmol·L⁻¹) did not affect EFS-induced contractions of the colon, although at 10 µmol·L⁻¹ the contractions were reduced (to 36 ± 8% of control, n= 4). RS-127445 (0.1–10 µmol·L⁻¹) concentration-dependently reduced peristaltic frequency (n= 4). RS-127445 (1–30 mg·kg⁻¹), dose-dependently reduced faecal output, reaching significance at 10 and 30 mg·kg⁻¹ (n= 6–11). In blood and brain, >98% of RS-127445 was protein-bound.

Conclusions and implications:

High-protein binding of RS-127445 indicates that relatively high doses are required for efficacy. The results suggest that 5-HT_2B receptors tonically regulate colonic motility.     

Mast cell degranulation – a mechanism for the anti-arrhythmic effect of endothelin-1?

Background and purpose:

The aim of this study was to investigate whether the previously reported anti-arrhythmic effect of endothelin-1 (ET-1) is mediated by degranulation of cardiac mast cells prior to myocardial ischaemia.

Experimental approach:

Male Sprague-Dawley rats received either ET-1 (1.6 nmol·kg⁻¹) in the presence or absence of disodium cromoglycate (DSCG; 20 mg·kg⁻¹·h⁻¹) prior to coronary artery occlusion (CAO). In separate experiments rats were given compound 48/80 (50 µg·kg⁻¹) to compare the effects of ET-1 with those of a known mast cell degranulator. Ischaemia-induced ventricular arrhythmias were detected through continuous monitoring of a lead I electrocardiogram. After 30 min of CAO, the hearts were removed and mast cell degranulation determined by histological analysis. A parallel series of sham groups were performed to determine the direct effects of ET-1 and compound 48/80 on mast cell degranulation in the absence of ischaemia.

Key results:

ET-1 and compound 48/80 both exerted profound anti-arrhythmic effects, significantly reducing the total number of ventricular ectopic beats (P < 0.001) and the incidence of ventricular fibrillation (P < 0.05). These anti-arrhythmic effects were abolished by concomitant DSCG infusion prior to CAO. In sham animals ET-1 and compound 48/80 both induced mast cell degranulation (P < 0.001), an effect which was abolished by DSCG, confirming their ability to induce degranulation of mast cells.

Conclusions and implications:

These results demonstrate for the first time that when given prior to ischaemia ET-1 mediates its anti-arrhythmic effects, at least in part, via cardiac mast cell degranulation.     

Antagonism of endogenous nociceptin/orphanin FQ inhibits infarction-associated ventricular arrhythmias via PKC-dependent mechanism in rats

BACKGROUND AND PURPOSE

Evidence indicates nociceptin/orphanin FQ (N/OFQ) may participate in the pathology of cardiac arrhythmias associated with myocardial infarction. But the role of N/OFQ in the arrhythmogenesis in acute myocardial infarction is unclear. The aim of this study was to investigate the effects of endogenous N/OFQ on infarction-associated arrhythmias.

EXPERIMENTAL APPROACH

The expression of N/OFQ, PKC activity and ventricular arrhythmias in presence and absence of UFP-101, a specific antagonist of N/OFQ receptor, were examined following permanent coronary artery occlusion in anaesthetized rats. The effect of N/OFQ on action potential duration was examined in isolated rat cardiomyocytes.

KEY RESULTS

It was observed that N/OFQ was increased by 41% in the myocardium after coronary artery occlusion (P < 0.01 vs. control). Pretreatment with UFP-101 (10⁻⁷ mol·kg⁻¹, i.v.) reduced the incidence of ventricular ectopic beats by 70% and ventricular tachycardia by 51% respectively (all P < 0.05 vs. control). Meanwhile, PKC activity was elevated in the rats treated with UFP-101 (by 35%, P < 0.05 vs. control). A selective PKC inhibitor, calphostin C, completely abolished the anti-arrhythmic effects of UFP-101 (P < 0.01). N/OFQ (at 10⁻¹¹, 10⁻⁹ and 1 × 10⁻⁷ mol·L⁻¹) shortened the action potential duration by 3% (P > 0.05), 10% (P < 0.05) and 22% (P < 0.01), respectively, via N/OFQ receptor.

CONCLUSIONS AND IMPLICATIONS

Antagonism of endogenous N/OFQ produces anti-arrhythmic effects on ventricular arrhythmias in acute myocardial infarction, possibly via modulating PKC activity and action potential of myocytes.     

The efficacy of parecoxib on systemic inflammatory response associated with cardiopulmonary bypass during cardiac surgery

Aims

Cardiopulmonary bypass (CPB) during cardiac surgery is well known to be associated with the development of a systemic inflammatory response. The efficacy of parecoxib in attenuating this systemic inflammatory response is still unknown.

Methods

Patients undergoing elective mitral valve replacement with CPB were assessed, enrolled and randomly allocated to receive parecoxib (80 mg) or placebo. Blood samples were collected in EDTA vials for measuring serum cytokine concentrations, troponin T, creatinekinase myocardial‐brain isoenzyme CK‐MB concentrations and white cell counts.

Results

Compared with the control group, IL‐6 and IL‐8‐values in the parecoxib group increased to a lesser extent, peaking at 2 h after the end of CPB (IL‐6 31.8 pg ml⁻¹ ± 4.7 vs. 77.0 pg ml⁻¹ ± 14.1, 95% CI −47.6, −42.8, P < 0.001; IL‐8 53.6 pg ml⁻¹ ± 12.6 vs. 105.7 pg ml⁻¹ ± 10.8, 95% CI −54.8, −49.4, P < 0.001). Peak concentrations of anti‐inflammatory cytokine IL‐10 occurred immediately after termination of CPB and were higher in the parecoxib group (115.7 pg ml⁻¹ ± 10.5 vs. 88.4 pg ml⁻¹ ± 12.3, 95% CI 24.7, 29.9, P < 0.001). Furthermore, the increase in neutrophil counts caused by CPB during cardiac surgery was inhibited by parecoxib. The increases in serum troponin T and CK‐MB concentrations were also significantly attenuated by parecoxib in the early post‐operative days. Peak serum concentrations of CK‐MB in both groups occurred at 24 h post‐CPB (17.4 μg l⁻¹ ± 5.2 vs. 26.9 μg l⁻¹ ± 6.9, 95% CI −10.9, −8.1, P < 0.001). Peak troponin T concentrations occurred at 6 h post‐bypass (2 μg l⁻¹ ± 0.62 vs. 3.5 μg l⁻¹ ± 0.78, 95% CI −1.7, −1.3, P < 0.001).

Conclusion

Intra‐operative parecoxib attenuated the systemic inflammatory response associated with CPB during cardiac surgery and lowered the biochemical markers of myocardial injury.     

CYP3A activity in severe liver cirrhosis correlates with Child–Pugh and model for end-stage liver disease (MELD) scores

Aims

Impaired liver function often necessitates drug dose adjustment to avoid excessive drug accumulation and adverse events, but a marker for the extent of the required adjustment is lacking. The aim of this study was to investigate whether Child–Pugh (CP) and model for end-stage liver disease (MELD) scores correlate with drug clearance.

Methods

Midazolam was used as a CYP3A probe and its pharmacokinetics were analyzed in 24 patients with mild to severe liver cirrhosis (n = 4, 10 and 10 with CP class A, B and C, respectively) and six patients without liver disease.

Results

Both scores correlated well with unbound midazolam clearance (CL_u), unbound midazolam fraction and half-life (all P < 0.01), whereas the unbound steady-state volume of distribution was not significantly changed. In patients with severe liver cirrhosis unbound midazolam clearance was only 14% of controls (CP C: CL_u = 843 ± 346 l h⁻¹, MELD ≥ 15: CL_u = 805 ± 474 l h⁻¹, controls: CL_u = 5815 ± 2649 l h⁻¹, P < 0.01).

Conclusion

The correlation with unbound midazolam clearance suggests that either score predicts the metabolic capacity of CYP3A, the most relevant drug metabolizing enzyme subfamily in humans.     

Oral rapamycin attenuates inflammation and enhances stability of atherosclerotic plaques in rabbits independent of serum lipid levels

Background and purpose:

Atherosclerotic plaque rupture and thrombosis are the main cause of acute coronary syndrome. The study was aimed to test the hypothesis that oral administration of rapamycin may attenuate inflammation, inhibit progression and enhance stability of atherosclerotic plaques.

Experimental approach:

Thirty New Zealand rabbits were subjected to balloon-induced endothelial injury of the abdominal aorta and were fed a diet of 1% cholesterol for 20 weeks. From week 9 to week 20, the animals were treated with oral rapamycin (0.5 mg·kg⁻¹·day⁻¹; group A), oral simvastatin (5 mg·kg⁻¹·day⁻¹; group B) and no drugs (group C). At the end of week 20, all rabbits were challenged with injection of Chinese Russell''s viper venom and histamine. Serological, ultrasonographic, pathological, immunohistochemical and gene expression studies were performed.

Key results:

Rapamycin significantly increased the thickness of the fibrous caps and decreased plaque vulnerability index in group A rabbits. Serum lipid levels were higher whereas plaque burden was lower in group A than in group B (P < 0.05). The incidence of plaque rupture in group A (0%) and group B (0%) was significantly lower than that in group C (56.0%, P < 0.05).

Conclusions and implications:

Oral administration of rapamycin effectively attenuated inflammation, inhibited progression and enhanced stability of atherosclerotic plaques in rabbits, without altering serum lipid levels. Our findings suggest a novel approach to the treatment of atherosclerosis.     

Pharmacokinetic and pharmacodynamic analysis of enteric-coated mycophenolate sodium: limited sampling strategies and clinical outcome in renal transplant patients

AIMS

Pharmacokinetic (PK) and pharmacodynamic (PD) monitoring strategies and clinical outcome were evaluated in enteric-coated mycophenolate sodium (EC-MPS)-treated renal allograft recipients.

METHODS

PK [mycophenolic acid (MPA)] and PD [inosine monophosphate dehydrogenase (IMPDH) activity] data were analysed in 66 EC-MPS and ciclosporin A (CsA)-treated renal allograft recipients. Adverse events were considered in a follow-up period of 12 weeks.

RESULTS

Analyses confirmed a limited sampling strategy (LSS) consisting of PK and PD data at predose, 1, 2, 3 and 4 h after oral intake as an appropriate sampling method (MPA r²= 0.812; IMPDH r²= 0.833). MPA AUC_0–12 of patients with early biopsy-proven acute rejection was significantly lower compared with patients without a rejection (median MPA AUC_0–12 28 µg*h ml⁻¹ (7–45) vs. 40 µg*h ml⁻¹ (16–130), P < 0.01), MPA AUC_0–12 of patients with recurrent infections was significantly higher compared with patients without infections (median MPA AUC_0–12 65 µg*h ml⁻¹ (range 37–130) vs. 37 µg*h ml⁻¹ (range 7–120), P < 0.005). Low 12-h IMPDH enzyme activity curve (AEC_0–12) was associated with an increased frequency of gastrointestinal side-effects (median IMPDH AEC_0–12 43 nmol*h mg⁻¹ protein h⁻¹[range 12–67) vs. 75 nmol*h mg⁻¹ protein h⁻¹ (range 15–371), P < 0.01].

CONCLUSIONS

Despite highly variable absorption data, an appropriate LSS might be estimated by MPA AUC_0–4 and IMPDH AEC_0–4 in renal transplant patients treated with EC-MPS and CsA. Regarding adverse events, the suggested MPA-target AUC_0–12 from 30 to 60 µg*h ml⁻¹ seems to be appropriate in renal allograft recipients.     

Neutrophils contribute to intracerebral haemorrhages after treatment with recombinant tissue plasminogen activator following cerebral ischaemia

Background and purpose:

Polymorphonuclear neutrophils (PMNs) contribute to the vascular damage caused by transient cerebral ischaemia. Here we have evaluated the role of PMNs in intracerebral haemorrhage (ICH) induced in a model of thrombolysis with recombinant tissue plasminogen activator (t-PA) during the acute phase of cerebral ischaemia.

Experimental approach:

The middle cerebral artery (MCA) of male spontaneously hypertensive rats was occluded for 1 h followed by reperfusion and, 5 h later, infusion of thrombolytic products (generated in vitro by t-PA on autologous clots). Effects of pretreatment (before the MCA occlusion) with vinblastine (4 days before; 0.5 mg·kg⁻¹), monoclonal anti-neutrophil antibody (mAbRP3; 12 h, 0.3 mg·kg⁻¹) or saline on ICH, neutrophil infiltration, MCA vascular reactivity and brain infarct volume were assessed, 24 h after the beginning of reperfusion.

Key results:

Depletion of circulating neutrophils significantly reduced t-PA-induced ICH (vinblastine, 4.6 ± 1.0; mAbRP3, 5.2 ± 1.0 vs. saline, 10.8 ± 2.7 haemorrhages; P < 0.05). This depletion was associated with a decrease in cerebral infiltration by neutrophils and a decrease of endothelium-dependent, vascular dysfunction in isolated MCA, induced by the ischaemia/reperfusion and t-PA treatment. Brain infarct volume was significantly decreased after vinblastine treatment (159 ± 13 mm³ vs. 243 ± 16 mm³ with saline; P < 0.01) but not after depletion with mAbRP3 (221 ± 22 mm³).

Conclusions and implications:

Our results showed that pharmacological depletion of PMNs prevented t-PA-induced ICH, in parallel with a decrease in cerebral infiltration by PMNs and a decreased endothelial dysfunction in cerebral blood vessels.     

Complex adrenergic and inflammatory mechanisms contribute to phase 2 ventricular arrhythmias in anaesthetized rats

Background and purpose:

The mechanisms responsible for phase 2 (infarct-related) ventricular arrhythmias remain unclear. We have investigated the role of α₁ and β₁ adrenoceptor activation and the interaction of this with infarct neutrophil accumulation, in anaesthetized rats.

Experimental approach:

Neutrophil-replete Sprague-Dawley rats (n = 8–9 per group) were anaesthetized and randomized to receive vehicle, prazosin (0.5 mg·kg⁻¹ i.v.), atenolol (4 mg·kg⁻¹ i.v.) or their combination prior to left main coronary artery occlusion. A further group was depleted of neutrophils and received both atenolol and prazosin. Coronary ligation in all groups was maintained for 240 min.

Key results:

Atenolol and prazosin treatment lowered heart rates and blood pressures respectively, but neither agent given alone affected the incidence of phase 2 ventricular tachycardia or fibrillation. However, co-administration of atenolol with prazosin reduced phase 2 ventricular premature beats (log₁₀-transformed totals were 1.25 ± 0.26 vs. 2.43 ± 0.18 in controls; P < 0.05). Neutrophil depletion attenuated this antiarrhythmic effect (log₁₀-transformed total ventricular premature beats were 1.66 ± 0.35; P > 0.05 vs. controls).

Conclusions and implications:

Phase 2 arrhythmias appear to depend in part on a complex interaction between catecholamines and neutrophils. A model of this interaction is proposed.     

Montelukast inhibits neutrophil pro-inflammatory activity by a cyclic AMP-dependent mechanism

Background and purpose

The objective of this study was to characterize the effects of the cysteinyl leukotriene receptor antagonist, montelukast (0.1–2 µmol·L⁻¹), on Ca²⁺-dependent pro-inflammatory activities, cytosolic Ca²⁺ fluxes and intracellular cAMP in isolated human neutrophils activated with the chemoattractants, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (1 µmol·L⁻¹) and platelet-activating factor (200 nmol·L⁻¹).

Experimental approach

Generation of reactive oxygen species was measured by lucigenin- and luminol-enhanced chemiluminescence, elastase release by a colourimetric assay, leukotriene B₄ and cAMP by competitive binding ELISA procedures, and Ca²⁺ fluxes by fura-2/AM-based spectrofluorimetric and radiometric (⁴⁵Ca²⁺) procedures.

Key results

Pre-incubation of neutrophils with montelukast resulted in dose-related inhibition of the generation of reactive oxygen species and leukotriene B₄ by chemoattractant-activated neutrophils, as well as release of elastase, all of which were maximal at 2 µmol·L⁻¹ (mean percentages of the control values of 30 ± 1, 12 ± 3 and 21 ± 3 respectively; P < 0.05). From a mechanistic perspective, treatment of chemoattractant-activated neutrophils with montelukast resulted in significant reductions in both post-peak cytosolic Ca²⁺ concentrations and store-operated Ca²⁺ influx. These montelukast-mediated alterations in Ca²⁺ handling by the cells were associated with a significant elevation in basal cAMP levels, which resulted from inhibition of cyclic nucleotide phosphodiesterases.

Conclusions and implications

Montelukast, primarily a cysteinyl leukotriene (CysLT₁) receptor antagonist, exhibited previously undocumented, secondary, neutrophil-directed anti-inflammatory properties, which appeared to be cAMP-dependent.