Exenatide at therapeutic and supratherapeutic concentrations does not prolong the QTc interval in healthy subjects

Aims

Exenatide has been demonstrated to improve glycaemic control in patients with type 2 diabetes, with no effect on heart rate corrected QT (QT_c) at therapeutic concentrations. This randomized, placebo- and positive-controlled, crossover, thorough QT study evaluated the effects of therapeutic and supratherapeutic exenatide concentrations on QT_c.

Methods

Intravenous infusion was employed to achieve steady-state supratherapeutic concentrations in healthy subjects within a reasonable duration (i.e. days). Subjects received exenatide, placebo and moxifloxacin, with ECGs recorded pre-therapy and during treatment. Intravenous exenatide was expected to increase heart rate to a greater extent than subcutaneous twice daily or once weekly formulations. To assure proper heart rate correction, a wide range of baseline heart rates was assessed and prospectively defined methodology was applied to determine the optimal QT correction.

Results

Targeted steady-state plasma exenatide concentrations were exceeded (geometric mean ± SEM 253 ± 8.5 pg ml⁻¹, 399 ± 11.9 pg ml⁻¹ and 627 ± 21.2 pg ml⁻¹). QT_cP, a population-based method, was identified as the most appropriate heart rate correction and was prespecified for primary analysis. The upper bound of the two-sided 90% confidence interval for placebo-corrected, baseline-adjusted QT_cP (ΔΔQT_cP) was <10 ms at all time points and exenatide concentrations. The mean of three measures assessed at the highest steady-state plasma exenatide concentration of ∼500 pg ml⁻¹ (ΔΔQT_cP^avg) was −1.13 [−2.11, −0.15). No correlation was observed between ΔΔQT_cP and exenatide concentration. Assay sensitivity was confirmed with moxifloxacin.

Conclusions

These results demonstrated that exenatide, at supratherapeutic concentrations, does not prolong QT_c and provide an example of methodology for QT assessment of drugs with an inherent heart rate effect.     

Regulation of the inducible cyclo-oxygenase pathway in human cultured airway epithelial (A549) cells by nitric oxide

1. In airway epithelium, nitric oxide (NO) is synthesized in the setting of inflammation by inducible nitric oxide synthase (iNOS). Although the role of epithelial derived NO in the regulation of human airways is unknown, prostaglandin E₂ (PGE₂) is recognised as an important inhibitory mediator in human airways. Cyclo-oxygenase (COX) is the rate limiting enzyme in the production of prostanoids and since inflammatory pathways enhance the expression of an inducible COX (COX-2), both COX-2 and iNOS may be co-expressed in response to an inflammatory stimulus. Although regulation of the COX-2 pathway by NO has been demonstrated in animal models, its potential importance in human airway epithelium has not been investigated.
2. The effect of endogenous and exogenous NO on the COX-2 pathway was investigated in the A549 human airway epithelial cell culture model. Activity of the COX-2 pathway was assessed by PGE₂ EIA, and iNOS pathway activity by nitrite assay. A combination cytokine stimulus of interferon gamma (IFNγ) 100 u ml⁻¹, interleukin-1β (IL-1β) 1 u ml⁻¹ and lipopolysaccharide (LPS) 10 μg ml⁻¹ induced nitrite formation which could be inhibited by the competitive NOS inhibitor N^G-nitro-L-arginine-methyl-ester (L-NAME). IL-1β alone (1–50 u ml⁻¹) induced PGE₂ formation without significant nitrite formation, a response which was inhibited by the COX-2 specific inhibitor nimesulide. Submaximal stimuli used for further experiments were IFNγ 100 u ml⁻¹, IL-1β 1 u ml⁻¹ and LPS 10 μg ml⁻¹ to induce both the iNOS and COX-2 pathways, and IL-1β 3 u ml⁻¹ to induce COX-2 without iNOS activity.
3. Cells treated with IFNγ 100 u ml⁻¹, IL-1β 1 u ml⁻¹ and LPS 10 μg ml⁻¹ for 48 h either alone, or with the addition of L-NAME (0 to 10⁻² M), demonstrated inhibition by L-NAME of PGE₂ (3.61±0.55 to 0.51±0.04 pg/10⁴ cells; P<0.001) and nitrite (34.33±8.07 to 0 pmol/10⁴ cells; P<0.001) production. Restoration of the PGE₂ response (0.187±0.053 to 15.46±2.59 pg/10⁴ cells; P<0.001) was observed after treating cells with the same cytokine stimulus and L-NAME 10⁻⁶ M, but with the addition of the NOS substrate L-arginine (0 to 10⁻⁵ M).
4. Cells incubated with IL-1β 3 u ml⁻¹ for 6 h, either alone or with addition of the NO donor S-nitroso-acetyl-penicillamine (SNAP) (0 to 10⁻⁴ M), demonstrated increased PGE₂ formation (1.23±0.03 to 2.92±0.19 pg/10⁴ cells; P< 0.05). No increase in PGE₂ formation was seen when the experiment was repeated in the presence of the guanylate cyclase inhibitor methylene blue (50 μM). Cells treated with SNAP alone did not demonstrate an increased PGE₂ formation. Cells incubated with IL-1β 3 u ml⁻¹ for 6 h in the presence of dibutyryl cyclic guanylate monophosphate (0 to 10⁻³ M) also demonstrated an increased PGE₂ response (2.56±0.21 to 4.53±0.64 pg/10⁴ cells; P<0.05).
5. These data demonstrate that in a human airway epithelial cell culture system, both exogenous and endogenous NO increase the activity of the COX-2 pathway in the setting of inflammatory cytokine stimulation, and that this effect is likely to be mediated by guanylate cyclase. This suggests a role for NO in the regulation of human airway inflammation.

     

Myrtucommulone,a natural acylphloroglucinol,inhibits microsomal prostaglandin E2 synthase-1

Background and purpose:

The selective inhibition of prostaglandin (PG)E₂ formation via interference with microsomal PGE₂ synthase (mPGES)-1 could have advantages in the treatment of PGE₂-associated diseases, such as inflammation, fever and pain, compared with a general suppression of all PG biosynthesis, provided by inhibition of cyclooxygenase (COX)-1 and 2. Here, we addressed whether the naturally occurring acylphloroglucinol myrtucommulone (MC) from Myrtus communis L. (myrtle) affected mPGES-1.

Experimental approach:

The effect of MC on PGE₂ formation was investigated in a cell-free assay by using microsomal preparations of interleukin-1β-stimulated A549 cells as the source of mPGES-1, in intact A549 cells, and in lipopolysaccharide-stimulated human whole blood. Inhibition of COX-1 and COX-2 activity in cellular and cell-free assays was assessed by measuring 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-oxo PGF_1α formation.

Key results:

MC concentration-dependently inhibited cell-free mPGES-1-mediated conversion of PGH₂ to PGE₂ (IC₅₀ = 1 µmol·L⁻¹). PGE₂ formation was also diminished in intact A549 cells as well as in human whole blood at low micromolar concentrations. Neither COX-2 activity in A549 cells nor isolated human recombinant COX-2 was significantly affected by MC up to 30 µmol·L⁻¹, and only moderate inhibition of cellular or cell-free COX-1 was evident (IC₅₀ > 15 µmol·L⁻¹).

Conclusions and implications:

MC is the first natural product to inhibit mPGES-1 that efficiently suppresses PGE₂ formation without significant inhibition of the COX enzymes. This provides an interesting pharmacological profile suitable for interventions in inflammatory disorders, without the typical side effects of coxibs and non-steroidal anti-inflammatory drugs.     

IL-1β reduces tonic contraction of mesenteric lymphatic muscle cells,with the involvement of cycloxygenase-2 and prostaglandin E2

Background and Purpose

The lymphatic system maintains tissue homeostasis by unidirectional lymph flow, maintained by tonic and phasic contractions within subunits, ‘lymphangions’. Here we have studied the effects of the inflammatory cytokine IL-1β on tonic contraction of rat mesenteric lymphatic muscle cells (RMLMC).

Experimental Approach

We measured IL-1β in colon-conditioned media (CM) from acute (AC-CM, dextran sodium sulfate) and chronic (CC-CM, T-cell transfer) colitis-induced mice and corresponding controls (Con-AC/CC-CM). We examined tonic contractility of RMLMC in response to CM, the cytokines h-IL-1β or h-TNF-α (5, 10, 20 ng·mL⁻¹), with or without COX inhibitors [TFAP (10⁻⁵ M), diclofenac (0.2 × 10⁻⁵ M)], PGE₂ (10⁻⁵ M)], IL-1-receptor antagonist, Anakinra (5 μg·mL⁻¹), or a selective prostanoid EP₄ receptor antagonist, GW627368X (10⁻⁶ and 10⁻⁷ M).

Key Results

Tonic contractility of RMLMC was reduced by AC- and CC-CM compared with corresponding control culture media, Con-AC/CC-CM. IL-1β or TNF-α was not found in Con-AC/CC-CM, but detected in AC- and CC-CM. h-IL-1β concentration-dependently decreased RMLMC contractility, whereas h-TNF-α showed no effect. Anakinra blocked h-IL-1β-induced RMLMC relaxation, and with AC-CM, restored contractility to RMLMC. IL-1β increased COX-2 protein and PGE₂ production in RMLMC.. PGE₂ induced relaxations in RMLMC, comparable to h-IL-1β. Conversely, COX-2 and EP₄ receptor inhibition reversed relaxation induced by IL-1β.

Conclusions and Implications

The IL-1β-induced decrease in RMLMC tonic contraction was COX-2 dependent, and mediated by PGE₂. In experimental colitis, IL-1β and tonic lymphatic contractility were causally related, as this cytokine was critical for the relaxation induced by AC-CM and pharmacological blockade of IL-1β restored tonic contraction.     

Tacrolimus placental transfer at delivery and neonatal exposure through breast milk

Aim(s)

The current investigation aims to provide new insights into fetal exposure to tacrolimus in utero by evaluating maternal and umbilical cord blood (venous and arterial), plasma and unbound concentrations at delivery. This study also presents a case report of tacrolimus excretion via breast milk.

Methods

Maternal and umbilical cord (venous and arterial) samples were obtained at delivery from eight solid organ allograft recipients to measure tacrolimus and metabolite bound and unbound concentrations in blood and plasma. Tacrolimus pharmacokinetics in breast milk were assessed in one subject.

Results

Mean (±SD) tacrolimus concentrations at the time of delivery in umbilical cord venous blood (6.6 ± 1.8 ng ml⁻¹) were 71 ± 18% (range 45–99%) of maternal concentrations (9.0 ± 3.4 ng ml⁻¹). The mean umbilical cord venous plasma (0.09 ± 0.04 ng ml⁻¹) and unbound drug concentrations (0.003 ± 0.001 ng ml⁻¹) were approximately one fifth of the respective maternal concentrations. Arterial umbilical cord blood concentrations of tacrolimus were 100 ± 12% of umbilical venous concentrations. In addition, infant exposure to tacrolimus through the breast milk was less than 0.3% of the mother''s weight-adjusted dose.

Conclusions

Differences between maternal and umbilical cord tacrolimus concentrations may be explained in part by placental P-gp function, greater red blood cell partitioning and higher haematocrit levels in venous cord blood. The neonatal drug exposure to tacrolimus via breast milk is very low and likely does not represent a health risk to the breastfeeding infant.     

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.     

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.     

Human microdose evaluation of the novel EP1 receptor antagonist GSK269984A

AIM

The primary objective was to evaluate the pharmacokinetics (PK) of the novel EP₁ antagonist GSK269984A in human volunteers after a single oral and intravenous (i.v.) microdose (100 µg).

METHOD

GSK269984A was administered to two groups of healthy human volunteers as a single oral (n= 5) or i.v. (n= 5) microdose (100 µg). Blood samples were collected for up to 24 h and the parent drug concentrations were measured in separated plasma using a validated high pressure liquid chromatography-tandem mass spectrometry method following solid phase extraction.

RESULTS

Following the i.v. microdose, the geometric mean values for clearance (CL), steady-state volume of distribution (V_ss) and terminal elimination half-life (t_1/2) of GSK269984A were 9.8 l h⁻¹, 62.8 l and 8.2 h. C_max and AUC(0,∞) were 3.2 ng ml⁻¹ and 10.2 ng ml⁻¹ h, respectively; the corresponding oral parameters were 1.8 ng ml⁻¹ and 9.8 ng ml⁻¹ h, respectively. Absolute oral bioavailability was estimated to be 95%. These data were inconsistent with predictions of human PK based on allometric scaling of in vivo PK data from three pre-clinical species (rat, dog and monkey).

CONCLUSION

For drug development programmes characterized by inconsistencies between pre-clinical in vitro metabolic and in vivo PK data, and where uncertainty exists with respect to allometric predictions of the human PK profile, these data support the early application of a human microdose study to facilitate the selection of compounds for further clinical development.     

Tachykinin receptor modulation of cyclooxygenase-2 expression in human polymorphonuclear leucocytes

Background and purpose:

We investigated the ability of natural and synthetic selective NK receptors agonists and antagonists to modulate cyclooxygenase-2 (COX-2) expression in human polymorphonuclear leucocytes (PMNs).

Experimental approach:

The presence of all three tachykinin in PMNs was assessed by Western blot and PCR techniques. Natural and synthetic ligands selective for the tachykinin receptors were used to modulate COX-2 protein (measured with Western blotting) and activity [as prostaglandin E₂ (PGE₂) output]. Effects of substance P (SP) on phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) activation were studied to analyse the signalling pathway involved in COX-2 up-regulation mediated by SP.

Key results:

Stimulation of NK receptors with the natural ligands SP, neurokinin A (NKA) and neurokinin B, in the pmol·L⁻¹-µmol·L⁻¹ concentration range, modulated COX-2 expression and PGE₂ release in a concentration- and time-dependent manner. Experiments with synthetic selective agonists [Sar⁹, Met(O₂)¹¹]SP, [β-Ala⁸] NKA(4-10), senktide or selective antagonists L703,606, SR48,968 or SR142801, confirmed that COX-2 up-regulation was mediated by NK receptors. We found that mainly p38, p42 and p46 MAPKs were phosphorylated by SP and SB202190, PD98059 and SP600125, which are selective inhibitors of these kinases, blocked SP-induced COX-2 expression. SP also induced nuclear translocation of NF-κB concentration-dependently, with a maximum effect at 1 nmol·L⁻¹.

Conclusions and implications:

Human PMNs possess functional NK₁, NK₂ and NK₃ receptors, which mediate the induction of COX-2 expression and NF-κB activation by SP.     

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.     

Diadenosine pentaphosphate is a potent activator of cardiac ryanodine receptors revealing a novel high-affinity binding site for adenine nucleotides

Background and purpose:

Diadenosine polyphosphates are normally present in cells at low levels, but significant increases in concentrations can occur during cellular stress. The aim of this study was to investigate the effects of diadenosine pentaphosphate (Ap5A) and an oxidized analogue, oAp5A on the gating of sheep cardiac ryanodine receptors (RyR2).

Experimental approach:

RyR2 channel function was monitored after incorporation into planar bilayers under voltage-clamp conditions.

Key results:

With10 µmol·L⁻¹ cytosolic Ca²⁺, a significant ‘hump’ or plateau at the base of the dose–response relationship to Ap5A was revealed. Open probability (Po) was significantly increased to a plateau of approximately 0.2 in the concentration range 100 pmol·L⁻¹–10 µmol·L⁻¹. High Po values were observed at >10 µmol·L⁻¹ Ap5A, and Po values close to 1 could be achieved. Nanomolar levels of ATP and adenosine also revealed a hump at the base of the dose–response relationships, although GTP did not activate at any concentration, indicating a common, high-affinity binding site on RyR2 for adenine-based compounds. The oxidized analogue, oAp5A, did not significantly activate RyR2 via the high-affinity binding site; however, it could fully open the channel with an EC₅₀ of 16 µmol·L⁻¹ (Ap5A EC₅₀ = 140 µmol·L⁻¹). Perfusion experiments suggest that oAp5A and Ap5A dissociate slowly from their binding sites on RyR2.

Conclusions and implications:

The ability of Ap5A compounds to increase Po even in the presence of ATP and their slow dissociation from the channel may enable these compounds to act as physiological regulators of RyR2, particularly under conditions of cellular stress.     

Toxicodynamic effects of ciclosporin are reflected by metabolite profiles in the urine of healthy individuals after a single dose

AIMS

The immunosuppressant ciclosporin is an efficient prophylaxis against transplant organ rejection but its clinical use is limited by its nephrotoxicity. Our previous systematic studies in the rat indicated urine metabolite pattern changes to be sensitive indicators of the negative effects of ciclosporin on the kidney. To translate these results, we conducted an open label, placebo-controlled, crossover study assessing the time-dependent toxicodynamic effects of a single oral ciclosporin dose (5 mg kg⁻¹) on the kidney in 13 healthy individuals.

METHODS

In plasma and urine samples, ciclosporin and 15-F_2t-isoprostane concentrations were assessed using HPLC-MS and metabolite profiles using ¹H-NMR spectroscopy.

RESULTS

The maximum ciclosporin concentrations were 1489 ± 425 ng ml⁻¹ (blood) and 2629 ± 1308 ng ml⁻¹ (urine). The increase in urinary 15-F_2t-isoprostane observed 4 h after administration of ciclosporin indicated an increase in oxidative stress. 15-F_2t-isoprostane concentrations were on average 2.9-fold higher after ciclosporin than after placebo (59.8 ± 31.2 vs. 20.9 ± 19.9 pg mg⁻¹ creatinine, P < 0.02). While there were no conclusive changes in plasma 15-F_2t-isoprostane concentrations or metabolite patterns, non-targeted metabolome analysis using principal components analysis and partial least square fit analysis revealed significant changes in urine metabolites typically associated with negative effects on proximal tubule cells. The major metabolites that differed between the 4 h urine samples after ciclosporin and placebo were citrate, hippurate, lactate, TMAO, creatinine and phenylalanine.

CONCLUSION

Changes in urine metabolite patterns as a molecular marker are sufficiently sensitive for the detection of the negative effects of ciclosporin on the kidney after a single oral dose.     

Effect of AeroChamber Plus™ on the lung and systemic bioavailability of beclometasone dipropionate/formoterol pMDI

AIM

To assess the effect of AeroChamber Plus™ on lung deposition and systemic exposure to extra-fine beclometasone dipropionate (BDP)/formoterol (100/6 µg) pMDI (Foster®). The lung deposition of the components of the combination given with the pMDI was also evaluated using the charcoal block technique.

METHODS

Twelve healthy male volunteers received four inhalations of extra-fine BDP/formoterol (100/6 µg) using (i) pMDI alone, (ii) pMDI and AeroChamber Plus™ and (iii) pMDI and charcoal ingestion.

RESULTS

Compared with pMDI alone, use of AeroChamber Plus™ increased the peak plasma concentrations (C_max) of BDP (2822.3 ± 1449.9 vs. 5454.9 ± 3197.1 pg ml⁻¹), its active metabolite beclometasone 17-monopropionate (17-BMP) (771.6 ± 288.7 vs. 1138.9 ± 495.6 pg ml⁻¹) and formoterol (38.4 ± 17.8 vs. 54.7 ± 20.0 pg ml⁻¹). For 17-BMP and formoterol, the AUC(0,30 min), indicative of lung deposition, was increased in the AeroChamber Plus™ group by 41% and 45%, respectively. This increase was mainly observed in subjects with inadequate inhalation technique. However, use of AeroChamber Plus™ did not increase the total systemic exposure to 17-BMP and formoterol. Results after ingestion of charcoal confirmed that AUC(0,30 min) can be taken as an index of lung bioavailability and that more than 30% of the inhaled dose of extra-fine BDP/formoterol 100/6 µg was delivered to the lung using the pMDI alone.

CONCLUSIONS

The use of AeroChamber Plus™ optimizes the delivery of BDP and formoterol to the lung in subjects with inadequate inhalation technique. The total systemic exposure was not increased, supporting the safety of extra-fine BDP/formoterol pMDI with AeroChamber Plus™.     

Prostaglandin E2 induces spontaneous rhythmic activity in mouse urinary bladder independently of efferent nerves

BACKGROUND AND PURPOSE

The acute effects of PGE₂ on bladder smooth muscle and nerves were examined to determine the origin of PGE₂-induced spontaneous rhythmic contractions.

EXPERIMENTAL APPROACH

Contraction studies, confocal Ca²⁺ imaging and electrophysiological recordings in strips of mouse urinary bladder were used to differentiate the effects of PGE₂ on bladder smooth muscle and efferent nerves.

KEY RESULTS

PGE₂ (50 µM) increased the tone and caused phasic contractions of detrusor smooth muscle strips. Confocal Ca²⁺ imaging showed that PGE₂ increased the frequency of whole-cell Ca²⁺ transients (WCTs) (72 ± 5%) and intracellular recordings showed it increased the frequency of spontaneous depolarizations, from 0.31·s⁻¹ to 0.90·s⁻¹. Non-selective inhibition of EP receptors using SC-51322 and AH-6809 (10 µM), or the L-type Ca²⁺ channel blocker nifedipine (1 µM), prevented these phasic contractions and WCTs, and reduced the tone (by 45 ± 7% and 59 ± 6%, respectively). Blocking P2X1 receptors with NF449 (10 µM) caused a small but significant reduction in the frequency of PGE₂-induced phasic contractions (24 ± 9%) and WCTs (28 ± 17%) but had no significant effect on spontaneous depolarizations or tone. Inhibiting muscarinic receptors with cyclopentolate (1 µM) had no significant effect on these measures. Spontaneous WCTs became synchronous in PGE₂, implying enhanced functional coupling between neighbouring cells. However, the electrical input resistance was unchanged.

CONCLUSIONS AND IMPLICATIONS

It was concluded that depolarization alone is sufficient to explain a functional increase in intercellular coupling and the ability of PGE₂ to increase detrusor spontaneous rhythmic activity does not require parasympathetic nerves.     

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.     

The effect of tafamidis on the QTc interval in healthy subjects

Aims

The transthyretin (TTR) stabilizer, tafamidis, has demonstrated efficacy and safety in the treatment of TTR familial amyloid polyneuropathy (20 mg day⁻¹). Tafamidis use in TTR cardiomyopathy led to the study of the potential effect of tafamidis on the QT_c interval in healthy subjects.

Methods

This randomized, three treatment, three period, six sequence crossover study with placebo, a positive control (moxifloxacin 400 mg) and tafamidis (400 mg, to achieve a supra-therapeutic C_max of ∽20 µg ml⁻¹) was conducted in healthy volunteers at three clinical research units. Oral dosing in each of the three treatment periods was separated by a washout period of  ≥ 14 days. Serial triplicate 12-lead electrocardiograms were performed. QT_c intervals were derived using the Fridericia correction method. Safety and tolerability were assessed by physical examination, vital signs measurement, laboratory analyses and monitoring of adverse events (AEs).

Results

A total of 42 subjects completed the study. The upper limit of the two-sided 90% confidence intervals (CIs) for the difference in baseline-adjusted QT_cF between tafamidis 400 mg and placebo was <10 ms (non-inferiority criterion) for all time points. The lower limit of the two-sided 90% CI between moxifloxacin 400 mg and placebo exceeded 5 ms at the pre-specified moxifloxacin t_max of 3 h post-dose, confirming assay sensitivity. C_max and AUC(0,24 h) for tafamidis were 20.36 µg ml⁻¹ and 305.4 µg ml⁻¹ h, respectively. There were no serious/severe AEs or treatment discontinuations due to AEs.

Conclusions

This thorough QT_c study suggests that a supra-therapeutic single 400 mg oral dose of tafamidis does not prolong the QT_c interval and is well-tolerated in healthy volunteers.     

Population pharmacokinetics of oral diclofenac for acute pain in children

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Diclofenac is an effective oral analgesic for acute postoperative pain. In adults 25 mg is half as effective as 50 mg, but 50 mg and 100 mg are similarly effective (ceiling effect). Diclofenac has linear pharmacokinetics in this range.
• Diclofenac is frequently used ‘off-label’ in children for acute pain but optimum dosing is unclear (dosing of diclofenac in clinical paediatric studies ranges from 0.5–2.5 mg kg⁻¹). There is currently no licensed oral paediatric formulation of diclofenac.

WHAT THIS STUDY ADDS

• Using a new diclofenac oral suspension, a dose of 1 mg kg⁻¹ in children aged 1 to 12 years gives a similar exposure to 50 mg in adults; paediatric patients are unlikely to benefit from higher doses.

AIMS

To develop a population pharmacokinetic model for a new diclofenac suspension (50 mg 5 ml⁻¹) in adult volunteers and paediatric patients, and recommend a dose for acute pain in children.

METHODS

Blood samples were drawn at the start and end of surgery, and on removal of the venous cannula from 70 children (aged 1 to 12 years, weight 9 to 37 kg) who received a preoperative oral 1 mg kg⁻¹ dose; these were pooled with rich (14 post-dose samples) data from 30 adult volunteers. Population pharmacokinetic modelling was undertaken with NONMEM. The optimum adult dose of diclofenac for acute pain is 50 mg. Simulation from the final model was performed to predict a paediatric dose to achieve a similar AUC to 50 mg in adults.

RESULTS

A total of 558 serum diclofenac concentrations from 100 subjects was used in the pooled analysis. A single disposition compartment model with first order elimination and dual absorption compartments was used. The estimates of CL/F and V_D/F were 53.98 l h⁻¹ 70 kg⁻¹ and 4.84 l 70 kg⁻¹ respectively. Allometric size models appeared to predict adequately changes in CL and V_D with age. Of the simulated doses investigated, 1 mg kg⁻¹ gave paediatric AUC_{(0, 12 h)} to adult 50 mg AUC_{(0, 12 h)} ratios of 1.00, 1.08 and 1.18 for ages 1–3, 4–6 and 7–12 years respectively.

CONCLUSIONS

This study has shown 1 mg kg⁻¹ diclofenac to produce similar exposure in children aged 1 to 12 years as 50 mg in adults, and is acceptable for clinical practice; patients are unlikely to obtain further benefit from higher doses.     

Inotropy and L-type Ca2+ current,activated by β1- and β2-adrenoceptors,are differently controlled by phosphodiesterases 3 and 4 in rat heart

Background and purpose

β₁- and β₂-adrenoceptors coexist in rat heart but β₂-adrenoceptor-mediated inotropic effects are hardly detectable, possibly due to phosphodiesterase (PDE) activity. We investigated the influence of the PDE3 inhibitor cilostamide (300 nmol·L⁻¹) and the PDE4 inhibitor rolipram (1 µmol·L⁻¹) on the effects of (−)-catecholamines.

Experimental approach

Cardiostimulation evoked by (−)-noradrenaline (ICI118551 present) and (−)-adrenaline (CGP20712A present) through β₁- and β₂-adrenoceptors, respectively, was compared on sinoatrial beating rate, left atrial and ventricular contractile force in isolated tissues from Wistar rats. L-type Ca²⁺-current (I_Ca-L) was assessed with whole-cell patch clamp.

Key results

Rolipram caused sinoatrial tachycardia. Cilostamide and rolipram did not enhance chronotropic potencies of (−)-noradrenaline and (−)-adrenaline. Rolipram but not cilostamide potentiated atrial and ventricular inotropic effects of (−)-noradrenaline. Cilostamide potentiated the ventricular effects of (−)-adrenaline but not of (−)-noradrenaline. Concurrent cilostamide + rolipram uncovered left atrial effects of (−)-adrenaline. Both rolipram and cilostamide augmented the (−)-noradrenaline (1 µmol·L⁻¹) evoked increase in I_Ca-L. (−)-Adrenaline (10 µmol·L⁻¹) increased I_Ca-L only in the presence of cilostamide but not rolipram.

Conclusions and implications

PDE4 blunts the β₁-adrenoceptor-mediated inotropic effects. PDE4 reduces basal sinoatrial rate in a compartment distinct from compartments controlled by β₁- and β₂-adrenoceptors. PDE3 and PDE4 jointly prevent left atrial β₂-adrenoceptor-mediated inotropy. Both PDE3 and PDE4 reduce I_Ca-L responses through β₁-adrenoceptors but the PDE3 component is unrelated to inotropy. PDE3 blunts both ventricular inotropic and I_Ca-L responses through β₂-adrenoceptors.     

Identification of an additional supraspinal component to the analgesic mechanism of action of buprenorphine

Background and purpose:

Buprenorphine displays attributes of opioids, but also some features distinct from them. We examined spinal and supraspinal signal transduction of buprenorphine-induced anti-nociception in mice compared with morphine and fentanyl.

Experimental approach:

The opioid receptor antagonist naloxone, Pertussis toxin (PTX), G_z protein antisense and nociceptin/orphanin-FQ receptor agonist nociceptin, and antagonist, JTC-801, were injected supraspinally (intracerebroventricular) and spinally (intrathecal). Also the cell-permeable Ser/Thr protein phosphatase inhibitor okadaic acid was given supraspinally.

Key results:

Spinal naloxone (20 µg) or PTX (1 µg) attenuated morphine, fentanyl and buprenorphine (s.c.) anti-nociception. Supraspinal naloxone or PTX attenuated morphine and fentanyl, but not buprenorphine anti-nociception. Spinal G_z protein antisense did not alter buprenorphine, morphine or fentanyl anti-nociception and supraspinal G_z-antisense did not alter morphine or fentanyl anti-nociception. However, supraspinal G_z-antisense (not random sense) reduced buprenorphine anti-nociception. Peripheral JTC-801 (1 mg·kg⁻¹, i.p.) enhanced the ascending (3 mg·kg⁻¹) and descending (30 mg·kg⁻¹) portions of buprenorphine''s dose–response curve, but only spinal, not supraspinal, nociceptin (10 nmol·L⁻¹) enhanced buprenorphine anti-nociception. Intracereboventricular okadaic acid (0.001–10 pg) produced a biphasic low-dose attenuation, high-dose enhancement of buprenorphine(3 or 30 mg·kg⁻¹, s.c.) anti-nociception, but did not affect morphine or fentanyl anti-nociception.

Conclusions and implications:

Buprenorphine has an opioid component to its supraspinal mechanism of analgesic action. Our present results reveal an additional supraspinal component insensitive to naloxone, PTX and nociceptin/orphanin-FQ, but involving G_z protein and Ser/Thr protein phosphatase. These data might help explain the unique preclinical and clinical profiles of buprenorphine.