Characterization of a critical role for CFTR chloride channels in cardioprotection against ischemia/reperfusion injury

Aim:

To further characterize the functional role of cystic fibrosis transmembrane conductance regulator (CFTR) in early and late (second window) ischemic preconditioning (IPC)- and postconditioning (POC)-mediated cardioprotection against ischemia/reperfusion (I/R) injury.

Methods:

CFTR knockout (CFTR^−/−) mice and age- and gender-matched wild-type (CFTR^+/+) and heterozygous (CFTR^+/-) mice were used. In in vivo studies, the animals were subjected to a 30-min coronary occlusion followed by a 40-min reperfusion. In ex vivo (isolate heart) studies, a 45-min global ischemia was applied. To evaluate apoptosis, the level of activated caspase 3 and TdT-mediated dUTP-X nick end labeling (TUNEL) were examined.

Results:

In the in vivo I/R models, early IPC significantly reduced the myocardial infarct size in wild-type (CFTR^+/+) (from 40.4%±5.3% to 10.4% ±2.0%, n=8, P<0.001) and heterozygous (CFTR^+/-) littermates (from 39.4%±2.4% to 15.4% ±5.1%, n=6, P<0.001) but failed to protect CFTR knockout (CFTR^−/−) mice from I/R induced myocardial infarction (46.9%±6.2% vs 55.5%±7.8%, n=6, P>0.5). Similar results were observed in the in vivo late IPC experiments. Furthermore, in both in vivo and ex vivo I/R models, POC significantly reduced myocardial infarction in wild-type mice, but not in CFTR knockout mice. In ex vivo I/R models, targeted inactivation of CFTR gene abolished the protective effects of IPC against I/R-induced apoptosis.

Conclusion:

These results provide compelling evidence for a critical role for CFTR Cl⁻ channels in IPC- and POC-mediated cardioprotection against I/R-induced myocardial injury.     

Discovery of novel PTP1B inhibitors with antihyperglycemic activity

Aim:

To discover and optimize a series of novel PTP1B inhibitors containing a thiazolidinone-substituted biphenyl scaffold and to further evaluate the inhibitory effects of these compounds in vitro and in vivo.

Methods:

A total of 36 thiazolidinone substituted biphenyl scaffold derivatives were prepared. An in vitro biological evaluation was done by Enzyme-based assay. The in vivo efficacy of 7Fb as an antihyperglycemic agent was evaluated in a BKS db/db diabetic mouse model with a dose of 50 mg·kg^-1·d^-1 for 4 weeks.

Results:

The in vitro biological evaluation showed that compounds 7Fb and 7Fc could increase the insulin-induced tyrosine phosphorylation of IRβ in CHO/hIR cells. In in vivo experiments, compound 7Fb significantly lowered the postprandial blood glucose, from 29.4±1.2 mmol/L with the vehicle to 24.7±0.6 mmol/L (P<0.01), and the fasting blood glucose from 27.3±1.5 mmol/L with the vehicle to 23.6±1.2 mmol/L (P<0.05).

Conclusion:

A novel series of compounds were discovered to be PTP1B inhibitors. Among them, compound 7Fb significantly lowered the postprandial and fasting glucose levels, and the blood glucose level declined more rapidly than in metformin-treated mice. Thus, 7Fb may be a potential lead compound for developing new agents for the treatment of type II diabetes.     

Atenolol blunts blood pressure increase during dynamic resistance exercise in hypertensives

AIMS

This study was conducted to determine whether atenolol was able to decrease BP level and mitigate BP increase during dynamic resistance exercise performed at three different intensities in hypertensives.

METHODS

Ten essential hypertensives (systolic/diastolic BP between 140/90 and 160/105 mmHg) were blindly studied after 6 weeks of placebo and atenolol. In each phase, volunteers executed, in a random order, three protocols of knee-extension exercises to fatigue: (i) one set at 100% of 1 RM; (ii) three sets at 80% of 1 RM; and (iii) three sets at 40% of 1 RM. Intra-arterial radial blood pressure was measured throughout the protocols.

RESULTS

Atenolol decreased systolic BP maximum values achieved during the three exercise protocols (100% = 186 ± 4 vs. 215 ± 7, 80% = 224 ± 7 vs. 247 ± 9 and 40% = 223 ± 7 vs. 252 ± 16 mmHg, P < 0.05). Atenolol also mitigated an increase in systolic BP in the first set of exercises (100% =+38 ± 5 vs.+54 ± 9; 80% =+68 ± 11 vs. +84 ± 13 and 40% =+69 ± 7 vs.+84 ± 14, mmHg, P < 0.05). Atenolol decreased diastolic BP values and mitigated its increase during exercise performed at 100% of 1 RM (126 ± 6 vs. 145 ± 6 and +41 ± 6 vs.+52 ± 6, mmHg, P < 0.05), but not at the other exercise intensities.

CONCLUSIONS

Atenolol was effective in both reducing systolic BP maximum values and mitigating BP increase during resistance exercise performed at different intensities in hypertensive subjects.     

Chemokine receptor CXCR3 is important for lung tissue damage and airway remodeling induced by short-term exposure to cigarette smoking in mice

Aim:

To investigate the role of chemokine receptor CXCR3 in cigarette smoking (CS)-induced pulmonary damage.

Methods:

CXCR3 knockout (CXCR3-/-) mice were used. Differences in airspace enlargement, mRNA expression of matrix metalloproteinases (MMPs), transforming growth factor (TGF) β1, CXCL10 in lung homogenates, and CXCL10 content in bronchoalveolar lavage (BAL) fluids and homogenates were compared between CXCR3-/- mice and wild-type (WT) mice three days after three-day CS exposures.

Results:

The linear intercept was significantly less in CXCR3-/- mice than in WT mice (30.1±0.9 μm vs 40.3±2.4 μm, P<0.01). Morphologically, collagen was deposited less around airways and vessels in CXCR3-/- mice. The lung hydroxyproline content was significantly lower in CXCR3-/- mice than in WT mice (6.0±1.0 μg/mL vs 12.0±1.6 μg/mL, P<0.05). Profoundly lower mRNA expression of MMP2, MMP12, TGFβ1, and CXCL10 was seen in lung homogenates from CXCR3-/- mice. CXCL10 concentrations in BAL fluid and lung homogenates were significantly lower in CXCR3-/- mice than in WT mice (BAL fluid: 19.3±1.4 pg/mL vs 24.8±1.6 pg/mL, P<0.05; lung homogenates: 76.6±7.0 pg/mL vs 119.5±15.9 pg/mL, P<0.05).

Conclusion:

CXCR3 is important in mediating lung tissue damage and airway remodeling following a short-term CS insult, possibly through up-regulation of CXCL10 and inducement of mRNA expression of MMPs. Targeting CXCR3 may be helpful for prevention of CS-induced pulmonary pathology.     

Different magnitude of resistance to nondepolarizing muscle relaxants in the denervated mouse skeletal muscle

Aim:

To test the hypothesis that different magnitude of resistance of denervated skeletal muscle to nondepolarizing muscle relaxants (NDMRs) is related to their varying potencies at ɛ-AChR and γ-AChR.

Methods:

Both innervated and denervated mouse muscle cells, and human embryonic kidney 293 (HEK293) cells expressing ɛ-AChR or γ-AChR were used. The effects of NDMRs on nAChR were explored using whole-cell patch clamp technique.

Results:

NDMRs vecuronium (VEC), atracurium (ATR) and rocuronium (ROC) produced reversible, dose-dependent inhibition on the currents induced by 30 μmol/L acetylcholine both in innervated and denervated skeletal muscle cells. Compared to those obtained in innervated skeletal muscle cells, denervation shifted the concentration-response curves rightward and significantly increased the 50% inhibitory concentration (IC₅₀) values (VEC: from 11.2 to 39.2 nmol/L, P<0.01; ATR: from 24.4 to 129.0 nmol/L, P<0.01; ROC: from 37.9 to 101.4 nmol/L, P<0.01). In HEK293 cell expression system, ATR was less potent at γ-AChR than ɛ-AChR (IC₅₀ values: 35.9 vs 22.3 nmol/L, P<0.01), VEC was equipotent at both receptor subtypes (IC₅₀ values: 9.9 vs 10.2 nmol/L, P>0.05), while ROC was more potent at γ-AChR than ɛ-AChR (IC₅₀ values: 22.3 vs 33.5 nmol/L, P<0.05).

Conclusion:

Magnitude differences of resistance to different NDMRs caused by denervation are associated with distinct potencies of NDMRs at nAChR subtypes.     

Paradoxical relationship between RAVE （relative activity versus endocytosis） values of several opioid receptor agonists and their liability to cause dependence

Aim:

To examine the relationship between the RAVE (relative activity versus endocytosis) values of opiate agonists and their dependence liability by studying several potent analgesics with special profiles in the development of physical and psychological dependence.

Methods:

The effects of (−)-cis-(3R,4S,2′R) ohmefentanyl (F9202), (+)-cis-(3R,4S,2′S) ohmefentanyl (F9204), dihydroetorphine (DHE) and morphine on [³⁵S]GTPγS binding, forskolin-stimulated cAMP accumulation, and receptor internalization were studied in CHO cells stably expressing HA-tagged μ-opioid receptors (CHO-HA-MOR). cAMP overshoot in response to the withdrawal of these compound treatments was also tested.

Results:

All four agonists exhibited the same rank order of activity in stimulation of [³⁵S]GTPγS binding, inhibition of adenylyl cyclase (AC) and induction of receptor internalization: DHE>F9204>F9202>morphine. Based on these findings and the previous in vivo analgesic data obtained from our and other laboratories, the RAVE values of the four agonists were calculated. The rank order of RAVE values was morphine>F9202>F9204>DHE. For the induction of cAMP overshoot, the rank order was F9202≥morphine>F9204≥DHE.

Conclusion:

Taken in combination with previous findings of these compounds'' liability to develop dependence, the present study suggests that the agonist with the highest RAVE value seems to have a relatively greater liability to develop psychological dependence relative to the agonist with the lowest RAVE value. However, the RAVE values of these agonists are not correlated with their probability of developing physical dependence or inducing cAMP overshoot, a cellular hallmark of dependence.     

The potent calcitonin gene-related peptide receptor antagonist, telcagepant, does not affect nitroglycerin-induced vasodilation in healthy men

AIMS

To assess the effect of the calcitonin gene-related peptide (CGRP) receptor antagonist, telcagepant, on the haemodynamic response to sublingual nitroglycerin (NTG).

METHODS

Twenty-two healthy male volunteers participated in a randomized, placebo-controlled, double-blind, two-period, crossover study. Subjects received 500 mg telcagepant or placebo followed, 1.5 h later, by 0.4 mg NTG. To assess the haemodynamic response the following vascular parameters were measured: blood pressure, aortic augmentation index (AIx) and brachial artery diameter (BAD). Data are presented as mean (95% confidence interval, CI).

RESULTS

The aortic AIx following NTG decreased by −18.50 (−21.02, −15.98) % after telcagepant vs. −17.28 (−19.80, −14.76) % after placebo. The BAD fold increase following NTG was 1.14 (1.12, 1.17) after telcagepant vs. 1.13 (1.10, 1.15) after placebo. For both AIx and BAD, the hypothesis that telcagepant does not significantly affect the changes induced by NTG is supported (P < 0.0001). In addition, no vasoconstrictor effect of telcagepant could be demonstrated.

CONCLUSIONS

Telcagepant did not affect NTG-induced haemodynamic changes. These data suggest that NTG-induced vasodilation is not CGRP dependent.     

Contribution of CYP2C19 and CYP3A4 to the formation of the active nortilidine from the prodrug tilidine

AIMS

To investigate in vivo the effect of the CYP2C19 genotype on the pharmacokinetics of tilidine and the contribution of CYP3A4 and CYP2C19 to the formation of nortilidine using potent CYP3A4 inhibition by ritonavir.

METHODS

Fourteen healthy volunteers (seven CYP2C19 poor and seven ultrarapid metabolizers) received ritonavir orally (300 mg twice daily) for 3 days or placebo, together with a single oral dose of tilidine and naloxone (100 mg and 4 mg, respectively). Blood samples and urine were collected for 72 h. Noncompartmental analysis was performed to determine pharmacokinetic parameters of tilidine, nortilidine, bisnortilidine and ritonavir.

RESULTS

Tilidine exposure increased sevenfold and terminal elimination half-life fivefold during ritonavir treatment, but no significant differences were observed between the CYP2C19 genotypes. During ritonavir treatment, nortilidine area under the concentration–time curve was on average doubled, with no differences between CYP2C19 poor metabolizers [2242 h ng ml⁻¹ (95% confidence interval 1811–2674) vs. 996 h ng ml⁻¹ (95% confidence interval 872–1119)] and ultrarapid metabolizers [2074 h ng ml⁻¹ (95% confidence interval 1353–2795) vs. 1059 h ng ml⁻¹ (95% confidence interval 789–1330)]. The plasma concentration–time curve of the secondary metabolite, bisnortilidine, showed a threefold increase of time to reach maximal observed plasma concentration; however, area under the concentration–time curve was not altered by ritonavir.

CONCLUSIONS

The sequential metabolism of tilidine is inhibited by the potent CYP3A4 inhibitor, ritonavir, independent of the CYP2C19 genotype, with a twofold increase in the exposure of the active nortilidine.     

Degarelix, a novel GnRH antagonist, causes minimal histamine release compared with cetrorelix, abarelix and ganirelix in an ex vivo model of human skin samples

AIMS

Early studies on gonadotrophin-releasing hormone (GnRH) antagonists pointed out histamine-mediated anaphylactic reactions as a potential adverse effect of these drug candidates. In this study we have compared the histamine-releasing potential of four approved and marketed antagonists, degarelix, cetrorelix, abarelix and ganirelix in an ex vivo model of human skin samples.

METHODS

Human skin samples were obtained during cosmetic plastic surgery and kept in oxygenated saline solution. The samples were incubated either without or at different concentrations of the antagonists (3, 30 or 300 µg ml⁻¹ for all, except for ganirelix 1, 10 or 100 µg ml⁻¹). The drug-induced effect was expressed as the increase relative to basal release. The histamine-releasing capacity of the skin was verified by a universal histamine releaser, compound 40/80.

RESULTS

Degarelix had no significant effect on basal histamine release in the 3 to 300 µg ml⁻¹ concentration range. The effect of ganirelix was moderate causing a nonsignificant increase of 81 ± 27% at the 100 µg ml⁻¹ concentration. At 30 and 300 µg ml⁻¹ concentrations abarelix (143 ± 29% and 362 ± 58%, respectively, P < 0.05) and cetrorelix (228 ± 111% and 279 ± 46%, respectively, P < 0.05) caused significantly increased histamine release.

CONCLUSIONS

In this ex vivo human skin model, degarelix displayed the lowest capacity to release histamine followed by ganirelix, abarelix and cetrorelix. These findings may provide indirect hints as to the relative likelihood of systemic anaphylactic reactions in clinical settings.     

Expression of a dominant-negative Rho-kinase promotes neurite outgrowth in a microenvironment mimicking injured central nervous system

Aim:

To investigate whether lentiviral vector (LV)-mediated expression of a dominant negative mutant Rho-kinase (DNROCK) could inhibit activation of the Rho/ROCK signaling pathway and promote neurite outgrowth in a hostile microenvironment mimicking the injured central nervous system (CNS) in vitro.

Methods:

Lentiviral stock was produced using the three-plasmid system by transfecting HEK293 cells. Myelin prepared from rat brain was purified by two rounds of discontinuous density gradient centrifugation and osmotic disintegration. Differentiated PC12 cells and dissociated adult rat dorsal root ganglion (DRG) neurons were transduced with either LV/DNROCK or LV/green fluorescent protein (GFP) and seeded on solubilized myelin proteins. The effect of DNROCK on growth cone morphology was tested by rhodamine-conjugated phalloidin staining. Expression of DNROCK was determined by immunoblotting. The length of the longest neurite, the percentage of neurite-bearing neurons, or the total process outgrowth for all transduced neurons were measured by using the Scion image analysis program.

Results:

Transduction of DNROCK inhibited serum-induced stress fiber formation in NIH 3T3 cells and induced enlargement of cell bodies and decreased the phosphorylation levels of MYPT1 in HeLa cells. LV/DNROCK blocked myelin-induced increase in ROCK translocation from cytosol to membrane in LV/GFP-treated PC12 cells. DNROCK promotes neurite outgrowth of differentiated PC12 cells and DRG neurons on myelin protein. LV/DNROCK-transduced PC12 cells had longer neurites than LV/GFP-transduced cells (39.18±2.19 μm vs 29.32±1.7 μm, P<0.01) on myelin-coated coverslips. Furthermore, a significantly higher percentage of LV/DNROCK-transduced cells had extended neurites than LV/GFP-transduced cells (63.75%±8.03% vs 16.3%±3.70%, P<0.01). LV/DNROCK-transduced DRG neurons had longer neurite length (325.22±10.8 μm vs 202.47±9.3 μm, P<0.01) and more primary neurites per cell than those in LV/GFP-transduced cells plated on myelin and laminin (7.8±1.25 vs 4.84±1.45, P<0.01) or on laminin alone (5.2±1.88). LV/DNROCK-transduced cells had significantly larger growth cones (33.12±1.06 μm²) than LV/GFP-pretreated cells (23.72±1.22 μm²).

Conclusion:

These results indicate that blocking the RhoA/ROCK signaling pathway by expression of DNROCK is effective in facilitating neurite outgrowth in a microenvironment mimicking injury of central nervous system.     

Equilibrative nucleoside transporter 1 genotype, cytidine deaminase activity and age predict gemcitabine plasma clearance in patients with solid tumours

AIM

Gemcitabine (GEM) enters normal and tumour cells via concentrative (CNT) and equilibrative nucleoside transporters (ENT) and is subsequently deaminated to the inactive difluorodeoxyurine (dFdU) by cytidine deaminase (CDA). The aim of our study was to ascertain whether the nucleoside transporter genotype and the CDA activity phenotype can predict total GEM plasma clearance.

METHODS

Forty-seven patients received GEM 1000–1250 mg m⁻² i.v. over 30 min. Plasma concentrations of GEM and dFdU were measured and individual pharmacokinetic profiles were determined. CDA activity was measured ex vivo in plasma samples. The two most common hENT1 and hCNT1 polymorphisms were determined from genomic DNA.

RESULTS

Multivariate analysis revealed that GEM plasma clearance (CL) was positively correlated with the end of infusion dFdU : GEM ratio (P < 0.0001), which is a marker of in vivo CDA activity. The ENT1 genotype characterized by high transport capacity (G/G) and age were inversely correlated with CL (P= 0.027 and 0.048, respectively). A strong correlation was found between end of infusion GEM concentration and area under the concentration–time curve from time 0 to infinity (AUC(0,∞)) (r²= 0.77).

CONCLUSIONS

Our results confirm the role of CDA and age on the interindividual variability of GEM CL and show the contribution of the hENT1 genotype for the first time.     

Effect of a proton pump inhibitor on the pharmacokinetics of imatinib

AIMS

Imatinib mesylate (Gleevec®/Glivec®), which has revolutionized the treatment of chronic myeloid leukemias (CML) and gastrointestinal stromal tumours (GIST), has been reported to cause gastric upset. Consequently, proton pump inhibitors (PPI) are frequently co-administered with imatinib. Because PPI can elevate gastric pH and delay gastric emptying or antagonize ATP-binding-cassette transporters, they could influence imatinib absorption and pharmacokinetics. We aimed to evaluate whether use of omeprazole has a significant effect on imatinib pharmacokinetics.

METHODS

Twelve healthy subjects were enrolled in a two-period, open-label, single-institution, randomized cross-over, fixed-schedule study. In one period, each subject received 400 mg imatinib orally. In the other period, 40 mg omeprazole (Prilosec®) was administered orally for 5 days, and on day 5 it was administered 15 min before 400 mg imatinib. Plasma concentrations of imatinib and its active N-desmethyl metabolite {"type":"entrez-protein","attrs":{"text":"CGP74588","term_id":"875877231"}}CGP74588 were assayed by LC-MS, and data were analyzed non-compartmentally.

RESULTS

PPI administration did not significantly affect the imatinib area under the plasma concentration vs time curve (AUC) (34.1 µg ml⁻¹ h alone vs 33.1 µg ml⁻¹ h with omeprazole, P= 0.64; 80% power), maximum plasma concentration (C_max) (2.04 µg ml⁻¹ alone vs 2.02 µg ml⁻¹ with omeprazole, P= 0.97), or half-life (13.4 h alone vs 14.1 h with omeprazole, P= 0.13).

CONCLUSIONS

Our results indicate that the use of omeprazole does not significantly affect the pharmacokinetics of imatinib, as opposed to, for example, dasatinib where PPI decreased AUC and C_max two-fold.     

Effect of the ethinylestradiol/levonorgestrel combined oral contraceptive on the activity of cytochrome P4503A in obese women

AIM(S)

While it is known that CYP3A4/5 activity is decreased with combined oral contraceptive (COC) use and obesity suppresses CYP expression, the combined effects of obesity and COC use on CYP3A4/5 activity are unclear. Therefore, our aim was to examine the effect of COC usage on CYP3A4/5 activity in obese women.

METHODS

Thirty-four, obese (body mass index, BMI > 30 kg m⁻²) women of reproductive age (18–35 years old) were placed on a COC pill containing 20 µg ethinylestradiol/100 µg levonorgestrel for 21 days starting at the onset of menses. A midazolam pharmacokinetic study was conducted prior to initiation and after 21 days of COC treatment. Serial blood samples were collected and plasma concentrations of midazolam were measured using liquid chromatography tandem mass spectrometry. Pharmacokinetic parameters were estimated using a non-compartmental method.

RESULTS

Midazolam clearance, a surrogate measure of CYP3A4/5 activity, was significantly decreased upon COC use (63.3 l h⁻¹vs. 53.9 l h⁻¹, P < 0.05). A median decrease of 5.6 l h⁻¹ (95% CI −4.1, 13.3 l h⁻¹) was observed. However, the magnitude of change was similar to that reported in women with normal BMI.

CONCLUSIONS

Although we hypothesized that obesity might amplify the impact on CYP3A4/5 activity in COC users, we found that this was not the case. This finding is reassuring regarding potential additional drug−drug interactions in obese COC users as CYP3A4/5 is a major enzyme in the metabolism of many marketed drugs.     

A pharmacokinetic evaluation of five H1 antagonists after an oral and intravenous microdose to human subjects

AIMS

To evaluate the pharmacokinetics (PK) of five H₁ receptor antagonists in human volunteers after a single oral and intravenous (i.v.) microdose (0.1 mg).

METHODS

Five H₁ receptor antagonists, namely NBI-1, NBI-2, NBI-3, NBI-4 and diphenhydramine, were administered to human volunteers as a single 0.1-mg oral and i.v. dose. Blood samples were collected up to 48 h, and the parent compound in the plasma extract was quantified by high-performance liquid chromatography and accelerator mass spectroscopy.

RESULTS

The median clearance (CL), apparent volume of distribution (V_d) and apparent terminal elimination half-life (t_1/2) of diphenhydramine after an i.v. microdose were 24.7 l h⁻¹, 302 l and 9.3 h, and the oral C_max and AUC_0–∞ were 0.195 ng ml⁻¹ and 1.52 ng h ml⁻¹, respectively. These data were consistent with previously published diphenhydramine data at 500 times the microdose. The rank order of oral bioavailability of the five compounds was as follows: NBI-2 > NBI-1 > NBI-3 > diphenhydramine > NBI-4, whereas the rank order for CL was NBI-4 > diphenhydramine > NBI-1 > NBI-3 > NBI-2.

CONCLUSIONS

Human microdosing provided estimates of clinical PK of four structurally related compounds, which were deemed useful for compound selection.     

CFTR chloride channel as a molecular target of anthraquinone compounds in herbal laxatives

Aim:

To clarify whether CFTR is a molecular target of intestinal fluid secretion caused by the anthraquinone compounds from laxative herbal plants.

Methods:

A cell-based fluorescent assay to measure I⁻ influx through CFTR chloride channel. A short-circuit current assay to measure transcellular Cl⁻ current across single layer FRT cells and freshly isolated colon mucosa. A closed loop experiment to measure colon fluid secretion in vivo.

Results:

Anthraquinone compounds rhein, aloe-emodin and 1,8-dihydroxyanthraquinone (DHAN) stimulated I⁻ influx through CFTR chloride channel in a dose-dependent manner in the presence of physiological concentration of cAMP. In the short-circuit current assay, the three compound enhanced Cl⁻ currents in epithelia formed by CFTR-expressing FRT cells with EC₅₀ values of 73±1.4, 56±1.7, and 50±0.5μmol/L, respectively, and Rhein also enhanced Cl⁻ current in freshly isolated rat colonic mucosa with a similar potency. These effects were completely reversed by the CFTR selective blocker CFTR_inh-172. In in vivo closed loop experiments, rhein 2 mmol/L stimulated colonic fluid accumulation that was largely blocked by CFTR_inh-172. The anthraquinone compounds did not elevate cAMP level in cultured FRT cells and rat colonic mucosa, suggesting a direct effect on CFTR activity.

Conclusion:

Natural anthraquinone compounds in vegetable laxative drugs are CFTR potentiators that stimulated colonic chloride and fluid secretion. Thus CFTR chloride channel is a molecular target of vegetable laxative drugs.     

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.

     

High dose methotrexate chemotherapy: pharmacokinetics, folate and toxicity in osteosarcoma patients

AIMS

To investigate the relationships between pretreatment folate concentrations, MTX pharmacokinetics and acute toxicities following high dose methotrexate (HD MTX) therapy.

METHODS

MTX and its major extracellular metabolite 7-OH-MTX were measured in eight serum samples per HD MTX cycle in 65 consecutive osteosarcoma patients (288 cycles) and AUC (area under the blood concentration–time curve) was calculated. Pretreatment concentrations of folate in serum (S) and erythrocytes (ER) were determined. Hepatic, renal and haematological toxicities, assessed by routine laboratory parameters, as well as mucositis were graded according to National Cancer Institute Common Terminology Criteria for adverse events (CTCAE v.3.0). Dermatitis and pleuritis were reported as occurred or not.

RESULTS

S- and ER-folate pretreatment concentrations increased significantly with increasing number of HD MTX cycles (P < 0.001). ER-folate pretreatment concentrations were higher among males (median 610 nmol l⁻¹, 95% CI 550, 680) compared with females (median 465 nmol l⁻¹, 95% CI 430, 520, P < 0.001), but showed no correlation with MTX or 7-OH-MTX pharmacokinetics. We found correlations between alanine aminotransferase peak concentration (ALAT_max) and clearance of MTX (P < 0.001), gender (P < 0.001), age (P < 0.001) and 7-OH-MTX concentrations (P < 0.001), the latter being the main factor influencing ALAT_max.

CONCLUSION

Our results suggest that 7-OH-MTX is involved in the development of HD MTX hepatic toxicity and that young female patients are most affected.     

In vitro and in vivo percutaneous absorption of seleno-L-methionine, an antioxidant agent, and other selenium species

Aim:

To investigate the in vitro and in vivo percutaneous absorption of seleno-L-methionine (Se-L-M), an ultraviolet (UV)-protecting agent, from aqueous solutions.

Methods:

Aqueous solutions of Se-L-M were prepared in pH 4, 8, and 10.8 buffers. The pH 8 buffer contained 30% glycerol, propylene glycol (PG) and polyethylene glycol (PEG) 400. The in vitro skin permeation of Se-L-M via porcine skin and nude mouse skin was measured and compared using Franz diffusion cells. The in vivo skin tolerance study was performed, which examined transepidermal water loss (TEWL), skin pH and erythema.

Results:

In the excised porcine skin, the flux was 0.1, 11.4 and 8.2 μg·cm⁻²·h⁻¹ for the pH 4, 8, and 10.8 buffers, respectively. A linear correlation between the flux and skin deposition was determined. According to permeation across skin with different treatments (stripping, delipidation, and ethanol treatments), it was determined that the intracellular route comprised the predominant pathway for Se-L-M permeation from pH 8 buffer. Aqueous solutions of seleno-DL-methionine (Se-DL-M), selenium sulfide and selenium-containing quantum dot nanoparticles were also used as donor systems. The DL form showed a lower flux (7.0 vs 11.4 μg·cm⁻²·h⁻¹) and skin uptake (23.4 vs 47.3 μg/g) as compared to the L form, indicating stereoselective permeation of this compound. There was no or only negligible permeation of selenium sulfide and quantum dots into and across the skin. With in vivo topical application for 4 and 8 h, the skin deposition of Se-L-M was about 7 μg/g, and values were comparable to each other. The topical application of Se-L-M for up to 5 d did not caused apparent skin irritation. However, slight inflammation of the dermis was noted according to the histopathological examination.

Conclusion:

Se-L-M was readily absorbed by the skin in both the in vitro and in vivo experiments. The established profiles of Se-L-M skin absorption will be helpful in developing topical products of this compound.     

Inhibition of the active principle of the weak opioid tilidine by the triazole antifungal voriconazole

AIMS

To investigate in vivo the influence of the potent CYP2C19 and CYP3A4 inhibitor voriconazole on the pharmacokinetics and analgesic effects of tilidine.

METHODS

Sixteen healthy volunteers received voriconazole (400 mg) or placebo together with a single oral dose of tilidine (100 mg). Blood samples and urine were collected for 24 h and experimental pain was determined by using the cold pressor test. Noncompartimental analysis was performed to determine pharmacokinetic parameters of tilidine, nortilidine and voriconazole, whereas pharmacodynamic parameters were analysed by nonparametric repeated measures anova (Friedman).

RESULTS

Voriconazole caused a 20-fold increase in exposition of tilidine in serum [AUC 1250.8 h*ng ml⁻¹, 95% confidence interval (CI) 1076.8, 1424.9 vs. 61 h*ng ml⁻¹, 95% CI 42.6, 80.9; P < 0.0001], whereas the AUC of nortilidine also increased 2.5-fold. After voriconazole much lower serum concentrations of bisnortilidine were observed. The onset of analgesic activity occurred later with voriconazole, which is in agreement with the prolonged t_max of nortilidine (0.78 h, 95% CI 0.63, 0.93 vs. 2.5 h, 95% CI 1.85, 3.18; P < 0.0001) due to the additional inhibition of nortilidine metabolism to bisnortilidine. After voriconazole the AUC under the pain withdrawal–time curve was reduced compared with placebo (149 s h⁻¹, 95% CI 112, 185 vs. 175 s h⁻¹, 95% CI 138, 213; P < 0.016), mainly due to the shorter withdrawal time 0.75 h after tilidine administration.

CONCLUSIONS

Voriconazole significantly inhibited the sequential metabolism of tilidine with increased exposure of the active nortilidine. Furthermore, the incidence of adverse events was almost doubled after voriconazole and tilidine.     

Influence of ABCB1 polymorphisms and haplotypes on tacrolimus nephrotoxicity and dosage requirements in children with liver transplant

AIMS

The aim of this study was to investigate the influence of genetic polymorphisms in ABCB1 on the incidence of nephrotoxicity and tacrolimus dosage-requirements in paediatric patients following liver transplantation.

METHODS

Fifty-one paediatric liver transplant recipients receiving tacrolimus were genotyped for ABCB1 C1236>T, G2677>T and C3435>T polymorphisms. Dose-adjusted tacrolimus trough concentrations and estimated glomerular filtration rates (EGFR) indicative of renal toxicity were determined and correlated with the corresponding genotypes.

RESULTS

The present study revealed a higher incidence of the ABCB1 variant-alleles examined among patients with renal dysfunction (≥30% reduction in EGFR) at 6 months post-transplantation (1236T allele: 63.3% vs 37.5% in controls, P= 0.019; 2677T allele: 63.3% vs. 35.9%, p = 0.012; 3435T allele: 60% vs. 39.1%, P= 0.057). Carriers of the G2677->T variant allele also had a significant reduction (%) in EGFR at 12 months post-transplant (mean difference = 22.6%; P= 0.031). Haplotype analysis showed a significant association between T-T-T haplotypes and an increased incidence of nephrotoxicity at 6 months post-transplantation (haplotype-frequency = 52.9% in nephrotoxic patients vs 29.4% in controls; P= 0.029). Furthermore, G2677->T and C3435->T polymorphisms and T-T-T haplotypes were significantly correlated with higher tacrolimus dose-adjusted pre-dose concentrations at various time points examined long after drug initiation.

CONCLUSIONS

These findings suggest that ABCB1 polymorphisms in the native intestine significantly influence tacrolimus dosage-requirement in the stable phase after transplantation. In addition, ABCB1 polymorphisms in paediatric liver transplant recipients may predispose them to nephrotoxicity over the first year post-transplantation. Genotyping future transplant recipients for ABCB1 polymorphisms, therefore, could have the potential to individualize better tacrolimus immunosuppressive therapy and enhance drug safety.