Ticagrelor binds to human P2Y12 independently from ADP but antagonizes ADP-induced receptor signaling and platelet aggregation

Summary. Background: P2Y₁₂ plays an important role in regulating platelet aggregation and function. This receptor is the primary target of thienopyridine antiplatelet agents, the active metabolites of which bind irreversibly to the receptor, and of newer agents that can directly and reversibly modulate receptor activity. Objective: To characterize the receptor biology of the first reversibly binding oral P2Y₁₂ antagonist, ticagrelor (AZD6140), a member of the new cyclopentyltriazolopyrimidine (CPTP) class currently in phase III development. Methods: Ticagrelor displayed apparent non‐competitive or insurmountable antagonism of ADP‐induced aggregation in human washed platelets. This was investigated using competition binding against [³H]ADP, [³³P]2MeS‐ADP and the investigational CPTP compound [¹²⁵I]AZ11931285 at recombinant human P2Y₁₂. Functional receptor inhibition studies were performed using a GTPγS‐binding assay, and further binding studies were performed using membranes prepared from washed human platelets. Results: Radioligand‐binding studies demonstrated that ticagrelor binds potently and reversibly to human P2Y₁₂ with K_on and K_off of (1.1 ± 0.2) × 10^?4 nm ^?1 s^?1 and (8.7 ± 1.4) × 10^?4 s^?1, respectively. Ticagrelor does not displace [³H]ADP from the receptor (K_i > 10 μm ) but binds competitively with [³³P]2MeS‐ADP (K_i = 4.3 ± 1.3 nm ) and [¹²⁵I]AZ11931285 (K_i = 0.33 ± 0.04 nm ), and shows apparent non‐competitive inhibition of ADP‐induced signaling but competitive inhibition of 2MeS‐ADP‐induced signaling. Binding studies on membranes prepared from human washed platelets demonstrated similar non‐competitive binding for ADP and ticagrelor. Conclusions: These data indicate that P2Y₁₂ is targeted by ticagrelor via a mechanism that is non‐competitive with ADP, suggesting the existence of an independent receptor‐binding site for CPTPs.     

Chemical molecular‐based approach to overcome multidrug resistance in cancer by targeting P‐glycoprotein (P‐gp)

Multidrug resistance (MDR) remains one of the major impediments for efficacious cancer chemotherapy. Increased efflux of multiple chemotherapeutic drugs by transmembrane ATP‐binding cassette (ABC) transporter superfamily is considered one of the primary causes for cancer MDR, in which the role of P‐glycoprotein (P‐gp/ABCB1) has been most well‐established. The clinical co‐administration of P‐gp drug efflux inhibitors, in combination with anticancer drugs which are P‐gp transport substrates, was considered to be a treatment modality to surmount MDR in anticancer therapy by blocking P‐gp‐mediated multidrug efflux. Extensive attempts have been carried out to screen for sets of nontoxic, selective, and efficacious P‐gp efflux inhibitors. In this review, we highlight the recent achievements in drug design, characterization, structure–activity relationship (SAR) studies, and mechanisms of action of the newly synthetic, potent small molecules P‐gp inhibitors in the past 5 years. The development of P‐gp inhibitors will increase our knowledge of the mechanisms and functions of P‐gp‐mediated drug efflux which will benefit drug discovery and clinical cancer therapeutics where P‐gp transporter overexpression has been implicated in MDR.     

Lack of significant food effect on the pharmacokinetics of ticagrelor in healthy volunteers

What is known and Objective: Ticagrelor is the first reversibly binding oral P2Y₁₂ receptor antagonist and has been approved in the European Union and the USA for the reduction of clinical thrombotic events in patients with acute coronary syndromes. This study aimed to assess the effect of food on ticagrelor pharmacokinetics. Methods: The study was an open‐label, randomized, 2‐period crossover single‐centre trial; 26 healthy volunteers received a single 270 mg (3 × 90 mg tablets) ticagrelor dose orally following: (i) a 10‐h overnight fast; and (ii) after a standard high‐fat, high‐calorie breakfast. Ticagrelor and AR‐C124910XX (a major pharmacologically active metabolite) plasma concentrations were quantified for pharmacokinetic analysis. Results: Ticagrelor median time to maximum concentration (t_max; 2·5 h vs. 1·5 h) was slightly delayed in the fed vs. fasting state. Maximum concentration of ticagrelor (C_max) was comparable between the two states with 95% confidence intervals (CI) of the geometric least‐squares (GLS) mean ratio (0·85–1·03) being within no‐effect limits (0·80–1·25). Ticagrelor exposure was slightly higher with food intake; area under the plasma concentration–time curve from zero to infinity (AUC) was 21% higher compared with fasting state (95% CI of GLS mean ratio = 1·13–1·30). For AR‐C124910XX, AUC (95% CI of GLS mean ratio = 0·93–1·07) was unaffected by food consumption. Median t_max of the metabolite was slightly longer in the fed than fasting state (3·5 h vs. 1·5 h). Mean C_max for AR‐C124910XX was slightly lower (22%) with food intake vs. fasting (95% CI of GLS mean ratio 0·69–0·88). What is new and Conclusion: Food effects on ticagrelor AUC and AR‐C124910XX C_max were small and are considered to be of minimal clinical significance. Thus, ticagrelor can be administered with or without food.     

β‐Cell subcellular localization of glucose‐stimulated Mn uptake by X‐ray fluorescence microscopy: implications for pancreatic MRI

Manganese (Mn) is a calcium (Ca) analog that has long been used as a magnetic resonance imaging (MRI) contrast agent for investigating cardiac tissue functionality, for brain mapping and for neuronal tract tracing studies. Recently, we have extended its use to investigate pancreatic β‐cells and showed that, in the presence of MnCl₂, glucose‐activated pancreatic islets yield significant signal enhancement in T₁‐weigheted MR images. In this study, we exploited for the first time the unique capabilities of X‐ray fluorescence microscopy (XFM) to both visualize and quantify the metal in pancreatic β‐cells at cellular and subcellular levels. MIN‐6 insulinoma cells grown in standard tissue culture conditions had only a trace amount of Mn, 1.14 ± 0.03 × 10^?11 µg/µm², homogenously distributed across the cell. Exposure to 2 m m glucose and 50 µ m MnCl₂ for 20 min resulted in nonglucose‐dependent Mn uptake and the overall cell concentration increased to 8.99 ± 2.69 × 10^?11 µg/µm². When cells were activated by incubation in 16 m m glucose in the presence of 50 µ m MnCl₂, a significant increase in cytoplasmic Mn was measured, reaching 2.57 ± 1.34 × 10^?10 µg/µm². A further rise in intracellular concentration was measured following KCl‐induced depolarization, with concentrations totaling 1.25 ± 0.33 × 10^?9 and 4.02 ± 0.71 × 10^?10 µg/µm² in the cytoplasm and nuclei, respectively. In both activated conditions Mn was prevalent in the cytoplasm and localized primarily in a perinuclear region, possibly corresponding to the Golgi apparatus and involving the secretory pathway. These data are consistent with our previous MRI findings, confirming that Mn can be used as a functional imaging reporter of pancreatic β‐cell activation and also provide a basis for understanding how subcellular localization of Mn will impact MRI contrast. Copyright © 2011 John Wiley & Sons, Ltd.     

Lack of clinically significant pharmacological interactions between ticagrelor and enoxaparin or unfractionated heparin in healthy subjects

What is known and Objective: Patients with acute coronary syndromes (ACS) receive several pharmacological therapies concomitantly, including antiplatelet and anticoagulant agents. As unfractionated heparin (UFH) activates platelets in vitro and in vivo, co‐administration with an antiplatelet agent may lead to decreased clinical effectiveness of the latter. The aim was therefore to determine any potential drug–drug interactions between the new oral antiplatelet agent ticagrelor, and UFH or enoxaparin. Methods: In two open‐label, three‐period, crossover trials, healthy subjects were randomized to receive ticagrelor alone or with enoxaparin (study 1) or UFH (study 2), or enoxaparin or UFH alone. Ticagrelor plasma concentrations, inhibition of platelet aggregation (IPA), anti‐factor Xa levels, activated partial thromboplastin time (aPTT) and activated coagulation time (ACT) were measured. Results: Thirty and 28 subjects completed studies 1 and 2, respectively. Study drugs were generally well tolerated, with no significant bleeding or serious adverse events. Co‐administration with enoxaparin or UFH had no significant effect on ticagrelor pharmacokinetics. The effect of ticagrelor on IPA was unimpaired by co‐administration of enoxaparin, except for a marginal (?2·9%; 908·7%.h, 881·9%.h) reduction in final extent area under the effect curve (AUEC)_2–12 (95% CI: ?51·6%.h, ?2·0%.h). Co‐administering UFH with ticagrelor caused small decreases in IPA_max (?3·8%; 94·6%, 91·0%) and AUEC_2–12 (?6·8%; 888·6%.h, 828·3%.h) vs. ticagrelor alone (95% CI: final extent IPA_max?5·7%, ?1·6%; AUEC_2–12?109·8%.h, ?10·8%.h). Ticagrelor had no clinically significant effects on enoxaparin as assessed by anti‐factor Xa (study 1), or UFH as assessed by aPTT or ACT (study 2). What is new and conclusions: Enoxaparin and UFH had no effect on the pharmacokinetics and no clinically significant effect on the pharmacodynamics of ticagrelor. Ticagrelor had no clinically significant effects on the pharmacodynamics of enoxaparin or UFH.     

Imaging integrin alpha‐v‐beta‐3 expression in tumors with an 18F‐labeled dimeric RGD peptide

Integrin α_vβ₃ receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth. Many radiolabeled probes utilize the tight and specific association between the arginine–glycine–aspartatic acid (RGD) peptide and integrin α_vβ₃, but one main obstacle for any clinical application of these probes is the laborious multistep radiosynthesis of ¹⁸F. In this study, the dimeric RGD peptide, E‐[c(RGDfK)]₂, was conjugated with NODAGA and radiolabeled with ¹⁸F in a simple one‐pot process with a radiolabeling yield of 20%, the whole process lasting only 45 min. NODAGA‐E‐[c(RGDfK)]₂ labeled with ¹⁸F at a specific activity of 1.8 MBq nmol^?1 and a radiochemical purity of 100% could be achieved. The logP value of ¹⁸F‐labeled NODAGA‐E‐[c(RGDfK)]₂ was ?4.26 ± 0.02. In biodistribution studies, ¹⁸F‐NODAGA‐E‐[c(RGDfK)]₂ cleared rapidly from the blood with 0.03 ± 0.01 percentage injected dose per gram (%ID g^?1) in the blood at 2 h p.i., mainly via the kidneys, and showed good in vivo stability. Tumor uptake of ¹⁸F‐NODAGA‐E‐[c(RGDfK)]₂ (3.44 ± 0.20 %ID g^?1, 2 h p.i.) was significantly lower than that of reference compounds ⁶⁸Ga‐labeled NODAGA‐E‐[c(RGDfK)]₂ (6.26 ± 0.76 %ID g^?1; p <0.001) and ¹¹¹In‐labeled NODAGA‐E‐[c(RGDfK)]₂ (4.99 ± 0.64 %ID g^?1; p < 0.01). Co‐injection of an excess of unlabeled NODAGA‐E‐[c(RGDfK)]₂ along with ¹⁸F‐NODAGA‐E‐[c(RGDfK)]₂ resulted in significantly reduced radioactivity concentrations in the tumor (0.85 ± 0.13 %ID g^?1). The α_vβ₃ integrin‐expressing SK‐RC‐52 tumor could be successfully visualized by microPET with ¹⁸F‐labeled NODAGA‐E‐[c(RGDfK)]₂. In conclusion, NODAGA‐E‐[c(RGDfK)]₂ could be labeled rapidly with ¹⁸F using a direct aqueous, one‐pot method and it accumulated specifically in α_vβ₃ integrin‐expressing SK‐RC‐52 tumors, allowing for visualization by microPET. Copyright © 2013 John Wiley & Sons, Ltd.     

Silencing of P‐glycoprotein increases mortality in temephos‐treated Aedes aegypti larvae

Re‐emergence of vector‐borne diseases such as dengue and yellow fever, which are both transmitted by the Aedes aegypti mosquito, has been correlated with insecticide resistance. P‐glycoproteins (P‐gps) are ATP‐dependent efflux pumps that are involved in the transport of substrates across membranes. Some of these proteins have been implicated in multidrug resistance (MDR). In this study, we identified a putative P‐glycoprotein in the Ae. aegypti database based on its significantly high identity with Anopheles gambiae, Culex quinquefasciatus, Drosophila melanogaster and human P‐gps. The basal ATPase activity of ATP‐binding cassette transporters in larvae was significantly increased in the presence of MDR modulators (verapamil and quinidine). An eightfold increase in Ae. aegypti P‐gp (AaegP‐gp) gene expression was detected in temephos‐treated larvae as determined by quantitative PCR. To analyse the potential role of AaegP‐gp in insecticide efflux, a temephos larvicide assay was performed in the presence of verapamil. The results showed an increase of 24% in temephos toxicity, which is in agreement with the efflux reversing effect. RNA interference (RNAi)‐mediated silencing of the AaegP‐gp gene caused a significant increase in temephos toxicity (57%). In conclusion, we have demonstrated for the first time in insects that insecticide‐induced P‐gp expression can be involved in the modulation of insecticide efflux.     

Hibiscus sabdariffa increases hydroxocobalamin oral bioavailability and clinical efficacy in vitamin B12 deficiency with neurological symptoms

The aim of the study was to evaluate the bioavailability and clinical benefits of oral new formulation (HB₁₂) of hydroxocobalamin (Hdrx) with Hibiscus sabdariffa (HS). First, in an observational study, a cohort of 30 vitamin B₁₂‐deficient patients (vit B₁₂ < 200 pg/mL) with neurological symptoms received oral fixed dose of Hdrx containing 15 mg Hdrx daily for 10 days followed by 15 mg monthly. Clinical benefits were evaluated on haematological and biochemical parameters, and neurological improvement at days 10 and 90 compared to day 0. To understand the mechanism, intestinal mucosa from mice were mounted in vitro in Ussing chambers to measure Hdrx Fluxes. In the clinical study, serum vitamin B₁₂ level increased from 55.1 ± 36.9 to 1330 ± 335.5 pg/mL at day 10 and 431.0 ± 24.27 pg/mL at day 90, without overt adverse effects. In mice ileum, (i) intestinal bioavailability of Hdrx increased in dose‐dependent manner with HB₁₂. The apparent permeability of Hdrx was P_app = 34.9 ± 4.6 × 10^?6 cm/s in the presence of 3 mg/mL (HB₁₂B) compared to the control P_app = 6.2 ± 0.7 × 10^?6 cm/s. (ii) Total transepithelial electrical conductance (G_t) increased in dose‐dependent manner with HB₁₂, G_t = 161.5 ± 10.8 mS/cm² with HB₁₂B (Hdrx 1 mg + HS 3 mg) compared to the control Hdrx, G_t = 28.7 ± 4.0 mS/cm². In conclusion, the clinical study suggests that injections are not required when Hdrx is given orally. Intestinal bioavailability of Hdrx increased in vitro when it was used concomitantly with HS.     

Plasminogen activator inhibitor‐1 inhibits factor VIIa bound to tissue factor

Summary. Background and objective: A growing body of experimental evidence supports broad inhibitory and regulatory activity of plasminogen activator inhibitor 1 (PAI‐1). The present study was designed to investigate whether PAI‐1 inhibits factor (F) VIIa complexed with tissue factor (TF), a well‐known procoagulant risk factor. Methods and results: The ability of PAI‐1 to inhibit FVIIa‐TF activity was evaluated in both clotting and factor X (FX) activation assays. PAI‐1 and its complex with vitronectin inhibit: (i) clotting activity of FVIIa‐TF (PAI‐1_IC50, 817 and 125 nm , respectively); (ii) FVIIa‐TF‐mediated FX activation (PAI‐1_IC50, 260 and 50 nm , respectively); and (iii) FVIIa bound to TF expressed on the surface of stimulated endothelial cells (PAI‐1_IC50, 260 and 120 nm , respectively). The association rate constant (k_a) for PAI‐1 inhibition of FVIIa‐TF was determined using a chromogenic assay. K_a for PAI‐1 inhibition of FVIIa bound to relipidated TF is 3.3‐fold higher than that for FVIIa bound to soluble TF (k_a = 0.09 ± 0.01 and 0.027 ± 0.03 μm ^?1 min^?1, respectively). Vitronectin increases k_a for both soluble and relipidated TF by 3.5‐ and 30‐fold, respectively (to 0.094 ± 0.020 and 2.7 ± 0.2 μm ^?1 min^?1). However, only a 3.5‐ to 5.0‐fold increase in the acylated FVIIa was observed on SDS PAGE in the presence of vitronectin for both relipidated and soluble TF, indicating fast formation of PAI‐1/vitronectin/FVIIa/relipidated TF non‐covalent complex. Conclusions: Our results demonstrate potential anticoagulant activity of PAI‐1 in the presence of vitronectin, which could contribute to regulation of hemostasis under pathological conditions such as severe sepsis, acute lung injury and pleural injury, where PAI‐1 and TF are overexpressed.     

Acute physiological and perceptual responses to high‐load resistance exercise in hypoxia

This study assessed whether hypoxia during high‐load resistance exercise could enhance the acute physiological responses related to muscular development. Twelve trained men performed exercise in three conditions: normoxia (fraction of inspired oxygen [F_IO₂] = 21%), moderate‐level hypoxia (F_IO₂ = 16%) and high‐level hypoxia (F_IO₂ = 13%). Exercise comprised high‐load squats and deadlifts (5 × 5 using 80% of 1‐repetition maximum with 180‐s rest). Muscle oxygenation and activation were monitored during exercise. Metabolic stress was estimated via capillary blood sampling. Perceived fatigue and soreness were also quantified following exercise. While the hypoxic conditions appeared to affect muscle oxygenation, significant differences between conditions were only noted for maximal deoxyhaemoglobin in the deadlift (P = 0·009). Blood lactate concentration increased from 1·1 to 1·2 mmol l^?1 at baseline to 9·5–9·8 mmol l^?1 after squats and 10·4–10·5 mmol l^?1 after deadlifts (P≤0·001), although there were no between‐condition differences. Perceived fatigue and muscle soreness were significantly elevated immediately and at 24 h following exercise, respectively, by similar magnitudes in all conditions (P≤0·001). Muscle activation did not differ between conditions. While metabolic stress is thought to moderate muscle activation and subsequent muscular development during hypoxic resistance training, it is not augmented during traditional high‐load exercise. This may be explained by the low number of repetitions performed and the long interset rest periods employed during this training. These findings suggest that high‐load resistance training might not benefit from additional hypoxia as has been shown for low‐ and moderate‐load training.     

Aspirin withdrawal in patients treated with ticagrelor presenting with non‐ST elevation myocardial infarction

Essentials

• Strong P2Y₁₂ blockade may cause platelet inhibition that is only minimally enhanced by aspirin.
• We evaluated aspirin withdrawal on platelet reactivity in ticagrelor treated patients.
• Aspirin withdrawal resulted in increased platelet reactivity to arachidonic acid.
• Aspirin withdrawal caused little difference in adenosine diphosphate‐induced platelet aggregation.

Summary

Background

Recent studies have shown that the thromboxane A₂‐dependent pathway is dependent on the ADP–P2Y₁₂ pathway, and that strong P2Y₁₂ receptor blockade alone causes inhibition of platelet aggregation that is minimally enhanced by aspirin. Data from the PLATO trial suggested that, among ticagrelor‐treated patients, high‐dose versus low‐dose (< 100 mg day^?1) aspirin is associated with an increased risk fof ischemic events.

Objectives

To evaluate the impact of aspirin withdrawal on platelet reactivity in acute coronary syndrome (ACS) patients treated with a potent P2Y₁₂ blocker.

Patients/Methods

This was a current prospective, randomized, placebo‐controlled, double‐blind, cross‐over study. The study population comprised 22 consecutive ACS patients who underwent percutaneous coronary intervention and were treated with aspirin (100 mg day^?1) and ticagrelor. Thirty days post‐ACS, open‐label aspirin was stopped, and patients were randomized to either blinded aspirin or placebo for 2 weeks, with each patient crossing over to the other arm for an additional 2 weeks. Platelet reactivity to arachidonic acid and ADP determined with light‐transmission aggregometry (LTA) and VerifyNow was evaluated at baseline, and 2 weeks and 4 weeks later.

Results

Aspirin withdrawal resulted in an increase in arachidonic‐acid induced platelet reactivity as determined with both LTA (77.0% ± 11.3% versus 20.8% ± 4.4%) and VerifyNow (607.7 ± 10.6 aspirin reaction units [ARU] versus 408.5 ± 14.4 ARU). Platelet response to ADP, as determined with both LTA and VerifyNow, did not differ with either aspirin or placebo (32.9% ± 2.6% versus 35.8% ± 3.6%, and 33.5 ± 6.4 P2Y₁₂ reaction units (PRU) versus 29.6 ± 5.7 PRU, respectively).

Conclusions

Aspirin withdrawal early post‐ACS results in increased platelet reactivity in response to arachidonic acid, despite concomitant treatment with the potent P2Y₁₂ blocker ticagrelor.

     

In vitro effects of the cyclooxygenase inhibitor indomethacin and of the phospholipase‐C inhibitor U‐73122 on carbachol‐induced contractions of porcine detrusor muscle

Prostaglandin synthetase inhibitors belong to one substance class additionally used in the treatment of bladder dysfunctions associated with involuntary bladder contractions. However, the mechanism of action of non‐steroidal anti‐inflammatory drugs (NSAIDs) on the detrusor muscle is not clear. In this study, it was examined in vitro whether the NSAID indomethacin exhibited an inhibitory effect on carbachol‐induced contractions of the porcine detrusor muscle. Additionally, the inhibitory effect of the phospholipase‐C inhibitor U‐73122 on carbachol‐induced contractions of the porcine detrusor muscle was investigated. Experiments were performed on the muscle strips of the porcine detrusor muscle suspended in a tissue bath. Effects of indomethacin at 10^?6 and 10^?5 m on the maximum carbachol‐induced contraction and on the carbachol–response curve were investigated. Additionally, the inhibitory influence of U‐73122 at a concentration of 10^?5.5 m on the carbachol–response curve was investigated. Pretreatment with indomethacin at both concentrations did not result in a significant reduction in the maximum contraction compared with the control. In the experiments in which carbachol concentration‐response curves were generated, indomethacin exhibited at both concentrations a very small but significant change at carbachol concentrations of 10^?8 and 10^?7.5 m . In the experiments with U‐73122, a significant change was found in the concentration–response curve of carbachol at all concentrations of carbachol from 10^?6.5 to 10^?4 m . The mean maximum carbachol‐induced contraction was 141.8 ± 6.8% after incubation with U‐73122 and 166.0 ± 6.4% in the control group (P < 0.05). Indomethacin did not inhibit the carbachol‐induced contractions of the porcine detrusor muscle. The cyclooxygenase does not play a significant role in the carbachol‐induced bladder contraction of the porcine detrusor muscle. The inhibitory action of the phospholipase‐C inhibitor U‐73122 on the carbachol‐induced contraction was significant, but small. The results point to an inferior role of this pathway.     

Radiolabelled GLP‐1 analogues for in vivo targeting of insulinomas

Internalizing agonists are usually selected for peptide receptor targeting. There is increasing evidence that non‐internalizing receptor antagonists can be used for this purpose. We investigated whether the glucagon‐like peptide‐1 receptor (GLP‐1R) antagonist exendin(9–39) can be used for in vivo targeting of GLP‐1R expressing tumours and compared the in vitro and in vivo characteristics with the GLP‐1R agonists exendin‐3 and exendin‐4. The binding and internalization kinetics of labelled [Lys⁴⁰(DTPA)]exendin‐3, [Lys⁴⁰(DTPA)]exendin‐4 and [Lys⁴⁰(DTPA)]exendin(9–39) were determined in vitro using INS‐1 cells. The in vivo targeting properties of [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3, [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 and [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) were examined in BALB/c nude mice with subcutaneous INS‐1 tumours. ^natIn‐labelled [Lys⁴⁰(DTPA)]exendin‐3, [Lys⁴⁰(DTPA)]exendin‐4 and [Lys⁴⁰(DTPA)]exendin(9–39) exhibited similar IC₅₀ values (13.5, 14.4 and 13.4 n m , respectively) and bound to 26 × 10³, 41 × 10³ and 37 × 10³ receptors per cell, respectively. [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3 and [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 showed rapid in vitro binding and internalization kinetics, whereas [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) showed lower binding and minimal internalization in vitro. In mice, high specific uptake of [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3 [25.0 ± 6.0% injected dose (ID) g^?1] in the tumour was observed at 0.5 h post‐injection (p.i.) with similar uptake up to 4 h p.i. [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 showed higher tumour uptake at 1 and 4 h p.i. (40.8 ± 7.0 and 41.9 ± 7.2% ID g^?1, respectively). Remarkably, [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) showed only low specific uptake in the tumour at 0.5 h p.i. (3.2 ± 0.7% ID g^?1), rapidly decreasing over time. In conclusion, the GLP‐1R agonists [Lys⁴⁰(DTPA)]exendin‐3 and [Lys⁴⁰(DTPA)]exendin‐4 labelled with ¹¹¹In could be useful for in vivo GLP‐1R targeting, whereas [Lys⁴⁰(DTPA)]exendin(9–39) is not suited for in vivo targeting of the GLP‐1R. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous quantification of myocardial perfusion,oxidative metabolism,cardiac efficiency and pump function at rest and during supine bicycle exercise using 1‐11C‐acetate PET – a pilot study

Background: PET using 1‐¹¹C‐acetate (ACE‐PET) applied at rest is used for measuring absolute myocardial blood flow (MBF) and oxidative metabolic rate (k_mono). We evaluated the feasibility of quantitative ACE‐PET during exercise. Methods: Five endurance athletes underwent dynamic PET scanning at rest and during supine bicycle stress. Exercise was maintained at a workload of 120 Watt for 17 min. The rate‐pressure product (RPP) was recorded repeatedly. MBF, k_mono in left (LV) and right (RV) ventricular wall, cardiac output (CO), cardiac efficiency and a lung uptake value reflecting left heart diastolic pressures were calculated from the PET data using previously validated models. Results: MBF increased from 0·71 ± 0·17 to 2·48 ± 0·25 ml min^?1 per ml, LV‐k_mono from 0·050 ± 0·005 to 0·146 ± 0·021 min^?1, RV‐k_mono from 0·023 + 0·006 to 0·087 + 0·014 min^‐1, RPP from 4·7 ± 0·8 to 13·2 ± 1·4 mmHg × min^?1 × 10³ and Cardiac Output from 5·2 ± 1·1 to 12·3 ± 1·2 l min ^?1 (all P < 0·001). Cardiac efficiency was unchanged (P = 0·99). Lung uptake decreased from 1·1 ± 0·2 to 0·6 ± 0·1 ml g^?1 (P < 0·001). Discussion: A number of important parameters related to cardiac function can be quantified non‐invasively and simultaneously with a short scanning protocol during steady state supine bicycling. This might open up new opportunities for studies of the integrated cardiac physiology in health and early asymptomatic disease.     

Apixaban, an oral, direct and highly selective factor Xa inhibitor: in vitro, antithrombotic and antihemostatic studies

Summary. Background: Apixaban is an oral, direct and highly selective factor Xa (FXa) inhibitor in late‐stage clinical development for the prevention and treatment of thromboembolic diseases. Objective: We evaluated the in vitro properties of apixaban and its in vivo activities in rabbit models of thrombosis and hemostasis. Methods: Studies were conducted in arteriovenous‐shunt thrombosis (AVST), venous thrombosis (VT), electrically mediated carotid arterial thrombosis (ECAT) and cuticle bleeding time (BT) models. Results: In vitro, apixaban is potent and selective, with a K_i of 0.08 nm for human FXa. It exhibited species difference in FXa inhibition [FXa K_i (nm ): 0.16, rabbit; 1.3, rat; 1.7, dog] and anticoagulation [EC_2× (μm , concentration required to double the prothrombin time): 3.6, human; 2.3, rabbit; 7.9, rat; 6.7, dog]. Apixaban at 10 μm did not alter human and rabbit platelet aggregation to ADP, γ‐thrombin, and collagen. In vivo, the values for antithrombotic ED₅₀ (dose that reduced thrombus weight or increased blood flow by 50% of the control) in AVST, VT and ECAT and the values for BT ED_3× (dose that increased BT by 3‐fold) were 0.27 ± 0.03, 0.11 ± 0.03, 0.07 ± 0.02 and > 3 mg kg^?1 h^?1 i.v. for apixaban, 0.05 ± 0.01, 0.05 ± 0.01, 0.27 ± 0.08 and > 3 mg kg^?1 h^?1 i.v. for the indirect FXa inhibitor fondaparinux, and 0.53 ± 0.04, 0.27 ± 0.01, 0.08 ± 0.01 and 0.70 ± 0.07 mg kg^?1 day^?1 p.o. for the oral anticoagulant warfarin, respectively. Conclusions: In summary, apixaban was effective in the prevention of experimental thrombosis at doses that preserve hemostasis in rabbits.     

Evaluation of the Pharmacokinetic Interaction Between Ticagrelor and Venlafaxine,a Cytochrome P-450 2D6 Substrate,in Healthy Subjects

Purpose

Ticagrelor is a reversibly binding P2Y₁₂ receptor antagonist used clinically for the prevention of atherothrombotic events in patients with acute coronary syndromes (ACS). Ticagrelor has been shown in vitro to be a weak inhibitor of cytochrome P-450 (CYP) 2D6, a clinically important enzyme for the metabolism of many drugs. This study assessed the effects of coadministration of ticagrelor on the pharmacokinetics of the CYP2D6 substrate venlafaxine. The impact of venlafaxine on ticagrelor pharmacokinetic parameters was also investigated.

Methods

Healthy subjects (N = 22) received a single 180-mg oral dose of ticagrelor on days 1 and 9 and oral doses of venlafaxine on day 4 (37.5 mg BID) and days 5 through 10 (75 mg BID). Plasma concentrations of ticagrelor, venlafaxine, and their metabolites (AR-C124910XX and O-desmethylvenlafaxine [ODV], respectively) were quantified for pharmacokinetic analyses. Safety and tolerability were assessed throughout the study.

Findings

Overall, 19 of 25 subjects were male; 14 were white, 10 were black, and 1 was Asian. Mean (SD) age was 26 (6) years, and mean (SD) body mass index was 24.3 (2.9) kg/m². Ticagrelor had no effect on overall exposure to venlafaxine, as assessed by the AUC_0–τ (geometric least squares mean ratio, 110.32 ng · h/mL [90% CI, 106.27–114.52]). Venlafaxine C_max was increased by 22% in the presence of ticagrelor (121.83 ng/mL [90% CI, 111.80–132.75]). ODV AUC_0–τ and C_max were unaffected by coadministration with ticagrelor (98.71 ng · h/mL [90% CI, 96.61–100.85] and 101.44 ng/mL [90% CI, 98.34–104.65], respectively). Venlafaxine had no effect on the C_max or AUC_0–∞ of ticagrelor (96.54 ng/mL [90% CI, 85.03–109.61] and 89.67 ng · h/mL [90% CI, 82.78–97.14]) or AR-C124910XX (106.39 ng/mL [90% CI, 96.10–117.78] and 106.32 ng · h/mL [90% CI, 97.28–116.21], respectively). Ticagrelor and venlafaxine were well tolerated whether given alone or in combination.

Implications

Ticagrelor had no clinically relevant effect on the plasma levels of venlafaxine and its CYP2D6-generated active metabolite, ODV. On the basis of these data, ticagrelor is not expected to affect CYP2D6-mediated drug metabolism to a clinically relevant extent. Venlafaxine had no effect on the pharmacokinetics of ticagrelor.     

Functional characterization of sex pheromone receptors in the purple stem borer,Sesamia inferens (Walker)

The sex pheromone communication system in moths is highly species‐specific and extremely sensitive, and pheromone receptors (PRs) are thought to be the most important factors in males. In the present study, three full‐length cDNAs encoding PRs were characterized from Sesamia inferens antennae. These three PRs were all male‐specific in expression, but their relative expression levels were very different; SinfOR29 was 17‐ to 23‐fold higher than the other two PRs. Phylogenetic and motif pattern analyses showed that these three PRs were allocated to different PR subfamilies with different motif patterns. Functional analysis using the heterologous expression system of Xenopus oocytes demonstrated that SinfOR29 specifically and sensitively responded to the major pheromone component, Z11‐16:OAc [concentration for 50% of maximal effect (EC₅₀) = 3.431 × 10^?7 M], while SinfOR21 responded robustly to a minor pheromone component Z11‐16:OH (EC₅₀ = 1.087 × 10^?6 M). SinfOR27, however, displayed no response to any of the three pheromone components, but, interestingly, it was sensitive to a non‐sex pheromone component Z9,E12‐14:OAc (EC₅₀ = 1.522 × 10^?6 M). Our results provide insight into the molecular mechanisms of specificity and sensitivity of the sex pheromone communication system in moths.     

Scaffold‐free 3D cellulose acetate membrane‐based cultures form large cartilaginous constructs

Scaffold‐free three‐dimensional (3D) cultures provide clinical potential in cartilage regeneration. The purpose of this study was to characterize a scaffold‐free 3D membrane‐based culture system, in which human articular chondrocytes were cultivated on a cellulose acetate membrane filter, and compare it to pellet and monolayer cultures. Chondrocytes were expanded in monolayer culture for up to 5 passages, transferred to membrane‐based or pellet cultures and harvested after 7 or 21 days. The chondrogenic potential was assessed by histology (toluidine blue, safranin‐O), immunohistochemistry for collagen type II and quantitative analysis of collagen type II α₁ (COL2A1). Membrane‐based cultures (P1) formed flexible disc‐like constructs (diameter 4000 µm, thickness 150 µm) with a large smooth surface after 7 days. Positive safranin‐O and collagen type II staining was found in membrane‐based and pellet cultures at P1–3. Expression of COL2A1 after 7 days was increased in both culture systems compared to monolayer culture up to P3, whereas cells from monolayer > P3 did not redifferentiate. The best results for COL2A1 expression were obtained from membrane‐based cultures at P1. After 21 days the membrane‐based cultures did not express COL2A1. We concluded that membrane‐based and pellet cultures showed the ability to promote redifferentiation of chondrocytes expanded in monolayer culture. The number of cell passages had an impact on the chondrogenic potential of cells. Membrane‐based cultures provided the highest COL2A1 expression and a large, smooth and cartilage‐like surface. As these are appropriate features for clinical applications, we assume that membrane‐based cultures might be of use in cartilage regeneration if they displayed similar results in vivo. Copyright © 2010 John Wiley & Sons, Ltd.     

Hyperosmolar glucose induces vasoconstriction through Rho/Rho‐kinase pathway in the rat aorta

Rho/Rho‐kinase signalling pathway plays a substantial role in vascular contractions. In this study, we investigated any roles of Rho/Rho‐kinase pathway in the vasoconstriction of the rat conductance and capacitance vessels by hyperosmolar glucose solution. Isolated aortic, mesenteric and renal rings were suspended and exposed to hyperosmolar glucose, sucrose and NaCl in the organ chambers filled with Krebs solution gassed with 95% O₂ and 5% CO₂ and maintained at 37 °C. The effect of a Rho‐kinase inhibitor, (+)‐(R)‐trans‐4‐(1‐aminoethyl)‐N‐(4‐pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y‐27632, 10^?5 m ), was tested on the contraction induced by hypertonic solutions. Endothelial integrity was also assessed after hyperosmolar glucose exposure. Moreover, the activity and expression of Rho‐kinase (ROCK‐2) as well as RhoA translocation were detected by Western blotting and enzyme‐linked immunosorbent assay‐based RhoA activity detection method detection kit. The vessels produced substantial contractions in response to hyperosmolar solutions. Y‐27632 significantly reduced hyperosmolarity‐induced vasoconstrictions (P < 0.05). Phosphorylation of myosin‐phosphatase target 1 increased after hyperosmolar glucose exposure, and this phosphorylation was significantly decreased by Y‐27632 (P < 0.05) in the aorta. Furthermore, RhoA translocation but not ROCK‐2 expression markedly increased by hyperosmolar glucose solution. These results may indicate that hyperosmolarity could induce vasoconstriction through Rho/Rho‐kinase signalling.     

MR imaging assessment of the kinetics of P846, a new gadolinium‐based MR contrast medium,in ischemically injured myocardium

The objectives of the study were: (1) to compare the kinetics of a new gadolinium‐based low‐diffusibility magnetic resonance (MR) contrast medium, P846 and Gd‐DOTA in left ventricular (LV) blood and in normal and ischemically injured myocardium using inversion recovery echo‐planar imaging (IR‐EPI) and (2) to compare the enhancement pattern after injection of P846 with Gd‐DOTA, using T₁‐weighted spin‐echo imaging (T₁‐SE). Sixteen rats were subjected to left descending artery (LAD) occlusion for 30 min, followed by 2.5 h reperfusion. MR imaging was performed before and after administration of the contrast medium in two different groups of animals: one group (n = 8) received 0.05 mmol kg^?1 P846 and the other (n = 8) 0.1 mmol kg^?1 Gd‐DOTA. A blipped IR‐EPI and a multislice T₁‐SE were performed before injection and for 90 min after injection. T₁‐values were derived by fitting regional signal intensity on the IR‐EPI images, the R₁, ΔR₁ (R_{1postcontrast} – R_1precontrast) and ΔR₁ ratios were calculated in LV blood, normal and injured myocardium. On SE‐T₁, the signal intensity ratio (SI) and extent of injury were measured. True infarct size was measured using histochemical staining. Changes in ΔR₁ were 4.8 times greater with 0.05 mmol kg^?1 P846 than with 0.1 mmol kg^?1 Gd‐DOTA in LV blood (6.3 ± 0.9 vs 0.9 ± 0.1 s^?1, p < 0.0001), normal (1.7 ± 0.2 vs 0.34 ± 0.03 s^?1, p < 0.0001) and ischemically injured myocardium (5.4 ± 0.4 vs 1.6 ± 0.1 s^?1, p < 0.0001). MR imaging experiments showed that the signal enhancement with P846 is longer (90 min), which might be explained by a lower diffusion of P846 compared with Gd‐DOTA (30 min). P846 differentiates viable and nonviable myocardium. Despite lower gadolinium dose, P846 permits differentiation of viable and nonviable myocardium owing to a high contrast and a long imaging window with conventional t₁‐weighted SE sequence. Copyright © 2008 John Wiley & Sons, Ltd.