Similar Maximal Effects Research Articles

Investigation of the Antinociceptive Activity of the Hydroethanolic Extract of Junglas nigra Leaf by the Tail-Immersion and Formalin Pain Tests in Rats

BackgroundJuglans (J.) nigra leaf is obtained from a plant that is used in traditional medicine in some countries to alleviate inflammatory diseases.AimThe aim of this study was to compare the effects of J. nigra extract on acute nociceptive and inflammatory pain in rats.MethodsAntinociceptive activity was examined in Wistar rats by the tail-immersion and formalin tests. Motor function was assessed using the rotarod test. Plant extract was administered intraperitoneally.ResultsIn the tail-immersion test, the maximal antinociceptive effect of the plant extract (100–330 mg/kg) was about 24–30% and is the result of the effect of a high concentration of ethanol. In the formalin test, the plant extract (41.3–330 mg/kg) significantly and dose-dependently inhibited nociception in both phases of the test with similar maximal effects of about 76% and 85%. Only the plant extract at the dose of 330 mg/kg caused a significant time-dependent reduction in time spent on the rotarod.ConclusionsIn rats, the preventive systemic administration of the hydroethanolic extract of J. nigra leaf reduced chemically but not thermally induced pain. Higher efficacy was obtained in pain associated with inflammation and tissue injury. The antinociceptive effect is dose-dependent and may be limited by motor impairment.

Pharmacology of Kappa Opioid Receptors: Novel Assays and Ligands.

The present study investigated the in vitro pharmacology of the human kappa opioid receptor using multiple assays, including calcium mobilization in cells expressing chimeric G proteins, the dynamic mass redistribution (DMR) label-free assay, and a bioluminescence resonance energy transfer (BRET) assay that allows measurement of receptor interaction with G protein and β-arrestin 2. In all assays, dynorphin A, U-69,593, and [D-Pro10]dyn(1-11)-NH2 behaved as full agonists with the following rank order of potency [D-Pro10]dyn(1-11)-NH2 > dynorphin A ≥ U-69,593. [Dmt1,Tic2]dyn(1-11)-NH2 behaved as a moderate potency pure antagonist in the kappa-β-arrestin 2 interaction assay and as low efficacy partial agonist in the other assays. Norbinaltorphimine acted as a highly potent and pure antagonist in all assays except kappa-G protein interaction, where it displayed efficacy as an inverse agonist. The pharmacological actions of novel kappa ligands, namely the dynorphin A tetrameric derivative PWT2-Dyn A and the palmitoylated derivative Dyn A-palmitic, were also investigated. PWT2-Dyn A and Dyn A-palmitic mimicked dynorphin A effects in all assays showing similar maximal effects but 3–10 fold lower potency. In conclusion, in the present study, multiple in vitro assays for the kappa receptor have been set up and pharmacologically validated. In addition, PWT2-Dyn A and Dyn A-palmitic were characterized as potent full agonists; these compounds are worthy of further investigation in vivo for those conditions in which the activation of the kappa opioid receptor elicits beneficial effects e.g. pain and pruritus.

Dual μ-opioid receptor and norepinephrine reuptake mechanisms contribute to dezocine- and tapentadol-induced mechanical antiallodynia in cancer pain

Dezocine is an opioid analgesic widely used in China, occupying over 45% of the domestic market of opioid analgesics. We have recently demonstrated that dezocine produced mechanical antiallodynia and thermal antihyperalgesia through spinal μ-opioid receptor activation and norepinephrine reuptake inhibition in neuropathic pain. This study further explored the dual μ-opioid receptor and norepinephrine reuptake mechanisms underlying dezocine-induced mechanical antiallodynia in bone cancer pain, compared with tapentadol, the first recognized analgesic in this class. Dezocine and tapentadol, given subcutaneously, exerted profound mechanical antiallodynia in bone cancer pain rats in a dose-dependent manner, yielding similar maximal effects but different potencies: ED50s of 0.6 mg/kg for dezocine and 7.5 mg/kg for tapentadol, respectively. Furthermore, their mechanical antiallodynia was partially blocked by intrathecal injection of the specific μ-opioid receptor antagonist CTAP, but not κ-opioid receptor antagonists GNTI and nor-BNI or δ-opioid receptor antagonist naltrindole. Intrathecal administrations of the specific norepinephrine depletor 6-OHDA (but not the serotonin depletor PCPA) for three consecutive days and single injection of the α-adrenoceptor antagonist phentolamine/α2-adrenoceptor antagonist yohimbine partially blocked dezocine- and tapentadol-induced mechanical antiallodynia. Strikingly, the combination of CTAP and yohimbine nearly completely blocked dezocine- and tapentadol-induced mechanical antiallodynia. Our results illustrate that both dezocine and tapentadol exert mechanical antiallodynia in bone cancer pain through dual mechanisms of μ-opioid receptor activation and norepinephrine reuptake inhibition, and suggest that the μ-opioid receptor and norepinephrine reuptake dual-targeting opioids are effective analgesics in cancer pain.

Preclinical Assessment of the Analgesic Pharmacology of NKTR-181 in Rodents.

Pharmacological evaluation of the mu-opioid receptor (MOR) agonist properties of NKTR-181 in rodent models. Graded noxious stimulus intensities were used in rats to establish the antinociceptive potency and efficacy of NKTR-181 relative to morphine, fentanyl, and oxycodone. Characteristics of MOR agonist actions, as measured by antinociceptive tolerance and cross-tolerance, as well as opioid-induced hyperalgesia (OIH) and naloxone-precipitated withdrawal in NKTR-181- and morphine-dependent in mice, were compared. NKTR-181 showed dose- and time-related antinociception with similar maximal effects to morphine in the rat and mouse hot-water tail-flick test. No sex or species differences were observed in NKTR-181 or morphine antinociception. Rats treated with NKTR-181 and morphine exhibited decreases in both potency and maximal efficacy as nociceptive stimulus intensity was increased from a water temperature of 50°C to 54°C. Evaluation of antinociception at a high stimulus intensity revealed that oxycodone and fentanyl exhibited greater efficacy than either NKTR-181 or morphine. The relative potency difference between NKTR-181 and morphine across all tail-flick studies was determined to be 7.6-fold (90% confidence interval, 2.6, 21.5). The peak antinociceptive effect of NKTR-181 was delayed compared to that of the other opioids and cumulative drug effects were not observed. Repeated treatment with escalating, approximately equi-analgesic doses of NKTR-181 or morphine, produced antinociceptive tolerance and cross-tolerance. Under these pharmacological conditions, OIH and naloxone-precipitated physical dependence were similar for NKTR-181 and morphine. NKTR-181 had a slower onset, but similar efficacy, to morphine in the models studied supporting reduced abuse potential while maintaining analgesic effect in comparison with current opioids.

Quercetin inhibits glucose transport by binding to an exofacial site on GLUT1

Quercetin, a common dietary flavone, is a competitive inhibitor of glucose uptake and is also thought to be transported into cells by GLUT1. In this study, we confirm that quercetin is a competitive inhibitor of GLUT1 and also demonstrate that newly synthesized compounds, WZB-117 and BAY-876 are robust inhibitors of GLUT1 in L929 cells. To measure quercetin interaction with L929 cells, we develop a new fluorescent assay using flow cytometry. The binding of quercetin and its inhibitory effects on 2-deoxyglucose (2DG) uptake showed nearly identical dose dependent effects, with both having maximum effects between 50 and 100 μM and similar half maximum effects at 8.9 and 8.5 μM respectively. The interaction of quercetin was rapid with t1/2 of 54 s and the onset and loss of its inhibitory effects on 2DG uptake were equally fast. This suggests that either quercetin is simply binding to surface GLUT1 or its transport in and out of the cell reaches equilibrium very quickly. If quercetin is transported, the co-incubation of quercetin with other glucose inhibitors should block quercetin uptake. However, we observed that WZB-117, an exofacial binding inhibitor of GLUT1 reduced quercetin interaction, while cytochalasin B, an endofacial binding inhibitor, enhanced quercetin interaction, and BAY-876 had no effect on quercetin interaction. Taken together, these data are more consistent with quercetin simply binding to GLUT1, but not actually being transported into L929 cells via the glucose channel in GLUT1.

Abstract 3485: Uncertainty envelope to evaluate drug interactivity with statistical analysis

Abstract Introduction. Development of new therapies for cancer and infections frequently involves combinations of two or more drugs. Existing methods to quantify drug interactivity are suitable for analyzing drugs with parallel concentration-effect curves and similar maximum drug effects. This study established a method for the remaining situations. Methods. Equations describing zero-interactivity effects of combination therapy were developed based on the principle that a drug cannot interact with itself (i.e., additivity only). These equations were used to convert the concentration-effect (C-E) curve of the combination to C-E curve for each of the two drugs in the combination as if they were used as single agents (single agent-equivalent curves). Confidence intervals (CI) of these curves were generated using the error propagation method. Effect data of drug combinations were analyzed by the new and existing methods. Results. The calculated single agent-equivalent curves plus 95% CI defined the boundaries of an envelope for additive drug effects (Uncertainty Envelope). The Envelope shape and size were determined by differences in the shapes of single agent C-E curves. The nature of drug interactivity is indicated by the location of experimental data points of the combination; data points located within the Envelope indicate additivity, whereas points above and below the Envelope respectively indicate statistically significant synergy and antagonism. In comparison with the existing methods, the Envelope method yielded more conservative results (additivity vs. synergy or antagonism). Conclusion. Uncertainty Envelope method enables the interactivity analysis between drugs with nonparallel C-E curves and unequal maximal effect, with statistical confidence. Citation Format: Liang Zhao, Jessie L.-s. Au, M. Guill Wientjes. Uncertainty envelope to evaluate drug interactivity with statistical analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3485. doi:10.1158/1538-7445.AM2015-3485

Faster-acting insulin aspart: earlier onset of appearance and greater early pharmacokinetic and pharmacodynamic effects than insulin aspart.

AimsTo evaluate the pharmacokinetics and pharmacodynamics of faster‐acting insulin aspart and insulin aspart in a randomized, single‐centre, double‐blind study.MethodsFifty‐two patients with type 1 diabetes (mean age 40.3 years) received faster‐acting insulin aspart, insulin aspart, or another faster aspart formulation (not selected for further development), each as a single 0.2 U/kg subcutaneous dose, under glucose‐clamp conditions, in a three‐way crossover design (3–12 days washout between dosing).ResultsFaster‐acting insulin aspart had a faster onset of exposure compared with insulin aspart, shown by a 57% earlier onset of appearance [4.9 vs 11.2 min; ratio 0.43, 95% confidence interval (CI) 0.36; 0.51], a 35% earlier time to reach 50% maximum concentration (20.7 vs 31.6 min; ratio 0.65, 95% CI 0.59; 0.72) and a greater early exposure within 90 min after dosing. The greatest difference occurred during the first 15 min, when area under the serum insulin aspart curve was 4.5‐fold greater with faster‐acting insulin aspart than with insulin aspart. Both treatments had a similar time to maximum concentration, total exposure and maximum concentration. Faster‐acting insulin aspart had a significantly greater glucose‐lowering effect within 90 min after dosing [largest difference: area under the curve for the glucose infusion rate (AUCGIR), 0–30 min ratio 1.48, 95% CI 1.13; 2.02] and 17% earlier time to reach 50% maximum glucose infusion rate (38.3 vs 46.1 min; ratio 0.83, 95% CI 0.73; 0.94). The primary endpoint (AUCGIR , 0–2 h) was 10% greater for faster‐acting insulin aspart, but did not reach statistical significance (ratio 1.10, 95% CI 1.00; 1.22). Both treatments had similar total and maximum glucose‐lowering effects, indicating similar overall potency.ConclusionsFaster‐acting insulin aspart was found to have earlier onset and higher early exposure than insulin aspart, and a greater early glucose‐lowering effect, with similar potency.

A novel and facile synthesis of tetra branched derivatives of nociceptin/orphanin FQ

Branched peptides have been found to be useful in several research fields however their synthesis and purification is complicated. Here we present a novel and facile synthesis of tetra branched derivatives of nociceptin/orphanin FQ (N/OFQ). Three N/OFQ tetra branched derivatives were prepared using novel cores (PWT1, PWT2 and PWT3) containing a maleimido moiety. [Cys18]N/OFQ-NH2 was linked to the cores via thiol-Michael reaction characterized by high yield and purity of the desired final product. In the electrically stimulated mouse vas deferens PWT-N/OFQ derivatives mimicked the inhibitory action of the natural sequence showing similar maximal effects and 3 fold higher potencies. The NOP selective antagonist SB-612111 antagonized the effects of N/OFQ and PWT derivatives with similar pKB values (8.02–8.48). In vivo after supraspinal administration PWT2-N/OFQ stimulated food intake in mice mimicking the action of N/OFQ. Compared to the natural peptide PWT2-N/OFQ was 40 fold more potent and elicited larger effects. These findings suggest that the PWT chemical strategy can be successfully applied to biologically active peptides to generate, with unprecedented high purity and yield, tetra branched derivatives displaying an in vitro pharmacological profile similar to that of the natural sequence associated, in vivo, to increased potency and effectiveness.

Polimeric nanoparticles contain a new NO donor (RuBPY) that induces rat aorta relaxation (700.5)

This work aimed to prepare the formulation containing free RuBPY and loaded in PNP and to verify if it induces relaxation in isolated rat aorta. We have analyzed the maximum effect (ME) and potency (pD2). P values 蠄0.05 were considered significant. Vascular reactivity experiments were performed on denuded phenylephrine‐contracted aorta and simultaneous amperometric measurements of NO were performed in time course experiments. It was evaluated the effect of the NO scavengers oxyhemoglobin and hydroxocobalamin on the relaxation induced by the formulation. Formulation containing RuBPY induced relaxation with similar ME (103.5±3.3%) as free RuBPY (100.2±0.7%) and similar pD2 values for formulation (7.15+0.33, n=5) and free RuBPY (7.76+0.20, n=5). Time‐course for free and in formulation RuBPY induced relaxation, and NO released was detected in the solution only by free RuBPY. The NO scavengers reduced the relaxing effect for both with greater inhibitory effect for the formulation. In conclusion, RuBPY formulation is a potential vasodilator and the formulation protects the complex RuBPY avoiding the NO leaking to the solution. Supported by FAPESP and CNPq.

A Pilot Study to Investigate the Efficacy of Nicotine Oral Soluble Film, Lozenge and Gum in Relief of Acute Smoking Cue-provoked Craving for Cigarette in Low Dependence Smokers

Introduction: A new nicotine film that releases nicotine quickly may lead to faster craving relief.Aims: This study compares the efficacy of 2.5 mg nicotine film with 2 mg nicotine lozenge and 2 mg nicotine gum on relieving provoked craving in low dependence smokers.Methods: A randomised, open-label, active comparators controlled study was conducted in 120 subjects. Subjects were abstinent from smoking for 4 hours prior to being provoked with smoking cues. After post-provocation craving assessment, subjects were administered one dose of the 3 treatments: nicotine film, lozenge, or gum. Craving intensity was then assessed at 50 seconds, 3, 5, 7, 15, 20, 25 and 30 minutes after administration.Results/Findings: Three treatments reduced craving with similar maximum effects. The effect was maintained up to 30 minutes. Nicotine film significantly reduced more craving than lozenge at 50 seconds, 3 and 5 minutes. It also significantly reduced more craving than gum at 50 seconds and 3 minutes. There was no significant difference between lozenge and gum.Conclusions: Nicotine film, lozenge and gum have similar maximum craving relief. Nicotine film significantly reduced more craving than lozenge and gum at early time points. Nicotine film may be particularly useful to provide acute craving relief.

Similar Maximum Systemic but not Local Cyclooxygenase-2 Inhibition by 50 mg Lumiracoxib and 90 mg Etoricoxib: A Randomized Controlled Trial in Healthy Subjects

Once daily doses of 100-400 mg lumiracoxib have been proposed to inhibit local prostaglandin synthesis longer than systemic prostaglandin synthesis due to local accumulation in inflamed, acidic tissue. Lower, less toxic doses, however, might still achieve the clinical goal and merit further contemplation. In a randomized, double-blind, placebo-controlled, three-way cross-over study, 18 healthy men received, with an interval of 24 h, two oral doses of 50 mg lumiracoxib or for comparison, 90 mg etoricoxib, for which local tissue accumulation has not been claimed as therapeutic component. Systemic and local drug concentrations, assessed by means of subcutaneous in-vivo microdialysis, were related to COX-2 inhibiting effects, quantified as inhibition of prostaglandin ex-vivo production in whole blood as well as local tissue prostaglandin (PG) concentrations. Twenty-four hours after the first dose, only etoricoxib was detectable in plasma and inhibited PGE2 production. In contrast, after the second dose, systemic PGE2 concentrations were significantly reduced by both coxibs, indicating similar maximum systemic effects of the selected doses. The local COX-2 inhibition by etoricoxib was most pronounced for PGD2. To the contrary, no indication was given of local inhibition of PG production by lumiracoxib at the dose tested. Doses of 50 mg lumiracoxib and 90 mg etoricoxib produced similar maximum inhibition of systemic COX-2 function whereas 50 mg lumiracoxib was ineffective in producing local COX-2 inhibition. At a 50 mg dosage, lumiracoxib does not provide peripheral effects that outlast its systemic actions in therapies of rheumatic diseases such as osteoarthritis.

Development of Capabilities for Simultaneous Measurement of Pulmonary and Systemic Pressure to Support Evaluation of Pulmonary Hypertension Therapies

Pulmonary arterial hypertension (PAH) is a life threatening disease with few proven treatment options. It is important that new therapies directed at decreasing pulmonary pressure demonstrate vascular selectivity on the pulmonary bed over the systemic bed. Therefore, we establised methods to directly measure pulmonary arterial pressure (PAP) and systemic blood pressure (systemic BP) simultaneously in conscious freely moving rats using novel HD‐S21 dual pressure transmitters from DSI. Transmitters were implanted in normotensive male Sprague Dawley rats, with one catheter of transmitter placed in the pulmonary artery and the other in abdominal aorta. To create a more clinically relevant PAH model and to increase the assay window, rats were exposed to hypoxic conditions (10% O2) over two weeks which increased baseline sPAP from ~25 mmHg to ~80 mmHg, with minimal effects on systemic BP. Validation studies were performed using standard of care PAH therapies: Tadalafil and Sildenafil (PDE5 inhibitors). Both compounds had similar maximal effect on systolic PAP (sPAP) reduction (~18–22 mmHg) and modest effects on the systemic BP (10–15 mmHg) on hypoxia‐induced PAH. In conclusion, innovative capabilities to directly measure PAP and systemic BP simultaneously have been established in conscious freely moving rat, and these methods provide a powerful approach to directly compare pulmonary vs. systemic hemodynamic activities minimizing cross‐study variability with different cohort of animals and to identify PAH therapies that demonstrate pulmonary selectivity.

Human 5-HT4 receptor stimulation in atria of transgenic mice

In human atrium, serotonin (5-HT) exerts pleiotropic effects, which are thought to be mediated via 5-HT4 receptors. Here, we used transgenic mice (TG) that overexpress the human 5-HT4(a) receptor under control of the heart-specific α-myosin heavy chain promoter in the atria (and ventricles). Contractile studies were performed in isolated electrically driven left atrial preparations and spontaneously beating right atrial preparation of TG and littermate control mice (wild type (WT)). 5-HT increased force of contraction and phospholamban phosphorylation on serine 16 only in left atrial preparations from TG but not from WT. In contrast, β-adrenoceptor stimulation of left atrial preparations by isoprenaline increased force of contraction with similar pEC50 values and to a similar maximum extent in both TG and WT. The contractile effects of 5-HT in left atrial preparations from TG could be blocked by the 5-HT4 receptor-specific antagonists GR125487 or GR113808. In right atrial preparations from WT and TG, the β-adrenoceptor agonist isoprenaline exerted a positive chronotropic effect with similar pEC50 values and similar maximum effects. Only in right atrial preparations from TG but not WT, 5-HT exerted a positive chronotropic effect that could be attenuated by 5-HT4 receptor-specific antagonists. Finally, in left atrial preparations of TG, a higher incidence of arrhythmias was noted compared to WT. The present data indicate that the human 5-HT4 receptors expressed in mouse atria are functional. This is the first transgenic model to study this human receptor in the atrium ex vivo or in vivo.

Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle by a new nitric oxide donor ruthenium compound

The aim of this study was to evaluate the relaxation in vitro of cavernous smooth muscle induced by a new NO donor of the complex nitrosil-ruthenium, named trans-[Ru(NH3)4(caffeine)(NO)]C13 (Rut-Caf) and sodium nitroprusside (SNP). The tissues, immersed in isolated bath systems, were pre-contracted with phenilephrine (PE) (1 µM) and then concentration-response curves (10 (-12) - 10(-4) M) were obtained. To clarify the mechanism of action involved, it was added to the baths ODQ (10 µM, 30 µM), oxyhemoglobin (10 µM), L-cysteine (100 µM), hydroxicobalamine (100 µM), glibenclamide, iberotoxin and apamine. Tissue samples were frozen in liquid nitrogen to measure the amount of cGMP and cAMP produced. The substances provoked significant relaxation of the cavernous smooth muscle. Both Rut-Caf and SNP determined dose-dependent relaxation with similar potency (pEC50) and maximum effect (E(max)). The substances showed activity through activation of the soluble guanylyl cyclase (sGC), because the relaxations were inhibited by ODQ. Oxyhemoglobin significantly diminished the relaxation effect of the substances. L-cysteine failed to modify the relaxations caused by the agents. Hydroxicobalamine significantly diminished the relaxation effect of Rut-Caf. Glibenclamide significantly increased the efficacy of Rut-Caf (pEC50 4.09 x 7.09). There were no alterations of potency or maximum effect of the substances with the addition of the other ion channel blockers. Rut-Caf induced production of significant amounts of cGMP and cAMP during the relaxation process. In conclusion, Rut-Caf causes relaxation of smooth muscle of corpus cavernosum by means of activation of sGC with intracellular production of cGMP and cAMP; and also by release of NO in the intracellular environment. Rut-Caf releases the NO free radical and it does not act directly on the potassium ion channels.

Endothelin-1 and -2: Two amino acids matter

AimsEndothelin-1 (ET-1) and endothelin-2 (ET-2; Trp6Leu7ET-1) are expressed by different cell types, but are considered to display identical pharmacological properties on endothelin receptors. We studied agonist-dependent aspects of endothelinA (ETA)-receptor function and the importance of amino acids 6 and 7 of ET-1 and ET-2 in this respect. Main methodsWe used isolated rat mesenteric resistance arteries in wire myographs, in a setting that minimizes influences of endothelium and sensorimotor nerves, to study arterial smooth muscle ETA-receptor-mediated vasomotor responses, to ET-1, ET-2 and chimeras thereof (Trp6ET-1 and Leu7ET-1). Key findingsET-1 and ET-2 cause arterial contractions with comparable sensitivities and maximal responses. BQ123 (ETA-antagonist) reduces sensitivity to ET-1 more potently than that to ET-2 (pKB: 7.1±0.2 versus 5.6±0.4). However, 1μM BQ123 relaxes maximal contractile responses to ET-2 more markedly than those to ET-1. Leu7ET-1 is a contractile agonist with lower potency and similar maximal effect compared to ET-1 and greater sensitivity to BQ123 than ET-2. Up to 256nM Trp6ET-1 did not cause contraction and did not antagonize arterial responses to ET-1. SignificanceArterial smooth muscle ETA-receptor function displays agonist-dependent aspects. This involves roles of amino acids on position 6 and 7 of the endothelin sequence. Agonist-dependent pathologies may benefit from the design of specific, agonist-selective ET-receptor antagonists.

Tamoxifen and Its Metabolites Cause Acute Vasorelaxation of Aortic Rings by Inducing Vasodilator Prostanoid Synthesis

The vascular effects of tamoxifen (Tam) and its metabolites are poorly known. We compared the vasorelaxation induced by Tam and its metabolites (N-desmethyl-Tam, 4-hydroxy-Tam, and endoxifen) in aortic rings from rats using standardized organ bath procedures, and we investigated the mechanisms involved in this effect. Tam and its metabolite-induced vasorelaxation in a concentration-dependent manner. Although 4-hydroxy-Tam and Tam had similar potency (pD2 = 8.5 ± 0.1 vs. 8.8 ± 0.1, respectively) and maximum effect (Emax = 88.5% ± 1.3% vs. 92.6% ± 1.3%, respectively), N-desmethyl-Tam and endoxifen were more potent and showed higher Emax than Tam did (pD2 = 9.0 ± 0.1 and 8.9 ± 0.1; Emax = 101.1% ± 1.8% and 101.0% ± 1.8% for N-desmethyl-Tam and endoxifen, respectively). Although preincubation of aortic rings with the estrogen receptor antagonist ICI 182780 or with the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester hydrochloride induced no changes in the vasorelaxation induced by Tam or 4-hydroxy-Tam, both drugs significantly reduced Emax in response to N-desmethyl-Tam or to endoxifen. Inhibition of cyclooxygenase with indomethacin or the incubation with the prostaglandin D2 and E2 receptor antagonist AH6809 reduced the vasorelaxation-induced Tam and its metabolites by approximately 50%. Preincubation with Nω-nitro-L-arginine methyl ester hydrochloride combined with indomethacin abolished the vasorelaxation-induced Tam and its metabolites. These results show that Tam and its metabolites cause acute vasorelaxation by inducing vasodilator prostanoids synthesis.

Differential adenosine uptake in mixed neuronal/glial or purified glial cultures of avian retinal cells: Modulation by adenosine metabolism and the ERK cascade

Adenosine is an important modulator of neuronal survival and differentiation in the CNS. Our previous work showed that nucleoside transporters (NTs) are present in cultures of chick retinal cells, but little is known about the mechanisms regulating adenosine transport in these cultures. Our aim in the present work was to study the participation of the adenosine metabolism as well as the ERK pathway on adenosine uptake in different types of retinal cultures (mixed and purified glial cultures). Kinetic analysis in both cultures revealed that the uptake reached equilibrium after 30min and presented two components. Incubation of cultures with S-(p-nitrobenzyl)-6-thioinosine (NBTI) or dipyridamole, different inhibitors of equilibrative nucleoside transporters (ENTs), produced a significant and concentration-dependent uptake reduction in both cultures. However, while dipyridamole presented similar maximal inhibitory effects in both cultures (although in different concentrations), the inhibition by NBTI was smaller in glial cultures than in mixed cultures, suggesting the presence of different transporters. Moreover, pre-incubation of [3H]-adenosine with adenosine deaminase (ADA) or adenosine kinase (ADK) inhibition with iodotubercidin promoted significant uptake inhibition in both cultures, indicating that the uptake is predominantly for adenosine and not inosine, and that taken up adenosine is preferentially directed to the synthesis of adenine nucleotides. In both cultures, the MEK inhibitors PD98059 or UO126, but not the inactive analog U0124, induced a significant and concentration-dependent uptake decrease. We have not observed any change in adenosine metabolism induced by MEK inhibitors, suggesting that this pathway is mediating a direct effect on NTs. Our results show the expression of different NTs in retinal cells in culture and that the activity of these transporters can be regulated by the ERK pathway or metabolic enzymes such as ADK which are then potential targets for regulation of Ado levels in normal or pathological conditions.

P.2.018 Behavioural and cognitive alterations of the young megencephaly (BALB/cByJ-Kv1.1 mceph/mceph) mouse

Two novel ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe4,Aib7, Aib11,Arg14,Lys15]N/OFQ-NH2 (peptide-1) and [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 (peptide-2), have been generated by combining different modifications of N/OFQ sequence. In the present study, we investigated the actions of two analogues and compared them with those of N/OFQ in four assays. Peptide-1 mimicked N/OFQ effects in mouse vas deferens and mouse colon and showed similar maximal effects but higher potency relative to N/OFQ. The effects of peptide-1 were sensitive to NOP receptor selective antagonist ([Nphe1]N/OFQ(1-13)-NH2) but not to naloxone in vitro. Peptide-1 (25 pmol, i.c.v.) mimicked the pronociceptive action of N/OFQ (2.5 nmol, i.c.v.) in mouse tail withdrawal assay, displaying higher potency and longer lasting effects. In anesthetized rats, peptide-1 (1 nmol/kg, i.v.) produced a marked decrease in mean arterial pressure, which was comparable to that evoked by i.v. N/OFQ (100 nmol/kg). Peptide-2 did not produce any effect per se but antagonized N/OFQ actions in mouse vas deferens and mouse colon assays. Peptide-2 is active in vivo where it prevented the pronociceptive effect induced by 2.5 nmol N/OFQ i.c.v. in the mouse tail withdrawal assay. Furthermore, peptide-2 at 5 nmol produced alone a robust and long lasting antinociceptive effect. Moreover, peptide-2 (10 and 40 nmol/kg i.v.) didn't produce any effect per se but antagonized hypotensive actions produced by i.v. administration of N/OFQ. Collectively, these findings demonstrate that [(pF)Phe4,Aib7,Aib11, Arg14,Lys15]N/OFQ-NH2 behaves as a highly potent NOP receptor agonist which produces long lasting effects in vivo and [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 acts as a pure and competitive antagonist of the NOP receptor.

Design, synthesis and in vitro evaluation of ( R)-4-(2-( tert-butylamino)-1-hydroxyethyl)-2-(hydroxymethyl)phenyl hydrogen phenylboronate: A novel salbutamol derivative with high intrinsic efficacy on the β 2 adrenoceptor

We tested a set of boron containing arylethanolamine derivatives on the human and guinea pig β 2 adrenoceptor (β 2AR) 3-D structures by docking methodology. The compound with the highest affinity based on docking analysis, ( R)-4-(2-( tert-butylamino)-1-hydroxyethyl)-2-(hydroxymethyl)phenyl hydrogen phenylboronate (boronterol) was synthesized, characterized and tested in guinea pig tracheal rings at basal tone and with histamine-induced contractions. Boronterol was at least eightfold more potent than salbutamol as a smooth muscle relaxant drug (judged by the EC 50 values) and showed a similar maximal relaxant effect as isoproterenol. ICI118,551 showed competitive antagonism on the relaxing effect of boronterol. These results suggest the β 2AR agonist action of boronterol.

RhBMP2/7 Heterodimer: An Osteoblastogenesis Inducer of Not Higher Potency but Lower Effective Concentration Compared with rhBMP2 and rhBMP7 Homodimers

Heterodimeric bone morphogenetic proteins (BMPs) were exhibited to be more potent than and thus potential substitutes for homodimeric BMPs whose clinical application is limited for the drawbacks resulted from their higher effective doses. This study aims to delineate the biofunctional characteristics of recombinant human BMP2/7 (rhBMP2/7) heterodimer in inducing osteoblastogenesis of MC3T3-E1 through in vitro time-course and dose-response studies. rhBMP2/7 heterodimer induced cell migration with a significantly lower optimal concentration and higher peak effect than the respective homodimers. rhBMP2/7 heterodimer induced cell differentiation with significantly lower threshold concentrations but similar maximum effects. On day 28, the area of calcium depositions induced by 50 ng/mL rhBMP2/7 was 12- or 38-fold more than that of 50 ng/mL rhBMP2 or 50 ng/mL rhBMP7, respectively. The results indicated that rhBMP2/7 heterodimer was an osteoblastogenesis inducer of not higher potency but lower effective concentration compared with rhBMP2 and rhBMP7 homodimers.