- Involvement of Nicotinic Receptor Subtypes in Behavioral Effects of Nicotinic Drugs in Squirrel Monkeys. [Journal Article]
- JPJ Pharmacol Exp Ther 2018 May 21
- Evidence suggests that the α4β2, but not the α7, subtype of nicotinic acetylcholine receptor (nAChR) plays a key role in mediating the behavioral effects of nicotine and related drugs. However, the i...
Evidence suggests that the α4β2, but not the α7, subtype of nicotinic acetylcholine receptor (nAChR) plays a key role in mediating the behavioral effects of nicotine and related drugs. However, the importance of other nAChR subtypes remains unclear. The present studies were conducted to examine the involvement of nAChR subtypes by determining the effects of selected nicotinic agonists and antagonists in squirrel monkeys: a) responding for food-reinforcement; or b) discriminating the nicotinic agonist (+)-epibatidine (0.001 mg/kg) from vehicle. In food-reinforcement studies, nicotine, (+)-epibatidine, varenicline, and cytisine all produced dose-dependent decreases in rates of food-maintained responding. The rate-decreasing effects of nicotine were antagonized by mecamylamine (non-selective), not appreciably altered by dihydro-β-erythroidine (α4β2-selective), and exacerbated by the nicotinic partial agonists, varenicline and cytisine. Results from discrimination studies show that non-nicotinic drugs did not substitute for (+)-epibatidine and that, excepting lobeline, nicotinic agonists produced either full [(+)-epibatidine, (-)-epibatidine, nicotine] or partial (varenicline, cytisine, anabaseine, isoarecolone) substitution for (+)-epibatidine. In interaction studies with antagonists differing in selectivity, (+)-epibatidine discrimination was substantively antagonized by mecamylamine, slightly attenuated by hexamethonium (peripherally restricted) or dihydro-β-erythroidine, and not altered by methyllycaconitine (α7-selective). Varenicline and cytisine enhanced (+)-epibatidine's discriminative-stimulus effects. Correlational analysis revealed a close correspondence between relative behavioral potencies of nicotinic agonists in both studies and their published relative binding affinities at α4β2 and α3β4, but not α7 nAChR, subtypes. Collectively, these results are consistent with the idea that the α4β2 and α3β4 but not α7 nAChR subtypes play a role in the behavioral effects of nicotinic agonists.
- Ibuprofen decreases spontaneous activity and enhances nerve evoked contractions to minimise mitomycin C induced bladder dysfunction. [Journal Article]
- JPJ Pharmacol Exp Ther 2018 May 21
- Inflammation may play a causal role in urological side effects reported following intravesical mitomycin C (MMC). Our aim was to investigate the effects of the non-steroidal anti-inflammatory drug ib...
Inflammation may play a causal role in urological side effects reported following intravesical mitomycin C (MMC). Our aim was to investigate the effects of the non-steroidal anti-inflammatory drug ibuprofen (IBU) on the cytotoxic potency of MMC and the potential for IBU to protect against bladder dysfunction. Malignant (RT4, T24) and normal (UROtsa) urothelial lines were treated with MMC followed by ibuprofen, with cell viability and caspase-3 activity assessed. Female C57BL/6JArc mice (Saline/Control, MMC, Saline + IBU and MMC + IBU), received intravesical treatment (1hr) with saline or MMC (2mg/mL), with IBU (1mg/mL) delivered in drinking water (for 7-days). Voiding pattern analysis was conducted prior to and following (1,3,7 days) treatment. A whole bladder preparation was used to assess compliance, contractile responses and urothelial mediator release. Ibuprofen selectively increased the cytotoxic potency of MMC and caspase-3 activity in both malignant cells lines but not in UROtsa. MMC significantly increased voiding frequency at 24 hours and 3 days, while administration of ibuprofen significantly reduced this effect. MMC significantly increased the frequency of spontaneous contractions from 2.3±0.5 contractions/min in saline controls to 4.8 ±0.16 contractions/min, with ibuprofen protecting against this change. Interestingly, while nerve evoked responses were not altered by MMC, they were increased in both IBU groups. Ibuprofen improved voiding dysfunction following MMC treatment through reducing spontaneous phasic activity and enhancing nerve mediated contractions. Ibuprofen use in bladder cancer patients may help to minimise the urological adverse effects associated with intravesical MMC.
- Linaclotide Attenuates Visceral Organ Crosstalk: Role of Guanylate Cyclase C Activation in Reversing Bladder-Colon Cross Sensitization. [Journal Article]
- JPJ Pharmacol Exp Ther 2018 May 21
- CONCLUSIONS: Our findings suggest a novel treatment paradigm for GC-C agonism in IBS-C and BPS mediated through visceral organ crosstalk, a mechanism not previously linked to linaclotide.
- OBE022, an oral and selective prostaglandin F2α receptor antagonist as an effective and safe modality for the treatment of preterm labor. [Journal Article]
- JPJ Pharmacol Exp Ther 2018 May 18
- Preterm birth is the major challenge in obstetrics affecting ~10% of pregnancies. Pan-prostaglandin synthesis inhibitors (NSAID) prevent preterm labor and prolong pregnancy but raise concerns about f...
Preterm birth is the major challenge in obstetrics affecting ~10% of pregnancies. Pan-prostaglandin synthesis inhibitors (NSAID) prevent preterm labor and prolong pregnancy but raise concerns about fetal renal and cardiovascular safety. We conducted preclinical studies examining the tocolytic effect and fetal safety of the oral prodrug candidate OBE022 and its parent OBE002, both potent and highly selective antagonist of the contractile PGF2α prostaglandin receptor (FP). Efficacy of OBE022 and OBE002, alone and in combination with other tocolytics, was assessed in human tissues and pregnant animal models for inhibition of uterine contraction and delay of parturition. Selective safety of OBE022 and/or OBE002, compared to NSAID indomethacin, was assessed on renal function, closure of the ductus arteriosus and inhibition of platelet aggregation. In in vitro studies, OBE002 inhibited spontaneous, oxytocin- and PGF2α-induced human myometrial contractions alone and was more effective in combination with atosiban or nifedipine. In in vivo studies, OBE022 and OBE002 reduced spontaneous contractions in near-term pregnant rat. In pregnant mice, OBE022 delayed RU486-induced parturition and exerted synergistic effects in combination with nifedipine. OBE022 and/or OBE002 did not show the fetal side effects of ductus arteriosus constriction, impairment of kidney function or inhibition of platelet aggregation observed with indomethacin. Orally active OBE022 and OBE002 exhibits potent tocolytic effects on human tissues ex vivo and animal models in vivo without causing the adverse fetal side effects seen with indomethacin. Selectively targeting the FP receptor in combination with existing tocolytics may be an effective strategy for preventing or delaying preterm delivery.
- Activation of Large Conductance, Calcium-Activated K Channels by Nitric Oxide Mediates Apelin-Induced Relaxation of Coronary Arteries. [Journal Article]
- JPJ Pharmacol Exp Ther 2018 May 17
- Apelin increases coronary blood flow, cardiac contractility and cardiac output. Based on these favorable hemodynamic effects, apelin and apelin-like analogs are being developed for treating heart fai...
Apelin increases coronary blood flow, cardiac contractility and cardiac output. Based on these favorable hemodynamic effects, apelin and apelin-like analogs are being developed for treating heart failure and related disorders; however, the molecular mechanisms underlying apelin-induced coronary vasodilation are unknown. This study aimed to elucidate the signaling pathways by which apelin causes smooth muscle relaxation in coronary arteries. Receptors for apelin (APJ receptors) were expressed in coronary arteries, as determined by Western blot and PCR analyses. Immunofluorescence imaging studies identified APJ receptors on endothelial and smooth muscle cells. In isolated endothelial cells, apelin caused an increase in DAF-2 fluorescence that was abolished by nitro-l-arginine (NLA) and F13A, an APJ receptor antagonist, consistent with increased NO production. In arterial rings, apelin caused endothelium-dependent relaxations that were abolished by NLA, F13A, and iberiotoxin. Neither ODQ nor DT-2, a protein kinase G inhibitor, had any effect on apelin-induced relaxations, and apelin itself had no effect on intracellular cGMP accumulation in coronary arteries. Patch clamp studies in isolated smooth muscle cells demonstrated that the NO donors, DEA NONOate and sodium nitroprusside, caused increases in large conductance, calcium-activated K (BKCa) currents, which were inhibited by iberiotoxin but not ODQ. Thus, apelin causes endothelium-dependent relaxation of coronary arteries by stimulating endothelial APJ receptors and releasing NO, which acts in a cGMP-independent manner and increases BKCa activity in the underlying smooth muscle cells. The results provide a mechanistic basis for apelin-induced coronary vasodilation and may provide guidance for the future development of novel apelin-like therapeutic agents.
- Molecular basis of brain RAS in cardiovascular and neurological disorders: Uncovering a key role for the astroglial AT1R. [Journal Article]
- JPJ Pharmacol Exp Ther 2018 May 11
- The central renin angiotensin system (RAS) is one of the most widely investigated cardiovascular systems in the brain. It is implicated in a myriad of cardiovascular diseases. However, studies from t...
The central renin angiotensin system (RAS) is one of the most widely investigated cardiovascular systems in the brain. It is implicated in a myriad of cardiovascular diseases. However, studies from the last decade have identified its involvement in several neurological abnormalities. Understanding the molecular functionality of the various RAS components can thus provide considerable insight into the phenotypic differences and mechanistic drivers of not just cardiovascular, but also neurological disorders. Since activation of one of its primary receptors, the angiotensin type-1 receptor (AT1R) results in an augmentation of oxidative stress and inflammatory cytokines, it becomes essential to investigate not just neuronal RAS, but also glial RAS as well. Glial cells are key homeostatic regulators in the brain, and are critical players in the resolution of overt oxidative stress and neuroinflammation. Designing better and effective therapeutic strategies that target the brain RAS, could well hinge on understanding the molecular basis of both neuronal and glial RAS. This review provides a comprehensive overview of the major studies that have investigated the mechanisms and regulation of brain RAS, and it also provides insight into the potential role of glial AT1Rs in the pathophysiology of cardiovascular and neurological disorders.
- Protein Engineering on Human Recombinant Follistatin: Enhancing Pharmacokinetic Characteristics for Therapeutic Application. [Journal Article]
- JPJ Pharmacol Exp Ther 2018 May 11
- Follistatin (FS) is an important regulatory protein, a natural antagonist for transforming growth factor beta family members activin and myostatin. The diverse biological roles of the activin and myo...
Follistatin (FS) is an important regulatory protein, a natural antagonist for transforming growth factor beta family members activin and myostatin. The diverse biological roles of the activin and myostatin signaling pathways make FS a promising therapeutic target for treating human diseases exhibiting inflammation, fibrosis and muscle disorders, such as Duchenne muscular dystrophy. However, rapid heparin-mediated hepatic clearance of FS limits its therapeutic potential. We targeted the heparin binding loop of FS for site-directed mutagenesis to improve clearance parameters. By generating a series of FS variants with one, two, or three negative amino acid substitutions, we demonstrated a direct and proportional relationship between the degree of heparin binding affinity in vitro and the exposure in vivo. The triple mutation K(76,81,82)E abolished heparin binding affinity, resulting in ~20-fold improved in vivo exposure. This triple mutant retains full functional activity and antibody-like pharmacokinetic profile, and shows a superior developability profile in physical stability and cell productivity compared to FS variants which substitute the entire heparin binding loop with alternative sequences. Our surgical approach to mutagenesis should also reduce the immunogenicity risk. To further lower this risk, we introduced a novel glycosylation site into the heparin-binding loop. This hyperglycosylated variant showed a 10-fold improved exposure and decreased clearance in mice compared an IgG1 Fc fusion protein containing the native FS sequence. Collectively, our data highlights the importance of improving pharmacokinetic properties by manipulating heparin binding affinity and glycosylation content and provide a valuable guideline to design desirable therapeutic FS molecules.
- Luminal polyethylene glycol alleviates intestinal preservation injury irrespective of molecular size. [Journal Article]
- JPJ Pharmacol Exp Ther 2018 May 08
- Intestinal preservation injury (IPI) and the resulting mucosal injury raise several serious challenges early after intestinal transplantation (ITx). The current clinical approach using only vascular ...
Intestinal preservation injury (IPI) and the resulting mucosal injury raise several serious challenges early after intestinal transplantation (ITx). The current clinical approach using only vascular perfusion allows the shortest preservation period among the abdominal organs. The experimental addition of luminal polyethylene glycol (PEG) solutions has been repeatedly suggested to alleviate preservation injury, improve graft quality and prolong the preservation time. We investigated if the size of PEG in the solution influences the development of intestinal preservation injury. Small intestines of Sprague Dawley rats were perfused with University of Wisconsin solution. Group 1 underwent vascular perfusion only (clinical control), group 2 received additional luminal PEG3350 Da, group 3 received luminal PEG10000 Da whereas group 4 received luminal PEG20000 Da (n=8/group). Tissue samples were obtained after 4h, 8h and 14h. We studied the tissue damage (Chiu/Park score, Goblet cells, apoptosis, tight junctions), activation of c-Jun NH2-terminal kinase (JNK) and p38-mitogen-activated protein kinase (MAPK) and performed Ussing chamber assessments. Mucosal morphological and electrophysiological parameters were significantly improved in the groups receiving luminal PEG. There was significantly less apoptotic activity in group 2, 3 and 4. Both MAPKs revealed an activation peak after 4h with group 3 showing lesser p38-MAPK activation. PEG 20kDa interfered with protein immunodetection. The results indicate that luminal solutions of PEG of medium and large molecular weight significantly delay the onset and development of IPI. Hence, the present study provides further evidence that luminal interventions may allow for longer cold storage intervals of the intestinal grafts.
- Survivin inhibitors mitigate chemotherapeutic resistance in breast cancer cells by suppressing genotoxic NF-kappaB activation. [Journal Article]
- JPJ Pharmacol Exp Ther 2018 May 07
- Therapeutic resistance developed after chemotherapy and aggressive metastasis are the major causes for cancer-related death in triple negative breast cancer (TNBC) patients. Survivin is the smallest ...
Therapeutic resistance developed after chemotherapy and aggressive metastasis are the major causes for cancer-related death in triple negative breast cancer (TNBC) patients. Survivin is the smallest member of Inhibitor-of-Apoptosis Proteins (IAPs) family, which plays critical roles in cell division and cell survival. High expression levels of survivin have been associated with therapeutic resistance in various cancers. We recently developed a novel small molecule survivin inhibitor mimicking IAP-binding motif of second mitochondria-derived activator of caspase, which showed high potency in promoting survivin degradation. Here, we show survivin inhibitors MX106/107 suppress TNBC cell proliferation. Moreover, MX106/107 synergized with chemotherapeutic drugs or radiation, and significantly enhanced tumoricidal efficacy of genotoxic treatments. Mechanistically, MX106/107 induced degradation of XIAP and/or cIAP1, which inhibited NF-κB activation by genotoxic agents. Treatment with MX106/107 alone did not activate alternative NF-κB signaling in breast cancer cells, which likely is owing to their selective potency in degrading survivin in these cells. Additionally, survivin degradation by MX106/107 dramatically increased abnormal mitotic spindle formation and cell division failure, which led to cell cycle arrest in breast cancer cells. Overall, our study suggests that combination treatment of TNBC using survivin inhibitors MX106/107 with cytotoxic chemotherapeutic drugs can achieve significantly improved therapeutic efficacy, which depends on MX106/107-mediated inhibition of genotoxic NF-κB activation.
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- Reduction in secretion of very low density lipoprotein-triacylglycerol by a matrix metalloproteinase inhibitor in a rat model of diet-induced hypertriglyceridemia. [Journal Article]
- JPJ Pharmacol Exp Ther 2018 May 07
- Matrix metalloproteinase inhibitors (MMPIs) reduced serum triacylglycerol (TAG) levels in streptozotocin-induced diabetic rats and Zucker fa/fa rats in our previous study. However, the mechanisms und...
Matrix metalloproteinase inhibitors (MMPIs) reduced serum triacylglycerol (TAG) levels in streptozotocin-induced diabetic rats and Zucker fa/fa rats in our previous study. However, the mechanisms underlying TAG reduction by MMPIs remain unclear. The present study aimed to elucidate the mechanism by which F81-1144b, a MMPI, lowers serum TAG levels in an animal model of high sucrose diet (HSD)-induced hypertriglyceridemia. F81-1144b was repeatedly administered to rats fed HSD, and its effects were evaluated on TAG levels in serum and the liver, very low density lipoprotein (VLDL) secretion, de novo fatty acid (FA) synthesis in the liver, and the expression of genes regulating the metabolism of FA, TAG, and VLDL in the liver and serum. F81-1144b lowered TAG levels in serum and the liver, VLDL-TAG secretion, de novo FA synthesis in the liver, and serum levels of insulin and glucose. F81-1144b suppressed the expression of genes related to the de novo synthesis of FA and TAG, key proteins (lipin 1 and apolipoprotein CIII) responsible for VLDL metabolism, and sterol regulatory element-binding protein-1c and carbohydrate response element-binding protein. F81-1144b little affected the expression of genes related directly to the degradation of TAG or FA, while it up-regulated that of gene for uncoupling protein 2 in the liver. These results suggest that MMPIs are a novel type of therapeutic agent for the treatment of hypertriglyceridemia because the metabolic effects of F81-1144b expected from changes in the expression of genes regulating lipid metabolism would alter metabolism differently from those induced by fibrates, niacin, or n-3 FAs.