Sphingosylphosphorylcholine acts in an anti-inflammatory manner in renal mesangial cells by reducing interleukin-1beta-induced prostaglandin E2 formation

Sphingosylphosphorylcholine (SPC) is a bioactive lipid that binds to G protein-coupled-receptors and activates various signaling cascades. Here, we show that in renal mesangial cells, SPC not only activates various protein kinase cascades but also activates Smad proteins, which are classical members of the transforming growth factor-beta (TGFbeta) signaling pathway. Consequently, SPC is able to mimic TGFbeta-mediated cell responses, such as an anti-inflammatory and a profibrotic response. Interleukin-1beta-stimulated prostaglandin E(2) formation is dose-dependently suppressed by SPC, which is paralleled by reduced secretory phospholipase A(2) (sPLA(2)) protein expression and activity. This effect is due to a reduction of sPLA(2) mRNA expression caused by inhibited sPLA(2) promoter activity. Furthermore, SPC upregulates the profibrotic connective tissue growth factor (CTGF) protein and mRNA expression. Blocking TGFbeta signaling by a TGFbeta receptor kinase inhibitor causes an inhibition of SPC-stimulated Smad activation and reverses both the negative effect of SPC on sPLA(2) expression and the positive effect on CTGF expression. In summary, our data show that SPC, by mimicking TGFbeta, leads to a suppression of proinflammatory mediator production and stimulates a profibrotic cell response that is often the end point of an anti-inflammatory reaction. Thus, targeting SPC receptors may represent a novel therapeutic strategy to cope with inflammatory diseases.

BIOCHEMICAL PROPERTIES OF GABA (B) RECEPTORS (BACLOFEN, ADENYLATE CYCLASE, CYCLIC-AMP, PHOSPHOLIPASE, PROTEIN KINASE C)

(gamma)-Aminobutyric acid (GABA), a neurotransmitter in the mammalian central nervous system, influences neuronal activity by interacting with at least two pharmacologically and functionally distinct receptors. GABA(,A) receptors are sensitive to blockade by bicuculline, are associated with benzodiazepine and barbiturate binding sites, and mediate chloride flux. The biochemical and pharmacolocal properties of GABA(,B) receptors, which are stereoselectively activated by (beta)-p-chlorophenyl GABA (baclofen), are less well understood. The aim of this study was to define these features of GABA(,B) receptors, with particular emphasis on their possible relationship to the adenylate cyclase system in brain.^ By themselves, GABA agonists have no effect on cAMP accumulation in rat brain slices. However, some GABA agonists markedly enhance the cAMP accumulation that results from exposure to norepinephrine, adenosine, VIP, and cholera toxin. Evidence that this response is mediated by the GABA(,B) system is provided by the finding that it is bicuculline-insensitive, and by the fact that only those agents that interact with GABA(,B) binding sites are active in this regard. GABA(,B) agonists are able to enhance neurotransmitter-stimulated cAMP accumulation in only certain brain regions, and the response is not influenced by phosphodiesterase inhibitors, although is totally dependent on the availability of extracellular calcium. Furthermore, data suggest that inhibition of phospholipase A(,2), a calcium-dependent enzyme, decreases the augmenting response to baclofen, although inhibitors of arachidonic acid metabolism are without effect. These findings indicate that either arachidonic acid or lysophospholipid, products of PLA(,2)-mediated degradation of phospholipids, mediates the augmentation. Moreover, phorbol esters, compounds which directly activate protein kinase C, were also found to enhance neurotransmitter-stimulated cAMP accumulation in rat brain slices. Since this enzyme is known to be stimulated by unsaturated fatty acids such as arachidonate, it is proposed that GABA(,B) agonists enhance cAMP accumulation by fostering the production of arachidonic acid which stimulates protein kinase C, leading to the phosphorylation of some component of the adenylate cyclase system. Thus, GABA, through an interaction with GABA(,B) receptors, modulates neurotransmitter receptor responsiveness in brain. The pharmocological manipulation of this response could lead to the development of therapeutic agents having a more subtle influence than current drugs on central nervous system function. ^

The role of self-monitoring of blood glucose in patients treated with SGLT-2 inhibitors: a European expert recommendation.

The role for the novel treatment approach of sodium-glucose cotransporter-2 (SGLT-2) in type 2 diabetes is increasing. Structured self-monitoring of blood glucose (SMBG), based on a less intensive and a more intensive scheme, may contribute to an optimization of SGLT-2 inhibitor based treatment. The current expert recommendation suggests individualized approaches of SMBG, using simple and clinically applicable schemes. Potential benefits of SMBG in SGLT-2 inhibitor based treatment approaches are early assessment of treatment success or failure, timely modification of treatment, detection of hypoglycemic episodes, assessment of glucose excursions, and support of diabetes management and education. The length and frequency of SMBG should depend on the clinical setting and the quality of metabolic control.

Regulation of cyclooxygenase-2 (COX-2) in rat renal cortex by adrenal glucocorticoids and mineralocorticoids

Production of prostaglandins involved in renal salt and water homeostasis is modulated by regulated expression of the inducible form of cyclooxygenase-2 (COX-2) at restricted sites in the rat renal cortex. Because inflammatory COX-2 is suppressed by glucocorticoids, and prostaglandin levels in the kidney are sensitive to steroids, the sensitivity of COX expression to adrenalectomy (ADX) was investigated. By 2 weeks after ADX in mature rats, cortical COX-2 immunoreactivity increased 10-fold in the cortical thick ascending limb and macula densa. The constitutive isoform, COX-1, was unchanged. The magnitude of the changes and specificity of COX-2 immunoreactivity were validated by in situ hybridization histochemistry of COX-2 mRNA and Western blot analysis. Increased COX-2 activity (>5-fold) was documented by using a specific COX-2 inhibitor. The COX-2 up-regulation in ADX rats was reversed by replacement therapy with either corticosterone or deoxycorticosterone acetate. In normal rats, inhibition of glucocorticoid receptors with RU486 or mineralocorticoid receptors with spironolactone caused up-regulation of renal cortical COX-2. These results indicate that COX-2 expression in situ is tonically inhibited by adrenal steroids, and COX-2 is regulated by mineralocorticoids as well as glucocorticoids.

Motivations and perceived influences on rural and urban general practitioners when prescribing conventional non-steroidal anti-inflammatory drugs or COX-2 inhibitors

Background and objective: Prescribers in rural and remote locations perceive that there are different influences on their prescribing compared with those experienced by urban prescribers. The aim of this study was to compare the motivations and perceived influences on general practitioners (GPs) when prescribing COX-2 inhibitors rather than conventional non-steroidal anti-inflammatory drugs (NSAIDs) between rural and urban-based GPs in Queensland, Australia. Methods: A questionnaire was administered to two geographically distinct groups of GPs, one urban (n = 67) and one rural (n = 67), investigating the reasons that the GP would prescribe a COX-2 inhibitor rather than a conventional NSAID or vice versa and also focusing on patients requesting a prescription for a COX-2 inhibitor. Results and discussion: A 51% response rate (n = 68) was achieved. The difference between the rural and the urban GPs was that the urban GPs were more likely to perceive that they were influenced to prescribe COX-2 inhibitors by their patients' knowledge of these new (at the time) drugs. GPs in both the rural and urban areas perceived the COX-2 selective inhibitors to be safer than conventional NSAIDs, and that there was little difference in terms of efficacy between the two drug classes. However, GPs from both of the study areas stated that conventional NSAIDs were preferred over COX-2 selective inhibitors, primarily due to their expense, if their patients were not at risk for developing a GI bleed. Conclusion: The motivations and perceived influences to prescribe a COX-2 inhibitor in rural and in urban areas of Queensland, Australia were very similar. Almost all surveyed GPs in rural and urban areas had patients request a prescription, or enquire about the COX-2 inhibitors. Urban GPs were more likely to feel pressured to prescribe a COX-2 inhibitor than their rural counterparts, agreeing with other research which found that patient pressure to prescribe appears to be greater in urban general practice.

Long-term loading inhibits ERK1/2 phosphorylation and increases FGFR3 expression in MC3T3-E1 osteoblast cells

Bone tissue homeostasis relies upon the ability of cells to detect and interpret extracellular signals that direct changes in tissue architecture. This study utilized a four-point bending model to create both fluid shear and strain forces (loading) during the time-dependent progression of MC3T3-E1 preosteoblasts along the osteogenic lineage. Loading was shown to increase cell number, alkaline phosphatase (ALP) activity, collagen synthesis, and the mRNA expression levels of Runx2, osteocalcin (OC), osteopontin, and cyclo-oxygenase-2. However, mineralization in these cultures was inhibited, despite an increase in calcium accumulation, suggesting that loading may inhibit mineralization in order to increase matrix deposition. Loading also increased fibroblast growth factor receptor-3 (FGFR3) expression coincident with an inhibition of FGFR1, FGFR4, FGF1, and extracellular signal-related kinase (ERK)1/2 phosphorylation. To examine whether these loading-induced changes in cell phenotype and FGFR expression could be attributed to the inhibition of ERK1/2 phosphorylation, cells were grown for 25 days in the presence of the MEK1/2 inhibitor, U0126. Significant increases in the expression of FGFR3, ALP, and OC were observed, as well as the inhibition of FGFR1, FGFR4, and FGF1. However, U0126 also increased matrix mineralization, demonstrating that inhibition of ERK1/2 phosphorylation cannot fully account for the changes observed in response to loading. in conclusion, this study demonstrates that preosteoblasts are mechanoresponsive, and that long-term loading, whilst increasing proliferation and differentiation of preosteoblasts, inhibits matrix mineralization. In addition, the increase in FGFR3 expression suggests that it may have a role in osteoblast differentiation.

Molecular diversity in venom from the Australian Brown Snake, Pseudonaja textilis

Venom from the Australian elapid Pseudonaja textilis (Common or Eastern Brown snake), is the second most toxic snake venom known and is the most common cause of death from snake bite in Australia. This venom is known to contain a prothrombin activator complex, serine proteinase inhibitors, various phospholipase A(2)s, and pre-and postsynaptic neurotoxins. In this study, we performed a proteomic identification of the venom using two- dimensional gel electrophoresis, mass spectrometry, and de novo peptide sequencing. We identified most of the venom proteins including proteins previously not known to be present in the venom. In addition, we used immunoblotting and post-translational modification-specific enzyme stains and antibodies that reveal the complexity and regional diversity of the venom. Modifications observed include phosphorylation, gamma-carboxylation, and glycosylation. Glycoproteins were further characterized by enzymatic deglycosylation and by lectin binding specificity. The venom contains an abundance of glycoproteins with N-linked sugars that include glucose/mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acids. Additionally there are multiple isoforms of mammalian coagulation factors that comprise a significant proportion of the venom. Indeed two of the identified proteins, a procoagulant and a plasmin inhibitor, are currently in development as human therapeutic agents.

Signal transduction pathways involved in proteolysis-inducing factor induced proteasome expression in murine myotubes

The proteolysis-inducing factor (PIF) is produced by cachexia-inducing tumours and initiates protein catabolism in skeletal muscle. The potential signalling pathways linking the release of arachidonic acid (AA) from membrane phospholipids with increased expression of the ubiquitin-proteasome proteolytic pathway by PIF has been studied using C2C12 murine myotubes as a surrogate model of skeletal muscle. The induction of proteasome activity and protein degradation by PIF was blocked by quinacrine, a nonspecific phospholipase A2 (PLA2) inhibitor and trifluroacetyl AA, an inhibitor of cytosolic PLA2. PIF was shown to increase the expression of calcium-independent cytosolic PLA2, determined by Western blotting, at the same concentrations as those inducing maximal expression of 20S proteasome α-subunits and protein degradation. In addition, both U-73122, which inhibits agonist-induced phospholipase C (PLC) activation and D609, a specific inhibitor of phosphatidylcholine-specific PLC also inhibited PIF-induced proteasome activity. This suggests that both PLA 2 and PLC are involved in the release of AA in response to PIF, and that this is important in the induction of proteasome expression. The two tyrosine kinase inhibitors genistein and tryphostin A23 also attenuated PIF-induced proteasome expression, implicating tyrosine kinase in this process. PIF induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) at the same concentrations as that inducing proteasome expression, and the effect was blocked by PD98059, an inhibitor of MAPK kinase, as was also the induction of proteasome expression, suggesting a role for MAPK activation in PIF-induced proteasome expression. © 2003 Cancer Research UK.

Effect of dapagliflozin in patients with type 2 diabetes who have inadequate glycaemic control with metformin:a randomised, double-blind, placebo-controlled trial

Correction of hyperglycaemia and prevention of glucotoxicity are important objectives in the management of type 2 diabetes. Dapagliflozin, a selective sodium-glucose cotransporter-2 inhibitor, reduces renal glucose reabsorption in an insulin-independent manner. We assessed the efficacy and safety of dapagliflozin in patients who have inadequate glycaemic control with metformin.

Dapagliflozin add-on to metformin in type 2 diabetes inadequately controlled with metformin:a randomized, double-blind, placebo-controlled 102-week trial

Background: Management of type 2 diabetes with metformin often does not provide adequate glycemic control, thereby necessitating add-on treatment. In a 24-week clinical trial, dapagliflozin, an investigational sodium glucose cotransporter 2 inhibitor, improved glycemic control in patients inadequately controlled with metformin. The present study is an extension that was undertaken to evaluate dapagliflozin as long-term therapy in this population.Methods: This was a long-term extension (total 102 weeks) of a 24-week phase 3, multicenter, randomized, placebo-controlled, double-blind, parallel-group trial. Patients were randomly assigned (1:1:1:1) to blinded daily treatment (placebo, or dapagliflozin 2.5 to 5, or 10 mg) plus open-label metformin (=1,500 mg). The previously published primary endpoint was change from baseline in glycated hemoglobin (HbA1c) at 24 weeks. This paper reports the follow-up to week 102, with analysis of covariance model performed at 24 weeks with last observation carried forward; a repeated measures analysis was utilized to evaluate changes from baseline in HbA1c, fasting plasma glucose (FPG), and weight.Results: A total of 546 patients were randomized to 1 of the 4 treatments. The completion rate for the 78-week double-blind extension period was lower for the placebo group (63.5%) than for the dapagliflozin groups (68.3% to 79.8%). At week 102, mean changes from baseline HbA1c (8.06%) were +0.02% for placebo compared with -0.48% (P = 0.0008), -0.58% (P

Efficacy and safety of dapagliflozin monotherapy in people with Type 2 diabetes:a randomized double-blind placebo-controlled 102-week trial

Aims: To assess initial pharmacotherapy of Type 2 diabetes with the sodium-glucose cotransporter-2 inhibitor dapagliflozin. Methods: This double-blind, placebo-controlled trial, randomly allocated people with Type 2 diabetes aged 18-77 years and inadequate glycaemic control on diet and exercise [HbA1c 53-86 mmol/mol (7.0-10.0%)] to receive placebo (n = 75) or dapagliflozin monotherapy 2.5 mg (n = 65), 5 mg (n = 64) or 10 mg (n = 70) once daily in the morning. After 24 weeks, low-dose double-blind metformin 500 mg/day was added to the placebo group regimen (placebo+low-dose metformin group). Changes in HbA1c level, fasting plasma glucose and body weight, as well as adverse events, were assessed over 102 weeks. Results: Of the 274 participants randomized, 167 completed the study (60.9%). At 102 weeks, significant differences vs placebo+low-dose metformin with dapagliflozin 5 and 10 mg were observed for HbA1c (-5.8 mmol/mol [-0.53%], P = 0.018; and -4.8 mmol/mol [-0.44%], P = 0.048), respectively); and for FPG (-0.69 mmol/L, P = 0.044; and -1.12 mmol/l, P = 0.001, respectively). For body weight, the difference between the dapagliflozin 10-mg group and the placebo+low-dose metformin group was significant (-2.60 kg; P = 0.016). Hypoglycaemic events were uncommon, with rates of 5.3% for placebo+low-dose metformin group and 0-4.6% for the dapagliflozin groups. Genital infections and urinary tract infections were more common in the dapagliflozin groups than in the placebo+low-dose metformin group. Conclusions: Dapagliflozin as monotherapy in treatment-naïve people with early Type 2 diabetes improved glycaemic control and reduced weight without increasing hypoglycaemia over 102 weeks. Dapagliflozin may provide an alternative initial pharmacotherapy in such people.

Do selective cyclo-oxygenase-2 inhibitors and traditional non-steroidal anti-inflammatory drugs increase the risk of atherothrombosis? Meta-analysis of randomised trials

Objective: To assess the effects of selective cyclo-oxygenase-2 (COX 2) inhibitors and traditional non-steroidal anti-inflammatory drugs (NSAIDs) on the risk of vascular events. Design: Meta-analysis of published and unpublished tabular data from randomised trials, with indirect estimation of the effects of traditional NSAIDs. Data sources: Medline and Embase (January 1966 to April 2005); Food and Drug Administration records; and data on file from Novartis, Pfizer, and Merck. Review methods: Eligible studies were randomised trials that included a comparison of a selective COX 2 inhibitor versus placebo or a selective COX 2 inhibitor versus a traditional NSAID, of at least four weeks' duration, with information on serious vascular events (defined as myocardial infarction, stroke, or vascular death). Individual investigators and manufacturers provided information on the number of patients randomised, numbers of vascular events, and the person time of follow-up for each randomised group. Results: In placebo comparisons, allocation to a selective COX 2 inhibitor was associated with a 42% relative increase in the incidence of serious vascular events (1.2%/year v 0.9%/year; rate ratio 1.42, 95% confidence interval 1.13 to 1.78; P = 0.003), with no significant heterogeneity among the different selective COX 2 inhibitors. This was chiefly attributable to an increased risk of myocardial infarction (0.6%/year v 0.3%/year; 1.86, 1.33 to 2.59; P = 0.0003), with little apparent difference in other vascular outcomes. Among trials of at least one year's duration (mean 2.7 years), the rate ratio for vascular events was 1.45 (1.12 to 1.89; P = 0.005). Overall, the incidence of serious vascular events was similar between a selective COX 2 inhibitor and any traditional NSAID (1.0%/year v 0.9/%year; 1.16, 0.97 to 1.38; P = 0.1). However, statistical heterogeneity (P = 0.001) was found between trials of a selective COX 2 inhibitor versus naproxen (1.57, 1.21 to 2.03) and of a selective COX 2 inhibitor versus non-naproxen NSAIDs (0.88, 0.69 to 1.12). The summary rate ratio for vascular events, compared with placebo, was 0.92 (0.67 to 1.26) for naproxen, 1.51 (0.96 to 2.37) for ibuprofen, and 1.63 (1.12 to 2.37) for diclofenac. Conclusions: Selective COX 2 inhibitors are associated with a moderate increase in the risk of vascular events, as are high dose regimens of ibuprofen and diclofenac, but high dose naproxen is not associated with such an excess.

A phase I pharmacokinetic and pharmacodynamic study of the oral mitogen-activated protein kinase kinase (MEK) inhibitor, WX-554, in patients with advanced solid tumours

Purpose: We performed a multi-centre phase I study to assess the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of the orally available small molecule mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, WX-554, and to determine the optimal biological dose for subsequent trials.
Experimental design: Patients with treatment-refractory, advanced solid tumours, with adequate performance status and organ function were recruited to a dose-escalation study in a standard 3 + 3 design. The starting dose was 25 mg orally once weekly with toxicity, PK and PD guided dose-escalation with potential to explore alternative schedules.
Results: Forty-one patients with advanced solid tumours refractory to standard therapies and with adequate organ function were recruited in eight cohorts up to doses of 150 mg once weekly and 75 mg twice weekly. No dose-limiting toxicities were observed during the study, and a maximum tolerated dose (MTD) was not established. The highest dose cohorts demonstrated sustained inhibition of extracellular signal-regulated kinase (ERK) phosphorylation in peripheral blood mononuclear cells following ex-vivo phorbol 12-myristate 13-acetate stimulation. There was a decrease of 70 ± 26% in mean phosphorylated (p)ERK in C1 day 8 tumour biopsies when compared with pre-treatment tumour levels in the 75 mg twice a week cohort. Prolonged stable disease (>6 months) was seen in two patients, one with cervical cancer and one with ampullary carcinoma.
Conclusions: WX-554 was well tolerated, and an optimal biological dose was established for further investigation in either a once or twice weekly regimens. The recommended phase 2 dose is 75 mg twice weekly.

Effect of the synthetic coumarin, ethyl 2-oxo-2H-chromene-3-carboxylate, on activity of Crotalus durissus ruruima sPLA2 as well as on edema and platelet aggregation induced by this factor

We show that ethyl 2-oxo-2H-chromene-3-carboxylate (EOCC), a synthetic coumarin, irreversibly inhibits phospholipase A(2) (sPLA2) from Crotalus durissus ruruima venom (sPLA2r) with an IC(50) of 3.1 +/- 0.06 nmol. EOCC strongly decreased the V(max) and K(m), and it virtually abolished the enzyme activity of sPLA2r as well as sPLA2s from other sources. The edema induced by 5PLA2r + EOCC was less than that induced by 5PLA2r treated with p-bromophenacyl bromide, which was more efficient at neutralizing the platelet aggregation activity of native 5PLA2r. Native 5PLA2r induced platelet aggregation of 91.54 +/- 9.3%, and sPLA2r +/- EOCC induced a platelet aggregation of 18.56 +/- 6.5%. EOCC treatment also decreased the myotoxic effect of sPLA2r. Mass spectrometry showed that EOCC formed a stable complex with sPLA2r, which increased the mass of native 5PLA2r from 14,299.34 da to 14,736.22 Da. Moreover, the formation of this complex appeared to be involved in the loss of 5PLA2r activity. Our results strongly suggest that EOCC can be used as a pharmacological agent against the 5PLA2 in Crotalus durissus sp. venom as well as other sPLA2s. (C) 2010 Elsevier Ltd. All rights reserved.

Treatment with paracetamol, ketorolac or etoricoxib did not hinder alveolar bone healing: a histometric study in rats

Prostaglandins control osteoblastic and osteoclastic function under physiological or pathological conditions and are important modulators of the bone healing process. The non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX) activity and consequently prostaglandins synthesis. Experimental and clinical evidence has indicated a risk for reparative bone formation related to the use of non-selective (COX-1 and COX-2) and COX-2 selective NSAIDs. Ketorolac is a non-selective NSAID which, at low doses, has a preferential COX-1 inhibitory effect and etoricoxib is a new selective COX-2 inhibitor. Although literature data have suggested that ketorolac can interfere negatively with long bone fracture healing, there seems to be no study associating etoricoxib with reparative bone formation. Paracetamol/acetaminophen, one of the first choices for pain control in clinical dentistry, has been considered a weak anti-inflammatory drug, although supposedly capable of inhibiting COX-2 activity in inflammatory sites. OBJECTIVE: The purpose of the present study was to investigate whether paracetamol, ketorolac and etoricoxib can hinder alveolar bone formation, taking the filling of rat extraction socket with newly formed bone as experimental model. MATERIAL AND METHODS: The degree of new bone formation inside the alveolar socket was estimated two weeks after tooth extraction by a differential point-counting method, using an optical microscopy with a digital camera for image capture and histometry software. Differences between groups were analyzed by ANOVA after confirming a normal distribution of sample data. RESULTS AND CONCLUSIONS: Histometric results confirmed that none of the tested drugs had a detrimental effect in the volume fraction of bone trabeculae formed inside the alveolar socket.