Effect of cyclooxygenase inhibitors on gentamicin-induced nephrotoxicity in rats

The frequent use of nonsteroidal anti-inflammatory drugs (NSAID) in combination with gentamicin poses the additional risk of nephrotoxic renal failure. Cyclooxygenase-1 (COX-1) is the main enzyme responsible for the synthesis of renal vasodilator prostaglandins, while COX-2 participates predominantly in the inflammatory process. Both are inhibited by non-selective NSAID such as indomethacin. Selective COX-2 inhibitors such as rofecoxib seem to have fewer renal side effects than non-selective inhibitors. The objective of the present study was to determine whether the combined use of rofecoxib and gentamicin can prevent the increased renal injury caused by gentamicin and indomethacin. Male Wistar rats (250-300 g) were treated with gentamicin (100 mg/kg body weight, ip, N = 7), indomethacin (5 mg/kg, orally, N = 7), rofecoxib (1.4 mg/kg, orally, N = 7), gentamicin + rofecoxib (100 and 1.4 mg/kg, respectively) or gentamicin + indomethacin (100 and 5 mg/kg, respectively, N = 8) for 5 days. Creatinine clearance and alpha-glutathione-S-transferase concentrations were used as markers of renal injury. Animals were anesthetized with ether and sacrificed for blood collection. The use of gentamicin plus indomethacin led to worsened renal function (0.199 ± 0.019 ml/min), as opposed to the absence of a nephrotoxic effect of rofecoxib when gentamicin plus rofexicob was used (0.242 ± 0.011 ml/min). These results indicate that COX-2-selective inhibitors can be used as an alternative treatment to conventional NSAID, especially in situations in which risk factors for nephrotoxicity are present.

6-Sulfatoxymelatonin as a predictor of clinical outcome in depressive patients

Objectives This study established the value of the 6-sulfatoxymelatonin (aMT6s) urine concentration as a predictor of the therapeutic response to noradrenaline reuptake inhibitors in depressive patients. Methods Twenty-two women aged 18-60 years were selected. Depressive symptoms were assessed by using the Hamilton Depression Scale. Urine samples were collected at 0600-1200 h, 1200-1800 h, 1800-2400 h, and 2400-0600 h intervals, 1 day before and 1 day after starting on the nortriptyline treatment. Urine aMT6s concentration was analyzed by a one-way analysis of variance/Bonferroni test. Spearman`s rank correlation coefficient was used to analyze the correlation between depressive symptoms after 2 weeks of antidepressant treatment and the increase in aMT6s urine concentration. Results Higher and lower size effect groups were compared by independent Student`s t-tests. At baseline, the 2400- to 0600-h interval differed from all other intervals presenting a significantly higher aMT6s urine concentration. A significant difference in aMT6s urine concentrations was found 1 day after treatment in all four intervals. Higher size effect group had lower levels of depressive symptoms 2 weeks after the treatment. A positive correlation between depressive symptoms and the delta of aMT6s in the 2400-0600h interval was observed. Conclusion Our results reinforce the hypothesis that aMT6s excretion is a predictor of clinical outcome in depression, especially in regard to noradrenaline reuptake inhibitors. Copyright (C) 2011 John Wiley & Sons, Ltd.

Influência do triptofano, da fluoxetina e da paraclorofenilalanina no desenvolvimento inicial e na sobrevivência de larvas de matrinxã

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Medial amygdaloid nucleus 5-HT2C receptors are involved in the hypophagic effect caused by zimelidine in rats

The medial amygdaloid nucleus (MeA) is a sub-region of the amygdaloid complex that has been described as participating in food intake regulation. Serotonin has been known to play an important role in appetite and food intake regulation. Moreover, serotonin 5-HT2C and 5-HT1A receptors appear to be critical in food intake regulation. We investigated the role of the serotoninergic system in the MeA on feeding behavior regulation in rats. The current study examined the effects on feeding behavior regulation of the serotonin reuptake inhibitor, zimelidine, administered directly into the MeA or given systemically, and the serotoninergic receptors mediating its effect. Our results showed that microinjection of zimelidine (0.2, 2 and 20 nmol/100 nL) into the MeA evoked dose dependent hypophagic effects in fasted rats. The selective 5-HT1A receptor antagonist WAY-100635 (18.5 nmol/100 nL) or the 5-HT1B receptor antagonist SB-216641 microinjected bilaterally into the MeA did not change the hypophagic effect evoked by local MeA zimelidine treatment. However, microinjection of the selective 5-HT2C receptor antagonist SB-242084 (10 nmol/100 nL) was able to block the hypophagic effect of zimelidine. Moreover, microinjection of the 5-HT2C receptor antagonist SB-242084 into the MeA also blocked the hypophagic effect caused by zimelidine administered systemically. These results suggest that MeA 5-HT2C receptors modulate the hypophagic effect caused by local MeA administration as well as by systemic zimelidine administration. Furthermore, 5-HT2C into the MeA could be a potential target for systemic administration of zimelidine. (C) 2012 Elsevier Ltd. All rights reserved.

Vitamin E reduces antidepressant-related beta-adrenoceptor down-regulation in cultured cells. Comparable effects on St. John's wort and tricyclic antidepressant treatment

The mode of action of antidepressants is still a matter of debate. Acute inhibition of neurotransmitter reuptake in central neuronal synapses, followed by a down-regulation of central postsynaptic beta-adrenoceptor (beta-AR) numbers were consistently observed in vivo, while a reduction in surface beta-AR density was found in cell cultures. Effects of the tricyclic antidepressant desipramine (DMI) were abolished by vitamin E (alpha-TOC) in vitro as well as in vivo. Alpha-TOC interfered with antidepressant-induced changes of cellular plasma membrane properties and with recycling of beta-AR. St. John's wort (SJW) extract reduced beta-AR numbers in cultured cells to a similar extent as DMI or the selective serotonin re-uptake inhibitor fluoxetine. We chronically co-exposed cell cultures to SJW extract and to alpha-TOC. Receptor down-regulation following exposure to the plant extract was inhibited in the presence of alpha-TOC suggesting a mode of action of SJW extract comparable to that of synthetic antidepressants. Inhibition of cell proliferation by the plant extract was also significantly reduced by alpha-TOC.

The discovery of new 11beta-hydroxysteroid dehydrogenase type 1 inhibitors by common feature pharmacophore modeling and virtual screening

11beta-Hydroxysteroid dehydrogenase (11beta-HSD) enzymes catalyze the conversion of biologically inactive 11-ketosteroids into their active 11beta-hydroxy derivatives and vice versa. Inhibition of 11beta-HSD1 has considerable therapeutic potential for glucocorticoid-associated diseases including obesity, diabetes, wound healing, and muscle atrophy. Because inhibition of related enzymes such as 11beta-HSD2 and 17beta-HSDs causes sodium retention and hypertension or interferes with sex steroid hormone metabolism, respectively, highly selective 11beta-HSD1 inhibitors are required for successful therapy. Here, we employed the software package Catalyst to develop ligand-based multifeature pharmacophore models for 11beta-HSD1 inhibitors. Virtual screening experiments and subsequent in vitro evaluation of promising hits revealed several selective inhibitors. Efficient inhibition of recombinant human 11beta-HSD1 in intact transfected cells as well as endogenous enzyme in mouse 3T3-L1 adipocytes and C2C12 myotubes was demonstrated for compound 27, which was able to block subsequent cortisol-dependent activation of glucocorticoid receptors with only minor direct effects on the receptor itself. Our results suggest that inhibitor-based pharmacophore models for 11beta-HSD1 in combination with suitable cell-based activity assays, including such for related enzymes, can be used for the identification of selective and potent inhibitors.

CHARACTERIZATION OF SELECTIVE INHIBITION OF ADENYLYL CYCLASE ACTIVITY BY SMALL MOLECULE INHIBITOR NKY80

The nine membrane-bound isoforms of adenylyl cyclase (AC), via synthesis of the signaling molecule cyclic AMP (cAMP), are involved in many isoform specific physiological functions. Decreasing AC5 activity has been shown to have potential therapeutic benefit, including reduced stress on the heart, pain relief, and attenuation of morphine dependence and withdrawal behaviors. However, AC structure is well conserved, and there are currently no isoform selective AC inhibitors in clinical use. P-site inhibitors inhibit AC directly at the catalytic site, but with an uncompetitive or noncompetitive mechanism. Due to this mechanism and nanomolar potency in cell-free systems, attempts at ligand-based drug design of novel AC inhibitors frequently use P-site inhibitors as a starting template. One small molecule inhibitor designed through this process, NKY80, is described as an AC5 selective inhibitor with low micromolar potency in vitro. P-site inhibitors reveal important ligand binding “pockets” in the AC catalytic site, but specific interactions that give NKY80 selectivity are unclear. Identifying and characterizing unique interactions between NKY80 and AC isoforms would significantly aid the development of isoform selective AC inhibitors. I hypothesized that NKY80’s selective inhibition is conferred by AC isoform specific interactions with the compound within the catalytic site. A structure-based virtual screen of the AC catalytic site was used to identify novel small molecule AC inhibitors. Identified novel inhibitors are isoform selective, supporting the catalytic site as a region capable of more potent isoform selective inhibition. Although NKY80 is touted commercially as an AC5 selective inhibitor, its characterization suggests strong inhibition of both AC5 and the closely related AC6. NKY80 was also virtually docked to AC to determine how NKY80 binds to the catalytic site. My results show a difference between NKY80 binding and the conformation of classic P-site inhibitors. The selectivity and notable differences in NKY80 binding to the AC catalytic site suggest a catalytic subregion more flexible in AC5 and AC6 that can be targeted by selective small molecule inhibitors.

Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry

Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC 1.14.13.39) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC50 values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (Kd ≈ 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor–heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein–protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED50 values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies.

Stress and antidepressants differentially regulate neurotrophin 3 mRNA expression in the locus coeruleus.

The mechanisms by which stress and anti-depressants exert opposite effects on the course of clinical depression are not known. However, potential candidates might include neurotrophic factors that regulate the development, plasticity, and survival of neurons. To explore this hypothesis, we examined the effects of stress and antidepressants on neurotrophin expression in the locus coeruleus (LC), which modulates many of the behavioral and physiological responses to stress and has been implicated in mood disorders. Using in situ hybridization, we demonstrate that neurotrophin 3 (NT-3) is expressed in noradrenergic neurons of the LC. Recurrent, but not acute, immobilization stress increased NT-3 mRNA levels in the LC. In contrast, chronic treatment with antidepressants decreased NT-3 mRNA levels. The effect occurred in response to antidepressants that blocked norepinephrine uptake, whereas serotonin-specific reuptake inhibitors did not alter NT-3 levels. Electroconvulsive seizures also decreased NT-3 expression in the LC as well as the hippocampus. Ntrk3 (neurotrophic tyrosine kinase receptor type 3; formerly TrkC), the receptor for NT-3, is expressed in the LC, but its mRNA levels did not change with stress or antidepressant treatments. Because, NT-3 is known to be trophic for LC neurons, our results raise the possibility that some of the effects of stress and antidepressants on LC function and plasticity could be mediated through NT-3. Moreover, the coexpression of NT-3 and its receptor in the LC suggests the potential for autocrine mechanisms of action.

Cost-effectiveness of cognitive-behavioural therapy and drug interventions for major depression

Objective: Antidepressant drugs and cognitive-behavioural therapy (CBT) are effective treatment options for depression and are recommended by clinical practice guidelines. As part of the Assessing Cost-effectiveness - Mental Health project we evaluate the available evidence on costs and benefits of CBT and drugs in the episodic and maintenance treatment of major depression. Method: The cost-effectiveness is modelled from a health-care perspective as the cost per disability-adjusted life year. Interventions are targeted at people with major depression who currently seek care but receive non-evidence based treatment. Uncertainty in model inputs is tested using Monte Carlo simulation methods. Results: All interventions for major depression examined have a favourable incremental cost-effectiveness ratio under Australian health service conditions. Bibliotherapy, group CBT, individual CBT by a psychologist on a public salary and tricyclic antidepressants (TCAs) are very cost-effective treatment options falling below $A10 000 per disability-adjusted life year (DALY) even when taking the upper limit of the uncertainty interval into account. Maintenance treatment with selective serotonin re-uptake inhibitors (SSRIs) is the most expensive option (ranging from $A17 000 to $A20 000 per DALY) but still well below $A50 000, which is considered the affordable threshold. Conclusions: A range of cost-effective interventions for episodes of major depression exists and is currently underutilized. Maintenance treatment strategies are required to significantly reduce the burden of depression, but the cost of long-term drug treatment for the large number of depressed people is high if SSRIs are the drug of choice. Key policy issues with regard to expanded provision of CBT concern the availability of suitably trained providers and the funding mechanisms for therapy in primary care.

Studies on the synthesis of novel PP2A inhibitors and a GPCR detergent

Chapter 1 While targeting kinases in oncology research has been explored extensively, targeting protein phosphatases is currently in its infancy. However, a number of pharmaceutical companies are currently looking to expand their research efforts in this area. PP2A has been shown to down-regulate ERK5, a mitogen-activated protein kinase (MAPK) that has been shown to be important in driving the invasive phenotype of prostate cancer. Fostriecin and its related structural analogues PD 113,270 and 113,271 have been shown to inhibit a mitotic entry checkpoint in cell growth through the potent and selective inhibition of protein phosphatases PP1, PP2A, and PP4 (IC50 of 45 μM, 1.5 nM, and 3 nM respectively). Fostriecin is one of the most selective protein phosphatase inhibitors disclosed to date with a 104 fold selectivity for PP2A/PP4 versus PP1. Unfortunately, fostriecin and its analogues are very unstable, and this instability has effectively prevented them from being used as effective therapeutic leads. The microcystins and nodularins on the other hand, exhibit significant inhibitory activity against PP1 and PP2A (IC50 = 26 pM and 1.8 nM respectively), but their high toxicity has prevented any therapeutic application. Truncation of the ADDA chain from these polypeptides completely attenuates PP inhibitory activity. Simpler analogues incorporating the N-acylated ADDA chain and D-Ala retain moderate activity against PP1 and PP2A (IC50 = 1.0 μM and 0.17 μM respectively). The generation of a new series of fostriecin analogues to further expand its structure-activity relationship is envisaged with a view to creating new more stable PP2A inhibitors. It was hoped that by incorporating some of the more stable structural features of ADDA into fostriecin that stability and activity could be reconciled. With that in mind a series of PP2A inhibitors were synthesised and biologically evaluated. Chapter 2 GPCRs are an important area of research and are the targets of a quarter of the drugs on the market (2005). As a result, GPCRs continue to be at the forefront of research in both small and large drug companies. However one of the difficulties in studying this diverse class of membrane proteins is their tendency to denature in aqueous solution. As a result there is a pressing need to develop new detergents to solubilise, stabilise and crystallise GPCRs in their native form for further study. Cholesterol analogues have been shown to be important for stabilising membrane proteins and preventing their thermal inactivation. In addition the β2-adrenergic receptor, a GPCR membrane protein, has been crystallised in the active state with two cholesterol molecules bound between the I, II, III and IV helices of the protein. This appears to represent a distinct cholesterol binding pocket on the membrane protein that is speculated to be conserved across up to 44% of the rhodopsin class of GPCRs. CHOBIMALT is a cholesterol-based detergent that has been shown to exhibit promising GPCR-stabilising properties. When benchmarked against other cholesterol based detergents it was found to be superior to all others tested except for cholesteryl hemisuccinate.1 CHOBIMALT has an aggregation number of roughly 200 and forms 210 ± 30 kDa micelles, which are significantly larger than those of most detergents used for biological systems which is likely due to the packing constraints associated with CHOBMALT’s large polar headgroup.2 As a result, CHOBIMALT is used mostly as an additive to other commercially available detergents in order to decrease micelle size. A branched dimaltoside motif is common in recently synthesised detergents by Chae and co-workers. These detergents have shown promising detergent properties, for example the maltose neopentyl glycol (MNG) detergent synthesised by Chae. This branched dimaltoside detergent was shown to be able to solubilise and stabilise the very labile light harvesting complex I (LHI) from Rhodopsin capsulatus in its active form for 20 days with little loss of protein conformation.3 A cholesterol-based detergent was envisaged that combines the cholesterol framework of CHOBIMALT but replaces its linear tetrasaccharide with a branched dimaltoside. This detergent would then be investigated to assess its ability to solubilise, stabilise and crystallise GPCR proteins. This cholesterol-based detergent (shown below) was eventually synthesised in 9 linear steps from cholesterol.

Fluoxetine induces preventive and complex effects against colon cancer development in epithelial and stromal areas in rats

Fluoxetine (FIX) is a drug commonly used as antidepressant. However, its effects on tumorigenesis remain controversial. Aiming to evaluate the effects of FIX treatment on early malignant changes, we analyzed serotonin (5-HT) metabolism and recognition, aberrant crypt foci (ACF), proliferative process, microvessels, vascular endothelial growth factor (VEGF), and cyclooxygenase-2 (COX-2) expression in colon tissue. Male Wistar rats received a daily FLX-gavage (30 mg kg(-1)) and, a single dose of 1.2 dimethylhydrazine (DMH; i.p., 125 mg kg(-1)). After 6 weeks of FIX-treatment, our results revealed that FIX and nor-fluoxetine (N-FIX) are present in colon tissue, which was related to significant increase in serotonin (5-HT) levels (P < 0.05) possibly through a blockade in SERT mRNA (serotonin reuptake transporter; P < 0.05) resulting in lower 5-hydroxyindoleacetic acid (5-HIAA) levels (P < 0.01) and, 5-HT2C receptor mRNA expressions. FIX-treatment decreased dysplastic ACF development (P < 0.01) and proliferative process (P < 0.001) in epithelia. We observed a significant decrease in the development of malignant microvessels (P < 0.05), VEGF (P < 0.001), and COX-2 expression (P < 0.01). These findings suggest that FIX may have oncostatic effects on carcinogenic colon tissue, probably due to its modulatory activity on 5-HT metabolism and/or its ability to reduce colonic malignant events. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

The plasma membrane calcium pump, its role and regulation: New complexities and possibilities

Significant progress has been achieved in elucidating the role of the plasma membrane Ca2+-ATPase in cellular Ca2+ homeostasis and physiology since the enzyme was first purified and physiology since the enzyme was first purified and cloned a number of years ago. The simple notion that the PM Ca2+-ATPase controls resting levels of [Ca2+](CYT) has been challenged by the complexity arising from the finding of four major isoforms and splice variants of the Ca2+ pump, and the finding that these are differentially localized in various organs and subcellular regions. Furthermore, the isoforms exhibit differential sensitivities to Ca2+, calmodulin, ATP, and kinase-mediated phosphorylation. The latter pathways of regulation can give rise to activation or inhibition of the Ca2+ pump activity, depending on the kinase and the particular Ca2+ pump isoform. Significant progress is being made in elucidating subtle and more profound roles of the PM Ca2+-ATPase in the control of cellular function. Further understanding of these roles awaits new studies in both transfected cells and intact organelles, a process that will be greatly aided by the development of new and selective Ca2+ pump inhibitors. (C) 1999 Elsevier Science Inc.

Evaluation of CSF Neurotransmitters and Folate in 25 Patients with Rett Disorder and Effects of Treatment

Background: Rett disorder (RD) is a progressive neurodevelopmental entity caused by mutations in the MECP2 gene. It has been postulated that there are alterations in the levels of certain neurotransmitters and folate in the pathogenesis of this disease. Here we re-evaluated this hypothesis. Patients and Methods: We evaluated CSF folate, biogenic amines and pterines in 25 RD patients. Treatment with oral folinic acid was started in those cases with low folate. Patients were clinically evaluated and videotaped up to 6 months after therapy. Results: CSF folate was below the reference values in 32% of the patients. Six months after treatment no clinical improvement was observed. Three of the four patients with the R294X mutation had increased levels of a dopamine metabolite associated to a particular phenotype. Three patients had low levels of a serotonin metabolite. Two of them were treated with fluoxetine and one showed clinical improvement. No association was observed between CSF folate and these metabolites, after adjusting for the patients age and neopterin levels. Conclusion: Our results support that folinic acid supplementation has no significant effects on the course of the disease. We report discrete and novel neurotransmitter abnormalities that may contribute to the pathogenesis of RD highlighting the need for further studies on CSF neurotransmitters in clinically and genetically well characterized patients.

Antidepressant induced excessive yawning and indifference

Introduction Antidepressant induced excessive yawning has been described as a possible side effect of pharmacotherapy. A syndrome of indifference has also been described as another possible side effect. The frequency of those phenomena and their physiopathology are unknown. They are both considered benign and reversible after antidepressant discontinuation but severe cases with complications as temporomandibular lesions, have been described. Methods We report two unprecedented cases in which excessive yawning and indifference occurred simultaneously as side effects of antidepressant therapy, discussing possible physiopathological mechanisms for this co-occurrence. Case 1: A male patient presented excessive yawning (approximately 80/day) and apathy after venlafaxine XR treatment. Symptoms reduced after a switch to escitalopram, with a reduction to 50 yawns/day. Case 2: A female patient presented excessive yawning (approximately 25/day) and inability to react to environmental stressors with desvenlafaxine. Conclusion Induction of indifference and excessive yawning may be modulated by serotonergic and noradrenergic mechanisms. One proposal to unify these side effects would be enhancement of serotonin in midbrain, especially paraventricular and raphe nucleus.