Submitted by Cristiane Chim (cristiane.chim@ucpel.edu.br) on 2016-11-11T11:14:08Z No. of bitstreams: 1 MARCO SALEH RIBEIRO.pdf: 1233321 bytes, checksum: aebd2e7f49d852825cab51345cf5eebc (MD5) / Made available in DSpace on 2016-11-11T11:14:08Z (GMT). No. of bitstreams: 1 MARCO SALEH RIBEIRO.pdf: 1233321 bytes, checksum: aebd2e7f49d852825cab51345cf5eebc (MD5) Previous issue date: 2015-08-31 / Bipolar disorder is a psychiatric illness associated with alternate states of depression and mania/hypomania episodes. Glycogen synthase kinase 3β (GSK-3β) and synaptosomal-associated protein 25 (SNAP-25) are associated respectively, with the modulation of gene expression and neural plasticity in the pathogenesis of mood disorders. Curcumin, active compound extracted from Curcuma longa, has been described as a neuroprotective agent in neurodegenerative disorders, although their mechanisms of action are not completely elucidated. In this study we investigate the impact of ketamine-induced model of mania on GSK-3β and SNAP-25 immunocontent, and assess in this model the neuroprotective effect of Curcumin pretreatment. Rats received peanut oil (vehicle), curcumin 20 or 50 mg/kg (p.o.), once a day for 14 days. From the 8th to the 14th day the animals also received saline or ketamine (25 mg/kg i.p.), once a day. On the 15th day of treatment, the animals received a single injection of ketamine or saline and the locomotor activity was assessed in the open-field apparatus after 30 min. Immunodetection of the proteins pGSK-3β and SNAP-25 were evaluated by Western Blotting in the prefrontal cortex (PFC) and the hippocampus (HP). The administration of Curcumin in doses of 20 and 50 mg/kg prevented the hyperlocomotion induced by ketamine in the open-field test. In addition, both doses of Curcumin prevented the decrease in density of pGSK-3β and SNAP-25 ketamine-induced in PFC and HP. In conclusion, our results show that Curcumin prevents hyperlocomotion and presents a neuroprotective effect by restoring SNAP-25 density probably via modulation of GSK-3β. / O transtorno bipolar é uma doença psiquiátrica associada a estados alternados de episódios de depressão e mania / hipomania. O glicogênio sintase quinase 3β (GSK-3β) e proteína associada a sinaptossomas de 25 (SNAP-25) estão associados, respectivamente, com a modulação da expressão gênica e a plasticidade neural na patogênese de perturbações do humor. A curcumina, composto ativo extraído de Curcuma longa, tem sido descrito como um agente neuroprotetor em desordens neurodegenerativas, embora o seu mecanismo de ação não está completamente elucidado. Neste estudo foi investigado o impacto do modelo induzida por cetamina de mania em GSK-3β e SNAP-25, e avaliado neste modelo, o efeito neuroprotetor da curcumina pré-tratamento. Os ratos receberam óleo de amendoim (veículo), curcumina 20 ou 50 mg / kg (p.o.), uma vez por dia durante 14 dias. A partir do dia 8 ao dia 14 os animais também receberam solução salina ou cetamina (25 mg / kg i.p.), uma vez por dia. No 15º dia de tratamento, os animais receberam uma única injeção de cetamina ou uma solução salina e a atividade locomotora foi avaliada no aparelho campo aberto após 30 min. Imunodetecção das proteínas pGSK-3p e SNAP-25 foram avaliadas por Western Blotting no córtex pré-frontal (PFC) eo hipocampo (HP). A administração da curcumina, em doses de 20 e 50 mg / kg preveniu o hiperlocomoção induzida por cetamina no teste de campo aberto. Além disso, ambas as doses de curcumina evitou a diminuição da densidade de pGSK-3β e SNAP-25 em PFC e HP induzida por cetamina. Em conclusão, os nossos resultados mostram que a curcumina impede hiperlocomoção e apresenta um efeito neuroprotector, restaurando SNAP-25, provavelmente, através da modulação da densidade de GSK-3β.

Lithium has been used for more than 50 years as a primary therapy for bipolar affective disorder (BPD) and has proven highly effective for both acute and long-term phases of the disease. Unfortunately, the molecular and cellular mechanisms underlying the mood-stabilizing action of lithium for the treatment of BPD remains largely unknown. In an effort towards understanding the complexities of lithium's action in the nervous system, I have utilized the Drosophila neurological mutant Shudderer (Shu). Previous findings have suggested that the adult Shu phenotypes may be improved by providing a diet containing millimolar concentrations of lithium. Using well-established genetic techniques and behavioral paradigms I thoroughly characterized the Shu mutant phenotypes. I found that the mutant displays morphological and behavioral abnormalities indicative of dysregulated neuronal excitability that include: down-turning wings and indented dorsal thorax, defects in negative geotaxis, deficits in locomotion, abnormal sleep architecture and unusual patterns of leg-shaking behaviors upon recovery of ether anesthetics. Furthermore, I confirmed that lithium was able to significantly improve many aspects of Shu behaviors. Recombination-based mutation mapping in Shu revealed that the genetic lesion lies somewhere within the gene CG9907, which encodes the voltage-gated sodium channel á-subunit paralytic (para). Subsequent genetic experiments using para hypomorphic mutant alleles as well as a UAS-RNAi/GAL4 system showed that a reduction in sodium channel levels resulted in a drastic improvement of the mutant defects. Together, these data suggest that the lithium-responsive Shu mutant is likely a gain-of-function allele of para. Sequencing of the entire para coding region identified a missense mutation in a highly conserved region of the para coding sequence, in transmembrane segment S2 of homology domain III ((M1350I). To date, this is the first known discovery of a sodium channel mutant allele in Drosophila which causes hyperactivity. These data suggest that the Shu phenotypes are somehow caused by an increase in sodium channel activation. Lastly, I identified a number of genes likely to functionally interact with the Shu mutation. Of note, the Ca2+/calmodulin-activated Ser/Thr protein phosphatase alpha subunit gene CanA-14F is up-regulated in Shu and reduction of its activity suppresses the mutant phenotypes. Furthermore, a large percentage of genes encoding anti-microbial peptides (AMP) were also significantly up-regulated in Shu, possibly acting downstream of CanA-14F. A genetic deficiency screen looking for genes that alter the Shu phenotypes has identified that the gene Glutathione S-transferase S1 (Gsts1) suppresses the morphological and behavioral defects in the lithium-responsive mutant. Overall, these genes will help decipher how the gain-of-function sodium channel Shu mutation alters nervous system function. In addition, they will shed light on those mechanisms responsible for lithium's mood-stabilizing effects in the brain.

No description available.

MLK3 est une ser/thr MAP3K qui active la voie de signalisation des MAPKs dans différents types cellulaires. GSK3β interagit et active MLK3 en la phosphorylant sur le residue ser 792. Cependant, le rôle de MLK3 ainsi que l’interaction entre MLK3 et GSK3β n’ont pas été précédemment étudiés dans le muscle squelettique. La croissance post-natale du muscle et la régénération musculaire chez l’adulte sont dépendantes de l’accrétion de myonoyaux, un processus médié par les cellules satellites qui prolifèrent, se différencient puis fusionnent aux fibres préexistantes. Durant ma thèse, j’ai démontré que GSK3β agit en amont de MLK3 pour induire la prolifération des cellules satellites, et cela par l’activation de la voie de signalisation MLK3-p38γ MAPK. In vivo, les muscles de souris déficientes injectés par la CTX montrent une diminution du nombre de cellules satellites prolifératrices Pax7+/ki67+, ainsi qu’une accélération du processus de régénération. En conclusion, mes résultats évoquent un nouveau rôle de MLK3 dans le muscle squelettique pouvant servir pour vaincre les dystrophies musculaire. / MLK3 is a Ser/Thr MAP3K, which activates MAPKs signalling pathways in different cell types. The Ser/Thr kinase GSK3-β directly phosphorylate Ser 792 residue and activate MLK3. Since neither the role of MLK3, nor GSK3-β -MLK3 interaction have been previously investigated in muscle, the aim of my thesis was to elucidate their contribution in the regulation of muscle mass and physiology.Skeletal muscle post-natal growth and adult regeneration relies on satellite cell-mediated myonuclear accretion, during which, activated satellite cells, proliferate, differentiate and fuse with preexisting myotubes.I have demonstrated that in skeletal muscle, GSK3-β acts upstream of MLK3 to induce satellite cells proliferation through the induction of MLK3-p38γ MAPK signalling. Similarly, in vivo CTX-induced TA damage in MLK3 KO mice resulted in decreased number of proliferating Pax7+/ki67+ satellite cells, with a rapid muscle regeneration ability.These data suggest provide a yet unknown role of MLK3 in skeletal muscle tissue that could help in curing age-related muscle dystrophies.

The goal of this study was to identify novel drug repurposing opportunities in cancer by utilizing the off-target profiles of clinically relevant kinase inhibitors. This was based on the observation that the global target profiles of compounds are largely ignored and that many compounds have activity that cannot be explained by their cognate target alone. Additionally, by utilizing clinically relevant compounds, any results would hold a high potential for eventual clinical implementation. We utilized a systems pharmacology approach utilizing cell viability-based drug screening to identify compounds with beneficial off-target activity and then using chemical and phosphoproteomics in order to elucidate the mechanisms of action of these compounds. We found that tivantinib has off-target activity in NSCLC cells through inhibition of GSK3. Based on tivantinib’s ability to inhibit GSK3, we hypothesized that tivantinib would therefore have activity in acute myeloid leukemia (AML). We found that tivantinib had potent activity in AML through inhibition of GSK3. We also identified a highly synergistic combination with ABT-199 by drug synergy screening which was effective in HL60 cells and patient derived AML cells. We also found that the anaplastic lymphoma kinase (ALK) inhibitor, ceritinib, had activity across several ALK-negative lung cancer cell lines. We utilized integrated functional proteomics to identify the new targets and network-wide signaling effects. Combining pharmacological inhibitors and RNA interference revealed a polypharmacology mechanism involving the noncanonical targets IGF1R, FAK1, RSK1 and RSK2. Mutating the downstream signaling hub YB1 protected cells from ceritinib. Consistent with YB1 signaling being known to cause taxol resistance, combination of ceritinib with paclitaxel displayed strong synergy, particularly in cells expressing high FAK autophosphorylation, which we show to be prevalent in lung cancer. Together, we present a systems chemical biology platform for elucidating multikinase inhibitor mechanisms, synergistic drug combinations, mechanistic biomarker candidates and identifying novel drug repurposing opportunities.

<p>Multiple myeloma (MM) is a hematological tumor of plasma blast/plasma cell origin heterogeneous with respect to the morphological differentiation stage of the tumor cells, genetic alterations and course of disease. A challenge in MM research is to overcome resistance to therapy, which inevitably arises. In this thesis, we have used different strategies to sensitize MM cells to apoptosis and explored possible mechanisms of apoptotic control by the insulin-like growth factor-1 receptor (IGF-1R) survival pathway.</p><p>mTOR is a key molecule in the regulation of translation activated by survival signaling pathways in MM. We demonstrate that the mTOR-inhibitor rapamycin alone induced apoptosis in primary MM cells. In addition, rapamycin sensitized MM cells to apoptosis induced by dexamethasone, a glucocorticoid frequently used in MM therapy. MM survival factors IGF-1 and IL-6 could neither restore phosphorylation of the mTOR target p70S6K, nor cell growth inhibited by rapamycin and dexamethasone.</p><p>To study the regulation of inhibitors of apoptosis (IAP), we induced apoptosis and cell cycle arrest with dexamethasone and simultaneously abrogated IGF-1R signaling using the antagonistic antibody αIR3 or the selective IGF-1R inhibitor picropodophyllin (PPP). Dexamethasone transiently up-regulated c-IAP2. The subsequent down-regulation of c-IAP2 and XIAP was associated with the onset of apoptosis. c-IAP2 and XIAP levels further decreased when enhancing dexamethasone-induced apoptosis using αIR3 or PPP indicating a role for IAPs in regulating resistance to apoptosis in MM.</p><p>Finally, we explored glycogen synthase kinase (GSK)3 as a possible pro-apoptotic molecule and its role in regulating sensitization to apoptosis. We show that inhibition of GSK3 counteracts growth inhibition induced by dexamethasone alone and in combinatorial treatments with inhibitors against PI 3-kinase, mitogen-activated protein kinase (MEK), mTOR and IGF-1R. CT99021 also reversed cell cycle arrest induced by LY294002 or rapamycin. Importantly, the GSK3 inhibitor CT99021 sustained viability in untreated and dexamethasone-treated primary MM cells.</p>

Multiple myeloma (MM) is a hematological tumor of plasma blast/plasma cell origin heterogeneous with respect to the morphological differentiation stage of the tumor cells, genetic alterations and course of disease. A challenge in MM research is to overcome resistance to therapy, which inevitably arises. In this thesis, we have used different strategies to sensitize MM cells to apoptosis and explored possible mechanisms of apoptotic control by the insulin-like growth factor-1 receptor (IGF-1R) survival pathway. mTOR is a key molecule in the regulation of translation activated by survival signaling pathways in MM. We demonstrate that the mTOR-inhibitor rapamycin alone induced apoptosis in primary MM cells. In addition, rapamycin sensitized MM cells to apoptosis induced by dexamethasone, a glucocorticoid frequently used in MM therapy. MM survival factors IGF-1 and IL-6 could neither restore phosphorylation of the mTOR target p70S6K, nor cell growth inhibited by rapamycin and dexamethasone. To study the regulation of inhibitors of apoptosis (IAP), we induced apoptosis and cell cycle arrest with dexamethasone and simultaneously abrogated IGF-1R signaling using the antagonistic antibody αIR3 or the selective IGF-1R inhibitor picropodophyllin (PPP). Dexamethasone transiently up-regulated c-IAP2. The subsequent down-regulation of c-IAP2 and XIAP was associated with the onset of apoptosis. c-IAP2 and XIAP levels further decreased when enhancing dexamethasone-induced apoptosis using αIR3 or PPP indicating a role for IAPs in regulating resistance to apoptosis in MM. Finally, we explored glycogen synthase kinase (GSK)3 as a possible pro-apoptotic molecule and its role in regulating sensitization to apoptosis. We show that inhibition of GSK3 counteracts growth inhibition induced by dexamethasone alone and in combinatorial treatments with inhibitors against PI 3-kinase, mitogen-activated protein kinase (MEK), mTOR and IGF-1R. CT99021 also reversed cell cycle arrest induced by LY294002 or rapamycin. Importantly, the GSK3 inhibitor CT99021 sustained viability in untreated and dexamethasone-treated primary MM cells.

Methylglyoxal (MG) is a reactive dicarbonyl metabolite formed during glucose, protein and fatty acid metabolism. In hyperglycemic conditions, an increased MG level has been linked to the development of diabetes and the accompanying vascular inflammation encountered at both macro- and microvascular levels. The present study explores the mechanisms of MG-induced leukocyte recruitment in mouse cremasteric microvasculature. Biochemical and intravital microscopy studies performed suggest that administration of MG (25 and 50 mg/kg) to mouse cremaster muscle tissue induces dose-dependent leukocyte recruitment in cremasteric vasculature with 84-92% recruited cells being neutrophils. MG treatment up-regulated the expression of endothelial cell (EC) adhesion molecules P-selectin, E-selectin and intercellular adhesion molecule-1 (ICAM-1) via the activation of nuclear factor-κB (NF-κB) signalling pathway and contributed to the increased leukocyte rolling flux, reduced leukocyte rolling velocity, and increased leukocyte adhesion, respectively. The inhibition of NF-κB blunted MG-induced endothelial adhesion molecule expression and thus attenuated leukocyte recruitment. Further study of signalling pathways revealed that MG induced Akt-regulated transient glycogen synthase kinase 3 (GSK3) activation in ECs, which was responsible for NF-κB activation at early time-points (< 1 h). After MG activation for 1 h, the endothelial GSK3 activity was decreased due to the up-regulation of serum- and glucocorticoid-regulated kinase 1 (SGK1), which was responsible for maintaining NF-κB activity at later time-points. Silencing GSK3 or SGK1 attenuated P-selectin, E-selectin and ICAM-1 expression in ECs, and abated MG-induced leukocyte recruitment. SGK1 also promoted cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) activity which was partially involved in ICAM-1 expression. Silencing CREB blunted ICAM-1 expression while P-selectin and E-selectin levels remained unaffected. MG also induced GSK3 activation in isolated neutrophils after 30 min treatment, an effect that was not responsible for MG-elicited Mac-1 expression. These data suggest the sequential activation of GSK3 and SGK1 in ECs as the pivotal signalling mechanism in MG-elicited leukocyte recruitment. Additionally, MG-treatment led to uncoupling of endothelial nitric oxide synthase (eNOS) following MG-induced superoxide generation in ECs. MG triggered eNOS uncoupling and hypophosphorylation associated with superoxide generation and biopterin depletion in EA.hy926 ECs. In cremaster muscle, as well as in cultured murine and human primary ECs, MG increased eNOS monomerization and decreased 5,6,7,8-tetrahydroboipterin (BH4)/total biopterin ratio, effects that were significantly mitigated by supplementation of BH4 or its precursor sepiapterin but not by NG-nitro-L-arginine methyl ester (L-NAME) or 5,6,7,8-tetrahydroneopterin (NH4). These observations confirm that MG administration triggers eNOS uncoupling. In murine cremaster muscle, MG triggered the reduction of leukocyte rolling velocity and the increases in rolling flux, adhesion, emigration and microvascular permeability. MG-induced leukocyte recruitment was significantly attenuated by supplementation of BH4 or sepiapterin or suppression of superoxide by L-NAME confirming the role of eNOS uncoupling in MG-elicited leukocyte recruitment. MG treatment further decreased the expression of guanosine triphosphate cyclohydrolase I in murine primary ECs, suggesting the impaired BH4 biosynthesis caused by MG. Taken together, these data suggest that vascular inflammation and endothelial dysfunction occurring in diabetes may be linked to GSK3/SGK1 regulated adhesion molecule expression, as well as the uncoupling of eNOS evoked by elevated levels of MG. These findings not only provide a better understanding of the role of MG in the development of diabetic vascular inflammation, but also suggest the potential therapeutic targets for MG-sensitive endothelial dysfunction in diabetes.

Synaptic vesicle (SV) recycling in the presynapse is essential for the maintenance of neurotransmission. During mild stimulation clathrin-mediated endocytosis (CME) dominates, however during intense stimulation activity-dependent bulk endocytosis (ADBE) is the dominant form of membrane retrieval. The aim of this thesis was to determine how the signalling molecule GSK3 controlled ADBE, with the hypothesis that this enzyme was required at multiple stages of this endocytosis mode. I also hoped to identify a specific cargo for ADBE. I found that during intense action potential stimulation, a localised calcium increase is necessary for the activation of Akt, which inhibited GSK3. This activation was mediated via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. Furthermore, I found that phosphatidylinositol 4-kinaseIIα (PI4KIIα), a molecule whose abundance is regulated by GSK3, had a key role in ADBE. Specifically, I found that the absence of PI4KIIα accelerated CME but inhibited ADBE and that PI4KIIα controls CME and ADBE via distinct mechanisms. The PI4KIIα study revealed potential cross-talk between CME and ADBE. To determine whether modulation of either endocytosis mode impacts on the other, the retrieval of genetically-encoded reporters of SV cargo was monitored during intense stimulation during inhibition of either CME or ADBE. The recovery of almost all SV cargo was unaffected by ADBE inhibition but was arrested by abolishing CME. In contrast, VAMP4-pHluorin retrieval was perturbed by inhibiting ADBE and not by blocking CME. Knockdown of VAMP4 also arrested ADBE, indicating that in addition to being the first identified ADBE cargo, it is also essential for this endocytosis mode to proceed.

The tachykinin NK-3 receptor is a G-protein coupled receptor activated by mammalian tachykinin neuropeptides, which can modulate dopaminergic neurotransmission, and alter dopamine-mediated behaviors. The NK-3 receptor is currently under investigation as a novel therapeutic target for cocaine addiction. Our studies, as outlined in this dissertation, sought to determine if NK-3 receptors have a functional role in the acute as well as long-term behavioral effects of cocaine. Administration of NK-3 receptor agonists or antagonists potentiates or attenuates dopamine-mediated behaviors, respectively. Based on these findings, we hypothesized that blockade of neurokinin-3 receptors would alter acute and long-term behavioral responses to cocaine. We investigated whether acute and repeated administration of the NK-3 receptor antagonist SB 222200 altered hyperactivity induced by cocaine, and determined a possible mechanism involving dopamine D1 receptors in the striatum. We also determined whether NK-3 receptor blockade altered the development and expression of behavioral sensitization after repeated cocaine administration. Lastly, we investigated whether modulation of behavioral effects of acute and repeated cocaine by NK-3 receptors involved GSK3 phosphorylation in the nucleus accumbens. As described in this dissertation, we show that acute administration of the NK-3 receptor antagonist SB 222200 before a cocaine injection attenuated stereotypic responses produced by cocaine. Repeated administration of SB 222200 enhanced stereotypic activity produced by either cocaine or a low dose of SKF 82958 (0.125 mg/kg, i.p.) when administered seven days later. Dopamine receptor binding studies were performed to determine the mechanism of enhanced stereotypic responses. Binding studies showed a 19.7% increase in dopamine D1 receptor density in the striatum seven days later after repeated SB 222200 administration. These findings demonstrate that acute blockade of NK-3 receptors attenuated cocaine-induced behaviors in agreement with previous studies. Furthermore, these studies also show novel effects of repeated blockade of NK-3 receptors, which causes subsequent enhancement of cocaine and dopamine D1 receptor-mediated behaviors, possibly resulting from dopamine D1 receptor up-regulation in the striatum. In order to determine a role of NK-3 receptors in the development of cocaine-induced behavioral sensitization, the NK-3 receptor antagonist SB 222200 (2.5 or 5 mg/kg, s.c.) was administered prior to daily cocaine injections for 5 days. After a 7-day drug-free period, behavioral responses to a cocaine challenge were measured. Repeated administration of cocaine for 5 days induced a sensitized response upon a cocaine challenge 7 days later. Administration of SB 222200 prior to daily cocaine attenuated the development of behavioral sensitization. Moreover, administration of SB 222200 prior to the cocaine challenge blocked the expression of behavioral sensitization. These findings demonstrate that NK-3 receptor activity is involved in the development and expression of behavioral sensitization to cocaine. Lastly, we examined GSK3 phosphorylation in the nucleus accumbens induced by acute and repeated cocaine administration and determined if phosphorylation was altered by NK-3 receptor blockade. Similar to the drug administration regimens used in the behavioral studies, the NK-3 receptor antagonist SB 222200 was administered 30 mins prior to an acute cocaine injection. The nucleus accumbens was examined for changes in GSK3 phosphorylation by Western blot analysis. Increases in phosphorylation of the isoforms, GSK3α and GSK3β in the nucleus accumbens were detected 20 mins after an acute injection of cocaine. NK-3 receptor blockade prior to cocaine administration did not alter the cocaine-induced increase in GSK3 phosphorylation. Similar to the behavioral sensitization studies, SB 222200 was administered prior to repeated cocaine for 5 days, and 7 days later GSK3 phosphorylation was measured after a subsequent cocaine challenge. In contrast to the increases in GSK3α and GSK3β in the nucleus accumbens after an acute cocaine injection, no regulation of GSK3 phosphorylation was found after prior repeated cocaine administration and cocaine challenge. Administration of SB 222200 prior to repeated cocaine produced an increase in GSK3α and GSK3β phosphorylation after a cocaine challenge. Collectively, these data point to involvement of NK-3 receptor activity in changes in the phosphorylation of GSK3 in the nucleus accumbens produced by cocaine. In summary, functional involvement of NK-3 receptors in acute and long-term behavioral effects of cocaine was investigated. In agreement with previous findings, studies in this dissertation demonstrate that acute blockade of NK-3 receptors attenuates cocaine-induced behaviors. In addition, we found novel effects of repeated blockade of NK-3 receptors on cocaine-induced hyperactivity. There is enhancement of subsequent cocaine and dopamine D1 receptor-mediated behaviors possibly due to dopamine D1 receptor up-regulation in the striatum. NK-3 receptor activity was shown to be involved in long-term behavioral effects of cocaine and molecular changes in GSK3 phosphorylation in the nucleus accumbens. Blockade of NK-3 receptors prevented the development and expression of behavioral sensitization to cocaine and also blocked the changes in the phosphorylation of GSK3 in the nucleus accumbens. This dissertation has demonstrated a role of NK-3 receptors in modulating acute as well long-term cocaine-induced behavioral hyperactivity. Therefore, there is potential clinical relevance of NK-3 receptors in cocaine abuse and dependence as a therapeutic target for treatment, which warrants further characterization in future preclinical and clinical investigations. / Pharmacology