Stefano Pieretti

Istituto Superiore di Sanità | ISS · National Centre for Drug Research and Evaluation

Objectives Interleukin (IL) 17s cytokines are key drivers of inflammation that are functionally dysregulated in several human immune-mediated inflammatory diseases (IMIDs), such as rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease (IBD). Targeting these cytokines has some therapeutic benefits, but issues associated with low therap...

Rubiscolin-6 (amino acid sequence: YPLDLF) is a selective δ-opioid receptor peptide isolated from spinach Rubisco. Its synthetic analogue, peptide YPMDIV is the most potent described so far for its increased opioid activity, thus in this work it was considered as lead compound for the design of twelve new analogues e.g. LMAS1-12. Firstly all the no...

In this study, 1H-pyrazole-3-carboxylic acids related to the cannabinoid type 1 (CB1) receptor antagonist rimonabant were amidated with valine or tert-leucine, and the resulting acids were further diversified as methyl esters, amides, and N-methyl amides. In vitro receptor binding and functional assays demonstrated a wide series of activities relat...

The discovery of efficacious and safe analgesics with reduced side effects is the foremost challenge in the pain field. In this work, we report the in vitro and in vivo evaluation of linear and cyclic analogues of biphalin with the aim to complete the series of structural modifications previously applied in the development of opioid peptides incorp...

In the quest for novel therapeutic agents from plants, the choice of extraction solvent and technique plays a key role. In this study, the possible differences in the phytochemical profile and bioactivity (antioxidant and enzyme inhibitory activity) of the Alstonia boonei leaves and stem bark extracted using water, ethyl acetate and methanol, and d...

Recently, mixed opioid/NOP agonists came to the spotlight for their favorable functional profiles and promising outcomes in clinical trials as novel analgesics. This study reports on two novel chimeric peptides incorporating the fragment Tyr-c[D-Lys-Phe-Phe]Asp-NH2 (RP-170), a cyclic peptide with high affinity for µ and κ opioid receptors (or MOP a...

Peripherally active tetrapeptides as selective κ opioid receptor (KOR) agonists have been prepared in good overall yields and high purity following solid-phase peptide synthesis via Fmoc protection strategy. Structural modifications at the first and second position of the lead compound FF(D-Nle)R-NH 2 (FE200041) were contemplated with aromatic side...

Marianecci C, Rinaldi F, Di Marzio L, et al. Int J Nanomedicine. 2014;9(1):635–651. The Editor and Publisher of International Journal of Nanomedicine wish to retract the published article. Concerns were raised regarding the alleged duplication of TEM images in Figure 1. Specifically, Figure 1A, F1, appears to have been duplicated with the same ima...

Common bean (Phaseolus vulgaris) represents one of the most famous foods with antiobesity activity showing a significant efficacy against fat accumulation, insulin resistance and dyslipidaemia. In this work, two Italian varieties of common bean, i.e., Tondino del Tavo and Cannellino Bio, from the centre of Italy were studied to characterise their p...

Introduction: Chronic pain is related to gastrointestinal (GI) functions because food components affect inflammation and pain through their action on the GI immune and/or neural system and because many analgesics interact with the gut to alter its structure and function. Immunoglobulin G4 (IgG4) are food-specific antibodies resulting from exposure...

Formyl peptide receptor type 2 (FPR2/ALX) belongs to the formyl peptide receptors (FPRs) family clustered on chromosome 19 and encodes a family of three Class A of G protein-coupled receptors (GPCRs). A short N-terminal region, an NPXXY motif in transmembrane (TM) region 7 and an E/DRY motif that bridges TM3 and TM6 stabilizing inactive receptor co...

Phenylpropanoid glycosides are a class of natural substances of plant origin with interesting biological activities and pharmacological properties. This study reports the antinociceptive and anti-inflammatory effects of calceolarioside A, a phenylpropanoid glycoside previously isolated from various Calceolaria species. In models of acute nociceptio...

Menopause is the last step in the reproductive history of a woman. The ovaries stop producing hormones and the body reacts by lowering its functions, including the cognitive one. Phytoestrogens are plant products with the estrogen-like activity which are able to mimic many of estrogen’s functions. The aim of the present experiment was to study the...

Grape pomace is commonly considered a waste product of monovarietal red wine production. Methods: HPLC-DAD analysis was performed to determine the polyphenol and flavonoid contents of all the extracts obtained from Montepulciano d’Abruzzo red wine and grape skins whereas, GC-MS was applied to the determination of fatty acid composition in grape see...

This study aims to investigate the pharmacokinetic properties, antinociceptive, and antioxidant activities of rubiscolin-6, soymorphin-6, and their C-terminal amides. Rubiscolin-6 and soymorphin-6 are two exhorphins derived from spinach and soybean, respectively. The four peptides were synthesized following Fmoc-SPPS strategy to give the final pept...

The kappa opioid receptor (KOR) represents an attractive target for the development of drugs as potential antidepressants, anxiolytics and analgesics. A robust computational approach may guarantee a reduction in costs in the initial stages of drug discovery, novelty and accurate results. In this work, a virtual screening workflow of a library consi...

The use of α-amino-γ lactam of Freidinger (Agl) may serve as an impressive method to increase the biological stability of peptides and an appropriate tool to elucidate their structure-activity relationships. The endomorphin-2 (EM-2) and [D-Ala², des-Leu⁵] enkephalin amide (DAPEA) are two linear opioid tetrapeptides agonists of MOR and MOR/DOR respe...

Glycyrrhiza glabra, commonly known as liquorice, contains several bioactive compounds such as flavonoids, sterols, triterpene, and saponins; among which, glycyrrhizic acid, an oleanane-type saponin, is the most abundant component in liquorice root. Diabetic peripheral neuropathy is one of the major complications of diabetes mellitus, leading to pai...

High-altitude exposure leads to many physiological challenges, such as weight loss and dehydration. However, little attention has been posed to the role of nutrition and ethnic differences. Aiming to fulfill this gap, five Italian trekkers and seven Nepalese porters, all males, recorded their diet in diaries during a Himalayan expedition (19 days),...

Menopause is the last step in the reproductive history of a woman. The ovaries stop producing hormones and the body reacts by lowering its functions, including the neuronal one. Phytoestrogens are plant products with estrogen-like activity able to affect many body functions. The aim of the present experiment was to study the effects of 30 days of r...

Crocus sativus L. (saffron) appears to own neuroprotective effects on cognitive impairment in patients with Alzheimer’s disease (AD). The purpose of this work is to review evidence and mechanisms of saffron-induced therapeutic outcomes and measureable cognitive benefits in AD. The literature was reviewed, and preclinical and clinical studies were...

Pyridazine derivatives, such as arylpiperazinylalkyl pyridazinones, display antinociceptive effects to thermal and chemical stimuli. Here, we extended our previous knowledge on the pharmacological profile of 4-amino-6-methyl-2-(3-(4-(4-methylcyclohexa-1,3-dien-1-yl)piperazin-1-yl)propyl)-5-vinylpyridazin-3(2H)-one, here referred as ET1, paving the...

The aim of this work is to investigate the pharmacokinetic properties, antinociceptive and antioxidant activities of rubiscolin-6, soymorphin-6 and their C-terminal amides; The four peptides were synthesized following Fmoc-SPPS strategy to give the final peptides in excellent overall yields and purity following analytical RP-HPLC analysis. None of...

Viscum album L. (Mistletoe) is one of the most famous plants in many countries utilized for several purposes. The current study aimed to describe chemical profiles and biological activities of homogenizer‐assisted extract (HAE) and ultrasound‐assisted extract (UAE)) of V. album parts (leaf, fruits, and seeds). Antioxidant (radical scavenging, reduc...

s Rutabaga (Brassica Napus L.) belonging to Brassicaceae family, is a rich source of polyphenols and glucosinolates. Its consumption in human diet is highly appreciated for its nutritional contribution and health benefits. Brassica Napus L. is recognized as the world's most widely grown temperate oilseed crop containing erucic acid for industrial a...

Sweet pepper is a typical type of Capsicum annuum from Abruzzo region, recognized as a traditional and local product, traditionally cultivated in the town of Altino (Abruzzo region, Italy). The aim of this study is to compare the sweet type of peppers from Altino with the hot pepper cultivated in the same area, in order to delineate their different...

Kynurenine (kyn) and kynurenic acid (kyna) are well-defined metabolites of tryptophan catabolism collectively known as "kynurenines", which exert regulatory functions in host-microbiome signaling, immune cell response, and neuronal excitability. Kynurenine containing peptides endowed with opioid receptor activity have been isolated from natural org...

Four novel fluorinated cyclic analogues of biphalin with excellent to modest binding affinity for μ-, δ- and κ-receptors were synthesized. The cyclic peptides have a combination of piperazine or hydrazine linker with or without a xylene bridge. Among the ligands, MACE3 demonstrated a better activity than biphalin after intravenous administration, a...

The endocannabinoid system represents an integrated neuronal network involved in the control of several organisms’ functions, such as feeding behavior. A series of hybrids of 5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (mimonabant), a well-known inverse agonist of the type-1 cannabinoid receptor...

The object of the study was to estimate the long-lasting effects induced by ammonium glycyrrhizinate (AG) after a single administration in mice using animal models of pain and inflammation together with biochemical and docking studies. A single intraperitoneal injection of AG was able to produce anti-inflammatory effects in zymosan-induced paw edem...

Gout is a paradigm of acute, self-limiting inflammation caused by the deposition of monosodium urate (MSU) crystals within intra-and/or peri-articular areas, leading to excruciating pain, joint swelling and stiffness. The infiltration of leukocytes drives the inflammatory response and remains an attractive target for therapeutic intervention. In th...

In order to obtain novel pharmacological tools and to investigate a multitargeting analgesic strategy, the CB1 and CB2 cannabinoid receptor agonist JWH-018 was conjugated with the opiate analgesic oxycodone or with an enkephalin related tetrapeptide. The opioid and cannabinoid pharmacophores were coupled via spacers of different length and chemical...

Here, we report the chemical synthesis of two DPDPE analogues 7a (NOVA1) and 7b (NOVA2). This entailed the solid-phase synthesis of two enkephalin precursor chains followed by a CuI-catalyzed azide-alkyne cycloaddition, with the aim of improving in vivo analgesic efficacy versus DPDPE. NOVA2 showed good affinity and selectivity for the μ-opioid rec...

Aging is related to a number of functional and morphological changes leading to progressive decline of the biological functions of an organism. Reactive Oxygen Species (ROS), released by several endogenous and exogenous processes, may cause important oxidative damage to DNA, proteins, and lipids, leading to important cellular dysfunctions. Te imbal...

In this work we report the application of the Ring-Closing Metathesis (RCM) to the preparation of two cyclic olefin bridged analogues of biphalin (Tyr-D-Ala-Gly-Phe-NH-NH<-Phe<-Gly<-D-Ala<-Tyr), using the second generation Grubbs’ catalyst. The resulting cis and trans cyclic isomers were identified, fully characterized and tested in vitro at μ (MOR...

Ibuprofen is one of the non-steroidal anti-inflammatory drugs (NSAIDs) widely used to treat pain conditions. NSAIDs encounter several obstacles to passing across biological membranes. To overcome these constraints, we decided to study the effects of a new pH-sensitive formulation of niosomes containing Polysorbate 20 derivatized by Glycine and load...

Opioid peptides and opiate drugs such as morphine, mediate their analgesic effects, but also undesired side effects, mostly through activation of the mu opioid receptor. However, delta- and kappa-opioid receptors can also contribute to the analgesic effects of opioids. Recent findings showed that simultaneous activation of multiple opioid receptors...

In this paper we reported the synthesis and the in vitro and the in vivo biological evaluation of linear pseudo-peptides incorporating the N-VGCC blocker tripeptide Phe-NMe-Leu-Tyr(OBz)-NtBu and the biphalin pharmacophore Tyr-d-Ala-Gly-Phe. The novel sequences have been designed by using amino acids of different length to join the two pharmacophore...

Nociceptin /Orphanin FQ Peptide" receptor (NOPr) is a G-protein-coupled receptor with the nociceptin/orphanin FQ peptide (N/OFQ) as endogenous agonist. It is expressed in the nervous system as well as in some non-neural tissues. Its activation has pronociceptive effect at the supraspinal level, whereas at the spinal level it produces nociceptive ef...

In a number of animal and human studies, males and females have been shown to differ in their responsiveness to noxious stimuli. Indeed, sex and gender are important factors in the modulation of pain since the neonatal age. Chronic pain is more common in women than in men, with some painful diseases commonly reported only among women. It is becomin...

In this work we enhanced the ring lipophilicity of biphalin introducing a xylene moiety, thus obtaining three cyclic regioisomers. Novel compounds have similar in vitro activity as the parent compound but one of these (6a) shows a remarkable increase of in vivo antinociceptive effect. Nociception tests have disclosed its significant high potency an...

Given the poor bioavailability of curcumin, its antinociceptive effects are produced after chronic intravenous administration of high doses, while poly (d,l-lactide-co-glycolide)-loaded vesicles (PLGA) can improve drug delivery. This paper investigates the antinociceptive effects of curcumin-loaded PLGA nanovesicles (PLGA-CUR) administered via intr...

D-Pen2,D-Pen5 enkephalin (DPDPE) is one of the most selective synthetic peptide agonist targeting the δ-opioid receptor. Three cyclic analogues of DPDPE containing a xylene bridge in place of disulphide bond have been synthesized and fully characterized as opioid receptors agonists. The in vitro activity was investigated showing a good affinity of...

pH-sensitive nonionic surfactant vesicles (niosomes) by polysorbate-20 (Tween-20) or polysorbate-20 derivatized by glycine (added as pH sensitive agent), were developed to deliver Ibuprofen (IBU) and Lidocaine (LID). For the physical-chemical characterization of vesicles (mean size, size distribution, zeta potential, vesicle morphology, bilayer pro...

Cannabinoid (CB) and opioid systems are both involved in analgesia, food intake, mood and behavior. Due to the co-localization of µ-opioid (MOR) and CB1 receptors in various regions of the central nervous system (CNS) and their ability to form heterodimers, bivalent ligands targeting to both these systems may be good candidates to investigate the e...

Background and purpose: The diterpenoids carnosol (CS) and carnosic acid (CA) from Salvia spp. exert prominent anti-inflammatory activities but their molecular mechanisms remained unclear. Here we investigated the effectiveness of CS and CA in inflammatory pain and the cellular interference with their putative molecular targets. Experimental appr...

Fentanyl is a powerful opiate analgesic typically used for the treatment of severe and chronic pain, but its prescription is strongly limited by the well-documented side-effects. Different approaches have been applied to develop strong analgesic drugs with reduced pharmacologic side-effects. One of the most promising is the design of multitarget dr...

There is much evidence to suggest that gender is an important lactor in the modulation of pain. Literature data strongly suggest that men and women differ in their responses to pain: They are more variable in women than men, with increased pain sensitivity and many more painful diseases commonly reported among women. Gender differences in pharmacol...

The behavioral analysis of laboratory mice plays a key role in several medical and scientific research areas, such as biology, toxicology, pharmacology, and so on. Important information on mice behavior and their reaction to a particular stimulus is deduced from a careful analysis of their movements. Moreover, behavioral analysis of genetically mod...

The analysis of locomotion in laboratory animals plays a crucial role in many scientific research areas. In fact, important information on animals' behavior and their reaction to a particular stimulus is deduced from a careful analysis of their movements. The techniques commonly adopted to support such analysis have many limitations, which make the...

N-type voltage-dependent Ca(2+) channels (CaV 2.2) are located at nerve endings in the central and peripheral nervous systems and are strongly associated with the pathological processes of cerebral ischemia and neuropathic pain. CaV 2.2 blockers such as the ω-conotoxin MVIIA (Prialt) are analgesic and have opioid-sparing effects. With the aim to de...

Two novel opioid analogues have been designed by substituting the native d-Ala residues in position 2,2' of biphalin with two residues of d-penicillamine or l-penicillamine and by forming a disulfide bond between the thiol groups. The so-obtained compound 9 containing d-penicillamines showed excellent μ/δ mixed receptor affinities (K i (δ) = 5.2 nM...

Evaluating the behavior of mice and rats has substantially contributed to the progress of research in many scientific fields. Researchers commonly observe recorded video of animal behavior and manually record their observations for later analysis, but this approach has several limitations. The authors developed an automated system for tracking and...

Radio frequency identification (RFID) technology is more and more adopted in a wide range of applicative scenarios. Nevertheless, in many applications, commercial and general-purpose solutions can be unsuitable as in the case of the tracking of small-size living animals for the behavior analysis. In such a case, the whole RFID hardware, as well as...

Marianecci C, Rinaldi F, Di Marzio L, et al. Int J Nanomedicine. 2014;9(1):635–651. The Editor and Publisher of International Journal of Nanomedicine wish to retract the published article. Concerns were raised regarding the alleged duplication of TEM images in Figure 1. Specifically, Figure 1A, F1, appears to have been duplicated with the same imag...

Panel Turbiscan. Transmission and back scattering profile of formulation 1 (A), formulation 1 loading AG (B), formulation 2 (C), formulation 2 loading AG (D), formulation 3 (E), and formulation 3 loading AG (F) using Turbiscan Lab® Expert. Abbreviation: AG, monoammonium glycyrrhizinate solution (1% w/v).

Natural residues of the dimeric opioid peptide Biphalin were replaced by the corresponding homo- β3 amino acids. The derivative 1 containing hβ3 Phe in place of Phe showed good μ- and δ- receptor affinities (Kiδ = 0.72 nM; Kiμ = 1.1 nM), and antinociceptive activity in vivo together with an increased enzymatic stability in human plasma.

AM94 is a fluorinated analog of biphalin with non-hydrazine linker that has an in vitro affinity for μ-opioid and δ-opioid receptors tenfold higher than biphalin. Furthermore, in vivo evaluation in rats showed that AM94 has in hot plate test - after both intracerebroventricular and intravenous administrations - a greater and more durable efficacy t...

Animal tracking and animal behavior analysis have a crucial impact in biomedical disciplines to study new pathologies and effects of new drugs. There are several solutions, based on different technologies such as GPS, radar, and vision, designed to obtain animals tracking systems, but they are effective mainly in presence of large size animals and...

Today there is a very great deal of interest among members of the global natural products community in investigating new plant constituents. Recent studies demonstrate that liquorice extracts are useful in the treatment of dermatitis, eczema, and psoriasis, with an efficacy comparable to that of corticosteroids. In this work, niosomes made up of su...

Purpose: Anabolic androgenic steroids (AAS) are synthetic androgen-like compounds that are abused in sport communities despite their adverse effects. Nerve growth factor (NGF) influences neuronal differentiation and survival, and it also mediates higher brain functions such as learning and memory. Changes in NGF expression have been implicated in...

The effect of ochratoxin A on growth and aflatoxin production by Aspergillus parasiticus was investigated. High concentrations had a significant effect, causing stimulation of aflatoxin production. Key words: Aspergillus parasiticus, aflatoxin production, ochratoxin A.

In guinea-pig ileum (GPI), the chemotactic peptide N-formyl-Met-Leu-Phe-OH (fMLF) possesses spasmogenic properties through the activation of formyl peptide receptors (FPRs). Despite this, the mediators involved remain to be elucidated. fMLF (1nM-1μM) induced a dose-dependent contraction of GPI (EC(50)=24nM), that is blocked by pre-treatment with th...

Pediatric pain management underwent many changes since the undertreatment of pain in children was reported in the literature in 1980. Increasing data also suggest that long-term behavioural effects can be observed in children, following pain episodes as early as in the neonatal period. Therefore, the knowledge about safe and effective management of...

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

A number of pyridazinone derivatives bearing an arylpiperazinylalkyl chain were synthesized and tested icv in a model of acute nociception induced by thermal stimuli in mice (tail flick). The most interesting and potent compound in this series was 6a, which showed an ED(50) = 3.5 microg, a value about 3-fold higher with respect to morphine by the s...

Following a ligand-based drug design approach, a potent mixed formyl peptide receptor 1 (FPR1) and formyl peptide receptor-like 1 (FPRL1) agonist (14a) and a potent and specific FPRL1 agonist (14x) were identified. These compounds belong to a large series of pyridazin-3(2H)-one derivatives substituted with a methyl group at position 6 and a methoxy...

The potential anxiolytic and anti-depressive activity of CMP1 was studied in the elevated plus-maze test and in the forced swimming test. Furthermore, CMP1 sedative activity was evaluated in pentobarbital treated animals; the effect of CMP1 on spontaneous motor activity (total locomotion) was also evaluated. Our data show that CMP1, at doses that d...

DMSO is one of the most common solvents used experimentally to dissolve hydrophobic substances for in vivo and in vitro purposes. A wide range of pharmacological effects exerted by DMSO has been documented in both animal and human experimental models. However, only a few and sometimes contrasting data about the effects of DMSO in animal models of n...

A series of 4-amino-5-vinyl-3(2H)-pyridazinones and analogues were synthesized and their antinociceptive effect was evaluated in the mouse abdominal constriction model. Several of the novel compounds showed ED(50) values in the range 6-20mg/kg/sc and demonstrated to be able to completely protect all the treated animals from the effect of the noxiou...

Reelin is an extracellular matrix protein, secreted by GABAergic interneurons, that provides a signal for neural plasticity. A downregulation of reelin may be a factor to be considered in the study of major psychiatric disorders. The heterozygous reeler mouse model, thus, may be important to reveal those alterations in behavioral phenotype produced...

The significance of behavioral neuroscience and the validity of its animal models of human pathology largely depend on the possibility to replicate a given finding across different laboratories. Under the present test and housing conditions, this axiom fails to resist the challenge of experimental validation. When several mouse strains are tested o...

Phosphodiesterase 4 (PDE4) inhibitors are effective anti-inflammatory drugs, although some adverse effects are observed in animals and humans. These effects have forced researchers to find new PDE4 inhibitors with less adverse effects. We recently reported the synthesis of novel heterocyclic-fused pyridazinones that inhibit PDE4. As a first step in...

In this study we investigated how the peptides derived from the glucocorticoid-inducible protein annexin 1 are able to alter the nociceptive threshold of mice. The effects of the annexin1 fragment 2-26 (Anxa1(2-26)) on nociceptive threshold were studied using both chemical (formalin test) and thermal (hot plate and tail flick test) nociceptive stim...

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A series of 1-methyl-5-(pyrazol-3- and -5-yl- and 1, 2, 4-triazol-3- and 5-yl)-1, 2, 3, 6-tetrahydropyridine derivatives structurally related to arecoline were synthesized and evaluated on M(1), M(2), and M(3) muscarinic receptors using [(3)H] pirenzepine and [(3)H] NMS as ligands. The binding affinity depended on the position and size of the subst...

Nociceptin/orphanin FQ (NC) and its receptor (OP4) have been implicated in the regulation of various functions including nociception. [Nphe1]NC(1–13)NH2 (Nphe) is a selective OP4 antagonist which prevents the pronociceptive effects of supraspinal NC and causes per se a naloxone-insensitive antinociceptive effect. In the present study, we tested Nph...

Previous data indicate that intracerebroventricular administration of agonists for mu- and delta-opioid receptors induces limbic seizures in rats, but no data are reported in rabbits. We found that the mu- and delta-opioid peptides [D-Ala(2)-N,Me-Phe(4)-Gly(5)-ol]enkephalin (DAMGO), beta-endorphin and deltorphin II, induced EEG non-convulsive hippo...

The neurobiological and behavioral facets of adolescence have been poorly investigated in relation to the vulnerability to psychostimulants. Periadolescent (33-43 days) and adult (>70 days) Sprague-Dawley rats underwent a 3-day treatment history with D-amphetamine (AMPH) at 0, 2, or 10 mg/kg (once a day). After a short 5-day-long withdrawal interva...

The effects induced by nociceptin on morphine-induced release of dopamine (DA), 3,4-dihydroxyphenilacetic acid (DOPAC) and homovanillic acid (HVA) in the nucleus accumbens and nucleus caudate were studied in rats by microdialysis with electrochemical detection. Nociceptin administered intracerebroventricularly (i.c.v.) at doses of 2, 5 and 10 nmol/...

The effects induced by orphanin FQ (OFQ) on morphine-induced dopamine (DA), 3,4-dihydroxyphenilacetic acid (DOPAC) and homovanillic acid (HVA) release in the nucleus accumbens were studied in rats by using microdialysis with electrochemical detection. Morphine administered intraperitoneally (i.p., 2, 5 and 10 mg/kg) dose-dependently increased DA an...