Search

The Lagovirus genus comprises both pathogenic viruses as European brown hare syndrome virus (EBHSV‐ GII.1) and rabbit hemorrhagic disease viruses (RHDV‐GI.1 and RHDV2‐GI.2), that principally infect European brown hares (Lepus europeaus) and European rabbits (Oryctolagus cuniculus), respectively, causing severe necrotic hepatitis, spleen enlargement and disseminated haemorrhage. This genus includes also non‐pathogenic agents, such as rabbit calicivirus (RCV‐E1 – GI.3) and the non‐pathogenic hare Lagovirus, provisionally named hare calicivirus (HaCV – GII.2). The latter had been identified for the first time in 2012 in the gut contents and faeces of healthy young hares raised in a breeding farm. In this study, we further investigated the presence of HaCV by testing the intestinal tract of 621 wild hares collected between 2010 and 2018 in Northern and Central Italy, and in 2011 in Austria, Germany and Spain. These wild hares were found dead for causes other than EBHS or were healthy hares shot during the hunting season. Forty‐three out of 322 hare samples from Italy and 14 out of 299 samples from Austria and Germany were positive for HaCV–GII.2 by RT‐PCR using universal primers for lagoviruses and primers specific for HaCV. Sequence analysis of the full capsid protein gene conducted on 12 strains representative of different years and locations indicated that these viruses belong to the same, single cluster as the prototype strain initially identified at the hares’ farm (HaCV_Bs12_1). The relatively high level of genetic variation (88% nt identity) within this cluster suggests HaCVs may have been circulating widely in Europe for some time.

ObjectivesThe outbreak of COVID-19 posed the issue of urgently identifying treatment strategies. Colchicine was considered for this purpose based on well-recognised anti-inflammatory effects and potential antiviral properties. In the present study, colchicine was proposed to patients with COVID-19, and its effects compared with ‘standard-of-care’ (SoC).MethodsIn the public hospital of Esine, northern Italy, 140 consecutive inpatients, with virologically and radiographically confirmed COVID-19 admitted in the period 5–19 March 2020, were treated with ‘SoC’ (hydroxychloroquine and/or intravenous dexamethasone; and/or lopinavir/ritonavir). They were compared with 122 consecutive inpatients, admitted between 19 March and 5 April 2020, treated with colchicine (1 mg/day) and SoC (antiviral drugs were stopped before colchicine, due to potential interaction).ResultsPatients treated with colchicine had a better survival rate as compared with SoC at 21 days of follow-up (84.2% (SE=3.3%)vs63.6% (SE=4.1%), p=0.001). Cox proportional hazards regression survival analysis showed that a lower risk of death was independently associated with colchicine treatment (HR=0.151 (95% CI 0.062 to 0.368), pConclusionThis proof-of-concept study may support the rationale of use of colchicine for the treatment of COVID-19. Efficacy and safety must be determined in controlled clinical trials.

Carnivores are potential carriers of agents that infect their prey species, even though they themselves are not susceptible, such as the lagovirus that causes European brown hare syndrome (EBHS), a severe disease of brown hares endemic in Europe. During our wildlife surveillance in Lombardy, we identified an EBHS outbreak in a protected area by both virological analyses (sandwich ELISA and RT-PCR) of the target organs from one dead hare and serological examinations (competitive ELISA) of captured animals. Since four red foxes were contemporarily hunted in the same area, we examined their organs by RT-PCR for the EBHS agent (EBHSv). The intestinal content of one fox tested positive, while the fox’ other organs (liver, spleen, and mesenteric lymph nodes), and all of the samples from the remaining three foxes, tested negative. Moreover, in the gastrointestinal content of the positive fox, we found food debris that was genetically identified as being of hare origin. The competitive ELISA test for EBHSv antibodies gave negative results in all of the fox sera. Genetic analyses of the EBHSv amplicons obtained by RT-PCR in the hare and the fox indicated a full homology (99.9 % nucleotide and 100 % amino acid identity). These results support the fact that red fox, as other predators, feeding on EBHSv infected hares may have genetic prints of the virus in their gut contents. Even if we did not prove that lagovirus particles remained infective in the excreted feces and, thus, contaminated the ground in the outbreak area, these eventualities cannot be excluded, and we could at least conclude that red foxes might assume a potential role in the indirect transmission of lagovirus, as EBHSv.

Branched peptides have been found to be useful in several research fields however their synthesis and purification is complicated. Here we present a novel and facile synthesis of tetra branched derivatives of nociceptin/orphanin FQ (N/OFQ). Three N/OFQ tetra branched derivatives were prepared using novel cores ( PWT1 , PWT2 and PWT3 ) containing a maleimido moiety. [Cys 18 ]N/OFQ-NH 2 was linked to the cores via thiol-Michael reaction characterized by high yield and purity of the desired final product. In the electrically stimulated mouse vas deferens PWT-N/OFQ derivatives mimicked the inhibitory action of the natural sequence showing similar maximal effects and 3 fold higher potencies. The NOP selective antagonist SB-612111 antagonized the effects of N/OFQ and PWT derivatives with similar p K B values (8.02–8.48). In vivo after supraspinal administration PWT2-N / OFQ stimulated food intake in mice mimicking the action of N/OFQ. Compared to the natural peptide PWT2-N / OFQ was 40 fold more potent and elicited larger effects. These findings suggest that the PWT chemical strategy can be successfully applied to biologically active peptides to generate, with unprecedented high purity and yield, tetra branched derivatives displaying an in vitro pharmacological profile similar to that of the natural sequence associated, in vivo, to increased potency and effectiveness.

Background and Purpose Nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor antagonists have been proposed as a novel therapeutic approach to Parkinson's disease. Main limitations of previous studies were the use of structurally similar compounds and the evaluation of their acute effects only. We report here on the acute and long-term antiparkinsonian effects of the novel compound 2-[3-[4-(2-chloro-6-fluoro-phenyl)-piperidin-1-ylmethyl]-2-(morpholine-4-carbonyl)-indol-1-yl]-acetamide (NiK-21273) in comparison with the potent and selective NOP receptor antagonist SB-612111. Experimental Approach Basic pharmacological properties of NiK-21273 were studied in cell lines and isolated tissues (mouse and rat vas deferens). Antiparkinsonian effects were studied in reserpinized mice and 6-hydroxydopamine hemilesioned rats under both acute and chronic administration protocols. Key Results In vitro, NiK-21273 behaved as a potent (pA2 7.7) and selective NOP receptor antagonist. In vivo, it reduced hypokinesia in reserpinized mice at 0.1 and 1 but not 10 mg·kg−1, whereas SB-612111 (0.01–1 mg·kg−1) provided a dose-dependent antiparkinsonian effect. NiK-21273 ameliorated motor performance in 6-hydroxydopamine hemilesioned rats at 0.5 and 5 but not 15 mg·kg−1. SB-612111 replicated these effects in the 0.01–1 mg·kg−1 range without loss of efficacy. Both antagonists synergized with L-DOPA at subthreshold doses. Chronic administration of NiK-21273 provided delayed improvement in baseline activity at 0.5 and 1.5 mg·kg−1, although tolerance to the higher dose was observed. Conversely, SB-612111 (1 mg·kg−1) maintained its effects over time without modifying baseline activity. Conclusions and Implications NOP receptor antagonists provide motor benefit in parkinsonism models although the ‘therapeutic’ window and long-term effects may vary between compounds.

Background and Purpose Intrathecally (i.t.) administered nociceptin/orphanin FQ (N/OFQ) evokes antinociceptive effects in rodents. Recent studies in monkeys demonstrated that i.t. co-application of N/OFQ and morphine elicits synergistic antinociceptive actions suggesting mixed N/OFQ peptide (NOP) and μ opioid receptor agonists as innovative spinal analgesics. Thus, novel N/OFQ related peptides were synthesized in order to identify and pharmacologically characterize a mixed NOP/ μ opioid receptor agonist. Experimental Approach The following in vitro assays were used: calcium mobilization in cells expressing the human NOP or classical opioid receptors and chimeric G proteins, receptor and [35S]-GTPγS binding, [35S]-GTPγS binding in rat spinal cord membranes, guinea pig ileum bioassay. In vivo experiments were performed in monkeys using the tail withdrawal assay. Key Results From calcium mobilization studies [Dmt1]N/OFQ(1–13)-NH2 was selected as the most potent and least selective compound. The mixed NOP/opioid full agonist activity and high affinity of [Dmt1]N/OFQ(1–13)-NH2 was confirmed at human recombinant receptors in receptor binding, calcium mobilization and/or [35S]-GTPγS binding studies, at rat spinal cord receptors in [35S]-GTPγS binding experiments, and at guinea pig receptors inhibiting neurogenic contractions in the ileum. In vivo in the tail withdrawal assay in monkeys i.t. [Dmt1]N/OFQ(1–13)-NH2 was able to elicit robust and long-lasting antinociceptive effects. Conclusions and Implications Collectively, these results demonstrate that [Dmt1]N/OFQ(1–13)-NH2 behaves as NOP/opioid receptor universal agonist and substantiate the suggestion that such mixed ligands are worthy of development as innovative spinal analgesics.

The aim of the present study was to investigate the pharmacological activity of Pronetupitant, a novel compound designed to act as prodrug of the NK1 antagonist Netupitant. In receptor binding experiments Pronetupitant displayed high selectivity for the NK1 receptor. In a calcium mobilization assay performed on CHONK1 cells Pronetupitant (100 nM, 15 min preincubation) behaved as an NK1 antagonist more potent than Netupitant (pK(B) 8.72 and 7.54, respectively). In the guinea pig ileum bioassay Pronetupitant antagonized the contractile effect of SP showing a similar potency as Netupitant (pK(B)≈9). Similar results were obtained with 5 min preincubation time while at 2 min only Pronetupitant produced significant effects. In vivo in mice the intrathecal injection of 0.1 nmol SP elicited the typical scratching, biting and licking (SBL) nociceptive response. This effect of SP was dose dependently (0.1-10 mg/kg) antagonized by Pronetupitant given intravenously 2 h before the peptide. Superimposable results were obtained using Netupitant. Pharmacokinetic studies performed in rats demonstrate that Pronetupitant, after i.v. administration, is quickly (few minutes) and completely converted to Netupitant. Collectively the present results indicated that Pronetupitant acts in vitro as selective NK1 antagonist more potent than Netupitant. However based on the short half-life measured for Pronetupitant in rats, the in vivo action of Pronetupitant can be entirely interpreted as due to its conversion to Netupitant.

The novel NK(1) receptor ligand Netupitant has been characterized in vitro and in vivo. In calcium mobilization studies CHO cells expressing the human NK receptors responded to a panel of agonists with the expected order of potency. In CHO NK(1) cells Netupitant concentration-dependently antagonized the stimulatory effects of substance P (SP) showing insurmountable antagonism (pK(B) 8.87). In cells expressing NK(2) or NK(3) receptors Netupitant was inactive. In the guinea pig ileum Netupitant concentration-dependently depressed the maximal response to SP (pK(B) 7.85) and, in functional washout experiments, displayed persistent (up to 5h) antagonist effects. In mice the intrathecal injection of SP elicited the typical scratching, biting and licking response that was dose-dependently inhibited by Netupitant given intraperitoneally in the 1-10mg/kg dose range. In gerbils, foot tapping behavior evoked by the intracerebroventricular injection of a NK(1) agonist was dose-dependently counteracted by Netupitant given intraperitoneally (ID(50) 1.5mg/kg) or orally (ID(50) 0.5mg/kg). In time course experiments in gerbils Netupitant displayed long lasting effects. In all the assays Aprepitant elicited similar effects as Netupitant. These results suggest that Netupitant behaves as a brain penetrant, orally active, potent and selective NK(1) antagonist. Thus this molecule can be useful for investigating the NK(1) receptor role in the control of central and peripheral functions. Netupitant has clinical potential in conditions such as chemotherapy induced nausea and vomiting, in which the blockade of NK(1) receptors has been demonstrated valuable for patients.

The novel NK(1) receptor ligand Netupitant has been characterized in vitro and in vivo. In calcium mobilization studies CHO cells expressing the human NK receptors responded to a panel of agonists with the expected order of potency. In CHO NK(1) cells Netupitant concentration-dependently antagonized the stimulatory effects of substance P (SP) showing insurmountable antagonism (pK(B) 8.87). In cells expressing NK(2) or NK(3) receptors Netupitant was inactive. In the guinea pig ileum Netupitant concentration-dependently depressed the maximal response to SP (pK(B) 7.85) and, in functional washout experiments, displayed persistent (up to 5h) antagonist effects. In mice the intrathecal injection of SP elicited the typical scratching, biting and licking response that was dose-dependently inhibited by Netupitant given intraperitoneally in the 1-10mg/kg dose range. In gerbils, foot tapping behavior evoked by the intracerebroventricular injection of a NK(1) agonist was dose-dependently counteracted by Netupitant given intraperitoneally (ID(50) 1.5mg/kg) or orally (ID(50) 0.5mg/kg). In time course experiments in gerbils Netupitant displayed long lasting effects. In all the assays Aprepitant elicited similar effects as Netupitant. These results suggest that Netupitant behaves as a brain penetrant, orally active, potent and selective NK(1) antagonist. Thus this molecule can be useful for investigating the NK(1) receptor role in the control of central and peripheral functions. Netupitant has clinical potential in conditions such as chemotherapy induced nausea and vomiting, in which the blockade of NK(1) receptors has been demonstrated valuable for patients.

Nociceptin/orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ peptide (NOP) receptor. Recently knockout rats for the NOP receptor gene (NOP(-/-)) have been generated; these animals were used in the present study to investigate their emotional (open field, elevated plus maze, and forced swimming test), locomotor (drag and rotarod test), and nociceptive (plantar and formalin test) phenotypes in comparison with their NOP(+/+) littermates. In addition, N/OFQ sensitivity has been assessed in electrically stimulated vas deferens tissues taken from NOP(+/+) and NOP(-/-) rats. In the elevated plus maze and forced swimming tests NOP(-/-) rats showed anxiety- and anti-depressant-like phenotype, respectively. No differences were found in the open field test. NOP(-/-) rats outperformed their NOP(+/+) littermates in two motor behaviour assays. Genetic ablation of the NOP receptor gene produced a statistically significant increase in nociceptive behaviour of the mutant rats in the formalin test. Finally, in the electrically stimulated rat vas deferens taken from NOP(+/+) tissues, N/OFQ inhibited in a concentration-dependent manner the electrically induced twitches while the peptide was inactive in tissues taken from NOP(-/-) animals. These results, in line with previous findings obtained with selective NOP receptor antagonists in mice and rats and with mouse knockout studies, clearly indicate that endogenous N/OFQ-NOP receptor signalling plays an important role in controlling anxiety- and mood-related behaviours, exercise-driven locomotor activity and nociception. These observations are relevant for defining the therapeutic indications (and contraindications) of NOP receptor antagonists.

In this study we provided a pharmacological characterization of the recently synthesized nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) antagonist 1-[1-Cyclooctylmethyl-5-(1-hydroxy-1-methyl-ethyl)-1,2,3,6-tetrahydro-pyridin-4-yl]-3-ethyl-1,3-dihydro-benzoimidazol-2-one (GF-4) and investigated its antiparkinsonian properties. GF-4 inhibited N/OFQ binding to CHO(hNOP) cell membranes (pK(i) 7.46), and antagonized N/OFQ effects in a calcium mobilization assay and electrically stimulated isolated tissues (pK(B) 7.27-7.82), showing a approximately 5-fold selectivity over classical opioid receptors. In vivo, GF-4 dually modulated stepping activity in wild-type mice, causing facilitation in the 0.01-10mg/kg dose range and inhibition at 30mg/kg. These effects were mediated by NOP receptors since GF-4 was ineffective in NOP receptor knock-out mice. Antiparkinsonian properties of GF-4 were investigated in 6-hydroxydopamine hemilesioned rats. GF-4 ameliorated akinesia, bradykinesia and overall gait ability in the 0.1-10mg/kg dose range, but inhibited motor activity at 30mg/kg. To investigate the circuitry underlying motor facilitating and inhibitory effects of GF-4, microdialysis coupled to behavioral testing (akinesia test) was performed. An anti-akinetic dose of GF-4 (1mg/kg) reduced glutamate (GLU) and enhanced GABA release in SNr, while the pro-akinetic dose of GF-4 (30mg/kg) evoked opposite effects. Moreover, the anti-akinetic dose of GF-4 reduced GABA and increased GLU release in ventro-medial thalamus, the pro-akinetic dose decreasing GABA without affecting GLU release in this area. We conclude that GF-4 is an effective NOP receptor antagonist able to attenuate parkinsonian-like symptoms in vivo via inhibition of the nigro-thalamic pathway.