Worsening of the Toxic Effects of (±) Cis -4,4′-DMAR Following Its Co-Administration with (±) Trans -4,4′-DMAR: Neuro-Behavioural, Physiological, Immunohistochemical and Metabolic Studies in Mice

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/4.0/).4,4’-Dimethylaminorex (4,4’-DMAR) is a new synthetic stimulant, and only a little information has been made available so far regarding its pharmaco-toxicological effects. The aim of this study was to investigate the effects of the systemic administration of both the single (±)cis (0.1–60 mg/kg) and (±)trans (30 and 60 mg/kg) stereoisomers and their co-administration (e.g., (±)cis at 1, 10 or 60 mg/kg + (±)trans at 30 mg/kg) in mice. Moreover, we investigated the effect of 4,4′-DMAR on the expression of markers of oxidative/nitrosative stress (8-OHdG, iNOS, NT and NOX2), apoptosis (Smac/DIABLO and NF-κB), and heat shock proteins (HSP27, HSP70, HSP90) in the cerebral cortex. Our study demonstrated that the (±)cis stereoisomer dose-dependently induced psychomotor agitation, sweating, salivation, hyperthermia, stimulated aggression, convulsions and death. Conversely, the (±)trans stereoisomer was ineffective whilst the stereoisomers’ co-administration resulted in a worsening of the toxic (±)cis stereoisomer effects. This trend of responses was confirmed by immunohistochemical analysis on the cortex. Finally, we investigated the potentially toxic effects of stereoisomer co-administration by studying urinary excretion. The excretion study showed that the (±)trans stereoisomer reduced the metabolism of the (±)cis form and increased its amount in the urine, possibly reflecting its increased plasma levels and, therefore, the worsening of its toxicity.Peer reviewedFinal Published versio

HelixComplex snail mucus exhibits pro-survival, proliferative and pro-migration effects on mammalian fibroblasts.

Snail mucus is a mixture of active substances commonly thought to have healthy properties for the treatment of skin disorders. Although snail mucus is an ingredient of several cosmetic and para-pharmaceutic products, a comprehensive characterization of chemical composition and biological effects are still missing. Crude purified extracts from Helix aspersa muller mucus (HelixComplex®) were prepared and, after chemical characterization, tested on in vitro experimental models. Differently from what expected, HelixComplex® was characterized by the presence of small amounts of glycolic acid and allantoin. By using different in vitro assays on fibroblast cultures, we found that HelixComplex® lacked of cytotoxicity, protected cells from apoptosis (p<0.05) and, importantly, was able to significantly induce cell proliferation and migration through direct and indirect mechanisms. These effects were associated to morphological changes, cytoskeleton re-organization and release of cytokines. In conclusion, our findings suggest that snail mucus biological effects are attributable to cell proliferation and migration, and pave the way for further investigating snail mucus potential as therapeutic agent

Gliomi a lento accrescimento del cervelletto. Aspetti clinici e prognostici

Analisi del rapporto tra diagnosi istopatologia e sopravvivenza nei gliomi a lento accrescimento in FCP in età pediatrica e nell'adutlo

Effects of glucosamine and nucleotide association on fibroblast: extracellular matrix gene expression

Glucosamine (Gluc) is a drug used as an anti-inflammatory in moderate forms of knee arthrosis. A further off label use of Gluc is in the anti-aging treatments associated with Polideoxirybonucleotide (PDRN) through intra-dermal injection for a procedure called bio-stimulation. An unexpected effect on cultured dermal fibroblasts, during an experimental study on the gene activation in aesthetic bio-stimulation, was observed. The results have potential application in orthopaedic medical therapy. Fibroblast primary cultures were carried out, seeding cells on a layer of Gluc or PDRN alone or in combination for 24 h. Real Time-PCR was performed to investigate several gene expressions. The MMP13 and the IGF-I gene expression in fibroblast cultures were strongly inhibited after 24 h of incubation with the association of Gluc and PDRN, whereas Gluc and PDRN alone produced a modest inhibition of IGF-I and an activation of MMP13. MMP13 is present in osteoarthritic cartilage and this enzyme plays a significant role in cartilage collagen degradation. IGF1 is involved in growth and development and is successfully used in tissue-engineering for cartilage repair. Based on the reported data, we infer that the association of Gluc and PDRN has a potential application in cartilage therapy. Additional basic science and clinical studies are needed to confirm this preliminary report

Circadian and circannual rhythmicity in the occurrence of subarachnoid hemorrhage

BACKGROUND AND PURPOSE: Inconsistent data are available on the temporal pattern of onset of subarachnoid hemorrhage (SAH). We investigated the possible influence of vascular risk factors. METHODS: Of a consecutive series of 217 cases of SAH, precise determination (within 30 minutes) of the time of symptom onset was possible in 199 (91.7%). Partial Fourier series with up to six harmonics were applied to hourly and monthly data, and the best-fitting curves for circadian and annual rhythmicity were calculated. The amplitude-MESOR (rhythm-adjusted mean over the time period analyzed) ratio was used as a measure of temporal variability. RESULTS: In the total population, a significant circadian pattern of occurrence was demonstrated with major peaks in the morning (approximately 9 AM) and evening (approximately 9 PM) hours and a nocturnal trough (approximately 3 AM). Younger, male, and hypertensive subjects had lower amplitude-MESOR ratios; smokers had no significant rhythmicity. The annual pattern showed a 6-month periodicity with two major peaks in March and September and minor differences in the subgroups studied. CONCLUSIONS: Our study indicates that the temporal distribution in onset of SAH may be influenced by variable combinations of environmental and vascular risk factors

Standard of care, controversies and innovations in the medical treatment of Severe Traumatic Brain Injury (STBI)

Severe traumatic brain injury (STBI) is characterized by a primary injury which cannot be reversed and a secondary injury that can be prevented or reversed. Management of STBI patients in intensive care mainly aims at preventing the secondary injury. Treatment aims to: reducing ICP pressure (that can result in an ischemic insult); avoiding hypotension, hyperthermia, or hypoxemia; maintaining a normal electrolytes homeostasis; treating the Autonomic dysfunction syndrome, coagulopathies, Acute Kidney Injury and maintaining an adequate nutrition. Many treatment protocols are already well established, while many others are still debated. Moreover, new frontiers in STBI management are represented by the neurovascular regeneration and neurorestoration which are showing very promising results even if most of them still need a clinical validation. In this paper we review standard of care, controversies and innovations in the medical treatment of STBI

JEDI (jugular entrapment, dilated ventricles, intracranial hypertension) syndrome: a new clinical entity? A case report

Patients with idiopathic intracranial hypertension are frequently obese women with normal/slit ventricles. Patients with high-pressure hydrocephalus, instead, present enlarged ventricles. We describe a 63-year-old woman with signs and symptoms of intracranial hypertension. Brain MRI revealed hydrocephalus. Venous Doppler ultrasound showed external compression of the omohyoid muscles on the internal jugular veins. During jugular vein decompression, intracranial pressure dropped from 18 to 6 mmHg. Patient is asymptomatic at 2-year follow-up, with decreased brain ventricles. These findings could represent a novel form of high-pressure hydrocephalus that can be successfully treated without a CSF shunt. We called this syndrome JEDI (jugular entrapment dilated ventricles intracranial hypertension)

The utility of in vitro metabolism studies coupled to liquid chromatography-tandem mass spectrometry for the selection of diagnostic markers of novel psychoactive substances: the case of 4,4’-DMAR

para-Methyl-4-methylaminorex (4,4’-DMAR) is a synthetic, substituted oxazoline derivative and analogue of the scheduled aminorex and 4-methylaminorex (4-MAR), both of which originally designed and synthesized as appetite suppressants. The psychostimulant properties of these substances are due to the interaction with dopamine transporter (DAT), serotonin transporter (SERT) and norepinephrine transporter (NET). Chemically, because of the two chiral centers in the oxazoline ring, 4,4’-DMAR exist as two different racemic (±) –cis and (±) –trans mixture or four distinct enantiomers; recent evidence suggests that only the cis isomers show a biological activity; furthermore, if cis and trans isomers are administered in combination, the effects of the cis isomer are enhanced. A recently validated method was used to detect the main metabolites in rat plasma and brain tissues and it allowed to identify four metabolites, due to hydroxylation, oxidation, hydrolysis and deamination. (Lucchetti, et al., 2017) Here we describe the in vitro metabolism of cis-4,4’-DMAR, investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on samples obtained after incubation with pooled human liver microsomes (HLM) and recombinant isoforms of cytochrome P450 and uridine diphospho glucuronosyl-transferase, with the aim of identifying the most suitable diagnostic marker(s) of intake, to be targeted in human urine in case of voluntary assumption. Following incubation with HLM, no metabolite was detected for the trans isomer, while six metabolites were found for cis-4,4’-DMAR as a result of mono-hydroxylation, di-hydroxylation and oxidation of the hydroxylated derivatives. The main cytochrome P450 isoforms involved in metabolism of this substance seems to be cytochrome P450 2D6 (CYP2D6), which is a polymorphic one. The chromatograms also shows the presence of a small amount of trans-4,4’-DMAR, probably due to the incubation with HLM or the exposure to heat. The same analysis was performed on the racemic mixture and it shows that this association leads to a lower amount of metabolites. This result suggests that the trans isomer could exert an inhibitory effect on the cis metabolism

C subunit of F1/FO-ATP synthase as target for preventing the detrimental effect of myocardial ischemia/reperfusion injury

Objective: The pathophysiological effects of coronary heart diseases are imputable to the hurtful consequences of ischemia-reperfusion injury (IRI). The opening of a large pore in the mitochondrial membrane, namely, the mitochondrial permeability transition pore (mPTP), is widely recognized as the final step of IRI and is responsible for mitochondrial and cardiomyocyte death. We provided evidences that c subunit of the F1/FO-ATP synthase (FFAS) owns a pivotal role in mPTP formation and activity and thus we sought to test a new mPTP opening inhibitor directed against the c subunit, namely IB13, for the treatment of IRI in ex-vivo model of myocardial infarction

Metabolic profile of the synthetic drug 4,4′-dimethylaminorex in urine by LC–MS-based techniques: selection of the most suitable markers of its intake

Purpose: In this study, the phase I and II metabolic pathways of 4,4′-dimethylaminorex were characterized to select the marker(s) of intake allowing the unequivocal identification of this novel psychoactive substance in urine samples. Methods: The metabolic profile of 4,4′-dimethylaminorex was characterized using both in vitro and in vivo models. In detail, for the in vitro experiments, either pooled human liver microsomes or recombinant cytochrome P450 isoforms were selected, whereas the in vivo investigation was performed on male mice ICR (CD-1®). Sample preparation included enzymatic hydrolysis followed by liquid/liquid extraction. The instrumental analysis was performed by ultra-high-performance liquid chromatography coupled to either high- or low-resolution tandem mass spectrometry. Results: Five metabolic products were isolated only for the cis-isomer: the phase I metabolic reactions included hydrolysis, carboxylation, hydroxylation, and carbonylation. CYP2D6 was the principal isoenzyme involved, and the incubation in the presence of different allelic variants showed significant alteration on the metabolic profile. Once formed, the phase I metabolites underwent extensive conjugation. Not only the most abundant compounds detected, but also those with the most extended window of detection, were the carboxylated and the hydroxylated metabolites. These analytes together with the parent compound were selected as the most suitable markers of intake. Conclusions: Knowledge of the metabolic profiles of the new drugs is essential for their fast identification. Phase I and phase II metabolites of 4,4′-dimethylaminorex were identified and selected as markers of intake, to be considered as the most suitable analytical targets in forensic toxicology