L. Di Cesare Mannelli

Local anaesthetic activity of β-caryophyllene ☆

Abstract In this work we studied the local anaesthetic activity of β-caryophyllene, one of the main components of clove oil obtained from the dried flower-buds of Syzygium aromaticum (Myrtaceae family). We compared its activity to a chemically related compound, caryophyllene oxide. Anaesthetic activity was evaluated in vivo in the rabbit conjunctival reflex test and in vitro in a rat phrenic nerve-hemidiaphragm preparation. β-Caryophyllene (10 −4 –1 μg/ml), but not caryophyllene oxide, was able to reduce drastically, in a dose-dependent manner, the electrically evoked contractions of the rat phrenic hemidiaphragm. In the rabbit, conjunctival reflex test treatment with a solution of β-caryophyllene (10–1000 μg/ml) allowed a dose-dependent increase in the number of stimuli necessary to provoke the reflex. As in the in vitro results, caryophyllene oxide was ineffective also in the in vivo test. In conclusion, these data evidence the local anaesthetic activity of β-caryophyllene, which appears to be strictly dependent on its chemical structure.

Palmitoylethanolamide is a disease-modifying agent in peripheral neuropathy : pain relief and neuroprotection share a PPAR-alpha-mediated mechanism

Neuropathic syndromes which are evoked by lesions to the peripheral or central nervous system are extremely difficult to treat, and available drugs rarely joint an antihyperalgesic with a neurorestorative effect. N-Palmitoylethanolamine (PEA) exerts antinociceptive effects in several animal models and inhibits peripheral inflammation in rodents. Aimed to evaluate the antineuropathic properties of PEA, a damage of the sciatic nerve was induced in mice by chronic constriction injury (CCI) and a subcutaneous daily treatment with 30 mg kg−1 PEA was performed. On the day 14, PEA prevented pain threshold alterations. Histological studies highlighted that CCI induced oedema and an important infiltrate of CD86 positive cells in the sciatic nerve. Moreover, osmicated preparations revealed a decrease in axon diameter and myelin thickness. Repeated treatments with PEA reduced the presence of oedema and macrophage infiltrate, and a significant higher myelin sheath, axonal diameter, and a number of fibers were observable. In PPAR-α null mice PEA treatment failed to induce pain relief as well as to rescue the peripheral nerve from inflammation and structural derangement. These results strongly suggest that PEA, via a PPAR-α-mediated mechanism, can directly intervene in the nervous tissue alterations responsible for pain, starting to prevent macrophage infiltration.

Acetyl-l-carnitine increases artemin level and prevents neurotrophic factor alterations during neuropathy

Damages to the nervous system are the primarily cause of neuropathy and chronic pain. Current pharmacological treatments for neuropathic pain are not able to prevent or revert morphological and molecular consequences of tissue injury. On the other hand, many neurotrophins, like nerve growth factor (NGF), paired off restorative effects with hyperalgesia. Interestingly, the glial cell line-derived neurotrophic factors GDNF and Artemin (ARTN) seem to support neuron survival and to normalize abnormal pain behaviour. In the present research protein levels of NGF, GDNF and ARTN were evaluated in a rat model of peripheral neuropathy, the chronic constriction injury (CCI). NGF was increased by CCI in the ipsilateral dorsal root ganglia (DRG), in the spinal cord and in the periaqueductal grey matter (PAG). On the contrary, ARTN was decreased bilaterally in DRG, spinal cord and PAG. GDNF levels decreased in ipsilateral DRG, whereas the constriction did not modify its expression in the central nervous system districts. Repeated treatments with the antihyperalgesic and neuroregenerative compound acetyl-l-carnitine (ALCAR; 100 mgkg(-1) i.p. twice daily for 15 days) was able to prevent the increase of NGF levels. In conditions of pain relief ALCAR normalized peripheral and central alterations of GDNF and ARTN levels. Characteristically, sham animals that underwent the same ALCAR treatment, showed increased levels of ARTN both in the DRG and in the spinal cord. These data offer a new point of view on the mechanism of the antihyperalgesic as well as the neuroprotective effect of ALCAR.

The neuropathy-protective agent acetyl-l-carnitine activates protein kinase C-γ and MAPKs in a rat model of neuropathic pain

The gamma isoform of protein kinase C (PKCgamma) is an injury-activated intracellular modulator that boosts neuronal activity in algesic and neuroregenerative signalling pathways. Acetyl-L-carnitine (ALCAR), a physiological compound with role in bioenergetic functions, shows an antihyperalgesic effect and at the same time can exert neuroregenerative and neuroprotective effects. Aimed to explore the link between pain and neuroregeneration, the effect of ALCAR treatment (100 mg kg(-1) i.p. twice daily for 15 days) on PKCgamma and mitogen-activated protein kinases (MAPKs) expression has been evaluated in CCI (chronic constriction injury) rats. The sciatic nerve and the lumbar tract of the spinal cord were processed to evaluate the levels of the phosphorylated form of PKCgamma, ERK 1,2, SAP/JNK, p-38 and c-Jun; furthermore, the mRNA expression of the early genes c-Jun and c-Fos has been investigated. Fifteen days after injury, the analysis in the sciatic nerves highlighted a bilateral increase of the activated forms of PKCgamma, ERK 1,2 and SAP/JNK, whereas c-Jun showed an increase only ipsilaterally. ALCAR completely prevented mechanical hyperalgesia and provoked in the nerve a c-Jun increment only. In the lumbar tract of the spinal cord, higher levels of activated PKCgamma, ERK 1,2, p38, SAP/JNK and c-Jun proteins were detected in the ipsilateral side in respect of sham. ALCAR was able to stimulate this expression profile. At the transcriptional level c-Jun mRNA was increased in the ipsilateral side of spinal cord of CCI saline-treated rats, whereas c-Fos mRNA was unchanged. ALCAR had a stimulatory effect on both these early genes. These findings may represent a different approach in the study of the complex balance between pain and neuroregeneration and could constitute the basis for developing new disease modifying agents in the treatment of neuropathic pain.

Development of solid lipid nanoparticles as carriers for improving oral bioavailability of glibenclamide.

A solid lipid nanoparticle (SLN) formulation was developed with the aim of improving the oral bioavailability and the therapeutic effectiveness of glibenclamide (GLI), a poorly water-soluble drug used in the treatment of type 2 diabetes. The SLN was prepared using different lipid components (Precirol® and Compritol®) and preparation procedures. Precirol-based SLN, obtained with the emulsion of solvent evaporation technique gave the best results and was selected for drug loading. Addition of lecithin to the SLN core or PEG coating was effective in increasing the nanoparticles stability in simulated gastric solution. Both such formulations were stable after one month storage at 5±3°C, exhibited the absence of in vitro cytotoxicity, and presented a similar in vitro prolonged-release, reaching 100% release after 24h. The lecithin-containing GLI-loaded SLN formulation, selected for in vivo studies in virtue of its higher EE% than the PEG-coated formulation (70.3% vs 19.6%), showed a significantly stronger hypoglycemic effect with respect to the drug alone, in terms of both shorter onset time and longer duration of the effect. These positive results indicated that the proposed SLN approach was successful in improving GLI oral bioavailability, confirming its potential as an effective delivery system for a suitable therapy of diabetes.

Neuropathy‐induced apoptosis: Protective effect of physostigmine

Traumatic, infectious, metabolic, and chemical noxa to the nervous system are the etiology of a crippling disease generally termed neuropathy. Motor disorders, altered sensibility, and pain are the pathognomonic traits. Cellular alterations induced by this chronic pathology include mitochondrial dysfunctions that lead to the activation of the apoptotic cascade. Energy imbalance can compromise the maintenance of mitochondrial membrane potential, furthering the release of cytochrome C and the subsequent cleavage and activation of caspases. Chronic constriction injury (CCI) of the rat sciatic nerve is a neuropathy model able to induce a strong mitochondrial impairment with a consequent apoptotic induction. In this model, the acetylcholinesterase inhibitor physostigmine is administered at 0.125 mg/kg i.p. (twice per day) starting from the operation and for 15 days after. The cholinergic activation reduces cytosolic levels of cytochrome C, suggesting an improved stability of the mitochondrial membrane, and the expression level of the active caspase 3 fragments (19, 16 kDa) is reduced significantly with respect to saline treatment. Accordingly, physostigmine impairs caspase 3 protease activity. In fact, the target of the activated caspase 3, the 89‐kDa PARP fragment, is significantly less expressed in the ligated nerve of physostigmine‐treated rats, reaching levels that are comparable to those in the contralateral unligated nerve. Finally, this natural acetylcholinesterase inhibitor reduces DNA fragmentation both in the proximal and in the distal parts of the nerve. This protection correlates with the induction of XIAP. Therefore, apoptosis, central to tissue degeneration, is prevented by repeated physostigmine treatment of CCI animals.

Influence of ring size on the cognition-enhancing activity of DM235 and MN19, two potent nootropic drugs

A series of analogs of DM235 and MN19, characterized by rings with different size, have been prepared and evaluated for their nootropic activity in the mouse passive-avoidance test. It was found that the optimal ring size for the analogs of DM235, showing endocyclic both amidic groups, is 6 or 7 atoms. For the compounds structurally related to MN19, carrying an exocyclic amide group, the piperidine ring is the moiety which gives the most interesting compounds.

Selective HCN1 block as a strategy to control oxaliplatin-induced neuropathy

ABSTRACT Chemotherapy‐Induced Peripheral Neuropathy (CIPN) is the most frequent adverse effect of pharmacological cancer treatments. The occurrence of neuropathy prevents the administration of fully‐effective drug regimen, affects negatively the quality of life of patients, and may lead to therapy discontinuation. CIPN is currently treated with anticonvulsants, antidepressants, opioids and non‐opioid analgesics, all of which are flawed by insufficient anti‐hyperalgesic efficacy or addictive potential. Understandably, developing new drugs targeting CIPN‐specific pathogenic mechanisms would dramatically improve efficacy and tolerability of anti‐neuropathic therapies. Neuropathies are associated to aberrant excitability of DRG neurons due to the alteration in the expression or function of a variety of ion channels. In this regard, Hyperpolarization‐activated Cyclic Nucleotide‐gated (HCN) channels are overexpressed in inflammatory and neuropathic pain states, and HCN blockers have been shown to reduce neuronal excitability and to ameliorate painful states in animal models. However, HCN channels are critical in cardiac action potential, and HCN blockers used so far in pre‐clinical models do not discriminate between cardiac and non‐cardiac HCN isoforms. In this work, we show an HCN current gain of function in DRG neurons from oxaliplatin‐treated rats. Biochemically, we observed a downregulation of HCN2 expression and an upregulation of the HCN regulatory beta‐subunit MirP1. Finally, we report the efficacy of the selective HCN1 inhibitor MEL57A in reducing hyperalgesia and allodynia in oxaliplatin‐treated rats without cardiac effects. In conclusion, this study strengthens the evidence for a disease‐specific role of HCN1 in CIPN, and proposes HCN1‐selective inhibitors as new‐generation pain medications with the desired efficacy and safety profile. HighlightsNociceptive neurons from oxaliplatin‐treated rats show Ih gain of function.Increased current density is mediated by HCN1‐specific kinetic enhancement.Acceleration of HCN1 kinetics is associated to increased MiRP1 expression.Pan HCN blockers effectively revert neuropathy in vivo but cause bradycardia.HCN1‐selective blocker MEL57A shows intact analgesic efficacy without cardiac effects.

Effects of a water extract of Lepidium meyenii root in different models of persistent pain in rats

Abstract Lepidium meyenii (Walp.), commonly called maca, is an Andean crop belonging to the Brassicaceae family. Maca hypocotils are habitually consumed as customary food as well as traditional remedies for pathological conditions such as infertility. Moreover, the characterization of maca extracts revealed the presence of compounds that are able to modulate the nervous system. Aimed to evaluate the efficacy of L. meyenii in persistent pain, the present study analyzed the effects of a commercial root extract from maca in different animal models reproducing the most common causes of chronic painful pathologies. A qualitative characterization of this commercial extract by high performance liquid chromatography-mass spectrometry and tandem mass spectrometry analyses allowed us to confirm the presence of some macamides known as bioactive constituents of this root and the absence of the main aromatic glucosinolates. The acute oral administration of maca extract is able to reduce mechanical hypersensitivity and postural unbalance induced by the intra-articular injection of monoiodoacetate and the chronic-constriction injury of the sciatic nerve. Furthermore, L. meyenii extract reverts pain threshold alterations evoked by oxaliplatin and paclitaxel. A good safety profile in mice and rats was shown. In conclusion, the present maca extract could be considered as a therapeutic opportunity to relieve articular and neuropathic pain.

Environmental training is beneficial to clinical symptoms and cortical presynaptic defects in mice suffering from experimental autoimmune encephalomyelitis

ABSTRACT The effect of “prophylactic” environmental stimulation on clinical symptoms and presynaptic defects in mice suffering from the experimental autoimmune encephalomyelitis (EAE) at the acute stage of disease (21 ± 1 days post immunization, d.p.i.) was investigated. In EAE mice raised in an enriched environment (EE), the clinical score was reduced when compared to EAE mice raised in standard environment (SE).Concomitantly, gain of weight and increased spontaneous motor activity and curiosity were observed, suggesting increased well‐being in mice. Impaired glutamate exocytosis and cyclic adenosine monophosphate (cAMP) production in cortical terminals of SE‐EAE mice were evident at 21 ± 1 d.p.i. Differently, the 12 mM KCl‐evoked glutamate exocytosis from cortical synaptosomes of EE‐EAE mice was comparable to that observed in SE and EE‐control mice, but significantly higher than that in SE‐EAE mice. Similarly, the 12 mM KCl‐evoked cAMP production in EE‐EAE mice cortical synaptosomes recovered to the level observed in SE and EE‐control mice. MUNC‐18 and SNAP25 contents, but not Syntaxin‐1a and Synaptotagmin 1 levels, were increased in cortical synaptosomes from EE‐EAE mice when compared to SE‐EAE mice. Circulating IL‐1&bgr; was increased in the spinal cord, but not in the cortex, of SE‐EAE mice, and it did not recover in EE‐EAE mice. Inflammatory infiltrates were reduced in the cortex but not in the spinal cord of EE‐EAE mice. Demyelination was observed in the spinal cord; EE significantly diminished it. We conclude that “prophylactic” EE is beneficial to synaptic derangements and preserves glutamate transmission in the cortex of EAE mice. This article is part of the Special Issue entitled “Neurobiology of Environmental Enrichment”. Graphical abstract Figure. No caption available. HighlightsEnriched environment (EE) ameliorates clinical score and behaviours in EAE mice.Inflammatory infiltrates and spinal demyelination are reduced in EE‐EAE mice.EE preserves glutamate exocytosis efficiency from cortical terminals.cAMP production from cortical synaptosomes is unaltered in trained EAE mice.EE increases the expression of MUNC‐18 and SNAP‐25 in the cortex.