Carla Ghelardini

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.

Role of histamine in rodent antinociception

1 Effects of substances which are able to alter brain histamine levels on the nociceptive threshold were investigated in mice and rats by means of tests inducing three different kinds of noxious stimuli: mechanical (paw pressure), chemical (abdominal constriction) and thermal (hot plate). 2 A wide range of i.c.v. doses of histamine 2HCl was studied. Relatively high doses were dose‐dependently antinociceptive in all three tests: 5–100 μg per rat in the paw pressure test, 5–50 μg per mouse in the abdominal constriction test and 50–100 μg per mouse in the hot plate test. Conversely, very low doses were hyperalgesic: 0.5 μg per rat in the paw pressure test and 0.1–1 μg per mouse in the hot plate test. In the abdominal constriction test no hyperalgesic effect was observed. 3 The histamine H3 antagonist, thioperamide maleate, elicited a weak but statistically significant dose‐dependent antinociceptive effect by both parenteral (10–40 mg kg−1) and i.c.v. (1.1–10 μg per rat and 3.4–10 μg per mouse) routes. 4 The histamine H3 agonist, (R)‐α‐methylhistamine dihydrogenomaleate was hyperalgesic, with a rapid effect (15 min after treatment) following i.c.v. administration of 1 μg per rat and 3 μg per mouse, or i.p. administration of 100 mg kg−1in mice. In rats 20 mg kg−1, i.p., elicited hyperalgesia only 4 h after treatment. 5 Thioperamide‐induced antinociception was completely prevented by pretreatment with a non‐hyperalgesic i.p. dose of (R)‐α‐methylhistamine in the mouse hot plate and abdominal constriction tests. Antagonism was also observed when both substances were administered i.c.v. in rats. 6 l‐Histidine HCl dose‐dependently induced a slowly occurring antinociception in all three tests. The doses of 250 and 500 mg kg−1, i.p. were effective in the rat paw pressure test, and those of 500 and 1500 mg kg−1, i.p. in the mouse hot plate test. In the mouse abdominal constriction test 500 and 1000 mg kg−1, i.p. showed their maximum effect 2 h after treatment. 7 The histamine N‐methyltransferase inhibitor, metoprine, elicited a long‐lasting, dose‐dependent antinociception in all three tests by both i.p. (10–30 mg kg−1) and i.c.v. (50–100 μg per rat) routes. 8 To ascertain the mechanism of action of the antinociceptive effect of l‐histidine and metoprine, the two substances were also studied in combination with the histamine synthesis inhibitor (S)‐α‐fluoro‐methylhistidine and with (R)‐α‐methylhistamine, respectively. l‐Histidine antinociception was completely antagonized in all three tests by pretreatment with (S)‐α‐fluoromethylhistidine HCl (50 mg kg−1, i.p.) administered 2 h before l‐histidine treatment. Similarly, metoprine antinociception was prevented by (R)‐α‐methylhistamine dihydrogenomaleate 20 mg kg−1, i.p. administered 15 min before metoprine. Both (S)‐α‐fluoromethylhistidine and (R)‐α‐methylhistamine were used at doses which did not modify the nociceptive threshold when given alone. 9 The catabolism product, 1‐methylhistamine, administered i.c.v. had no effect in either rat paw pressure or mouse abdominal constriction tests. 10 These results indicate that the antinociceptive action of histamine may take place on the postsynaptic site, and that its hyperalgesic effect occurs with low doses acting on the presynaptic receptor. This hypothesis is supported by the fact that the H3 antagonist, thioperamide is antinociceptive and the H3 agonist, (R)‐α‐methylhistamine is hyperalgesic, probably modulating endogenous histamine release. l‐Histidine and metoprine, which are both able to increase brain histamine levels, are also able to induce antinociception in mice and rats. Involvement of the histaminergic system in the modulation of nociceptive stimuli is thus proposed.

Role of muscarinic receptor subtypes in central antinociception

1 The ability to modify the pain threshold by the two M1‐muscarinic agonists: McN‐A‐343 and AF‐102B and by the specific M2‐agonist arecaidine was examined in mice and rats by using three different noxious stimuli: chemical (writhing test), thermic (hot‐plate test) and mechanical (paw pressure test). 2 In the mouse hot‐plate test McN‐A‐343 (20–50 μg per mouse i.c.v.) and AF‐102B (1–10 mg kg−1 i.p.) produced significant antinociception which was prevented by atropine (1 μg per mouse i.c.v.) and by the two selective M1 antagonists: pirenzepine (0.01 μg per mouse i.c.v.) and dicyclomine (0.08 μg per mouse i.c.v. or 10 mg kg−1 i.p.) but not by the specific M2‐antagonist AFDX‐116 (0.1 μg per mouse i.c.v.), naloxone (1 mg kg−1 i.p.) or by the acetylcholine (ACh) depletor hemicholinium‐3 (HC‐3) (1 μg per mouse i.c.v.). McN‐A‐343 and AF‐102B were able to increase the pain threshold also in the mouse acetic acid writhing test and in rat paw pressure test. These antinociceptive effects were completely prevented by dicyclomine (0.08 μg per mouse i.c.v. or 10 mg kg−1 i.p.) but not by AFDX‐116 (0.1 μg per mouse or rat i.c.v.). 3 In contrast with the M1‐agonists, the M2‐agonist arecaidine (0.1–2 μg per mouse or rat i.c.v.) did not induce antinociception in all three analgesic tests. However, arecaidine, at the same i.c.v. doses, was able to reduce the pain threshold in the hot‐plate and paw pressure tests. 4 The site of muscarinic control of the pain threshold is localized in the CNS since drugs which do not cross the blood‐brain barrier such as McN‐A‐343, pirenzepine and arecaidine exerted their effects only if injected i.c.v. 5 On the basis of the above findings and existing literature we suggest that the postsynaptic muscarinic receptors involved in antinociception belong to the M1 subtype. Nevertheless, presynaptic autoreceptors (M2 subtype) may play a role in pain regulation since they are involved in modulation of endogenous ACh release.

Design and Study of Piracetam-like Nootropics, Controversial Members of the Problematic Class of Cognition-Enhancing Drugs

Cognition enhancers are drugs able to facilitate attentional abilities and acquisition, storage and retrieval of information, and to attenuate the impairment of cognitive functions associated with head traumas, stroke, age and age-related pathologies. Development of cognition enhancers is still a difficult task because of complexity of the brain functions, poor predictivity of animal tests and lengthy and expensive clinical trials. After the early serendipitous discovery of first generation cognition enhancers, current research is based on a variety of working hypotheses, derived from the progress of knowledge in the neurobiopathology of cognitive processes. Among other classes of drugs, piracetam-like cognition enhancers (nootropics) have never reached general acceptance, in spite of their excellent tolerability and safety. In the present review, after a general discussion of the problems connected with the design and development of cognition enhancers, the class is examined in more detail. Reasons for the problems encountered by nootropics, compounds therapeutically available and those in development, their structure activity relationships and mechanisms of action are discussed. Recent developments which hopefully will lead to a revival of the class are reviewed.

A Smart Platform for Hyperthermia Application in Cancer Treatment: Cobalt-Doped Ferrite Nanoparticles Mineralized in Human Ferritin Cages

Magnetic nanoparticles, MNPs, mineralized within a human ferritin protein cage, HFt, can represent an appealing platform to realize smart therapeutic agents for cancer treatment by drug delivery and magnetic fluid hyperthermia, MFH. However, the constraint imposed by the inner diameter of the protein shell (ca. 8 nm) prevents its use as heat mediator in MFH when the MNPs comprise pure iron oxide. In this contribution, we demonstrate how this limitation can be overcome through the controlled doping of the core with small amount of Co(II). Highly monodisperse doped iron oxide NPs with average size of 7 nm are mineralized inside a genetically modified variant of HFt, carrying several copies of α-melanocyte-stimulating hormone peptide, which has already been demonstrated to have excellent targeting properties toward melanoma cells. HFt is also conjugated to poly(ethylene glycol) molecules to increase its in vivo stability. The investigation of hyperthermic properties of HFt-NPs shows that a Co doping of 5% is enough to strongly enhance the magnetic anisotropy and thus the hyperthermic efficiency with respect to the undoped sample. In vitro tests performed on B16 melanoma cell line demonstrate a strong reduction of the cell viability after treatment with Co doped HFt-NPs and exposure to the alternating magnetic field. Clear indications of an advanced stage of apoptotic process is also observed from immunocytochemistry analysis. The obtained data suggest this system represents a promising candidate for the development of a protein-based theranostic nanoplatform.

Oxaliplatin-Induced Neuropathy: Oxidative Stress as Pathological Mechanism. Protective Effect of Silibinin

UNLABELLED Oxaliplatin is the standard treatment for advanced colorectal cancer. Its dose-limiting toxicity is the development of a painful neuropathic syndrome sustained by unclear mechanisms. Although the oxidative hypothesis is a matter of debate, direct data about oxidative damage induced in vivo by anticancer agents are lacking and the efficacy of the available antioxidant compounds are unsatisfactory. In a rat model of painful oxaliplatin-induced neuropathy (2.4 mgkg(-1) i.p., daily for 21 days), we described an important component of oxidative stress. In the plasma of oxaliplatin-treated rats, the increases in carbonylated protein and thiobarbituric acid reactive substances were the index of the resultant protein oxidation and lipoperoxidation, respectively. The same pattern of oxidation was revealed also in the sciatic nerve, and in the spinal cord where the damage reached the DNA level. The antioxidant compound silibinin (100 mgkg(-1) per os), administered once a day, starting from the first day of oxaliplatin injection until the 20th, prevented oxidative damage as did α-tocopherol. Repetitive administration of silibinin, as well as α-tocopherol, reduced oxaliplatin-dependent pain induced by mechanical and thermal stimuli. Antioxidants were also able to improve motor coordination. The antineuropathic effect of both molecules improved by about 50% oxaliplatin-induced behavioral alterations. PERSPECTIVE This study characterizes oxidative stress parameters in a rat model of oxaliplatin-induced neuropathy. A relationship between the improvement of oxidative alterations and pain relief is established in rats treated with natural antioxidant compounds like α-tocopherol and silibinin. Silibinin could be a valid therapeutic option for chemotherapy-induced neuropathy.

CGP 35348, a new GABAB antagonist, prevents antinociception and muscle-relaxant effect induced by baclofen.

1 CGP 35348, a new GABAB antagonist, was examined on antinociception induced by (±)‐baclofen by use of the hot plate and writhing tests in mice and the paw pressure test in rats. CGP 35348 was also studied in mice on (±)‐baclofen‐induced impairment of rota‐rod performance. 2 CGP 35348, injected either i.p. (60–100 mg kg−1 in mouse) or intracerebroventricularly (i.c.v.) (0.5–2.5 μg per mouse; 25 μg per rat) prevented (±)‐baclofen‐induced antinociception. 3 CGP 35348 did not modify oxotremorine‐ and morphine‐induced antinociception in mice and rats. 4 CGP 35348 (2.5 μg i.c.v. per mouse) also prevented (±)‐baclofen‐induced impairment of the rota‐rod test. 5 Two other GABAB antagonists, phaclofen (50 μg i.c.v. per mouse) and 2‐OH‐saclofen (2.5 μg‐10 μg i.c.v. per mouse) did not modify (±)‐baclofen‐induced antinociception. 7 These results suggest that, at present, CGP 35348 is the only compound able to antagonize (±)‐baclofen‐induced antinociception.

Morphologic Features and Glial Activation in Rat Oxaliplatin-Dependent Neuropathic Pain

UNLABELLED Neurotoxicity is the limiting side effect of the anticancer agent oxaliplatin. A tangled panel of symptoms, sensory loss, paresthesia, dysesthesia, and pain may be disabling for patients and adversely affect their quality of life. To elucidate the morphologic and molecular alterations that occur in the nervous system during neuropathy, rats were daily injected with 2.4 mg kg(-1) oxaliplatin intraperitoneally. A progressive decrease in the pain threshold and hypersensitivity to noxious and nonnoxious stimuli were evidenced during the treatment (7, 14, 21 days). On day 21, morphometric alterations were detectable exclusively in the dorsal root ganglia, whereas the activating transcription factor 3 and neurofilament (heavy-chain) expression changed dramatically in both the nerves and ganglia. Inflammatory features were not highlighted. Interestingly, satellite cells exhibited signs of activation. Glial modulation was characterized in the spinal cord and brain areas involved in pain signaling. On the 21st day, spinal astrocytes increased numerically whereas the microglial population was unaltered. The number of glial cells in the brain differed according to the zone and treatment time points. In particular, on day 21, a significant astrocyte increase was measured in the anterior cingulate cortex, somatosensory area 1, neostriatum, ventrolateral periaqueductal gray, and nucleus raphe magnus. PERSPECTIVES These data highlight the relevance of glial cells in chemotherapy-induced neurotoxicity as part of the investigation of the role that specific brain areas play in neuropathy.

Signaling pathway of morphine induced acute thermal hyperalgesia in mice

Abstract Systemic administration of morphine induced a hyperalgesic response in the hot plate test, at an extremely low dose (1–10 μg/kg). We have examined in vivo whether morphine, at an extremely low dose, induces acute central hypernociception following activation of the opioid receptor‐mediated PLC/PKC inositol‐lipid signaling pathway. The PLC inhibitor U73122 and the PKC blocker, calphostin C, dose dependently prevented the thermal hypernociception induced by morphine. This effect was also prevented by pretreatment with aODN against PLCβ3 at 2 nmol/mouse and PKCγ at 2–3 nmol/mouse. Low dose morphine hyperalgesia was dose dependently reversed by selective NMDA antagonist MK801 and ketamine. This study demonstrates the presence of a nociceptive PLCβ3/PKCγ/NMDA pathway stimulated by low concentrations of morphine, through μOR1 receptor, in mouse brain. This signaling pathway appears to play an opposing role in morphine analgesia. When mice were treated with a morphine analgesic dose (7 mg/kg), the downregulation of PLCβ3 or PKCγ at the same aODN doses used for the prevention of the hyperalgesic effect induced, respectively, a 46% and 67% potentiation in analgesic response. Experimental and clinical studies suggest that opioid may activate pronociceptive systems, leading to pain hypersensitivity and short‐term tolerance, a phenomenon encountered in postoperative pain management by acute opioid administration. The clinical management of pain by morphine may be revisited in light of the identification of the signaling molecules of the hyperalgesic pathway.

Antinociception induced by systemic administration of local anaesthetics depends on a central cholinergic mechanism.

1 The antinociceptive effects of systemically‐administered procaine, lignocaine and bupivacaine were examined in mice and rats by using the hot‐plate, writhing and tail flick tests. 2 In both species all three local anaesthetics produced significant antinociception which was prevented by atropine (5 mg kg−1, i.p.) and by hemicholinium‐3 (1 μg per mouse, i.c.v.), but not by naloxone (3 mg kg−1, i.p.), α‐methyl‐p‐tyrosine (100 mg kg−1, s.c.), reserpine (2 mg kg−1, i.p.) or atropine methylbromide (5.5 mg kg−1, i.p.). 3 Atropine (5 mg kg−1, i.p.) which totally antagonized oxotremorine (40 μg kg−1, s.c.) antinociception did not modify morphine (5 mg kg−1, s.c.) or baclofen (4 mg kg−1, s.c.) antinociception. On the other hand, hemicholinium, which antagonized local anaesthetic antinociception, did not prevent oxotremorine, morphine or baclofen antinociception. 4 Intracerebroventricular injection in mice of procaine (200 μg), lignocaine (150 μg) and bupivacaine (25 μg), doses which were largely ineffective by parenteral routes, induced an antinociception whose intensity equalled that obtainable subcutaneously. Moreover, the i.c.v. injection of antinociceptive doses did not impair performance on the rota‐rod test. 5 Concentrations below 10−10 m of procaine, lignocaine and bupivacaine did not evoke any response on the isolated longitudinal muscle strip of guinea‐pig ileum, or modify acetylcholine (ACh)‐induced contractions. On the other hand, they always increased electrically‐evoked twitches. 6 The same concentrations of local anaesthetics which induced antinociception did not inhibit acetylcholinesterase (AChE) in vitro. 7 On the basis of the above findings and the existing literature, a facilitation of cholinergic transmission by the local anaesthetics is postulated; this could be due to blockade of presynaptic muscarinic receptors.