Letizia Antonilli

Activity of Adenosine Receptors Type 1 Is Required for CX3CL1-Mediated Neuroprotection and Neuromodulation in Hippocampal Neurons

The chemokine fractalkine (CX3CL1) is constitutively expressed by central neurons, regulating microglial responses including chemotaxis, activation, and toxicity. Through the activation of its own specific receptor, CX3CR1, CX3CL1 exerts both neuroprotection against glutamate (Glu) toxicity and neuromodulation of the glutamatergic synaptic transmission in hippocampal neurons. Using cultured hippocampal neuronal cell preparations, obtained from CX3CR1−/− (CX3CR1GFP/GFP) mice, we report that these same effects are mimicked by exposing neurons to a medium conditioned with CX3CL1-treated mouse microglial cell line BV2 (BV2-st medium). Furthermore, CX3CL1-induced neuroprotection from Glu toxicity is mediated through the adenosine receptor 1 (AR1), being blocked by neuronal cell preparations treatment with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a specific inhibitor of AR1, and mimicked by both adenosine and the specific AR1 agonist 2-chloro-N6-cyclopentyladenosine. Similarly, experiments from whole-cell patch-clamped hippocampal neurons in culture, obtained from CX3CR1+/+ mice, show that CX3CL1-induced depression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- (AMPA-) type Glu receptor-mediated current (AMPA-current), is associated with AR1 activity being blocked by DPCPX and mimicked by adenosine. Furthermore, BV2-st medium induced a similar AMPA-current depression in CX3CR1GFP/GFP hippocampal neurons and this depression was again blocked by DPCPX. We also report that CX3CL1 induced a significant release of adenosine from microglial BV2 cells, as measured by HPLC analysis. We demonstrate that (i) CX3CL1, along with AR1, are critical players for counteracting Glu-mediated neurotoxicity in the brain and (ii) AR1 mediates neuromodulatory action of CX3CL1 on hippocampal neurons.

Adenosine A1 receptors and microglial cells mediate CX3CL1-induced protection of hippocampal neurons against Glu-induced death.

Fractalkine/CX3CL1 is a neuron-associated chemokine, which modulates microglia-induced neurotoxicity activating the specific and unique receptor CX3CR1. CX3CL1/CX3CR1 interaction modulates the release of cytokines from microglia, reducing the level of tumor necrosis factor-α, interleukin-1-β, and nitric oxide and induces the production of neurotrophic substances, both in vivo and in vitro. We have recently shown that blocking adenosine A1 receptors (A1R) with the specific antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) abolishes CX3CL1-mediated rescue of neuronal excitotoxic death and that CX3CL1 induces the release of adenosine from microglia. In this study, we show that the presence of extracellular adenosine is mandatory for the neurotrophic effect of CX3CL1 as reducing adenosine levels in hippocampal cultures, by adenosine deaminase treatment, strongly impairs CX3CL1-mediated neuroprotection. Furthermore, we confirm the predominant role of microglia in mediating the neuronal effects of CX3CL1, because the selective depletion of microglia from hippocampal cultures treated with clodronate-filled liposomes causes the complete loss of effect of CX3CL1. We also show that hippocampal neurons obtained from A1R−/− mice are not protected by CX3CL1 whereas A2AR−/− neurons are. The requirement of functional A1R for neuroprotection is not unique for CX3CL1 as A1R−/− hippocampal neurons are not rescued from Glu-induced cell death by other neurotrophins such as brain-derived neurotrophic factor and erythropoietin, which are fully active on wt neurons.

High levels of morphine-6-glucuronide in street heroin addicts

RationaleIn the body, heroin is rapidly transformed to 6-acetylmorphine (6-AM) and then to morphine, that in turn is mainly metabolized to morphine-3-glucuronide (M3G) and, at lesser extent, to morphine-6-glucuronide (M6G). Unlike M3G, M6G is a potent opioid agonist. Intravenous heroin abusers (IHU) are exposed to a wide array of drugs and contaminants that might affect glucuronidation.ObjectivesWe assessed plasma and urine concentrations of M3G and M6G in four groups of subjects: the first two included long-term IHU either exposed to street heroin (n=8) or receiving a single IV injection of morphine (n=4), while the other two groups included non-IHU patients receiving acute IV (n=8) or chronic oral (n=6) administrations of morphine.MethodsAfter solid phase extraction plasma and urine concentrations of morphine metabolites were determined by HPLC analyses.ResultsM3G accounted for the greater part of morphine glucuronides detected in body fluids of non-IHU patients treated with morphine. This pattern of metabolism remained stable across 15 days of oral administration of incremental doses of morphine. In contrast, the two groups of IHU (street heroin taking or morphine-treated subjects) showed a reduction of blood and urine M3G concentrations in favor of M6G. Consequently, M6G/M3G ratio was significantly higher in the two IHU groups in comparison with the non-IHU groups.ConclusionsChronic exposure to street heroin causes a relative increase in concentrations of the active metabolite, M6G. Since the pattern of M6G action seems closer to heroin than to morphine, the increased synthesis of M6G observed in IHU may prolong the narrow window of heroin effects.

Development and validation of an analytical method based on high performance thin layer chromatography for the simultaneous determination of lamotrigine, zonisamide and levetiracetam in human plasma.

Methods based on HPLC technology are the most frequently adopted for monitoring blood levels of novel antiepileptics. Here a rapid method based on HPTLC was developed for quantitative determination of lamotrigine (LTG), zonisamide (ZNS) and levetiracetam (LVT) in human plasma and compared with HPLC and LC-MS/MS methods. Chromatographic separation was achieved on silical gel 60F(254) plates using ethylacetate:methanol:ammonia (91:10:15v/v/v) as mobile phase. Quantitative analysis was carried out by densitometry at a wavelength of 312, 240 and 210nm for LTG, ZNS and LVT, respectively. Calibration curves were linear over range of 0-200ng for LTG and ZNS and 0-400ng for and LVT. The limit of quantification of LTG, ZNS and LTV was found to be 3.69, 3.7 and 6.85μg/ml, respectively. Intra and inter-assay precision provided relative standard deviations lower than 10% for all three analytes. Correlation and Bland-Altman plot showed general agreement between HPTLC and LC-MS/MS quantification, with a mean bias of -0.25, -0.46 and 0.5μg/ml for LTG ZNS and LVT, respectively. Likewise, comparison between HPLC-UV and LC-MS/MS showed good agreement for all the three compounds analyzed. In conclusion, the proposed HPTLC method is simple, rapid, precise and accurate. It therefore is appropriate for the routine quantification of therapeutic levels of LTG, ZNS and LVT in human plasma.

Analysis of cocaethylene, benzoylecgonine and cocaine in human urine by high-performance thin-layer chromatography with ultraviolet detection: a comparison with high-performance liquid chromatography

Cocaine and ethanol are frequently used at the same time, resulting in the formation of cocaethylene by transesterification. We studied the capability of high-performance thin-layer chromatography (HPTLC) to simultaneously detect cocaethylene, cocaine and benzoylecgonine in 16 urine specimens of drug addicts, previously tested as positive for benzoylecgonine at immunoenzymatic screening. Accuracy and precision, as well as detection and quantitation limits of the method, were evaluated by comparison with high-performance liquid chromatography (HPLC). HPTLC limit of quantitation was 1.0 microg/ml for the three compounds, whereas HPLC limits were 0.2 microg/ml for benzoylecgonine and cocaine, and 0.1 microg/ml for cocaethylene. The relative standard deviation (RSD) ranged from 1.03 to 12.60% and from 1.56 to 16.6% for intra- and inter-day HPTLC analysis, respectively. In the case of the HPLC method, the RSD for the intra-day precision ranged from 0.79 to 5.05%, whereas it ranged from 1.19 to 10.64% for the inter-day precision. In comparison with HPLC, HPTLC is less expensive and faster, requiring 2-3 h to analyze 10-12 samples on a single plate. In conclusion, HPTLC is suitable for determinations of the three analytes only for samples with high concentrations.

Cardiovascular and Hepatic Toxicity of Cocaine: Potential Beneficial Effects of Modulators of Oxidative Stress

Oxidative stress (OS) is thought to play an important role in the pharmacological and toxic effects of various drugs of abuse. Herein we review the literature on the mechanisms responsible for the cardiovascular and hepatic toxicity of cocaine with special focus on OS-related mechanisms. We also review the preclinical and clinical literature concerning the putative therapeutic effects of OS modulators (such as N-acetylcysteine, superoxide dismutase mimetics, nitroxides and nitrones, NADPH oxidase inhibitors, xanthine oxidase inhibitors, and mitochondriotropic antioxidants) for the treatment of cocaine toxicity. We conclude that available OS modulators do not appear to have clinical efficacy.

In vitro morphine metabolism by rat microglia

Morphine is mainly transformed to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in the liver. Glucuronidation is also performed by rat brain homogenates and UDP-glucuronosyltransferases (UGTs) are present in the brain. Here we investigated the possibility that microglia transforms morphine into its metabolites M3G and M6G. Primary cultures of neonatal rat microglia were incubated for different intervals of time in basal conditions or with different concentrations of morphine. The following measures were performed on these cultures and/or in the medium: (i) morphine as well as M3G and M6G concentrations; (ii) levels of mRNA coding for UGT1A1, UGT1A6, UGT1A7, and UGT2B1 as well as their protein levels; (iii) released prostaglandin (PG)E2 and nitrite concentrations. Results show that in basal conditions morphine and M3G are produced by microglia; accordingly, these cells expressed UGT1A1, UGT1A6 and UGT1A7, but not UGT2B1. When cultures were exposed to different concentrations of exogenous morphine, M6G was also synthesized. This shift in the glucuronidation was associated with variations in the expression of UGT isozymes. In particular, UGT1A7 expression was rapidly upregulated and this event was translated into enhanced protein levels of UGT1A7; lesser effects were exerted on UGT1A1 and UGT1A6. Upon prolonged exposure to morphine, microglial cell UGT expression returned to baseline conditions or even to reduced levels of expression. Morphine exposure did not affect the synthesis of both PGE2 and nitrites, ruling out a generalized priming of microglia by morphine. In conclusion, this study suggests that morphine glucuronides found in the cerebrospinal liquor upon peripheral morphine administration may at least in part be brain-born, reconciling the conceptual gap between the high hydrophilic features of morphine glucuronides and their presence beyond the blood-brain barrier.

Non-opioid induction of morphine-6-glucuronide synthesis is elicited by prolonged exposure of rat hepatocytes to heroin.

BACKGROUND Liver metabolism of morphine leads to the formation of morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G), the latter possessing strong opioid activity that however differs from that of the parent compound. In previous studies conducted in rats we have shown that repeated in vivo exposure to phenanthrene class of mu opioid receptor (MOR) agonists or antagonists (heroin, morphine, and naltrexone), but not to non-phenanthrene class of MOR agonist methadone, affects morphine glucuronidation by liver microsomes. METHODS In the present study, we measured the in vitro formation of M3G and M6G by rat hepatocytes incubated for 120 min with morphine (0.1-1.0 mM) after 72h pre-incubation with one of the following MOR agonists: heroin (3.3 or 6.6 microM), morphine (7.8 microM), or methadone (12 microM). The MOR antagonist naltrexone (10 or 25 microM) was also tested, alone or in combination with heroin. The amount of M3G and M6G synthesized was then measured by HPLC method. RESULTS Heroin inhibited M3G synthesis and induced the formation of M6G, which under basal conditions is not synthesized in rats. Heroin effects were not blocked by naltrexone. Morphine, but not methadone, produced effects similar to those of heroin but more modest in intensity. Pre-incubation with naltrexone alone slightly increased M3G synthesis, but had no effect on M6G formation. CONCLUSIONS These results are in agreement with those of previous ex vivo studies and indicate that exposure to heroin or, to a lesser extent, morphine, can affect morphine glucuronidation via direct non-opioid actions on the hepatocytes.

In vivo chronic exposure to heroin or naltrexone selectively inhibits liver microsome formation of estradiol-3-glucuronide in the rat

We have previously found that repeated exposure to heroin reduces liver synthesis of morphine-3-glucuronide (M3G) and increases the production of morphine-6-glucuronide (M6G), which normally is not formed in the rat. By contrast repeated exposure to naltrexone does not activate M6G synthesis but increases the V(max) of M3G formation. M3G synthesis depends on the activity of two isoforms of the UDP-glucuronosyltransferase (UGT), UGT1A1 and UGT2B1. These isozymes also activate the formation of estradiol-3-glucuronide (E3G) and estradiol-17-glucuronide (E17G), respectively. The goal of the present study was to investigate the role of UGT1A1 and UGT2B1 in the effects of heroin and naltrexone by determining their influence on the synthesis of E3G and E17G. Estradiol glucuronidation was performed using microsomes of rats treated daily, for 10 days, with saline, heroin (10mg/kg, i.p.), or naltrexone (40mg/kg, i.p.). Moreover, liver expression of both UGT1A1 and UGT2B1 was studied in the same experimental conditions by polymerase chain reaction analysis. Kinetic analysis showed that the V(max) for E3G formation was significantly reduced by both heroin (168.82+/-9.73nmol/mg/min) and naltrexone (194.60+/-16.6) relative to saline (624.60+/-17.6). Moreover, homotropic kinetic of E3G formation (Hill coefficient: 1.8) was transformed in Michaelis-Menten kinetic by both heroin (0.88) and naltrexone (1.15). The synthesis of E17G was not affected by either opioid. The expression of liver UGT1A1 and UGT2B1 did not differ across groups. The present results suggest that heroin and naltrexone can reduce estradiol glucuronidation via a specific interaction with UGT1A1 isoform.

Cannabis in epilepsy: From clinical practice to basic research focusing on the possible role of cannabidivarin

Cannabidivarin (CBDV) and cannabidiol (CBD) have recently emerged among cannabinoids for their potential antiepileptic properties, as shown in several animal models. We report the case of a patient affected by symptomatic partial epilepsy who used cannabis as self‐medication after the failure of countless pharmacological/surgical treatments. Clinical and video electroencephalogram (EEG) evaluations were periodically performed, and the serum levels of CBDV, CBD, and Δ9‐tetrahydrocannabinol were repeatedly measured. After cannabis administration, a dramatic clinical improvement, in terms of both decrease in seizure frequency and recovery of cognitive functions, was observed, which might parallel high CBDV plasma concentrations. To widen the spectrum of CBDV possible mechanisms of action, electrophysiological methods were applied to investigate whether it could exert some effects on γ‐aminobutyric acid (GABA)A receptors. Our experiments showed that, in human hippocampal tissues of four patients affected by drug‐resistant temporal lobe epilepsy (TLE) transplanted in Xenopus oocytes, there is decrease of current rundown (i.e., reduction of use‐dependent GABAA current) after prolonged exposure to CBDV. This result has been confirmed using a single case of Rasmussen encephalitis (RE). Our patients electroclinical improvement supports the hypothesis that cannabis could actually represent an effective, well‐tolerated antiepileptic drug. Moreover, the experimental data suggest that CBDV may greatly contribute to cannabis anticonvulsant effect through its possible GABAergic action.