Letizia Antonilli

Publications of Letizia Antonilli

• Erratum to: Induction of morphine-6-glucuronide synthesis by heroin self-administration in the rat.

  Authors: Maria Meringolo, Valentina Brusadin, Maria T De Luca, Christian Montanari, Letizia Antonilli, Paolo Nencini, Aldo Badiani

  Psychopharmacology. 03/2012;

• 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.

  Authors: Letizia Antonilli, Valentina Brusadin, Francesca Filipponi, Renzo Guglielmi, Paolo Nencini

  Journal of pharmaceutical and biomedical analysis. 12/2011; 56(4):763-70.

  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 determinationMethods 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.

• Induction of morphine-6-glucuronide synthesis by heroin self-administration in the rat.

  Authors: Maria Meringolo, Valentina Brusadin, Maria T De Luca, Christian L Montanari, Letizia Antonilli, Paolo Nencini, Aldo Badiani

  Psychopharmacology. 10/2011;

  RATIONALE: Heroin is rapidly metabolized to morphine that in turn is transformed into morphine-3-glucuronide (M3G), an inactive metabolite at mu-opioid receptor (MOR), and morphine-6-glucuronideRATIONALE: Heroin is rapidly metabolized to morphine that in turn is transformed into morphine-3-glucuronide (M3G), an inactive metabolite at mu-opioid receptor (MOR), and morphine-6-glucuronide (M6G), a potent MOR agonist. We have found that rats that had received repeated intraperitoneal injections of heroin exhibit measurable levels of M6G (which is usually undetectable in this species). OBJECTIVE: The goal of the present study was to investigate whether M6G synthesis can be induced by intravenous (i.v.) heroin self-administration (SA). MATERIALS AND METHODS: Rats were trained to self-administer either heroin (50 μg/kg per infusion) or saline for 20 consecutive 6-h sessions and then challenged with an intraperitoneal challenge of 10 mg/kg of heroin. Plasma levels of heroin, morphine, 6-mono-acetyl morphine, M3G, and M6G were quantified 2 h after the challenge. In vitro morphine glucuronidation was studied in microsomal preparations obtained from the liver of the same rats. RESULTS: Heroin SA induced the synthesis of M6G, as indicated by detectable plasma levels of M6G (89.7 ± 37.0 ng/ml vs. 7.35 ± 7.35 ng/ml after saline SA). Most important, the in vitro V (max) for M6G synthesis was correlated with plasma levels of M6G (r (2) = 0.78). Microsomal preparations from saline SA rats produced negligible amounts of M6G. CONCLUSION: Both in vivo and in vitro data indicate that i.v. heroin SA induces the synthesis of M6G. These data are discussed in the light of previous studies conducted in heroin addicts indicating that in humans heroin enhances the synthesis of the active metabolite of heroin and morphine.

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

  Authors: Clotilde Lauro, Raffaela Cipriani, Myriam Catalano, Flavia Trettel, Giuseppina Chece, Valentina Brusadin, Letizia Antonilli, Nico van Rooijen, Fabrizio Eusebi, Bertil B Fredholm, Cristina Limatola

  Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 03/2010; 35(7):1550-9.

  Fractalkine/CX3CL1 is a neuron-associated chemokine, which modulates microglia-induced neurotoxicity activating the specific and unique receptor CX3CR1. CX3CL1/CX3CR1 interaction modulates theFractalkine/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-alpha, interleukin-1-beta, and nitric oxide and induces the production of neurotrophic substances, both in vivo and in vitro. We have recently shown that blocking adenosine A(1) receptors (A(1)R) 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 A(1)R(-/-) mice are not protected by CX3CL1 whereas A(2A)R(-/-) neurons are. The requirement of functional A(1)R for neuroprotection is not unique for CX3CL1 as A(1)R(-/-) 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.

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

  Authors: Letizia Antonilli, Valentina Brusadin, Michele S Milella, Fabrizia Sobrero, Aldo Badiani, Paolo Nencini

  Biochemical pharmacology. 09/2008; 76(5):672-9.

  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 notWe 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.

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

  Authors: Manuela Graziani, Letizia Antonilli, Anna Rita Togna, Valentina Brusadin, Stefania Viola, Giuseppina Togna, Aldo Badiani, Paolo Nencini

  Drug and alcohol dependence. 07/2008;

  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 differsBACKGROUND: 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 120min with morphine (0.1-1.0mM) after 72h pre-incubation with one of the following MOR agonists: heroin (3.3 or 6.6muM), morphine (7.8muM), or methadone (12muM). The MOR antagonist naltrexone (10 or 25muM) 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.

• Activity of adenosine receptors type 1 Is required for CX3CL1-mediated neuroprotection and neuromodulation in hippocampal neurons.

  Authors: Clotilde Lauro, Silvia Di Angelantonio, Raffaela Cipriani, Fabrizia Sobrero, Letizia Antonilli, Valentina Brusadin, Davide Ragozzino, Cristina Limatola

  Journal of immunology (Baltimore, Md. : 1950). 07/2008; 180(11):7590-6.

  The chemokine fractalkine (CX(3)CL1) is constitutively expressed by central neurons, regulating microglial responses including chemotaxis, activation, and toxicity. Through the activation of its ownThe chemokine fractalkine (CX(3)CL1) is constitutively expressed by central neurons, regulating microglial responses including chemotaxis, activation, and toxicity. Through the activation of its own specific receptor, CX(3)CR1, CX(3)CL1 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 CX(3)CR1(-/-) (CX(3)CR1(GFP/GFP)) mice, we report that these same effects are mimicked by exposing neurons to a medium conditioned with CX(3)CL1-treated mouse microglial cell line BV2 (BV2-st medium). Furthermore, CX(3)CL1-induced neuroprotection from Glu toxicity is mediated through the adenosine receptor 1 (AR(1)), being blocked by neuronal cell preparations treatment with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a specific inhibitor of AR(1), and mimicked by both adenosine and the specific AR(1) agonist 2-chloro-N(6)-cyclopentyladenosine. Similarly, experiments from whole-cell patch-clamped hippocampal neurons in culture, obtained from CX(3)CR1(+/+) mice, show that CX(3)CL1-induced depression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- (AMPA-) type Glu receptor-mediated current (AMPA-current), is associated with AR(1) activity being blocked by DPCPX and mimicked by adenosine. Furthermore, BV2-st medium induced a similar AMPA-current depression in CX(3)CR1(GFP/GFP) hippocampal neurons and this depression was again blocked by DPCPX. We also report that CX(3)CL1 induced a significant release of adenosine from microglial BV2 cells, as measured by HPLC analysis. We demonstrate that (i) CX(3)CL1, along with AR(1), are critical players for counteracting Glu-mediated neurotoxicity in the brain and (ii) AR(1) mediates neuromodulatory action of CX(3)CL1 on hippocampal neurons.

• Short-term efficacy of Disulfiram or Naltrexone in reducing positive urinalysis for both cocaine and cocaethylene in cocaine abusers: a pilot study.

  Authors: Maria Caterina Grassi, Anna Maria Cioce, Franco Dei Giudici, Letizia Antonilli, Paolo Nencini

  Pharmacological research : the official journal of the Italian Pharmacological Society. 03/2007; 55(2):117-21.

  Cocaine abusers frequently report taking the drug in association with alcohol. This combined intake leads to the synthesis of cocaethylene, an active metabolite with effects similar to those ofCocaine abusers frequently report taking the drug in association with alcohol. This combined intake leads to the synthesis of cocaethylene, an active metabolite with effects similar to those of cocaine, but more prolonged. Since pharmacological effects of cocaethylene may partially account for the habit of cocaine abusers to take the drug in combination with ethanol, a main therapeutic goal in these patients should be making body fluids negative for cocaethylene. This randomized controlled open study conducted on 12 subjects co-abusers of cocaine and alcohol, evaluates the efficacy of a 12-week pharmacological treatment with Disulfiram (DIS) 400mg daily or Naltrexone (NTX) 50mg daily associated with Cognitive Behaviour Therapy (CBT), as compared to CBT alone, in terms of: (i) stay in treatment; (ii) drug-free urinalyses for cocaine and cocaethylene; (iii) reduction of alcohol and cocaine craving. Data presented in this study are restricted to the first 4 weeks of treatment when all the enrolled subjects were still available for examination. In fact, of the 12 subjects enrolled in the study only 4 (33%) completed the 12-week treatment. Of these, three were in the CBT group and one in the NTX/CBT group. Results show that CBT treated subjects remained in treatment longer than those assigned to either DIS/CBT or NTX/CBT therapies. However, during the first 4 weeks of treatment, CBT-group urine tested positive almost always for both cocaine and cocaethylene. In contrast, both DIS/CBT and NTX/CBT treatments were associated to a statistically significant reduction, of positive urinalysis for both cocaine and cocaethylene, with respect to CBT alone. Moreover, across the first 4 weeks of treatment DIS/CBT and NTX/CBT treated subjects maintained lower scores at Visual Analogue Scales (VAS) for both cocaine and alcohol craving than subjects receiving CBT alone. This pilot study suggests that the transient efficacy of pharmacological treatments in maintaining subjects drug free, does not add to the capability of CBT to retain them in treatment.

• Effect of repeated administrations of heroin, naltrexone, methadone, and alcohol on morphine glucuronidation in the rat.

  Authors: Letizia Antonilli, Emma Petecchia, Daniele Caprioli, Aldo Badiani, Paolo Nencini

  Psychopharmacology. 11/2005; 182(1):58-64.

  RATIONALE: Heroin is rapidly metabolized to morphine that in turn is transformed in morphine-3-glucuronide (M3G), an inactive metabolite, and morphine-6-glucuronide (M6G), a potent mu-opioid receptorRATIONALE: Heroin is rapidly metabolized to morphine that in turn is transformed in morphine-3-glucuronide (M3G), an inactive metabolite, and morphine-6-glucuronide (M6G), a potent mu-opioid receptor (MOR) agonist. We have found that heroin addicts exhibit higher M6G/M3G ratios relative to morphine-treated control subjects. We have also shown that heroin-treated rats exhibit measurable levels of M6G (which is usually undetectable in this species) and reduced levels of M3G. OBJECTIVE: We investigated the role of MOR in these effects of heroin, by examining the effects of methadone, a MOR agonist, and of naltrexone, a MOR antagonist, on morphine glucuronidation. We also investigated the effects of alcohol, which is known to alter drug metabolism and is frequently coabused by heroin addicts. METHODS: Morphine glucuronidation was studied in liver microsomes obtained from rats exposed daily for 10 days to saline, heroin (10 mg/kg, i.p.), naltrexone (20-40 mg/kg, i.p.), heroin + naltrexone (10 mg/kg+20-40 mg/kg, i.p.), methadone (5-20 mg/kg, i.p.), or 10% ethanol. RESULTS: Heroin induced the synthesis of M6G and decreased the synthesis of M3G. Naltrexone exhibited intrinsic modulatory activity on morphine glucuronidation, increasing the synthesis of M3G via a low-affinity/high-capacity reaction characterized by positive cooperativity. The rate of M3G synthesis in the heroin + naltrexone groups was not different from that of the naltrexone groups. Methadone and ethanol induced a modest increase in M3G synthesis and had no effect on M6G synthesis. CONCLUSION: The effects of heroin on morphine glucuronidation are not shared by methadone or alcohol (two drugs that figure prominently in the natural history of heroin addiction) and do not appear to depend on the activation of MOR.

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

  Authors: Letizia Antonilli, Federico Semeraro, Carmen Suriano, Luciano Signore, Paolo Nencini

  Psychopharmacology. 12/2003; 170(2):200-4.

  RATIONALE: In 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, toRATIONALE: In 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. OBJECTIVES: We 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. METHODS: After solid phase extraction plasma and urine concentrations of morphine metabolites were determined by HPLC analyses. RESULTS: M3G 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. CONCLUSIONS: Chronic 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.

• Repeated exposures to heroin and/or cadmium alter the rate of formation of morphine glucuronides in the rat.

  Authors: Letizia Antonilli, Carmen Suriano, Giovanna Paolone, Aldo Badiani, Paolo Nencini

  The Journal of pharmacology and experimental therapeutics. 12/2003; 307(2):651-60.

  After absorption, heroin is transformed into mono-acetyl-morphine and then into morphine. Morphine, in turn, is metabolized to morphine-3-glucuronide (M3G), an inactive compound, andAfter absorption, heroin is transformed into mono-acetyl-morphine and then into morphine. Morphine, in turn, is metabolized to morphine-3-glucuronide (M3G), an inactive compound, and morphine-6-glucuronide (M6G), a potent opioid agonist. Thus, changes in the rate of formation of M6G may alter the pharmacological consequences of a treatment with heroin or morphine. In this study, we investigate the effect of repeated exposures (10 daily i.p. injections) to heroin, morphine, cadmium (which has been previously shown to inhibit M3G formation in vitro), or heroin + cadmium on morphine glucuronidation both in vivo and ex vivo (i.e., microsomal preparation obtained from rats treated in vivo). Repeated heroin (2.5, 5.0, and 10 mg/kg) increased plasma levels of M6G (which was undetectable in all other groups) and reduced those of M3G. Also, the microsomal preparations obtained from the liver of repeated heroin rats, when incubated with morphine, yielded significant amounts of M6G (which was undetectable in all other groups) and decreased levels of M3G relative to the control groups. These effects were reversible upon discontinuation of heroin administration. In contrast, repeated morphine (10, 20, and 40 mg/kg) only slightly reduced M3G formation at the dose of 40 mg/kg. Repeated cadmium (5, 15, and 45 microg/kg) reduced the rate of M3G formation without inducing M6G synthesis. The effects of the repeated coadministration of heroin (10 mg/kg) and cadmium (15 microg/kg) were virtually identical to those of repeated heroin alone. In summary, repeated exposure of rats to heroin can shift morphine glucuronidation toward the formation of the active metabolite M6G.

• 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

  Authors: Letizia Antonilli, Carmen Suriano, Maria Caterina Grassi, Paolo Nencini

  Journal of Chromatography B: Biomedical Sciences and Applications.

  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 chromatographyCocaine 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 μg/ml for the three compounds, whereas HPLC limits were 0.2 μg/ml for benzoylecgonine and cocaine, and 0.1 μg/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.