Daniela Melisi

Glycosyl Derivatives of Dopamine and l-dopa as Anti-Parkinson Prodrugs: Synthesis, Pharmacological Activity and In Vitro Stability Studies

Novel glycosyl derivatives of dopamine and l-dopa (I–IV) are synthesized in order to overcome the problem of blood–brain barrier low permeability of dopamine and of low bioavailability of its precursor l-dopa. Esters synthesized link dopamine and l-dopa, by a succinyl linker, to C-3 position of glucose (I and III) and to C-6 of galactose (II and IV). The chemical and enzymatic stabilities of esters synthesized were evaluated in order to determine both their stability in aqueous medium and their feasibility in undergoing enzymatic cleavage by rat plasma to regenerate the original drug. Furthermore, we have shown the central effects of esters I–IV on classic dopaminergic models, such as morphine induced locomotion and reserpine-induced hypolocomotion. From the result obtained compounds I–IV appeared moderately stable in a pH 7.4 buffered solution and in rat plasma. Furthermore, pharmacological studies showed that both dopamine derivatives (I and II) were equiactive in reversing reserpine-induced hypolocomotion in rats, and both were more active than l-dopa or ester III and IV, while II and III were more potent in reducing morphine-induced locomotion than I and IV. The minimal vascular effects of these derivatives allow us to underline the possibility to use them in pathologies, such as Parkinson disease, characterised by an evident decreasing of dopamine concentration in the brain.

Novel sol-gel organic-inorganic hybrid materials for drug delivery

PURPOSE The aim of the present study was to synthetize and characterize novel sol-gel organic-inorganic hybrid materials to be used for controlled drug delivery application. MATERIALS AND METHODS Organic-inorganic hybrid class I materials based on poly(epsilon-caprolactone) (PCL 6, 12, 24 and 50 wt%) and zirconia-yttria (ZrO2-5%Y2O3) were synthesized by a sol-gel method, from a multicomponent solution containing zirconium propoxide [Zr(OC2H7)4], yttrium chloride (YCl3), PCL, water and chloroform (CHCl3). The structure of the hybrids was obtained by means of hydrogen bonds between the Zr-OH group (H-donor) in the sol-gel intermediate species and the carboxylic group (H-acceptor) in the repeating units of the polymer. RESULTS The presence of hydrogen bonds between organic-inorganic components of the hybrid materials was suggested by Fourier transform infrared (FTIR) analysis, and strongly supported by solid-state NMR. A single-step, sol-gel process was then used to precipitate microspheres containing ketoprofen or indomethacin for controlled drug delivery applications. Release kinetics in a simulated body fluid (SBF) were subsequently investigated. The amount of drug released was detected by UV-VIS spectroscopy. Pure anti-inflammatory agents exhibited linear release with time, in contrast drugs entrapped in the organic-inorganic hybrids were released with a logarithmic time dependence, starting with an initial burst effect followed by a gradual decrease. CONCLUSIONS The synthesis of amorphous materials containing drugs, obtained by sol-gel methods, helps to devise new strategies for controlled drug delivery system design.

D-galactose as a vector for prodrug design.

D-galactose is a simple and natural compound that has mainly been exploited in prodrug strategies. Galactosyl prodrugs can be considered a good approach to reach different goals in clinical drug application, especially when traditional drugs are likely to fail therapeutically owing to reasons such as the lack of site specificity, toxicity, and chemical instability. Indeed, of paramount importance is their ability to increase the selectivity of the parent compound, a phenomenon that helps to reduce the incidence of adverse effects, while preserving intact the pharmacodynamic features of the parent drug. Study results have varied according to the type of linkage between the drug and the hydroxyl group exploited. By working with these parameters, researchers have been able not only to generate selective pharmacological targeting of brain, liver, and cancerous cells, but also to improve cellular permeability as well as the pharmacokinetic profile of parent drugs. This review describes the broad spectrum of possibilities for exploiting D-galactose as a vector for prodrug design and the synthetic strategies that allow its realization.

Molecular Pharmacology of the Amiloride Analog 3-Amino-6-chloro-5-[(4-chloro-benzyl)amino]-N-[[(2,4-dimethylbenzyl)-amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) as a Pan Inhibitor of the Na+-Ca2+ Exchanger Isoforms NCX1, NCX2, and NCX3 in Stably Transfected Cells

With the help of single-cell microflorimetry, 45Ca2+ radiotracer fluxes, and patch-clamp in whole-cell configuration, we examined the effect of the amiloride derivative 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]-N-[[(2,4-dimethylbenzyl)amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) on the activity of the three isoforms of the Na+/Ca2+ exchanger (NCX) and on several other membrane currents including voltage- and pH-sensitive ones. This amiloride analog suppressed the bidirectional activity of all NCX isoforms in a concentration-dependent manner. The IC50 values of CB-DMB were in the nanomolar range for the outward and the inward components of the bidirectional NCX1, NCX2, and NCX3 activity. Deletion mutagenesis showed that CB-DMB inhibited NCX activity mainly at level of the f-loop but not through the interaction with Gly833 located at the level of the α2 repeat. On the other hand, CB-DMB suppressed in the micromolar range the other plasma membrane currents encoded by voltage-dependent Ca2+ channels, tetrodotoxin-sensitive Na+ channels, and pH-sensitive ASIC1a. Collectively, the data of the present study showed that CB-DMB, when used in the nanomolar range, is one of the most potent compounds that can block the activity of the three NCX isoforms when they work both in the forward and in the reverse modes of operation without interfering with other ionic channels.

T-type channel blocking properties and antiabsence activity of two imidazo (1,2-b)pyridazine derivatives structurally related to indomethacin

It is presently unclear whether the antiseizure effects exerted by NSAIDs are totally dependent on COX inhibition or not. To clarify this point we investigated whether 7-methyl-2-phenylimidazo[1,2-b]pyridazine-3-carboxylic acid (DM1) and 6-methoxy-2-phenylimidazo[1,2-b]pyridazine-3-carboxylic acid (DM2), two imidazo[1,2-b]pyridazines structurally related to indomethacin (IDM) but ineffective in blocking COXs, retain IDM antiabsence activity. When administered by intraperitoneal injection in WAG/Rij rats, a rat strain which spontaneously develops SWDs, both DM1 and DM2 dose-dependently suppressed the occurrence of these seizures. Importantly, these compounds were both more potent in suppressing SWD occurrence than IDM. As T-type channel blockade is considered a mechanism of action common to many antiabsence drugs we explored by whole cell patch clamp electrophysiology in stably transfected HEK-293 the effect of DM1 and DM2 on Ca(V)3.1 channels, the T-type channel subtype preferentially expressed in ventrobasal thalamic nuclei. Both these compounds dose-dependently suppressed the currents elicited by membrane depolarization in these cells. A similar T-type blocking effect was also observed when the cells were exposed to IDM. In conclusion, DM1 and DM2 whilst inactive on COXs, are potent antiabsence drugs. This suggests that compounds with structural features typical of NSAIDs may exert antiepileptic activity independently from COX inhibition and possibly by a direct interaction with T-type voltage-dependent Ca(2+) channels.

Ibuprofen delivered by poly(lactic-co-glycolic acid) (PLGA) nanoparticles to human gastric cancer cells exerts antiproliferative activity at very low concentrations

Purpose Epidemiological, clinical, and laboratory studies have suggested that ibuprofen, a commonly used nonsteroidal anti-inflammatory drug, inhibits the promotion and proliferation of certain tumors. Recently, we demonstrated the antiproliferative effects of ibuprofen on the human gastric cancer cell line MKN-45. However, high doses of ibuprofen were required to elicit these antiproliferative effects in vitro. The present research compared the antiproliferative effects of ibuprofen delivered freely and released by poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) in MKN-45 cells. Methods MKN-45 human gastric adenocarcinoma cells were treated with ibuprofen-loaded PLGA NPs. The proliferation of MKN-45 cells was then assessed by cell counting. The uptake of NPs was imaged by fluorescence microscopy and flow cytometry. The release of ibuprofen from ibuprofen-loaded PLGA NPs in the cells was evaluated by gas chromatography–mass spectrometry. Results Dramatic inhibition of cellular proliferation was observed in cells treated with ibuprofen-loaded PLGA NPs versus those treated with free ibuprofen at the same concentration. The localization of NPs was cytoplasmic. The initiation of ibuprofen release was rapid, commencing within 2 hours, and then increased slowly over time, reaching a maximum concentration at 24 hours. The inhibition of proliferation was confirmed to be due to the intracellular release of ibuprofen from the NPs. Using PLGA NPs as carriers, ibuprofen exerted an antiproliferative activity at concentrations > 100 times less than free ibuprofen, suggesting greater efficiency and less cellular toxicity. In addition, when carried by PLGA NPs, ibuprofen more quickly induced the expression of transcripts involved in proliferation and invasiveness processes. Conclusion Ibuprofen exerted an antiproliferative effect on MKN-45 cells at low concentrations. This effect was achieved using PLGA NPs as carriers of low doses of ibuprofen.

Galactosyl prodrug of ketorolac: synthesis, stability, and pharmacological and pharmacokinetic evaluations.

Although ketorolac is one of the most potent anti-inflammatory and analgesic drugs, its use has been strongly limited owing to the high incidence of adverse effects reported, particularly in the gastrointestinal tract. Using the prodrug approach, which allows the reduction of toxicological features of the parent drug without altering its pharmacological properties, we synthesized an orally administrable prodrug of ketorolac by means of its reversible conjugation to D-galactose (ketogal). In a single dose study, its pharmacokinetic profile was compared with that of ketorolac. Moreover, we found that this prodrug was able to maintain the anti-inflammatory and the analgesic activity of the drug without giving rise to gastric ulcer formation. Thus, these results indicate that ketogal is a highly effective and valid therapeutic alternative to ketorolac itself.

Galactosylated dopamine enters into the brain, blocks the mesocorticolimbic system and modulates activity and scanning time in Naples high excitability rats

Pathological conditions, such as Parkinsons disease and attention deficit hyperactivity disorder, have been linked to alterations of specific dopamine (DA) pathways. However, since exogenous DA does not cross the blood-brain barrier, DA levels can be modulated e.g. by DA precursors or DA reuptake blockers. Hereby histochemical, analytical and behavioral evidence shows that a galactosylated form of DA (GAL-DA) carries DA into the brain, thus modulating activity and nonselective attention in rats. To this aim adult male rats of the Naples high-excitability (NHE) and random bred controls (NRB) lines were given a single i.p. injection of GAL-DA (10 or 100 mg/kg). Three hours later the behavior was videotaped and analyzed for horizontal activity, orienting frequency and scanning duration. The dose of 100 mglkg of GAL-DA reduced by 25% the horizontal activity in NHE rats, mainly in the first part of the testing period. No effect was observed on orienting frequency or on scanning duration. However, GAL-DA 100 mg/kg was associated with longer rearing episodes in the second part of the testing period in NHE rats. In parallel experiments histochemistry with a galactose-specific lectin showed 10% increase in galactose residues into the striatum between 0.5 and 3.0 h. To quantify the level of GAL-DA, its metabolite DA-succinate and DA in the prefrontal cortex, neostriatum, and cerebellum, rats were killed 2.0 h after the injection of prodrug. Mass high performance liquid chromatography (HPLC) was used for analysis of GAL-DA and DA succinate whereas electrochemical HPLC for DA. Both HPLC techniques demonstrate that GAL-DA carries and releases DA into the brain. Specifically 100 mg/kg of GAL-DA increased DA level in the striatum in the NHE rats only. Moreover, DA in the mesencephalon (MES) was correlated positively with striatal and prefrontal cortex DA in NHE rats. In contrast DA in the MES was negatively correlated with striatal DA in NRB. GAL-DA disrupted these correlations in both rat lines. Thus, this new DA prodrug may modify DA neurotransmission and might have a potential clinical application.

The galactosylation of Nω-nitro-L-arginine enhances its anti-nocifensive or anti-allodynic effects by targeting glia in healthy and neuropathic mice

This study has investigated whether the galactosyl ester prodrug of N(ω)-nitro-L-arginine (NAGAL), shows enhanced analgesic efficacy in healthy mice and in models of visceral and neuropathic pain: the writhing test and the spared nerve injury (SNI), respectively. NAGAL was compared to methyl ester pro-drug of N(ω)-nitro-l-arginine (L-NAME), a widely exploited non-specific nitric oxide synthase (NOS) inhibitor, for analgesic potential. The writhing test revealed that the ED(50) value, along with the 95% confidence limit (CL) was 3.82 (1.77-6.04) mg/kg for NAGAL and, 36.75 (20.07-68.37) mg/kg for L-NAME. Notably, NAGAL elicited a greater anti-allodynic effect than L-NAME did in neuropathic mice. Biomolecular and morphological studies revealed that spared nerve injury increased the expressions of pro-inflammatory enzymes (caspase-1) and two glial cell biomarkers: integrin alpha M (ITGAM) and glial fibrillary acidic protein (GFAP) in the spinal cord. Finally, GLUT-3, an isoform of the hexose transporters capable to bind NAGAL and inducible NOS (iNOS), were found to be over-expressed in the activated astrocytes of the spinal cord of neuropathic mice. NAGAL administration normalized expression levels of these biomarkers. NAGAL showed a greater efficacy in inhibiting visceral pain and allodynia than L-NAME possibly by a greater cell permeation through the hexose transporter which is highly over-expressed by activated glia.

Prepuberal subchronic methylphenidate and atomoxetine induce different long-term effects on adult behaviour and forebrain dopamine, norepinephrine and serotonin in Naples High-Excitability rats

The psychostimulant methylphenidate and the non-stimulant atomoxetine are two approved drugs for attention-deficit hyperactivity disorder (ADHD) therapy. The aim of this study was to investigate the long-term effects of prepuberal subchronic methylphenidate and atomoxetine on adult behaviour and the forebrain neurotransmitter and metabolite content of Naples High-Excitability (NHE) rats, a genetic model for the mesocortical variant of ADHD. Male NHE rats were given a daily intraperitoneal injection (1.0mg/kg) of methylphenidate, atomoxetine or vehicle from postnatal day 29 to 42. At postnatal day 70-75, rats were exposed to spatial novelty in the Làt and radial (Olton) mazes. Behavioural analysis for indices of horizontal, vertical, non-selective (NSA) and selective spatial attention (SSA) indicated that only methylphenidate significantly reduced horizontal activity to a different extent, i.e., 39 and 16% respectively. Moreover methylphenidate increased NSA as assessed by higher leaning duration. The high-performance liquid chromatography (HPLC) tissue content assessment of dopamine, norepinephrine, serotonin and relative metabolites in the prefrontal cortex (PFC), cortical motor area (MC), dorsal striatum (DS), ventral striatum (VS), hippocampus and mesencephalon indicated that methylphenidate decreased (i) dopamine, DOPAC, norepinephrine, MHPG, 5-HT and 5-HIAA in the PFC, (ii) dopamine, DOPAC, HVA, serotonin, 5-HIAA in the DS, (iii) dopamine, DOPAC, HVA and MHPG (but increased norepinephrine) in the VS and (iv) norepinephrine, MHPG, serotonin and 5-HIAA in the hippocampus. Atomoxetine increased dopamine and decreased MHPG in the PFC. Like methylphenidate, atomoxetine decreased dopamine, DOPAC, HVA, serotonin and 5-HIAA in the DS, but decreased MHPG in the VS. These results suggest that methylphenidate determined long-term effects on behavioural and neurochemical parameters, whereas atomoxetine affected only the latter.