Francesco Impagnatiello

Isolation and Characterization of Neural Stem Cells from the Adult Human Olfactory Bulb

We have recently isolated stem cells deriving from the olfactory bulbs of adult patients undergoing particularly invasive neurosurgery. After improving our experimental conditions, we have now obtained neural stem cells according to clonal analysis. The cells can be expanded, established in continuous cell lines and differentiated into the three classical neuronal phenotypes (neurons, astrocytes, and oligodendrocytes). Also, after exposition to leukemia inhibitory factor, we are able to improve the number of neurons, an ideal biological source for transplantation in various neurodegenerative disorders.

Persistent Behavioral Sensitization to Chronic l-DOPA Requires A2A Adenosine Receptors

To investigate the role of A2A adenosine receptors in adaptive responses to chronic intermittent dopamine receptor stimulation, we compared the behavioral sensitization elicited by repeated l-DOPA treatment in hemiparkinsonian wild-type (WT) and A2A adenosine receptor knock-out (A2AKO) mice. Although the unilateral nigrostriatal lesion produced by intrastriatal injection of 6-hydroxydopamine was indistinguishable between WT and A2A KO mice, they developed strikingly different patterns of behavioral sensitization after daily treatment with low doses of l-DOPA for 3 weeks. WT mice initially displayed modest contralateral rotational responses and then developed progressively greater responses that reached a maximum within 1 week and persisted for the duration of the treatment. In contrast, any rotational behavioral sensitization in A2A KO mice was transient and completely reversed within 2 weeks. Similarly, the time to reach the peak rotation was progressively shortened in WT mice but remained unchanged in A2A KO mice. Furthermore, dailyl-DOPA treatment produced gradually sensitized grooming in WT mice but failed to induce any sensitized grooming in A2AKO mice. Finally, repeated l-DOPA treatment reversed the 6-OHDA-induced reduction of striatal dynorphin mRNA in WT but not A2A KO mice, raising the possibility that the A2A receptor may contribute to l-DOPA-induced behavioral sensitization by facilitating adaptations within the dynorphin-expressing striatonigral pathway. Together these results demonstrate that the A2A receptor plays a critical role in the development and particularly the persistence of behavioral sensitization to repeated l-DOPA treatment. Furthermore, they raise the possibility that the maladaptive dyskinetic responses to chronic l-DOPA treatment in Parkinsons disease may be attenuated by A2A receptor inactivation.

A new approach to antiglaucoma drugs: carbonic anhydrase inhibitors with or without NO donating moieties. Mechanism of action and preliminary pharmacology

The clinically used sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor dorzolamide (DRZ), a new sulfonamide CA inhibitor also incorporating NO-donating moieties, NCX250, and isosorbide mononitrate (ISMN) (an NO-donating compound with no CA inhibitory properties) were investigated for their intraocular pressure (IOP) lowering effects in rabbits with carbomer-induced glaucoma. NCX250 was more effective than DRZ or ISMN on lowering IOP, increasing ocular hemodynamics, decreasing the inflammatory processes and ocular apoptosis in this animal model of glaucoma. NO participate to the regulation of IOP in glaucoma, having also antiapoptotic and anti-inflammatory effects. The ophthalmic artery, both systolic and diastolic velocities, were significantly reduced in NCX250-treated eyes in comparison to DRZ treated ones, suggesting thus a beneficial effect of NCX250 on the blood supply to the optic nerve. Combining CA inhibition with NO-donating moieties in the same compound offers an excellent approach for the management of glaucoma.

Characterization of the Potent and Highly Selective A2A Receptor Antagonists Preladenant and SCH 412348 [7-[2-[4-2,4-Difluorophenyl]-1-piperazinyl]ethyl]-2-(2-furanyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] in Rodent Models of Movement Disorders and Depression

The adenosine A2A receptor has been implicated in the underlying biology of various neurological and psychiatric disorders, including Parkinsons disease (PD) and depression. Preladenant and SCH 412348 [7-[2-[4-2,4-difluorophenyl]-1-piperazinyl]ethyl]-2-(2-furanyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] are potent competitive antagonists of the human A2A receptor (Ki = 1.1 and 0.6 nM, respectively) and have >1000-fold selectivity over all other adenosine receptors, making these compounds the most selective A2A receptor antagonists reported to date. Both compounds attenuate hypolocomotion induced by the A2A receptor agonist CGS-21680 [2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine], suggesting that they inhibit A2A receptor activity in vivo. Their high degree of selectivity and robust in vivo activity make preladenant and SCH 412348 useful tools to investigate the role of the A2A receptor system in animal models of PD and depression. Oral administration of preladenant and SCH 412348 (0.1–1 mg/kg) to rats potentiated 3,4-dihydroxy-l-phenylalanine (l-Dopa)-induced contralateral rotations after 6-hydroxydopamine lesions in the medial forebrain bundle and potently attenuated the cataleptic effects of haloperidol. Preladenant (1 mg/kg) inhibited l-Dopa-induced behavioral sensitization after repeated daily administration, which suggests a reduced risk of the development of dyskinesias. Finally, preladenant and SCH 412348 exhibited antidepressant-like profiles in models of behavioral despair, namely the mouse tail suspension test and the mouse and rat forced swim test. These studies demonstrate that preladenant and SCH 412348 are potent and selective A2A receptor antagonists and provide further evidence of the potential therapeutic benefits of A2A receptor inhibition in PD (with reduced risk of dyskinesias) and depression (one of the primary nonmotor symptoms of PD).

Nitric Oxide (NO): An Emerging Target for the Treatment of Glaucoma

The predominant risk factor for the progression of glaucoma is an increase in IOP, mediated via a reduction in aqueous outflow through the conventional (trabecular meshwork and Schlemms canal) outflow pathway. Current IOP lowering pharmacological strategies target the uveoscleral (nonconventional) outflow pathway or aqueous humor production; however, to date no therapy that primarily targets the conventional pathway exists. Nitric oxide (NO) is an intracellular signaling molecule produced by endogenous NO synthases, well-known for its key role in vasodilation, through its action on smooth muscle cells. Under physiological conditions, NO mediates a multitude of diverse ocular effects, including maintenance of IOP. Nitric oxide donors have been shown to mediate IOP-lowering effects in both preclinical models and clinical studies, primarily through cell volume and contractility changes in the conventional outflow tissues. This review is focused on evaluating the current knowledge of the role and mechanism of action of endogenous NO and NO donors in IOP regulation. Data on key additional functions of NO in glaucoma pathology (i.e., ocular blood flow and effects on optic neuropathy) are also summarized. The potential for future therapeutic application of NO in the treatment of glaucoma is then discussed.

Aging-induced Nrf2-ARE pathway disruption in the subventricular zone drives neurogenic impairment in parkinsonian mice via PI3K-Wnt/β-catenin dysregulation.

Aging and exposure to environmental toxins including MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) are strong risk factors for developing Parkinsons disease (PD), a common neurologic disorder characterized by selective degeneration of midbrain dopaminergic (DAergic) neurons and astrogliosis. Aging and PD impair the subventricular zone (SVZ), one of the most important brain regions for adult neurogenesis. Because inflammation and oxidative stress are the hallmarks of aging and PD, we investigated the nature, timing, and signaling mechanisms contributing to aging-induced SVZ stem/neuroprogenitor cell (NPC) inhibition in aging male mice and attempted to determine to what extent manipulation of these pathways produces a functional response in the outcome of MPTP-induced DAergic toxicity. We herein reveal an imbalance of Nrf2-driven antioxidant/anti-inflammatory genes, such as Heme oxygenase1 in the SVZ niche, starting by middle age, amplified upon neurotoxin treatment and associated with an exacerbated proinflammatory SVZ microenvironment converging to dysregulate the Wingless-type MMTV integration site (Wnt)/β-catenin signaling, a key regulatory pathway for adult NPCs. In vitro experiments using coculture paradigms uncovered aged microglial proinflammatory mediators as critical inhibitors of NPC proliferative potential. We also found that interruption of PI3K (phosphatidylinositol3-kinase)/Akt and the Wnt/Fzd/β-catenin signaling cascades, which switch glycogen synthase kinase 3β (GSK-3β) activation on and off, were causally related to the impairment of SVZ-NPCs. Moreover, a synergy between dysfunctional microglia of aging mice and MPTP exposure further inhibited astrocyte proneurogenic properties, including the expression of key Wnts components. Last, pharmacological activation/antagonism studies in vivo and in vitro suggest the potential that aged SVZ manipulation is associated with DAergic functional recovery.

A novel nitric oxide releasing prostaglandin analog, NCX 125, reduces intraocular pressure in rabbit, dog, and primate models of glaucoma.

PURPOSE Nitric oxide (NO) is involved in a variety of physiological processes including ocular aqueous humor dynamics by targeting mechanisms that are complementary to those of prostaglandins. Here, we have characterized a newly synthesized compound, NCX 125, comprising latanoprost acid and NO-donating moieties. METHODS NCX 125 was synthesized and tested in vitro for its ability to release functionally active NO and then compared with core latanoprost for its intraocular pressure (IOP)-lowering effects in rabbit, dog, and nonhuman primate models of glaucoma. RESULTS NCX 125 elicited cGMP formation (EC(50) = 3.8 + or - 1.0 microM) in PC12 cells and exerted NO-dependent iNOS inhibition (IC(50) = 55 + or - 11 microM) in RAW 264.7 macrophages. NCX 125 lowered IOP to a greater extent compared with equimolar latanoprost in: (a) rabbit model of transient ocular hypertension (0.030% latanoprost, not effective; 0.039% NCX 125, Delta(max) = -10.6 + or - 2.3 mm Hg), (b) ocular hypertensive glaucomatous dogs (0.030% latanoprost, Delta(max)= -6.7 + or - 1.2 mm Hg; 0.039% NCX 125, Delta(max) = -9.1 + or - 3.1 mm Hg), and (c) laser-induced ocular hypertensive non-human primates (0.10% latanoprost, Delta(max) = -11.9 + or - 3.7 mm Hg, 0.13% NCX 125, Delta(max) = -16.7 + or - 2.2 mm Hg). In pharmacokinetic studies, NCX 125 and latanoprost resulted in similar latanoprost-free acid exposure in anterior segment ocular tissues. CONCLUSIONS NCX 125, a compound targeting 2 different mechanisms, is endowed with potent ocular hypotensive effects. This may lead to potential new perspectives in the treatment of patients at risk of glaucoma.

Nitric oxide-donating carbonic anhydrase inhibitors for the treatment of open-angle glaucoma.

Novel bi-functional compounds with a nitric oxide (NO)-releasing moiety bound to a dorzolamide scaffold were investigated. Several compounds were synthesized and their activity as selective carbonic anhydrase inhibitors (CAI) evaluated in vitro on recombinant hCA type I, II and IV enzyme isoforms where they showed different degrees of potency and selectivity to hCA II. A high resolution X-ray crystal structure for the CA II adduct with 8 confirmed the high affinity of this class of compounds for the enzyme. Compounds 4, 6, and 8 showed highly potent and efficacious NO-mediated properties as assessed by their vascular relaxant effect on methoxamine-precontracted rabbit aortic rings. Finally, compounds 4 and 6 exerted potent intraocular pressure (IOP) lowering effects in vivo in normotensive rabbits thereby anticipating their potential for the treatment of hypertensive glaucoma.

Adenosine receptors in neurological disorders

Adenosine is present in the extacellular space of organs and tissues in the mammalian organism where it modulates a variety of physiological processes through specific cell membrane receptors which have been identified as members of the G-protein coupled receptors (GPCRs). Currently, four receptors have been cloned and characterised, namely A1, A2A, A2B, A3 adenosine receptors. The different distribution of receptor populations in organs and tissues together with their importance in normal and altered conditions make each individual adenosine receptor an attractive target for the discovery of new pharmacological agents. Major efforts have involved the A1 and A2A receptors with the central nervous (and cardiovascular) systems being the medical areas of highest interest. Thus, a variety of selective receptor agonists and antagonists have been discovered and some are currently under development for the treatment of neurological disorders. Treatment of pain, neurodegenerative diseases, such as Parkinson’s (PD) and Huntington’s disease (HD), cognitive impairment and epilepsy are all potential therapy areas of future drug development. This review describes the scientific background information currently available together with the rationale underlying the innovative strategies associated with the modulation of each individual adenosine receptor.

A nitric oxide (NO)-releasing derivative of gabapentin, NCX 8001, alleviates neuropathic pain-like behavior after spinal cord and peripheral nerve injury

Nitric oxide (NO) participates, at least in part, to the establishment and maintenance of pain after nerve injury. Therefore, drugs that target the NO/cGMP signaling pathway are of interest for the treatment of human neuropathic pain. Various compounds endowed with NO‐releasing properties modulate the expression and function of inducible nitric oxide synthase (iNOS), the key enzyme responsible for sustained NO production under pathological conditions including neuropathic pain. With this background, we synthesized a new chemical entity, [1‐(aminomethyl)cyclohexane acetic acid 3‐(nitroxymethyl)phenyl ester] NCX8001, which has a NO‐releasing moiety bound to gabapentin, a drug currently used for the clinical management of neuropathic pain. We examined the pharmacological profile of this drug with respect to its NO‐releasing properties in vitro as well as to its efficacy in treating neuropathic pain conditions (allodynia) consequent to experimental sciatic nerve or spinal cord injuries. NCX8001 (1–30 μM) released physiologically relevant concentrations of NO as it induced a concentration‐dependent activation of soluble guanylyl cyclase (EC50=5.6 μM) and produced consistent vasorelaxant effects in noradrenaline‐precontracted rabbit aortic rings (IC50=1.4 μM). NCX8001, but not gabapentin, counteracted in a concentration‐dependent fashion lipopolysaccharide‐induced overexpression and function of iNOS in RAW264.7 macrophages cell line. Furthermore, NCX8001 also inhibited the release of tumor necrosis factor alpha (TNFα) from stimulated RAW264.7 cells. NCX8001 (28–280 μmol kg−1, i.p.) reduced the allodynic responses of spinal cord injured rats in a dose‐dependent fashion while lacking sedative or motor effects. In contrast, gabapentin (170–580 μmol kg−1, i.p.) resulted less effective and elicited marked side effects. NCX8001 alleviated the allodynia‐like responses of rats to innocuous mechanical or cold stimulation following lesion of the sciatic nerve. This effect was not shared by equimolar doses of gabapentin. Potentially due to the slow releasing kinetics of NO, NCX8001 alleviated pain‐like behaviors in two rat models of neuropathic pain in a fashion that is superior to its parent counterpart gabapentin. This new gabapentin derivative, whose mechanism deserves to be explored further, offers new hopes to the treatment of human neuropathic pain.