Elena Bastia

Forebrain Adenosine A2A Receptors Contribute to l-3,4-Dihydroxyphenylalanine-Induced Dyskinesia in Hemiparkinsonian Mice

Adenosine A2A receptor antagonists provide a promising nondopaminergic approach to the treatment of Parkinsons disease (PD). Initial clinical trials of A2A antagonists targeted PD patients who had already developed treatment complications known as l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID) in an effort to improve symptoms while reducing existing LID. The goal of this study is to explore the effect of A2A antagonists and targeted A2A receptor depletion on the actual development of sensitized responses to l-DOPA in mouse models of LID in PD. Hemiparkinsonian mice (unilaterally lesioned with 6-OHDA) were treated daily for 3 weeks with a low dose of l-DOPA (2 mg/kg) preceded by a low dose of selective A2A antagonist (KW-6002 [(E)-1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione] at 0.03 or 0.3 mg/kg, or SCH58261 [5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine] at 0.03 mg/kg) or vehicle intraperitoneally. In control mice, contralateral rotational responses to daily l-DOPA gradually increased over the initial week before reaching a persistent maximum. Both A2A antagonists inhibited the development of sensitized contralateral turning, with KW-6002 pretreatment reducing the sensitized rotational responses by up to threefold. The development of abnormal involuntary movements (a measure of LID) as well as rotational responses was attenuated by the postnatal depletion of forebrain A2A receptors in conditional (Cre/loxP system) knock-out mice. These pharmacological and genetic data provide evidence that striatal A2A receptors play an important role in the neuroplasticity underlying behavioral sensitization to l-DOPA, supporting consideration of early adjunctive therapy with an A2A antagonist to reduce the risk of LID in PD.

A Crucial Role for Forebrain Adenosine A 2A Receptors in Amphetamine Sensitization

Adenosine A2A receptors (A2ARs) are well positioned to influence the maladaptive CNS responses to repeated dopaminergic stimulation in psychostimulant addiction. Expression of A2ARs in brain is largely restricted to the nucleus accumbens and striatum, where molecular adaptations mediate chronic effects of psychostimulants such as behavioral sensitization. Using a novel forebrain-specific conditional (Cre/loxP system) knockout of the A2AR in coordination with classical pharmacological approaches, we investigated the involvement of brain A2ARs in amphetamine-induced behavioral sensitization. Tissue-specific, functional disruption of the receptor was confirmed by autoradiography, PCR, and the loss of A2A antagonist-induced motor stimulation. Daily treatment with amphetamine for 1 week markedly enhanced locomotor responses on day 8 in control mice and the sensitization remained robust after a week of washout. Their conditional knockout littermates however showed no sensitization to amphetamine on day 8 and only a modest sensitization following the washout. Pharmacological blockade of adenosine A2ARs also was able to block the development (but not the expression) of sensitization in multiple mouse strains. Thus activation of brain A2ARs plays a critical role in developing augmented psychomotor responses to repeated psychostimulant exposure.

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.

Inactivation of adenosine A2A receptors selectively attenuates amphetamine-induced behavioral sensitization

Repeated treatment with the psychostimulant amphetamine produces behavioral sensitization that may represent the neural adaptations underlying some features of psychosis and addiction in humans. In the present study we investigated the role of adenosine A2A receptors in psychostimulant-induced locomotor sensitization using an A2A receptor knockout (A2A KO) model. Daily treatment with amphetamine for 1 week resulted in an enhanced motor response on day 8 (by two-fold compared to that on day 1), and remained enhanced at day 24 upon rechallenge with amphetamine. By contrast, locomotor sensitization to daily amphetamine did not develop in A2A KO mice on day 8 or 24, and this absence was not the result of a nonspecific threshold effect. The absence of behavioral sensitization was selective for amphetamine since daily treatment with the D1 agonist SKF81297 (2.5 mg/kg) or the D2 agonist quinpirole (1.0 mg/kg) produced similar behavioral sensitization in both WT and A2A KO mice. Furthermore, coinjection of SKF81297 and quinpirole also resulted in indistinguishable locomotor sensitization in A2A KO and WT mice, suggesting normal D1 and D2 receptor responsiveness. Finally, at the cellular level A2A receptor inactivation abolished the increase in striatal dynorphin mRNA induced by repeated amphetamine administration. The selective absence of amphetamine-induced behavioral sensitization in A2A KO mice suggests a critical role of the A2A receptor in the development of psychostimulant-induced behavioral sensitization, and supports the pharmacological potential of A2A adenosinergic agents to modulate adaptive responses to repeated psychostimulant exposure.

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.

Tetanus toxin fragment C as a vector to enhance delivery of proteins to the CNS

The non-toxic neuronal binding domain of tetanus toxin (tetanus toxin fragment C, TTC) has been used as a vector to enhance delivery of potentially therapeutic proteins to motor neurons from the periphery following an intramuscular injection. The unique binding and transport properties of this 50-kDa polypeptide suggest that it might also enhance delivery of proteins to neurons after direct injection into the CNS. Using quantitative fluorimetry, we found that labeled TTC showed vastly superior retention within brain tissue after intracerebral injection compared to a control protein (bovine serum album). Fluorescence microscopy revealed that injected TTC was not retained solely in a restricted deposit along the needle track, but was distributed through gray matter in a pattern not previously described. The distribution of injected protein within the extracellular space of the gray matter and neuropil was also seen after injection of a recombinant fusion protein comprised of TTC linked to the enzyme superoxide dismutase (TTC-SOD-1). Injections of native SOD-1 in contrast showed only minimal retention of protein along the injection track. Immunohistochemistry demonstrated that both TTC and TTC-SOD-1 were distributed in a punctate perineuronal and intraneuronal pattern similar to that seen after their retrograde transport, suggesting localization primarily in synaptic boutons. This synaptic distribution was confirmed using HRP-labeled TTC with electron microscopy along with localization within neuronal endosomes. We conclude that TTC may be a useful vector to enhance neuronal delivery of potentially therapeutic enzymes or trophic factors following direct injection into the brain.

Adenosine A2A receptors in neuroadaptation to repeated dopaminergic stimulation: Implications for the treatment of dyskinesias in Parkinson's disease

The A2A receptor has recently attracted considerable interest as a potential target for Parkinson’s disease (PD) therapy based on the motor-enhancing and neuroprotective effects of A2A antagonists in animal models of PD. The unique neuronal localization of the adenosine A2A receptor in the basal ganglia and its extensive interactions with dopaminergic and glutamatergic systems led the authors to investigate a potential role of the A2A receptor in the development of behavioral sensitization in response to repeated dopaminergic stimulation. Because dopamine-induced behavioral sensitization shares several neurochemical and behavioral features with dyskinesia, characterizing this novel aspect of A2A receptor function may enhance understanding and management of dyskinesia in PD. Recent studies from several laboratories suggest that the A2A receptor may be an important mediator of maladaptive changes in response to long-term dopamine stimulation. The authors summarize their investigation of the role of A2A receptors in two paradigms of behavioral sensitization elicited by daily treatment with either l-dopa in hemiparkinsonian mice or amphetamine in naive mice. The results demonstrate that the A2A receptor is required for the development of behavioral sensitization in response to repeated l-dopa treatment in hemiparkinsonian mice and repeated amphetamine administration in normal mice. Together with pharmacologic studies, these results raise the possibility that the maladaptive dyskinetic responses to long-term l-dopa management of PD may be attenuated by A2A receptor blockade. Potential presynaptic, postsynaptic (cellular), and trans-synaptic (network) mechanisms are discussed.

Tetanus toxin fragment C fusion facilitates protein delivery to CNS neurons from cerebrospinal fluid in mice

To improve protein delivery to the CNS following intracerebroventricular administration, we compared the distribution of a human Cu/Zn superoxide dismutase:tetanus toxin fragment C fusion protein (SOD1:TTC) in mouse brain and spinal cord with that of tetanus toxin fragment C (TTC) or human SOD1 (hSOD1) alone, following continuous infusion into the lateral ventricle. Mice infused with TTC or SOD1:TTC showed intense anti‐TTC or anti‐hSOD1 labeling, respectively, throughout the CNS. In contrast, animals treated with hSOD1 revealed moderate staining in periventricular tissues. In spinal cord sections from animals infused with SOD1:TTC, the fusion protein was found in neuron nuclear antigen‐positive (NeuN+) neurons and not glial fibrillary acidic protein‐positive (GFAP+) astrocytes. The percentage of NeuN+ ventral horn cells that were co‐labeled with hSOD1 antibody was greater in mice treated with SOD1:TTC (cervical cord = 73 ± 8.5%; lumbar cord = 62 ± 7.7%) than in mice treated with hSOD1 alone (cervical cord = 15 ± 3.9%; lumbar cord = 27 ±4.7%). Enzyme‐linked immunosorbent assay for hSOD1 further demonstrated that SOD1:TTC‐infused mice had higher levels of immunoreactive hSOD1 in CNS tissue extracts than hSOD1‐infused mice. Following 24 h of drug washout, tissue extracts from SOD1:TTC‐treated mice still contained substantial amounts of hSOD1, while extracts from hSOD1‐treated mice lacked detectable hSOD1. Immunoprecipitation of SOD1:TTC from these extracts using anti‐TTC antibody revealed that the recovered fusion protein was structurally intact and enzymatically active. These results indicate that TTC may serve as a useful prototype for development as a non‐viral vehicle for improving delivery of therapeutic proteins to the CNS.

Intraocular Pressure–Lowering Activity of NCX 470, a Novel Nitric Oxide–Donating Bimatoprost in Preclinical Models

PURPOSE The prostaglandin F2alpha (PGF2α) analogue bimatoprost lowers intraocular pressure (IOP) by increasing uveoscleral outflow at doses shown to elicit redness of the eye. With the aim to enhance the IOP-lowering effect of bimatoprost we studied NCX 470 [(S,E)-1-((1R,2R,3S,5R)-2-((Z)-7-(ethylamino)-7-oxohept-2-enyl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate], a dual-acting compound combining bimatoprost with nitric oxide (NO) known to mainly act via relaxation of trabecular meshwork and Schlemms canal. METHODS New Zealand white rabbits with transient hypertonic saline-induced IOP elevation (tOHT-rabbits), cynomolgus monkeys with laser-induced ocular hypertension (OHT-monkeys), and normotensive dogs (ONT-dogs) were used. The levels of NCX 470, bimatoprost, and bimatoprost acid were determined in aqueous humor (AH), cornea (CR), and iris/ciliary body (ICB) by liquid chromatography-mass spectrometry/mass (LC-MS/MS), while cGMP in AH and ICB was monitored using an enzyme immunoassay (EIA) kit in pigmented Dutch Belted rabbits. RESULTS NCX 470 (0.14%, 30 μL) lowered IOP in tOHT-rabbits with an E(max) of -7.2 ± 2.8 mm Hg at 90 minutes. Bimatoprost at equimolar dose (0.1%, 30 μL) was noneffective in this model. NCX 470 (0.042%, 30 μL) was more effective than equimolar (0.03%, 30 μL) bimatoprost in ONT-dogs (IOP change, -5.4 ± 0.7 and -3.4 ± 0.7 mm Hg, respectively, P < 0.05) and in OHT-monkeys (IOP change, -7.7 ± 1.4 and -4.8 ± 1.7 mm Hg, respectively, P < 0.05) at 18 hours post dosing. NCX 470 (0.042%, 30 μL) or bimatoprost (0.03%, 30 μL) resulted in similar bimatoprost acid exposure in AH, CR, and ICB while cGMP was significantly increased in AH and ICB at 18 and 24 hours after NCX 470 dosing. CONCLUSIONS NCX 470 lowers IOP more than equimolar bimatoprost in three animal models of glaucoma by activating PGF2α and NO/cGMP signaling pathways.

New furoxan derivatives for the treatment of ocular hypertension

A small series of water-soluble NO-donor furoxans bearing a basic center at the 4-position, having a wide lipophilic-hydrophilic balance range, and endowed with different NO-release capacities, were synthesized and characterized. Selected members were studied for their IOP-lowering activity in the transient ocular hypertensive rabbit model at 1% dose. The most effective IOP-lowering products were compounds 3 and 7, whose activity 60min after administration was similar to that of Timolol. Notably, 7 was characterized by a long-lasting action. The IOP-lowering activity in this series of products appeared to be modulated by the lipophilic-hydrophilic balance rather than by the NO-donor capacity.