CNS Actions Research Articles

N6-cyclohexyladenosine is better than meperidine and buspirone at suppressing metabolism during TTM32 but does not improve outcome after cardiac arrest

N6-clyclohexyladenosine (CHA) is an adenosine A1 receptor agonist that inhibits thermogenesis. Cardiovascular side effects however, limit use of CHA as a therapeutic. We and others have shown that this can be reversed by administering 8-p-(sulfophenyl)theophylline (8-SPT), a nonspecific antagonist that does not cross the BBB. Other evidence shows that CNS actions of CHA may contribute to bradycardia through enhanced vagal tone and other mechanisms. Here we test the hypothesis that 8-SPT pretreatment alone is sufficient to prevent hypotension caused by CHA. To test this hypothesis, we pretreated rats with 8-SPT alone, and in combination with other antagonists to test the hypothesis that direct action of CHA on the heart is the primary mechanism by which CHA induces bradycardia and hypotension. Results show that pretreatment with 8-SPT alone is not sufficient to prevent CHA-induced hypotension. Pretreatment with 8-SPT or atropine alone did not prevent the fall in mean arterial pressure (MAP) and heart rate (HR), however, pretreatment with 8-SPT (25 mg/kg) and atropine (1 mg/kg) 15 min before CHA (1 mg/kg) preserves MAP and HR baseline values after CHA administration. We next asked if blood pressure was managed during the transition into a hypometabolic state, would prolong CHA-mediated inhibition of metabolism after cardiac arrest improve outcome better than anti-shivering medications meperidine and buspirone. We found that CHA-mediated hypotension can be mitigated by pretreatment with atropine and 8-SPT. This combination administered after cardiac arrest facilitated temperature management and metabolic suppression better than meperidine and buspirone, however, did not improve survival.

Loperamide, a peripheral Mu-Opioid receptor agonist, attenuates chemotherapy-induced neuropathic pain in rats

Opioids are employed in the management of chemotherapy-induced neuropathic pain (CINP) when other pain management approaches have failed and proven ineffective. However, their use in CINP is generally considered as a second-line or adjunctive therapy owing to their central side effects and development of tolerance with their long-term usage. Targeting peripheral sites may offer several advantages over the conventional CNS-based approaches as peripheral targets modulate pain signals at their source, thereby relieving pain with higher specificity, efficacy and minimizing adverse effects associated with off-site CNS actions. Therefore, present study was designed with an aim to investigate the effect of loperamide, a peripherally acting mu-opioid receptor agonist, on paclitaxel-induced neuropathic pain in rats and elucidate its underlying mechanism. Loperamide treatment significantly attenuated mechanical, and cold hypersensitivity and produced significant place preference behaviour in neuropathic rats indicating its potential to treat both evoked and spontaneous pain. More importantly, loperamide treatment in naïve rats did not produce place preference to drug-paired chamber pointing towards its non-addictive analgesic potential. Further, molecular investigations revealed increased expression of ion channels such as TRPA1, TRPM8; voltage-gated sodium channels (VGSCs) and neuroinflammatory markers in the dorsal root ganglion (DRG) and lumbar (L4-L5) spinal cord of neuropathic rats, which was significantly downregulated upon loperamide treatment. These findings collectively suggest that activation of peripheral mu-opioid receptors contributes to the amelioration of both evoked and spontaneous pain in neuropathic rats by downregulating TRP channels and VGSCs along with suppression of oxido-nitrosative stress and neuroinflammatory cascade.

Marco Capogna, a pioneering neuroscientist and true European

Marco Capogna, a pioneering neuroscientist and true European

Sodium Oxybate: a Substitute for Alcohol?

Gamma-hydroxybutyrate (GHB) is a neurotransmitter found naturally in the human brain. Sodium oxybate (SO) is the sodium salt of GHB. In 2000 GHB was classified a Schedule I controlled substance, while SO became a Schedule III controlled substance for medicinal use under the Controlled Substances Act. SO and alcohol share a similar pharmacological profile. GHB acts on GABAB receptors and extrasynaptic GABAA receptors resulting in alcohol-mimetic effects in several CNS actions. It substitutes the discriminative stimulus effects of alcohol in rats, and has cross tolerance with alcohol. All together, this leads to think of SO as a substitution therapy for alcohol use disorders. SO was initially studied in the prevention of alcohol withdrawal, and it showed similar efficacy to benzodiacepines. The studies on relapse prevention were developed later and the results are mixed and more complex to understand. While open label studies show a positive effect, RCTs have not been able to show a significant effect for the whole sample. Nevertheless, post-hoc analysis show a robust effect in the subsample of patients with high risk drinking levels, that would be the preferred target for a substitution treatment. The potential for abuse of GHB is well documented, which should be no surprise for a substitution treatment. Nevertheless, when correctly prescribed the risk of abuse of SO remains very low, as shown both in clinical trials and in the pharmacovigilance database, with more than 260000 cases documented. SO can be considered a substitution treatment, effective in patients with high risk drinking levels.DisclosureI was investigator in the SMO study on sodium oxybate, funded by D&A Pharma.

The Scaffold Protein PICK1 as a Target in Chronic Pain.

Well-tolerated and effective drugs for treating chronic pain conditions are urgently needed. Most chronic pain patients are not effectively relieved from their pain and suffer from debilitating drug side effects. This has not only drastic negative consequences for the patients’ quality of life, but also constitute an enormous burden on society. It is therefore of great interest to explore new potent targets for effective pain treatment with fewer side effects and without addiction liability. A critical component of chronic pain conditions is central sensitization, which involves the reorganization and strengthening of synaptic transmission within nociceptive pathways. Such changes are considered as maladaptive and depend on changes in the surface expression and signaling of AMPA-type glutamate receptors (AMPARs). The PDZ-domain scaffold protein PICK1 binds the AMPARs and has been suggested to play a key role in these maladaptive changes. In the present paper, we review the regulation of AMPARs by PICK1 and its relation to pain pathology. Moreover, we highlight other pain-relevant PICK1 interactions, and we evaluate various compounds that target PICK1 and have been successfully tested in pain models. Finally, we evaluate the potential on-target side effects of interfering with the action of PICK1 action in CNS and beyond. We conclude that PICK1 constitutes a valid drug target for the treatment of inflammatory and neuropathic pain conditions without the side effects and abuse liability associated with current pain medication.

Approaches of Prodrug Designing to Treat Cancer, Neurodegenerative Disorders and Viral Diseases

Classical prodrug design is a sweeping approach to throw away futile side effects related to drug therapy. The main purpose of prodrug designing is to ameliorate physicochemical, pharmaceutical, and pharmacokinetic characteristics of particular compounds to resolve issues like formulation, delivery to the target site, and toxicity limitations. To fabricate the pharmacological action in CNS, drugs must cross the blood-brain barrier (BBB). Therefore, prodrug strategies are designed which include lipidization and the use of carriers and transporters. In this article, we have reviewed different anticancer, neuroprotective, and antiviral prodrugs. Flurbiprofen prodrugs, glycosylated prodrugs, resveratrol prodrugs, levodopa, etc., are mapped out for neurodegenerative disorders in CNS. Due to the poor oral pharmacokinetic properties of antiviral agents, drug design methods are performed by combining the parent drug molecule with a number of active components such as dipeptide esters, amino acids, nucleosides, and macromolecular-based prodrugs.

Alcohol and IL-6 Alter Expression of Synaptic Proteins in Cerebellum of Transgenic Mice with Increased Astrocyte Expression of IL-6

Recent studies indicate that neuroimmune factors, including the cytokine interleukin-6 (IL-6), play a role in the CNS actions of alcohol. The cerebellum is a sensitive target of alcohol, but few studies have examined a potential role for neuroimmune factors in the actions of alcohol on this brain region. A number of studies have shown that synaptic transmission, and in particular inhibitory synaptic transmission, is an important cerebellar target of alcohol. IL-6 also alters synaptic transmission, although it is unknown if IL-6 targets are also targets of alcohol. This is an important issue because alcohol induces glial production of IL-6, which could then covertly influence the actions of alcohol. The persistent cerebellar effects of both IL-6 and alcohol typically involve chronic exposure and, presumably, altered gene and protein expression. Thus, in the current studies we tested the possibility that proteins involved in inhibitory and excitatory synaptic transmission in the cerebellum are common targets of alcohol and IL-6. We used transgenic mice that express elevated levels of astrocyte produced IL-6 to model persistently elevated expression of IL-6, as would occur in alcohol use disorders, and a chronic intermittent alcohol exposure/withdrawal paradigm (CIE/withdrawal) that is known to produce alcohol dependence. Multiple cerebellar synaptic proteins were assessed by Western blot. Results show that IL-6 and CIE/withdrawal have both unique and common actions that affect synaptic protein expression. These common targets could provide sites for IL-6/alcohol exposure/withdrawal interactions and play an important role in cerebellar symptoms of alcohol use such as ataxia.

Alcohol Enhances Responses to High Frequency Stimulation in Hippocampus from Transgenic Mice with Increased Astrocyte Expression of IL-6.

Recent studies show that alcohol exposure can induce glial production of neuroimmune factors in the CNS. Of these, IL-6 has gained attention because it is involved in a number of important physiological and pathophysiological processes that could be affected by alcohol-induced CNS production of IL-6, particularly under conditions of excessive alcohol use. For example, IL-6 has been shown to play a role in hippocampal behaviors and synaptic plasticity (long-term potentiation; LTP) associated with memory and learning. Surprisingly, in our in vitro studies of LTP at the Schaffer collateral to CA1 pyramidal neuron synapse in hippocampus from transgenic mice that express elevated levels of astrocyte produced IL-6 (TG), LTP was not altered by the increased levels of IL-6. However, exposure to acute alcohol revealed neuroadaptive changes that served to protect LTP against the alcohol-induced reduction of LTP observed in hippocampus from non-transgenic control mice (WT). Here we examined the induction phase of LTP to assess if presynaptic neuroadaptive changes occurred in the hippocampus of TG mice that contributed to the resistance of LTP to alcohol. Results are consistent with a role for IL-6-induced neuroadaptive effects on presynaptic mechanisms involved in transmitter release in the resistance of LTP to alcohol in hippocampus from the TG mice. These actions are important with respect to a role for IL-6 in physiological and pathophysiological processes in the CNS and in CNS actions of alcohol, especially when excessive alcohol used is comorbid with conditions associated with elevated levels of IL-6 in the CNS.

A Systematic Review on Analytical Methods to Determine Chiral and Achiral Forms of Venlafaxine and its Metabolite O-desmethylvenlafaxine

Background: Venlafaxine (VEN) is a bicyclic phenylethylamine derivative and possesses a marked structural difference from other antidepressant drugs present in the market. It works by eliciting the neurotransmitter action in CNS. It occurs in two enantiomeric forms i.e. R and S VEN. After the first pass metabolism, it gets metabolized into more active form O-desmethylvenlafaxine (ODV) which also exist in the enantiomeric forms. So it is important to develop a suitable analytical and bioanalytical method for the determination of VEN and its metabolite to quantify them accurately. Methods and Results: The current review summarizes methods to determine chiral and achiral forms of VEN and ODV. According to the literature, it is clear that most widely used method for the determination of VEN and ODV is liquid chromatography-mass spectroscopy, other methods used for routine analysis include UV spectroscopy, reverse phase high-performance liquid chromatography with PDA detector. For the determination of enantiomeric forms of VEN and ODV, different chiral columns have been utilized. Capillary electrophoresis with charged cyclodextrins is also used to determine the enantiomeric forms. Conclusion: Various analytical methods for determining VEN and its metabolite in different matrices have been discussed thoroughly in the present review.

Effects of acute and chronic arecoline in adult zebrafish: Anxiolytic-like activity, elevated brain monoamines and the potential role of microglia

Arecoline is a naturally occurring psychoactive alkaloid with partial agonism at nicotinic and muscarinic acetylcholine receptors. Arecoline consumption is widespread, making it the fourth (after alcohol, nicotine and caffeine) most used substance by humans. However, the mechanisms of acute and chronic action of arecoline in-vivo remain poorly understood. Animal models are a valuable tool for CNS disease modeling and drug screening. Complementing rodent studies, the zebrafish (Danio rerio) emerges as a promising novel model organism for neuroscience research. Here, we assessed the effects of acute and chronic arecoline on adult zebrafish behavior and physiology. Overall, acute and chronic arecoline treatments produced overt anxiolytic-like behavior (without affecting general locomotor activity and whole-body cortisol levels), with similar effects also caused by areca nut water extracts. Acute arecoline at 10 mg/L disrupted shoaling, increased social preference, elevated brain norepinephrine and serotonin levels and reduced serotonin turnover. Acute arecoline also upregulated early protooncogenes c-fos and c-jun in the brain, whereas chronic treatment with 1 mg/L elevated brain expression of microglia-specific biomarker genes egr2 and ym1 (thus, implicating microglial mechanisms in potential effects of long-term arecoline use). Finally, acute 2-h discontinuation of chronic arecoline treatment evoked withdrawal-like anxiogenic behavior in zebrafish. In general, these findings support high sensitivity of zebrafish screens to arecoline and related compounds, and reinforce the growing utility of zebrafish for probing molecular mechanisms of CNS drugs. Our study also suggests that novel anxiolytic drugs can eventually be developed based on arecoline-like molecules, whose integrative mechanisms of CNS action may involve monoaminergic and neuro-immune modulation.

Brain Distribution of Drugs: Pharmacokinetic Considerations.

It is crucial to understand the basic principles of drug transport, from the site of delivery to the site of action within the CNS, in order to evaluate the possible utility of a new drug candidate for CNS action, or possible CNS side effects of non-CNS targeting drugs. This includes pharmacokinetic aspects of drug concentration-time profiles in plasma and brain, blood-brain barrier transport and drug distribution within the brain parenchyma as well as elimination processes from the brain. Knowledge of anatomical and physiological aspects connected with drug delivery is crucial in this context. The chapter is intended for professionals working in the field of CNS drug development and summarizes key pharmacokinetic principles and state-of-the-art experimental methodologies to assess brain drug disposition. Key parameters, describing the extent of unbound (free) drug across brain barriers, in particular blood-brain and blood-cerebrospinal fluid barriers, are presented along with their application in drug development. Special emphasis is given to brain intracellular pharmacokinetics and its role in evaluating target engagement. Fundamental neuropharmacokinetic differences between small molecular drugs and biologicals are discussed and critical knowledge gaps are outlined.

The Effects of Chronic Amitriptyline on Zebrafish Behavior and Monoamine Neurochemistry.

Amitriptyline is a commonly used tricyclic antidepressant (TCA) inhibiting serotonin and norepinephrine reuptake. The exact CNS action of TCAs remains poorly understood, necessitating new screening approaches and novel model organisms. Zebrafish (Danio rerio) are rapidly emerging as a promising tool for pharmacological research of antidepressants, including amitriptyline. Here, we examine the effects of chronic 2-week exposure to 10 and 50μg/L amitriptyline on zebrafish behavior and monoamine neurotransmitters. Overall, the drug at50μg/Levoked pronounced anxiolytic-like effects in the novel tank test (assessed by more time in top, fewer transition and shorter latency to enter the top). Like other TCAs, amitriptyline reduced serotonin turnover, but also significantly elevated whole-brain norepinephrine and dopamine levels. The latter effect was not reported in this model previously, and accompanied higher brain expression of tyrosine hydroxylase (a rate-limiting enzyme of catecholamine biosynthesis), but unaltered expression of dopamine-β-hydroxylase and monoamine oxidase (the enzymes of dopamine metabolism). This response may underlie chronic amitriptyline action on dopamine and norepinephrine neurotransmission, and contribute to the complex CNS profile of this drug observed both clinically and in animal models. Collectively, these findings also confirm the important role of monoamine modulation in the regulation of anxiety-related behavior in zebrafish, and support the utility of this organism as a promising in-vivo model for CNS drug screening.

Altered hippocampal synaptic function in transgenic mice with increased astrocyte expression of CCL2 after withdrawal from chronic alcohol

CNS actions of the chemokine CCL2 are thought to play a role in a variety of conditions that can have detrimental consequences to CNS function, including alcohol use disorders. We used transgenic mice that express elevated levels of CCL2 in the CNS (CCL2-tg) and their non-transgenic (non-tg) littermate control mice to investigate long-term consequences of CCL2/alcohol/withdrawal interactions on hippocampal synaptic function, including excitatory synaptic transmission, somatic excitability, and synaptic plasticity. Two alcohol exposure paradigms were tested, a two-bottle choice alcohol (ethanol) drinking protocol (2BC drinking) and a chronic intermittent alcohol (ethanol) (CIE/2BC) protocol. Electrophysiological measurements of hippocampal function were made ex vivo, starting ∼0.6 months after termination of alcohol exposure. Both alcohol exposure/withdrawal paradigms resulted in CCL2-dependent interactions that altered the effects of alcohol on synaptic function. The synaptic alterations differed for the two alcohol exposure paradigms. The 2BC drinking/withdrawal treatment had no apparent long-term consequences on synaptic responses and long-term potentiation (LTP) in hippocampal slices from non-tg mice, whereas synaptic transmission was reduced but LTP was enhanced in hippocampal slices from CCL2-tg mice. In contrast, the CIE/2BC/withdrawal treatment enhanced synaptic transmission but reduced LTP in the non-tg hippocampus, whereas there were no apparent long-term consequences to synaptic transmission and LTP in hippocampus from CCL2-tg mice, although somatic excitability was enhanced. These results support the idea that alcohol-induced CCL2 production can modulate the effects of alcohol exposure/withdrawal on synaptic function and indicate that CCL2/alcohol interactions can vary depending on the alcohol exposure/withdrawal protocol used.

Photooxidized Vespa orientalis venom Improves Memory and Learning Activities in Rats

The work was aimed to study the effect of photooxidized Vespa orientalis venom on memory and learning activity on rats in presence of scopolamine and ondansetron using T maze, Elevated plus maze and passive avoidance. UV radiation exposure of Vespa orientalis venom (VOV) for 15, 30, 45 and 60 min intervals in presence to methylene blue to detoxify venom and studied by change in UV spectrum. Antigenicity study and in in-vivo and in-vitro neutralization study of Photooxidized Vespa orientalis venom (PVOV) against immunoglobulin from hyperimmunized rabbit was performed. The memory and learning activity of PVOV in presence and absence of scopolamine and ondansetron was studied. Forty five minute UV Radiation exposed VOV showed shift in λmax and increase absorbance indicated alternations in venom protein concentration, this in supported when PVOV showed loss of toxicity and decrease in mortality time in neutralization study in mice. Administration of PVOV for 28 days produced a notable improvement in spatial and long memory in rats when subjected in several tasks. When PVOV administered with scopolamine and ondansetron, all the parameters of spatial and long term memory tasks were significantly reduced inferred that PVOV acted by modulating either muscarinic or serotonergic receptors. However, other possibility of low-molecular weight protein and peptides or enzymes, which might also act by serotonergic / cholinergic system that affect CNS action. We concluded that although there is a possibility of employing PVOV in the treatment of depressive and chronic degenerative illnesses as a nonherbal and nonsynthetic alternative for patients not responding to the available therapy.

Effects of an Inhibitor of Monocyte Recruitment on Recovery from Traumatic Brain Injury in Mice Treated with Granulocyte Colony-Stimulating Factor.

Administration of the hematopoietic growth factor granulocyte-colony stimulating Factor (G-CSF) has been reported to enhance recovery from controlled cortical impact (CCI) in rodent models. G-CSF exerts actions in both the periphery (stimulation of hematopoiesis) and in the brain, where it serves as a neurotrophic factor, promoting neuronal survival and stimulating neural stem/progenitor cell proliferation in the hippocampus. In order to distinguish the direct CNS actions of G-CSF from its peripheral actions, experiments were designed to block the recruitment of peripheral monocytes to the site of the lesion produced by CCI. The selective C-C motif receptor 2 (CCR2) antagonist (RS504303) was co-administered with G-CSF for three days after CCI in a chimeric mouse previously transplanted with GFP-expressing (GFP+) blood stem-progenitor cells. Results: The drug significantly impaired infiltration of GFP+ bone marrow-derived cells to the frontal cortex and striatum without impeding recovery performance and hippocampal neurogenesis in the behavioral test, the Radial Arm Water Maze (RAWM). Administration of the CCR2 antagonist alone, without G-CSF, was effective in promoting recovery in RAWM. These results support the hypothesis that the direct action of G-CSF on neural cells, independent of its hematopoietic effects, is primarily responsible for enhanced recovery from CCI. In addition, this study confirms the importance of CCR2 and its ligand, monocyte chemotactic protein-1 (MCP-1), in mediating the inflammatory response following CCI.

Role of neurons and glia in the CNS actions of the renin-angiotensin system in cardiovascular control

Despite tremendous research efforts, hypertension remains an epidemic health concern, leading often to the development of cardiovascular disease. It is well established that in many instances, the brain plays an important role in the onset and progression of hypertension via activation of the sympathetic nervous system. Further, the activity of the renin-angiotensin system (RAS) and of glial cell-mediated proinflammatory processes have independently been linked to this neural control and are, as a consequence, both attractive targets for the development of antihypertensive therapeutics. Although it is clear that the predominant effector peptide of the RAS, ANG II, activates its type-1 receptor on neurons to mediate some of its hypertensive actions, additional nuances of this brain RAS control of blood pressure are constantly being uncovered. One of these complexities is that the RAS is now thought to impact cardiovascular control, in part, via facilitating a glial cell-dependent proinflammatory milieu within cardiovascular control centers. Another complexity is that the newly characterized antihypertensive limbs of the RAS are now recognized to, in many cases, antagonize the prohypertensive ANG II type 1 receptor (AT1R)-mediated effects. That being said, the mechanism by which the RAS, glia, and neurons interact to regulate blood pressure is an active area of ongoing research. Here, we review the current understanding of these interactions and present a hypothetical model of how these exchanges may ultimately regulate cardiovascular function.

Anxiogenic-like effects of chronic nicotine exposure in zebrafish

Nicotine is one of the most widely used and abused legal drugs. Although its pharmacological profile has been extensively investigated in humans and rodents, nicotine CNS action remains poorly understood. The importance of finding evolutionarily conserved signaling pathways, and the need to apply high-throughput in vivo screens for CNS drug discovery, necessitate novel efficient experimental models for nicotine research. Zebrafish (Danio rerio) are rapidly emerging as an excellent organism for studying drug abuse, neuropharmacology and toxicology and have recently been applied to testing nicotine. Anxiolytic, rewarding and memory-modulating effects of acute nicotine treatment in zebrafish are consistently reported in the literature. However, while nicotine abuse is more relevant to long-term exposure models, little is known about chronic effects of nicotine on zebrafish behavior. In the present study, chronic 4-day exposure to 1–2mg/L nicotine mildly increased adult zebrafish shoaling but did not alter baseline cortisol levels. We also found that chronic exposure to nicotine evokes robust anxiogenic behavioral responses in zebrafish tested in the novel tank test paradigm. Generally paralleling clinical and rodent data on anxiogenic effects of chronic nicotine, our study supports the developing utility of zebrafish for nicotine research.

Restless Eating, Restless Legs, and Sleep Related Eating Disorder.

Restless legs syndrome (RLS) often presents with a primary complaint of sleep initiation difficulty with only ambiguous allusions to motor symptoms. This may result in the condition being misdiagnosed as a psychophysiological insomnia. Further, nocturnal eating is common in RLS and like the classic motor symptoms, patients will describe an inability to initiate sleep until their urge (to eat) is addressed. Restless nocturnal eating arises, intensifies, and subsides in parallel to motor symptoms. Once misdiagnosed as psychophysiological insomnia, RLS patients are frequently treated with benzodiazepine receptor agonists. The CNS actions of these sedating agents, suppression of memory and executive function, unleash predisposed amnestic behaviors. In the case of RLS this would be expected to include the inappropriate ambulatory and eating behaviors of sleep related eating disorder (SRED). The evidence and implications of a link between the restless eating of RLS and SRED is presented here.

FGF19: How gut talks to brain to keep your sugar down

FGF19: How gut talks to brain to keep your sugar down

Rational design of sulfonated A3 adenosine receptor-selective nucleosides as pharmacological tools to study chronic neuropathic pain.

(N)-Methanocarba(bicyclo[3.1.0]hexane)adenosine derivatives were probed for sites of charged sulfonate substitution, which precludes diffusion across biological membranes, e.g., blood-brain barrier. Molecular modeling predicted that sulfonate groups on C2-phenylethynyl substituents would provide high affinity at both mouse (m) and human (h) A3 adenosine receptors (ARs), while a N(6)-p-sulfophenylethyl substituent would determine higher hA3AR vs mA3AR affinity. These modeling predictions, based on steric fitting of the binding cavity and crucial interactions with key residues, were confirmed by binding/efficacy studies of synthesized sulfonates. N(6)-3-Chlorobenzyl-2-(3-sulfophenylethynyl) derivative 7 (MRS5841) bound selectively to h/m A3ARs (Ki(hA3AR) = 1.9 nM) as agonist, while corresponding p-sulfo isomer 6 (MRS5701) displayed mixed A1/A3AR agonism. Both nucleosides administered ip reduced mouse chronic neuropathic pain that was ascribed to either A3AR or A1/A3AR using A3AR genetic deletion. Thus, rational design methods based on A3AR homology models successfully predicted sites for sulfonate incorporation, for delineating adenosine's CNS vs peripheral actions.