Adenosine A1 Receptor Agonist Research Articles

Pharmacology of Adenosine A1 Receptor Agonist in a Humanized Esterase Mouse Seizure Model Following Soman Intoxication

Recently a novel genetically modified mouse strain with serum carboxylesterase knocked-out and the human acetylcholinesterase gene knocked-in (KIKO) was created to simulate human responses to nerve agent (NA) exposure and its standard medical treatment. A1 adenosine receptor (A1AR) agonist N-bicyclo-(2.2.1)-hept-2-yl-5'-chloro-5'-deoxyadenosine (ENBA) alone is a potent anticonvulsant and neuroprotectant (A/N) in both rat and KIKO mouse soman (GD) seizure models. In this study we utilized the KIKO mouse to evaluate further the basic pharmacologic A/N effects of ENBA as an adjunct to standard NA medical treatments (i.e., atropine sulfate, pralidoxime chloride [2-PAM], and midazolam). Male mice, implanted with cortical electroencephalographic (EEG) electrodes, were pretreated with asoxime (HI-6) and exposed to an epileptogenic dose of GD (33 µg/kg, s.c.) or saline (sham exposure) and then treated 15 min after seizure onset with ENBA at 15 mg/kg, i.p. (a minimum efficacy dose in suppressing NA-induced seizure) alone or as an adjunct to standard medical treatments. We collected EEG activity, seizure suppression outcomes, daily body temperature and weight, heart rate, toxic signs, neuropathology, and lethality data for up to 14 days. Without ENBA, death from NA exposure was 45%, while with ENBA, either alone or in combination with midazolam, the survival improved to 80% and 90%, respectively. Additionally, seizure was suppressed quickly and permanently, toxic signs, hypothermia, and bradycardia recovered by 48 h, and no neuropathology was evident. Our findings confirmed that ENBA is a potent A/N adjunct for delayed medical treatments of NA exposure.

Cantharidin and sodium fluoride attenuate the negative inotropic effect of the A1-adenosine receptor agonist N6-(R)-phenylisopropyl adenosine in isolated human atria.

Cantharidin and sodium fluoride inhibit the activity of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A) and increase the force of contraction in human atrial preparations. R-phenylisopropyl adenosine (R-PIA) acts as an agonist at A1-adenosine receptors. R-PIA exerts a negative inotropic effect on human atria. The effect of R-PIA-and its various manifestations-are currently explained as a function of the inhibition of sarcolemmal adenylyl cyclase activity and/or opening of sarcolemmal potassium channels. We hypothesise that cantharidin and sodium fluoride may attenuate the negative inotropic effect of R-PIA. During open heart surgery, trabeculae carneae from the right atrium were obtained for human atrial preparations (HAPs). These trabeculae were mounted in organ baths and electrically stimulated at 1Hz. Furthermore, we studied isolated electrically stimulated left atrial (LA) preparations from female wild-type mice (CD1). The force of contraction was recorded under isometric conditions. R-PIA (1µM) exerted a rapid negative inotropic effect in the HAPs and mice LA preparations. These negative inotropic effects of R-PIA were attenuated by pre-incubation for 30min with 100-µM cantharidin in HAPs, but not in mice LA preparations. Adenosine signals via A1 receptors in a species-specific pathway in mammalian atria. We postulate that R-PIA, at least in part, exerts a negative inotropic effect via activation of serine/threonine phosphatases in the human atrium.

Effect of simultaneous application of adenosine A1 receptor agonist and A2A receptor antagonist on memory, inflammatory factors, and PSD-95 in lipopolysaccharide-induced memory impairment

The potential role of adenosine, a natural neuroprotective agent, and its receptors in the pathogenesis of Alzheimer's disease has been proposed. The present study aims to examine the effect of administering both an A1 receptor agonist and an A2A adenosine receptor antagonist simultaneously on memory, inflammatory factors, and PSD-95 in an LPS-induced Alzheimer's disease model in rats. Fifty-six male Wistar rats were randomly divided into seven groups: Saline, LPS, Saline + Vehicle, LPS + Vehicle, LPS + SCH58261 (A2A receptor antagonist), LPS + CPA (A1 receptor agonist), LPS + SCH58261+CPA. LPS (3 mg/kg/ip) was used to cause memory impairment. Treatment was performed by intraventricular injection of CPA at a dose of 700 μg and SCH-58261 at 40 μg for ten days. Passive avoidance and Y-maze tests were performed to examine animals’ memories. IL-10, TNF-α, and PSD-95 levels were measured in the brain using ELISA and western blot, respectively. Compared to the groups receiving each medication separately, the simultaneous administration of CPA and SCH58261 improved memory (P<0.05). Additionally, compared to the single medication groups, there was a significant increase in IL-10, PSD-95, and a significant decrease in TNF-α in the brain tissue (P<0.05). These findings suggest that the activation of A1 receptors along with A2A receptor inhibition could be a potential therapeutic strategy for Alzheimer's disease. These findings suggest that A1 receptor activation combined with A2A receptor inhibition may be a promising therapeutic approach for Alzheimer's disease.

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.

Lipid Trolling to Optimize A3 Adenosine Receptor-Positive Allosteric Modulators (PAMs).

A3 adenosine receptor (A3AR) positive allosteric modulators (PAMs) (2,4-disubstituted-1H-imidazo[4,5-c]quinolin-4-amines) allosterically increase the Emax of A3AR agonists, but not potency, due to concurrent orthosteric antagonism. Following mutagenesis/homology modeling of the proposed lipid-exposed allosteric binding site on the cytosolic side, we functionalized the scaffold, including heteroatom substitutions and exocyclic phenylamine extensions, to increase allosteric binding. Strategically appended linear alkyl-alkynyl chains with terminal amino/guanidino groups improved allosteric effects at both human and mouse A3ARs. The chain length, functionality, and attachment position were varied to modulate A3AR PAM activity. For example, 26 (MRS8247, p-alkyne-linked 8 methylenes) and homologues increased agonist Cl-IB-MECA's Emax and potency ([35S]GTPγS binding). The putative mechanism involves a flexible, terminally cationic chain penetrating the lipid environment for stable electrostatic anchoring to cytosolic phospholipid head groups, suggesting "lipid trolling", supported by molecular dynamic simulation of the active-state model. Thus, we have improved A3AR PAM activity through rational design based on an extrahelical, lipidic binding site.

2-Substituted (N)-Methanocarba A3 Adenosine Receptor Agonists: In Silico, In Vitro, and In Vivo Characterization.

2-Arylethynyl (N)-methanocarba adenosine 5'-methylamides are selective A3 adenosine receptor (AR) agonists containing a preestablished receptor-preferred pseudoribose conformation. Here, we compare analogues having bulky 2-substitution, either containing or lacking an ethynyl spacer between adenine and a cyclic group. 2-Aryl compounds 9-11, 13, 14, 19, 22, 23, 27, 29, 31, and 34, lacking a spacer, had human (h) A3AR K i values of 2-30 nM, and others displayed lower affinity. Mouse (m) A3AR affinity varied, with 2-arylethynyl having a higher affinity than 2-aryl analogues (7, 8 > 3c, 3d > 3b). However, 2-aryl-4'-truncated derivatives had greatly reduced hA3AR affinity, even containing affinity-enhancing N 6-dopamine-derived substituents. Molecular modeling, including molecular dynamics simulation, predicted stable poses in the canonical A3AR agonist binding site, but 2-aryl (ECL2 interactions) and 2-arylethynyl (TM2 interactions) substituents have different conformations and environments. In a hA3AR miniGαi recruitment assay, 31 (MRS8062) was (slightly) more potent compared to a β-arrestin2 recruitment assay, both in engineered HEK293T cells, and its maximal efficacy (E max) was much higher (165%) than reference agonist NECA's. Thus, in the 2-aryl series, A3AR affinity and selectivity were variable and generally reduced compared to the 2-arylethynyl series, with a greater dependence on the specific aryl group present. Selected compounds were studied in vivo in an ischemic model of peripheral artery disease (PAD). Rigidified 2-arylethynyl analogues 3a-3c were protective in this model of skeletal muscle ischemia-reperfusion injury/claudication, as previously shown only for moderately A3AR-selective ribosides or (N)-methanocarba derivatives. Thus, we have expanded the A3AR agonist SAR for (N)-methanocarba adenosines.

Seizure suppression and neuroprotection in soman-exposed rats following delayed intramuscular treatment of adenosine A1 receptor agonist as an adjunct to standard medical treatment

Soman produces excitotoxic effects by inhibiting acetylcholinesterase in the cholinergic synapses and neuromuscular junctions, resulting in soman-induced sustained status epilepticus (SSE). Our previous work showed delayed intramuscular (i.m.) treatment with A1 adenosine receptor agonist N-bicyclo-[2.2.1]-hept-2-yl-5′-chloro-5′-deoxyadenosine (ENBA) alone suppressed soman-induced SSE and prevented neuropathology. Using this same rat soman seizure model, we tested if delayed therapy with ENBA (60 mg/kg, i.m.) would terminate seizure, protect neuropathology, and aid in survival when given in conjunction with current standard medical countermeasures (MCMs): atropine sulfate, 2-PAM, and midazolam (MDZ). Either 15- or 30-min following soman-induced SSE onset, male rats received atropine and 2-PAM plus either MDZ or MDZ + ENBA. Electroencephalographic (EEG) activity, physiologic parameters, and motor function were recorded. Either 2- or 14-days following exposure surviving rats were euthanized and perfused for histology. All animals treated with MDZ + ENBA at both time points had 100% EEG seizure termination and reduced total neuropathology compared to animals treated with MDZ (2-day, p = 0.015 for 15-min, p = 0.002 for 30-min; 14-day, p < 0.001 for 15-min, p = 0.006 for 30-min), showing ENBA enhanced MDZ's anticonvulsant and neuroprotectant efficacy. However, combined MDZ + ENBA treatment, when compared to MDZ treatment groups, had a reduction in the 14-day survival rate regardless of treatment time, indicating possible enhancement of MDZ's neuronal inhibitory effects by ENBA. Based on our findings, ENBA shows promise as an anticonvulsant and neuroprotectant in a combined treatment regimen following soman exposure; when given as an adjunct to standard MCMs, the dose of ENBA needs to be adjusted.

Learning and memory function preserved by delayed A1 adenosine receptor agonist treatment following soman intoxication in rats and a humanized esterase mouse model

Exposure to organophosphorus compounds, such as soman (GD), cause widespread toxic effects, sustained status epilepticus, neuropathology, and death. The A1 adenosine receptor agonist N-bicyclo-(2.2.1)-hept-2-yl-5′-chloro-5′-deoxyadenosine (ENBA), when given 1 min after GD exposure, provides neuroprotection and prevents behavioral impairments. Here, we tested the ability of ENBA at delayed treatment times to improve behavioral outcomes via a two-way active avoidance task in two male animal models, each consisting of saline and GD exposure groups. In a rat model, animals received medical treatments (atropine sulfate [A], 2-PAM [P], and midazolam [MDZ]) or AP + MDZ + ENBA at 15 or 30 min after seizure onset and were subjected to behavioral testing for up to 14 days. In a human acetylcholinesterase knock-in serum carboxylesterase knock-out mouse model, animals received AP, AP + MDZ, AP + ENBA, or AP + MDZ + ENBA at 15 min post seizure onset and were subjected to the behavioral task on days 7 and 14. In rats, the GD/AP + MDZ + ENBA group recovered to saline-exposed avoidance levels while the GD/AP + MDZ group did not. In mice, in comparison with GD/AP + MDZ group, the GD/AP + MDZ + ENBA showed decreases in escape latency, response latency, and pre-session crossings, as well as increases in avoidances. In both models, only ENBA-treated groups showed control level inter-trial interval crossings by day 14. Our findings suggest that ENBA, alone and as an adjunct to medical treatments, can improve behavioral and cognitive outcomes when given at delayed time points after GD intoxication.

Crosstalk Inhibition Between 5-HT2A and Adenosine 2A Receptor-Induced Phrenic Motor Facilitation

Episodic spinal serotonin receptor activation elicits phrenic motor facilitation (pMF), a form of respiratory motor plasticity. Although both intermittent 5-HT2 and 5-HT7 receptor activation elicit pMF, they do so via distinct cellular mechanisms known as the Q (5-HT2A/B) and S (5-HT7) pathways. The Q and S pathways interact via powerful crosstalk inhibition; thus, concurrent activation of both receptor subtypes diminishes/abolishes pMF. At least some of the molecules giving rise to mutually inhibitory interactions between 5-HT2 and 5-HT7 receptors have been identified. Although episodic spinal adenosine 2A (A2A) receptor activation also elicits pMF via the S pathway, we do not know: 1) if 5-HT2 and A2A receptors interact via similar crosstalk inhibition, nor 2) if they do so via the same signaling mechanisms. Thus, we tested the hypothesis that concurrent 5-HT2 and A2A receptor activation diminishes pMF via crosstalk inhibition. Selective 5-HT2A (DOI) and A2A receptor agonists (CGS21680) were delivered to the C4 intrathecal space (3, 6 μl injections, 5-min intervals) in urethane anesthetized, artificially ventilated, vagotomized and paralyzed Sprague Dawley rats. Integrated phrenic nerve burst activity was measured before (baseline), during and 90 minutes post-drug injection. Spinal 5-HT2A and A2A receptor activation both increased phrenic nerve burst amplitude when delivered alone (i.e., pMF); however, pMF was no longer apparent with concurrent receptor activation. The magnitude of pMF, expressed as a % change in phrenic burst amplitude from baseline to 90-min post-injection, was significantly lower with concurrent receptor activation (5-HT2A + A2A= 15 ± 7.7%; n=5) versus 5-HT2A (84 ± 12%; n=5, p=0.0047) and A2A (88 ± 16%; n=5, p=0.0085) alone. Thus, 5-HT2A and A2A receptors interact via crosstalk inhibition similar to 5-HT2 and 5-HT7 receptors. However, the mechanism underlying this crosstalk inhibition remains unknown. NIH HL148030. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The A1 Adenosine Receptor Agonist N6 Cyclohexyl Adenosine Suppresses the Cold-Defense Threshold to a Surface Temperature Below 4°C in Sprague Dawley Rats

A1 adenosine receptor (A1AR) agonists have applications in targeted temperature management (TTM) for neurogenic fever, cardiac arrest and neonatal ischemic encephalopathy. By mitigating the cold-defense response the A1AR agonist N6 cyclohexyl adenosine (CHA) blocks shivering and cold-defense metabolic stress that limits the application and benefit of TTM. Stimulating CNS A1AR inhibits cold-induced shivering and nonshivering thermogenesis in rats and induces a torpor-like response in rats and hibernating species [1,2]. Dynamic control of surface temperature (Ts) in rats, designed to mimic conductive cooling used clinically, is an effective means to regulate body temperature after CHA [3]. In prior studies 17°C was the minimum Ts tested while in the clinic a fast rate of cooling is facilitated by setting cooling devices to 4°C. Unknown is if CHA suppresses the cold-defense response at a Ts of 4°C. The present study tests the hypothesis that the cold-defense threshold of CHA (1.0mg/kg, IP) is less than a Ts of 4°C, and we ask if the threshold differs between sexes. To define the cold-defense threshold, male and female Sprague-Dawley rats fed ad lib and housed at 20°C and 12:12 L:D were held overnight in a metabolic cage with a temperature-controlled surface set to 29°C and a video camera. The following day after 4h at 29°C with lights on, rats were injected with vehicle (week 1) or CHA (week 2) and the Ts was decreased to 28, 20, 16, 12, 8 and 4°C for 30min each. Cold-defense was assessed by an increase in the rate of oxygen consumption (VO2) and by behaviors suggestive of escape. Based on prior work showing a bimodal distribution of rats that respond to CHA and those that do not respond [4], we a priori chose to assess the proportion of nonresponders in the study population and to analyze responders and nonresponders separately. Of 9 rats (5 M, 4 F), one male did not respond to CHA. In rats that responded to CHA, vehicle (veh) treatment increased VO2 at Ts of 16°C to 4°C in both sexes (p&lt;0.0001 drug x Ts, 2-way RM ANOVA; p&lt;0.01, veh-29°C (lights on) vs veh-16, 12, 8, and 4°C, Tukey, n=8,8). CHA treatment decreased VO2 at all Ts tested (p&lt;0.05 CHA vs Veh, Tukey, n=8,8). Females trended towards higher VO2 at 29°C than males at all Ts, but sex differences decreased after treatment with CHA. Blinded analysis of behavior is in progress. Results show that CHA lowers the threshold of Ts induced cold-defense from 16°C to below 4°C. Suppressed VO2 at Ts above 16°C illustrates CHA-induced metabolic suppression unrelated to thermogenesis. Results inform optimal Ts to minimize time to reach target temperature without a cold-defense response. 1. Jinka et al., 2011, J Neurosci 31 (30):10752-10758. 2. Tupone et al., 2013, J Neurosci 33 (36):14512-14525. 3. Laughlin et al., 2018, Ther Hypothermia Temp Manag 8 (2):108-116. 4. Bailey et., 2017, J Pharm Exp Ther 362 (3):424-430. NNX13AB28A, 22-22EPSCoR-0018, P20GM130443, P20GM103395. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hypothermia as potential therapeutic approach to attenuating soman-induced seizure, neuropathology, and mortality with an adenosine A1 receptor agonist and body cooling

Organophosphorus nerve agents, such as soman (GD), produce excitotoxic effects resulting in sustained status epilepticus (SSE) and brain damage. Previous work shows that neuronal inhibitory effects of A1 adenosine receptor (A1AR) agonists, such as N6- Bicyclo (2.2.1)-hept-2-yl-5′-chloro-5′-deoxyadenosine (Cl-ENBA), suppresses GD-induced SSE and improves neuropathology. Some other physiologic effects of these agonists are hypothermia, hypotension, and sedation. Hypothermia may also shield the brain from injury by slowing down chemical insults, lessening inflammation, and contributing to improved neurological outcomes. Therefore, we attempted to isolate the hypothermic effect from ENBA by assessing the neuroprotective efficacy of direct surface body cooling in a rat GD-induced SSE model, and comparing the effects on seizure termination, neuropathology, and survival. Male rats implanted with a body temperature (Tb) transponder and electroencephalographic (EEG) electrodes were primed with asoxime (HI-6), exposed to GD 30 min later, and then treated with Cl-ENBA or had Tb lowered directly via body cooling at 30 min after the onset of seizure activity. Afterwards, they were either allowed to develop hypothermia as expected, or received thermal support to maintain normothermic Tb for a period of 6-h. Neuropathology was assessed at 24 h. Regardless of Cl-ENBA or surface cooling, all hypothermic GD-exposed groups had significantly improved 24-h survival compared to rats with normothermic Tb (81% vs. 39%, p < 0.001). Cl-ENBA offered neuroprotection independently of hypothermic Tb. While hypothermia enhanced the overall efficacy of Cl-ENBA by improving survival outcomes, body cooling didn't reduce seizure activity or neuropathology following GD-induced SSE.

Activation of hepatic adenosine A1 receptor ameliorates MASH via inhibiting SREBPs maturation

Metabolic (dysfunction)-associated steatohepatitis (MASH) is the advanced stage of metabolic (dysfunction)-associated fatty liver disease (MAFLD) lacking approved clinical drugs. Adenosine A1 receptor (A1R), belonging to the G-protein-coupled receptors (GPCRs) superfamily, is mainly distributed in the central nervous system and major peripheral organs with wide-ranging physiological functions; however, the exact role of hepatic A1R in MAFLD remains unclear. Here, we report that liver-specific depletion of A1R aggravates while overexpression attenuates diet-induced metabolic-associated fatty liver (MAFL)/MASH in mice. Mechanistically, activation of hepatic A1R promotes the competitive binding of sterol-regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) to sequestosome 1 (SQSTM1), rather than protein kinase A (PKA) leading to SCAP degradation in lysosomes. Reduced SCAP hinders SREBP1c/2 maturation and thus suppresses de novo lipogenesis and inflammation. Higher hepatic A1R expression is observed in patients with MAFL/MASH and high-fat diet (HFD)-fed mice, which is supposed to be a physiologically adaptive response because A1R agonists attenuate MAFL/MASH in an A1R-dependent manner. These results highlight that hepatic A1R is a potential target for MAFL/MASH therapy.

Therapeutic potency of A1 adenosine receptor antagonists in the treatment of cardiovascular diseases, current status and perspectives.

Cardiomyocytes form, transport, and metabolize the omnipresent metabolite adenosine. Depending upon the adenosine concentrations and the pharmacological properties of receptor subtypes, adenosine exerts (patho)physiological responses in the cardiovascular system. The objective of this review is to present different protective mechanisms of A1-adenosine receptor inhibitors in cardiovascular diseases. Literature references were collected and sorted using relevant keywords and key phrases as search terms in scientific databases such as Web of Science, PubMed and Google Scholar. A1 adenosine receptor regulates free fatty acid metabolism, lipolysis, heart rate, blood pressure, and cardiovascular toxicity. The evidence clearly supporting the therapeutic potency of pharmacological A1 adenosine receptors agonists and antagonists in modulating cardiovascular risk factor parameters and treatment of cardiovascular diseases. This review summarizes the protective role of pharmacological A1-adenosine receptor regulators in the pathogenesis of cardiovascular diseases for a better management of cardiovascular diseases.

Efficacy and safety of piclidenoson in plaque psoriasis: Results from a randomized phase 3 clinical trial (COMFORT-1).

A3 adenosine receptor (A3AR) is overexpressed in the skin and peripheral blood mononuclear cells of psoriasis patients. We investigated the efficacy/safety of piclidenoson (CF101), an orally bioavailable A3AR agonist that inhibits IL-17 and IL-23 production in keratinocytes, in moderate-to-severe plaque psoriasis. The randomized, placebo- and active-controlled, double-blind phase 3 COMFORT-1 trial randomized patients (3:3:3:2) to piclidenoson 2 mg BID, piclidenoson 3 mg BID, apremilast 30 mg BID or placebo. At Week 16, patients in the placebo arm were re-randomized (1:1:1) to piclidenoson 2 mg BID, piclidenoson 3 mg BID or apremilast 30 mg BID. The primary end point was the proportion of patients achieving ≥75% improvement in Psoriasis Area and Severity Index (PASI) from baseline (PASI-75) at Week 16 versus placebo. A total of 529 patients were randomized and received ≥1 dose of study medication (safety population). The efficacy analysis population for the primary end point included 426 patients (piclidenoson 2 mg BID, 127; piclidenoson 3 mg BID, 103; apremilast, 118; placebo, 78). Piclidenoson at 2 and 3 mg BID exhibited similar efficacy. The primary end point was met with the 3 mg BID dose: PASI 75 rate of 9.7% versus 2.6% for piclidenoson versus placebo, p = 0.037. The PASI responses with piclidenoson continued to increase throughout the study period in a linear manner. At week 32, analysis in the per-protocol population showed that a greater proportion of patients in the piclidenoson 3 mg BID arm (51/88, 58.0%) achieved improvement from baseline in Psoriasis Disability Index (PDI) compared to apremilast (59/108, 55.1%), and the test for noninferiority trended towards significance (p = 0.072). The safety/tolerability profile of piclidenoson was excellent and superior to apremilast. Piclidenoson demonstrated efficacy responses that increased over time alongside a favourable safety profile. These findings support its continued clinical development as a psoriasis treatment (ClinicalTrials.gov identifier: NCT03168256).

Abstract A074: Namodenoson inhibits the growth of pancreatic carcinoma via de-regulation of the Wnt/β-catenin signaling pathway

Abstract Background: The Wnt/β-catenin pathway controls the growth of pancreatic carcinoma by regulating processes such as cell cycle progression, apoptosis, and the tumor immune microenvironment. Treating pancreatic cancer with Wnt pathway inhibitors was found to be toxic in clinical studies. Namodenoson, an A3 adenosine receptor (A3AR) agonist, is under clinical development as a treatment of advanced liver cancer, where namodenoson was shown to have an excellent safety profile in phase II studies and is a phase III study is ongoing. The mechanism of action of namodenoson involves de-regulation of the Wnt and NF-κB signaling pathways, followed by an increase in pro-apoptotic proteins and Fas-ligand, resulting in tumor growth inhibition. The objectives of the currents study were to examine the anti-growth effect of namodenoson on pancreatic carcinoma cells both in vitro and in vivo and to investigate the molecular mechanisms involved. Methods: BxPC-3 human pancreatic carcinoma cells were cultured in the presence and absence of 0.01, 0.1, and 1 nM namodenoson, or in the presence of a combination of namodenoson (0.1 nM) and gemcitabine (0.2 µM). Cell growth was monitored via 3[H]-thymidine proliferation, MTT, and PrestoBlue assays. Western blot analyses were conducted to identify the regulatory cell growth proteins impacted by the treatment. A xenograft model with human BxPC-3 cells in nude mice was established to investigate the in-vivo effect of namodenoson. Results: A significant dose-dependent growth inhibition of BxPC-3 cells was observed with namodenoson in the3[H]-thymidine proliferation assay (1 nM: 67.4% ± 1.7%, p &amp;lt; 0.001; 0.1 nM: 53.7% ± 6.3%, p&amp;lt; 0.05; 0.01 nM: 27.9 % ± 2.3%, p&amp;lt; 0.005). The combination of namodenoson and gemcitabine showed an additive inhibitory effect in the MTT assay (namodenoson: 48.6% ± 1.4%; gemcitabine: 44.4% ± 0.7%; namodenoson plus gemcitabine: 65.4% ± 1.4%; p&amp;lt;0.001 for all). Western blot analyses demonstrated that namodenoson treatment was associated with de-regulation of the Wnt pathway regulatory proteins including p-Akt, NF- κB, GSK-3β, β-catenin and up-regulation of the BAD and BAX apoptotic proteins. In the xenograft model, daily oral treatment with 10 µg/kg of namodenoson led to a significant progressive inhibition of tumor growth. Conclusions: Nanomolar concentrations of namodenoson inhibited the growth of pancreatic carcinoma in cell culture and in a xenograft model. This effect involved A3AR activation and de-regulation of the Wnt/β-catenin pathway. Our results support a potential clinical utility for namodenoson in pancreatic cancer, a disease where an unmet clinical need exists, and novel therapeutic opportunities are required. Citation Format: Pnina Fishman, Avital Bareket-Samish, Inbal Izhak. Namodenoson inhibits the growth of pancreatic carcinoma via de-regulation of the Wnt/β-catenin signaling pathway [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A074.

Emerging Oral Therapies for the Treatment of Psoriasis: A Review of Pipeline Agents.

The introduction of biologic agents for the treatment of psoriasis has revolutionized the current treatment landscape, targeting cytokines in the interleukin (IL)-23/IL-17 pathway and demonstrating strong efficacy and safety profiles in clinical trials. These agents however are costly, are associated with a risk of immunogenicity, and require administration by intravenous or subcutaneous injection, limiting their use among patients. Oral therapies, specifically small molecule and microbiome therapeutics, have the potential to be more convenient and cost-effective agents for patients and have been a focus of development in recent years, with few targeted oral medications available for the disease. In this manuscript, we review pipeline oral therapies for psoriasis identified through a search of ClinicalTrials.gov (30 June 2022-1 October 2023). Available preclinical and clinical trial data on each therapeutic agent are discussed. Small molecules under development include tumor necrosis factor inhibitors, IL-23 inhibitors, IL-17 inhibitors, phosphodiesterase-4 inhibitors, Janus kinase inhibitors, A3 adenosine receptor agonists, and sphingosine-1-phosphate receptor 1 agonists, several of which are entering phase III trials. Oral microbials have also demonstrated success in early phase studies. As new oral therapies emerge for the treatment of psoriasis, real-world data and comparative trials are needed to better inform their use among patients.

Extrahelical Binding Site for a 1H-Imidazo[4,5-c]quinolin-4-amine A3 Adenosine Receptor Positive Allosteric Modulator on Helix 8 and Distal Portions of Transmembrane Domains 1 and 7.

This study describes the localization and computational prediction of a binding site for the A3 adenosine receptor (A3AR) positive allosteric modulator 2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-(3,4-dichlorophenyl)amine (LUF6000). The work reveals an extrahelical lipid-facing binding pocket disparate from the orthosteric binding site that encompasses transmembrane domain (TMD) 1, TMD7, and Helix (H) 8, which was predicted by molecular modeling and validated by mutagenesis. According to the model, the nearly planar 1H-imidazo[4,5-c]quinolinamine ring system lies parallel to the transmembrane segments, inserted into an aromatic cage formed by π-π stacking interactions with the side chains of Y2847.55 in TMD7 and Y2938.54 in H8 and by π-NH bonding between Y2847.55 and the exocyclic amine. The 2-cyclohexyl group is positioned "upward" within a small hydrophobic subpocket created by residues in TMDs 1 and 7, while the 3,4-dichlorophenyl group extends toward the lipid interface. An H-bond between the N-1 amine of the heterocycle and the carbonyl of G291.49 further stabilizes the interaction. Molecular dynamics simulations predicted two metastable intermediates, one resembling a pose determined by molecular docking and a second involving transient interactions with Y2938.54; in simulations, each of these intermediates converges into the final bound state. Structure-activity-relationships for replacement of either of the identified exocyclic or endocyclic amines with heteroatoms lacking H-bond donating ability were consistent with the hypothetical pose. Thus, we characterized an allosteric pocket for 1H-imidazo[4,5-c]quinolin-4-amines that is consistent with data generated by orthogonal methods, which will aid in the rational design of improved A3AR positive allosteric modulators. SIGNIFICANCE STATEMENT: Orthosteric A3AR agonists have advanced in clinical trials for inflammatory conditions, liver diseases, and cancer. Thus, the clinical appeal of selective receptor activation could extend to allosteric enhancers, which would induce site- and time-specific activation in the affected tissue. By identifying the allosteric site for known positive allosteric modulators, structure-based drug discovery modalities can be enabled to enhance the pharmacological properties of the 1H-imidazo[4,5-c]quinolin-4-amine class of A3AR positive allosteric modulators.

A3 receptor as a target for the treatment of Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disease with clinical features including memory loss, cognitive impairment and dementia that affects over six million Americans. Current treatments for AD have limited efficacy due, in part, to the poorly understood multifactorial nature of the mechanisms driving AD. Our data suggests that alteration of adenosine kinase‐dependent adenosine metabolism and inadequate A3 adenosine receptor (A3AR) signaling in the central nervous system (CNS) are key to these processes. The SAMP8 mouse are a mouse model of sporadic AD exhibiting many key characteristics of AD including age‐associated impairment in learning and memory, which corresponds to age‐related increase in beta amyloid, phosphorylated tau, oxidative stress, neuroinflammation, impaired efflux across the blood‐brain barrier and increased regulatory T‐cells in the brain.MethodWe first measured expressions of ADK and A3AR in the hippocampus of 12 month old. SAMP8 mice. We then treated 11 month old SAMP8 mice with MRS5980 an A3AR receptor agonist MLN5980 every other day for 6 weeks. At the end of the 4th week of treatment, behavior tests were conducted for two weeks. Mice were tested in T‐maze, Novel Object Recognition (NORPT), elevated plus maze and open field.Result12 month old SAMP8 mice treated with the A3AR agonist MRS5980 had improved learning and memory in the T‐maze and NORPT compared to the vehicle treated control mice. There was no difference between the young vehicle treated SAMP8 mice and the MRS5980 treated mice. MRS5980 did not affect activity or anxiety in the open field or elevated plus mazes indicating these factors did nor contribute to improvement in learning and memory.ConclusionHere we identify A3AR in key cognitive centers of the brain and its expression increases with cognitive impairment in the SAMP8 AD mouse model. Moreover, adenosine kinase, an enzyme that reduces extracellular adenosine, also increased. We discovered that supplemental A3AR signaling with highly selective, orally bioavailable, CNS‐penetrant A3AR agonists reversed cognitive impairment without any confounding influence on locomotor activity or anxiety. Our results indicate that A3AR is a useful pathway to give additional insight into changes in the brain with AD potential novel therapies.

ADENOSINE INFLUENCES FOXP3 EXPRESSION OF T REGS VIA THE A2AR/CREB PATHWAY IN A MOUSE MODEL OF SEPSIS.

The adenosine concentration and forkhead box protein (Foxp3) expression in T regulatory cells (T regs ) are increased during sepsis. However, the mechanism by which adenosine induces Foxp3 expression is incompletely understood. A cecal ligation and puncture (CLP) model was constructed using C57BL/J mice. The plasma adenosine concentration and Foxp3 expression in splenic T regs were increased consistently for 15 days after sepsis onset. Analysis of the mean fluorescence intensity of Foxp3 and adenosine concentration in the same mice revealed a linear correlation. In the CLP model, adenosine 2a receptor (A2aR) blockade inhibited Foxp3 expression in T regs . In vitro activation of A2aR promoted Foxp3 expression in T regs and facilitated secretion of extracellular vesicles. Transcriptome sequencing revealed that A2aR blockade led to changes in cyclic adenosine monophosphate response element-binding protein (CREB) transcription in T regs in our sepsis model. Use of adenosine or A2aR agonists promoted CREB expression, CREB phosphorylation at S133, T reg expression of Foxp3, and enhanced inhibition of proliferation of cluster of differentiation (CD)4+ lymphocytes. A2aR blockade or inhibition of CREB expression inhibited Foxp3 expression in T regs . In the CLP model, use of CREB inhibitors could inhibit Foxp3 expression and reduce the bacterial load. In summary, adenosine in sepsis promotes CREB phosphorylation via A2aR which, in turn, upregulates Foxp3 expression in T regs .

2-Amino-5-arylethynyl-thiophen-3-yl-(phenyl)methanones as A1 Adenosine Receptor Positive Allosteric Modulators.

A1 adenosine receptor (A1AR) agonists have cerebroprotective, cardioprotective, antinociceptive, and other pharmaceutical applications. We explored the structure-activity relationship of 5-arylethynyl aminothiophenes as A1AR positive allosteric modulators (PAMs). The derivatives were compared in binding and functional assays at the human A1AR, indicating that some fluoro-substituted analogues have enhanced PAM activity. We identified substitution of the terminal phenyl ring in 12 (2-F-Ph), 15 (3,4-F2-Ph, MRS7935), and 21 (2-CF3-Ph) as particularly enhancing the PAM activity. 15 was also shown to act as an A1 ago-PAM with EC50 ≈ 2 μM, without activity (30 μM) at other ARs. Molecular modeling indicated that both the 5-arylethynyl and the 4-neopentyl groups are located in a region outside the receptor transmembrane helix bundle that is in contact with the phospholipid bilayer, consistent with the preference for nonpolar substitution of the aryl moiety. Although they are hydrophobic, these PAMs could provide potential drug candidate molecules for engaging protective A1ARs.