257 Combination of adenosine antagonists A2AR (TT-10) and A2BR (TT-4) with checkpoint inhibitors demonstrate anti-tumor activity in CT-26 murine colon tumor allograft model

BackgroundTT-10 and TT-4 are potent and selective antagonists of adenosine A2A receptor (A2AR) and A2B receptor (A2BR) respectively. Both agents are being developed for the treatment of advanced cancers initially as monotherapy, using high levels of adenosine receptor expression in tumor tissue as biomarker.MethodsBalb/c mice were implanted with CT-26 cells and randomly assigned to 8 groups per study; (1) vehicle control, (2) adenosine antagonist - 1 mg/kg A2AR (TT-10) 1 mg/kg or 3 mg/kg A2BR (TT-4), (3) 10 mg/kg Anti-mPD-1, (4) 5 mg/kg Anti-mCTLA-4, (5) 100 mg/kg Irinotecan, (6) adenosine antagonist + Anti-mPD-1, (7) adenosine antagonist + Anti-mCTLA-4, (8) adenosine antagonist+ Irinotecan. Adenosine antagonists and control were given daily by oral gavage, Anti-mPD-1, Anti-mCTLA-4 and Irinotecan were administered Intraperitoneal. Treatment was started on day 1 post implant and mice were followed until individual tumor volume reached 2,000 or 3000 mm3 (as defined by protocol) or moribund. Tumor measurements and weights were taken every 2 to 3 days. In addition, a subset of mice were investigated for changes in peripheral whole blood and intra-tumor analysis on days 3 and 10 via flow cytometry. The populations of interest included CD223+, CD3+, CD4+, CD8+, CD25+FoxP3+, CD25-CD69+ and CD44+CD62L.ResultsAll implanted mice developed measurable tumors. Mean suppression of tumor growth was observed to be greater in single agent adenosine antagonists TT-10 and TT-4 when compared to the vehicle control and was observed to show overall greater suppression of tumor growth when combined with anti-mPD-1 or anti-m-CTLA-4. Tumor infiltrating lymphocyte analysis by flow cytometry, showed higher amounts of CD25+FoxP3+ present in control mice at day 3, than was observed in mice that were treated with A2AR alone, A2AR + anti-mPD-1 and A2AR + anti-m-CTLA-4.ConclusionsTT-10 and TT-4 alone was superior to vehicle control in slowing tumor growth. However, the combination of TT-10 + Anti-mPD-1 and TT-4 + Anti-mCTLA-4 showed the greatest tumor response and growth suppression. Furthermore, a striking reduction of CD25+FoxP3+ within the tumor was observed at day 3 in mice treated with A2AR alone, A2AR + anti-mPD-1 and A2AR + anti-m-CTLA-4 when compared to the vehicle control.

“The path is smooth that leadeth on to danger”: caffeine and psychosis

AimsTo review literature on the importance of caffeine intake with regard to psychosis. The need for intervention with regard to caffeine intake hinges on effectively recognizing potential risks.BackgroundCaffeine is the most widely consumed psychoactive substance worldwide and as such is generally considered acceptable but as a competitive adenosine antagonist, it affects dopamine transmission. Patients with serious mental illness are known to have higher caffeine intakes than the general population. The hierarchy of needs for this patient population is complex, frequently leaving the intake of caffeine under the radar of clinical priorities.MethodPubMed and Google Scholar search for caffeine/coffee and psychosis/schizophreniaResultOf the 43 articles that were considered relevant for clinical practice, caffeine consumption was associated with 1) appearance of psychotic symptoms and episodes (caffeine-induced psychosis) and chronic psychosis in high intake 2) exacerbation of psychosis in schizophrenic patients even in lower intakes, 3) treatment resistance possibly due to interference with antipsychotics (ex. clozapine), 4) abuse and addiction, 5) comorbidity with tobacco smoking and other addictions. Caffeine in low doses was associated with ameliorating cognitive and extrapyramidal side-effects of medication and as a potential treatment strategy for treatment-resistant schizophrenia.ConclusionCaffeine consumption may have a greater impact on psychotic symptoms and episodes than is recognized with negative effects outweighing any potential benefits. Greater awareness of the necessity to quantify caffeine intake and implementation of interventions to curb intake may contribute to better quality of care of serious mental illness. Further research is warranted.

Development and Charecterization of Caffeine and Quercetin Loaded Nasal Niosomal In-Situ Gel for Treatment of Depression

Caffeine and Quercetin both are used in combination for the treatment of patient with depression as they are adenosine antagonist due to which they elevate the level of neurotransmitters. The study was designed with two aims. First, is to enhance the solubility and bioavailability of BCS Class II i.e. Quercetin; secondly to ease administration of the formulation to the patient with depression. The culmination of this study, the caffeine and quercetin loaded niosomal in-situ gel for nose-to-brain delivery was formulated. Niosomes were prepared and optimized by using definitive screening design whereas, the Niosomal in-situ gel were prepared and optimized using central composite design. The vesicle size of the optimized batch was found to be 0.281±0.26µm. The % EE of all niosomal batches was found to be in a range of 81.52±0.21% to 98.72±0.16% for Caffeine and 94.3±0.31 to 99.73±0.23 for Quercetin and the cumulative % release was found to be in a range of 72.09±0.18% to 103.3±0.26% for Caffeine and 10.9±0.31% to 37.06±0.15% for Quercetin at 4hrs. DSC, FTIR studies were performed for pure drug and optimized niosomal batch. All the gels as per design were formulated, where the Spreadability was found to be in range of 5.1±0.26cm to 7.9±0.16cm and viscosity after gelation in range of 1800±0.11cps to 4780±0.26cps. The % drug permeated was found to be in range of 85.86±0.015% to 98.61±0.024% for Caffeine and 22.65±0.19% to 33.23±0.34% for Quercetin at 6hrs. These results indicated that niosomal In-situ gel can be used to enhance the bioavailability of drug by directly delivering the drug to the brain by avoiding first pass effect

Morphological and Motility Features of the Stable Bleb-Driven Monopodial Form of Entamoeba and Its Importance in Encystation

ABSTRACT Entamoeba histolytica and its reptilian counterpart and encystation model Entamoeba invadens formed a polarized monopodial morphology when treated with pentoxifylline. This morphology was propelled by retrograde flow of the cell surface resulting from a cyclic sol-gel conversion of cytoplasm and a stable bleb at the leading edge. Pentoxifylline treatment switched the unpolarized, adherent trophozoites to the nonadherent, stable bleb-driven form and altered the motility pattern from slow and random to fast, directionally persistent, and highly chemotactic. Interestingly, exogenously added adenosine produced multiple protrusions and random motility, an opposite phenotype to that of pentoxifylline. Thus, pentoxifylline, an adenosine antagonist, may be inducing the monopodial morphology by preventing lateral protrusions and restricting the leading edge to one site. The polarized form of E. invadens was aggregation competent, and time-lapse microscopy of encystation revealed its appearance during early hours, mediating the cell aggregation by directional cell migration. The addition of purine nucleotides to in vitro encystation culture prevented the formation of polarized morphology and inhibited the cell aggregation and, thus, the encystation, which further showed the importance of the polarized form in the Entamoeba life cycle. Cell polarity and motility are essential in the pathogenesis of Entamoeba parasites, and the stable bleb-driven polarized morphology of Entamoeba may also be important in invasive amoebiasis.

Purinergic Signaling and Related Biomarkers in Depression

It is established that purinergic signaling can shape a wide range of physiological functions, including neurotransmission and neuromodulation. The purinergic system may play a role in the pathophysiology of mood disorders, influencing neurotransmitter systems and hormonal pathways of the hypothalamic-pituitary-adrenal axis. Treatment with mood stabilizers and antidepressants can lead to changes in purinergic signaling. In this overview, we describe the biological background on the possible link between the purinergic system and depression, possibly involving changes in adenosine- and ATP-mediated signaling at P1 and P2 receptors, respectively. Furthermore, evidence on the possible antidepressive effects of non-selective adenosine antagonist caffeine and other purinergic modulators is reviewed. In particular, A2A and P2X7 receptors have been identified as potential targets for depression treatment. Preclinical studies highlight that both selective A2A and P2X7 antagonists may have antidepressant effects and potentiate responses to antidepressant treatments. Consistently, recent studies feature the possible role of the purinergic system peripheral metabolites as possible biomarkers of depression. In particular, variations of serum uric acid, as the end product of purinergic metabolism, have been found in depression. Although several open questions remain, the purinergic system represents a promising research area for insights into the molecular basis of depression.

Equine poisoning by coffee husk (Coffea canephora) in northern Espírito Santo, Brazil

Background: Brazil is the largest coffee (Coffea canephora) producer in the world, and Espírito Santo state is the second largest national producer of this commodity. Caffeine poisoning has been described in several animal and human species and is generally associated with accidental and/or intentional ingestion of caffeine-containing products. In horses, there are few reports in the literature about coffee poisoning, and most animals show clinical signs of excitability, involuntary muscle tremors, and chewing movements. Therefore, the objectives of the present study are to describe the clinical and epidemiological aspects of coffee (Coffea canephora) poisoning in horses in northern Espírito Santo, Brazil.Cases: Two horses from northern Espírito Santo presented with clinical signs of excessive sweating, reluctance to enter the trailer, muscle tremors, aggression, incoordination, constant tremors of the lips and tongue, chewing movements, and falling. Clinical signs began after the animals were confined in stalls containing coffee husk (Coffea canephora) for at least one week. After three days in the stall, the horse began to show clinical signs characterized by excessive sweating, reluctance to enter the trailer, aggression, and incoordination. On physical examination, there was marked dehydration, tachycardia (120 bpm), tachypnea (80 mpm) and a body temperature of 39.1ºC. In addition, the animal had cecum and ventral colon hypomotility. The horse was treated as soon as it was admitted to the hospital with a 10 mL / h intravenous drip of Ringer lactate solution; 100 mL intravenous mercepton every 24 h; 10 mL intramuscular vitamin B1 every 24 h, and 1.1 mL intravenous acepromazine 1%; when it showed increased excitability, it was treated for neurological signs and recovered four days after admission. The second animal was a 3-year-old female Mangalarga Marchador horse, weighing 280 kg. The animal was confined for 30 days with coffee husk bedding. The horse was fed 8 kg of corn silage and 4 kg of granulated feed per day. After two weeks in confinement, the animal began to show severe incoordination, extremely aggressive behavior, muscle tremors, constant tremors of the lips and tongue, chewing movements, excessive sweating, and falling.Discussion: The diagnosis of coffee husk poisoning was based on the epidemiological and clinical characteristics of the disease. In Espírito Santo, it seems to be common to use coffee husk as bedding material for horses, mostly as a substitute for sand and wood shavings. In the properties where the animals lived, the coffee was planted to sell, and the remains from production, especially the husks, were used as bedding for the animals. It has been reported that when horses are placed in stalls with coffee husks, they tend to eat the husks spontaneously, resulting in intoxication. The clinical signs observed in this study were similar to those described in horses experimentally intoxicated by the plant. The neurological effects observed were due to the action of caffeine as an adenosine antagonist. The observed neurological clinical signs observed in these cases were nonspecific, and other disorders of the equine central nervous system, such as rabies and leukoencephalomalacia, should be considered in the differential diagnosis of coffee poisoning. Coffee husks should not be used as bedding for horses, as it can cause animal poisoning and death due to the excitatory effects of caffeine, which can lead to spontaneous falls and serious trauma.

From Bench to Bedside and Back Again

Dexmedetomidine Diminishes Halothane Anesthetic Requirements in Rats Through a Postsynaptic Alpha 2 Adrenergic Receptor. By Segal IS, Vickery RG, Walton JK, Doze VA, and Maze M. Anesthesiology 1988; 125:590–4. Abstract reprinted with permission. The effect of 4(5)-[1-(2,3-dimethylphenyl)ethyl]imidazole (medetomidine), the α2 adrenergic agonist, on anesthetic requirements was investigated in rats anesthetized with halothane. Halothane MAC was determined before and after either dexmedetomidine (d-enantiomer) or levomedetomidine (l-enantiomer) 10, 30, and 100 μg/kg, or vehicle intraperitoneally. There was a dose-dependent increase in MAC with the d-, but not the l-, stereoisomer. At the highest dose of dexmedetomidine (100 μg/kg), halothane could be discontinued for up to 30 min with no response to tail clamping. To determine whether α2 adrenoreceptors mediated this effect of dexmedetomidine on MAC, cohorts of rats were pretreated with idazoxan, 10 mg/kg intraperitoneally, a highly selective α2 antagonist. This completely prevented the reduction of MAC caused by dexmedetomidine. To determine whether the reduction of MAC caused by dexmedetomidine was mediated in part through either opiate or adenosine receptors, groups of rats were pretreated with either naltrexone, 5 mg/kg intraperitoneally, an opiate antagonist, or 8-phenyltheophylline, 2.5 mg/kg intraperitoneally, an A1 adenosine antagonist. These two pretreatments did not alter the reduction of MAC by dexmedetomidine. To determine whether postsynaptic mechanisms mediate the anesthetic effect of dexmedetomidine, rats were depleted of central catecholamine stores with either n-(2-chloroethyl)-n-ethyl-2-bromobenzylamine or reserpine and α-methyl-para-tyrosine, and MAC was determined before and after each dose of dexmedetomidine. While the catecholamine-depleted rats had a lower basal MAC than the vehicle controls, there was still a profound reduction in halothane MAC after administration of dexmedetomidine. The reduction of MAC by dexmedetomidine was blocked with idazoxan in the catecholamine-depleted rats. These data indicate that the reduction of MAC caused by dexmedetomidine is mediated through α2 adrenoreceptors with no apparent involvement of either opiate or A1 adenosine receptors. Data from catecholamine-depleted rats suggest that the mediating mechanism must involve site(s) other than or in addition to the presynaptic α2 adrenergic receptors on noradrenergic neurons. The authors conclude that central postsynaptic α2 adrenergic receptors mediate a significant part of the reduction of anesthetic requirements caused by dexmedetomidine.

Hindbrain cytoglucopenia-induced increases in systemic blood glucose levels by 2-deoxyglucose depend on intact astrocytes and adenosine release

The hindbrain contains critical neurocircuitry responsible for generating defensive physiological responses to hypoglycemia. This counter-regulatory response (CRR) is evoked by local hindbrain cytoglucopenia that causes an autonomically mediated increase in blood glucose, feeding behavior, and accelerated digestion; that is, actions that restore glucose homeostasis. Recent reports suggest that CRR may be initially triggered by astrocytes in the hindbrain. The present studies in thiobutabarbital-anesthetized rats show that exposure of the fourth ventricle (4V) to 2-deoxyglucose (2DG; 15 μmol) produced a 35% increase in circulating glucose relative to baseline levels. While the 4V application of the astrocytic signal blocker, fluorocitrate (FC; 5 nmol), alone, had no effect on blood glucose levels, 2DG-induced increases in glucose were blocked by 4V FC. The 4V effect of 2DG to increase glycemia was also blocked by the pretreatment with caffeine (nonselective adenosine antagonist) or a potent adenosine A1 antagonist (8-cyclopentyl-1,3-dipropylxanthine; DPCPX) but not the NMDA antagonist (MK-801). These results suggest that CNS detection of glucopenia is mediated by astrocytes and that astrocytic release of adenosine that occurs after hypoglycemia may cause the activation of downstream neural circuits that drive CRR.