Purine Intake and All-Cause Mortality in Ovarian Cancer: Results from a Prospective Cohort Study.

BACKGROUND: Current biological evidence suggests that purine involvement in purine metabolism may contribute to the development and progression of ovarian cancer (OC), but the epidemiological association is currently unknown. METHODS: A total of 703 newly diagnosed patients with OC aged 18-79 years were included in this prospective cohort study. Utilizing a verified food-frequency questionnaire, the participants' dietary consumption was gathered. Using medical records and ongoing follow-up, the deaths up until 31 March 2021 were determined. To assess the hazard ratios (HRs) and 95% confidence intervals (CIs) of purine intake with OC mortality, Cox proportional-hazard models were utilized. RESULTS: During the median follow-up of 31 months (interquartile: 20-47 months), 130 deaths occurred. We observed an improved survival for the highest tercile of total purine intake compared with the lowest tercile (HR = 0.39, 95% CI = 0.19-0.80; p trend < 0.05), and this protective association was mainly attributed to xanthine intake (HR = 0.52, 95% CI = 0.29-0.94, p trend < 0.05). Additionally, we observed a curving relationship in which OC mortality decreased with total purine intake, and the magnitude of the decrease was negatively correlated with intake (p non-linear < 0.05). Significant inverse associations were also observed in subgroup analyses and sensitivity analyses according to demographic and clinical characteristics. Moreover, we observed that xanthine intake and hypoxanthine intake had a multiplicative interaction with ER and PR expression (p < 0.05), respectively. CONCLUSION: A high total purine and xanthine intake was linked to a lower risk of OC mortality. Further clarification of these findings is warranted.

The role of adenosine A1 receptor in the peripheral tramadol's effect in the temporomandibular joint of rats.

Peripheral tramadol's delivery in the temporomandibular joint (TMJ) leads to significant analgesic outcomes and inflammatory process's resolvent actions. Mechanistically, these properties are apart from the opioid system. Nevertheless, the molecular mechanisms behind these effects are still unclear. Therefore, the present study investigated the hypothesis that adenosine A1 receptors are involved in the tramadol-induced analgesic and anti-inflammatory effects in the TMJ. Animals were pretreated with an intra-TMJ injection of DPCPX (antagonist of A1 receptor) or tramadol and subsequent nociceptive challenge with an intra-TMJ injection of 1.5% formalin. For over 45 min, the nociceptive behavior was quantitated, and by the end of this assessment, the animals were euthanized, and the periarticular tissue was collected. Lastly, an in vitro assay of BMDM (Bone Marrow-Derived Macrophages) was performed to investigate tramadol activity in macrophages. The intra-TMJ injection of tramadol ameliorates formalin-induced hypernociception along with inhibiting leukocyte migration. The tramadol's peripheral anti-inflammatory effect was mediated by the adenosine A1 receptor and was associated with increased protein expression of α2a-adrenoceptor in the periarticular tissues (p < 0.05: ANOVA, Tukey's test). Also, tramadol inhibits formalin-induced leukocyte migration and protein expression of P2X7 receptors in the periarticular tissue (p < 0.05); however, DPCPX did not alter this effect (p > 0.05). Moreover, DPCPX significantly reduced the protein expression of the M2 macrophage marker, MRC1. In BMDM, tramadol significantly reduces inflammatory cytokines release, and DPCPX abrogated this effect (p < 0.05). We identify tramadol's peripheral effect is mediated by adenosine A1 receptor, possibly expressed in macrophages in the TMJ tissue. We also determined an important discovery related to the activation of A1R/α2a receptors in the tramadol action.

Methylxanthines and Neurodegenerative Diseases: An Update.

Methylxanthines (MTX) are purine derived xanthine derivatives. Whereas naturally occurring methylxanthines like caffeine, theophylline or theobromine are widely consumed in food, several synthetic but also non-synthetic methylxanthines are used as pharmaceuticals, in particular in treating airway constrictions. Besides the well-established bronchoprotective effects, methylxanthines are also known to have anti-inflammatory and anti-oxidative properties, mediate changes in lipid homeostasis and have neuroprotective effects. Known molecular mechanisms include adenosine receptor antagonism, phosphodiesterase inhibition, effects on the cholinergic system, wnt signaling, histone deacetylase activation and gene regulation. By affecting several pathways associated with neurodegenerative diseases via different pleiotropic mechanisms and due to its moderate side effects, intake of methylxanthines have been suggested to be an interesting approach in dealing with neurodegeneration. Especially in the past years, the impact of methylxanthines in neurodegenerative diseases has been extensively studied and several new aspects have been elucidated. In this review we summarize the findings of methylxanthines linked to Alzheimer´s disease, Parkinson's disease and Multiple Sclerosis since 2017, focusing on epidemiological and clinical studies and addressing the underlying molecular mechanisms in cell culture experiments and animal studies in order to assess the neuroprotective potential of methylxanthines in these diseases.

Effects of 7-Methylxanthine on Deprivation Myopia and Retinal Dopamine Release in Chickens.

INTRODUCTION: Intake of 7-methylxanthine (7-MX), an adenosine receptor (AR) antagonist, has been shown to inhibit school myopia in children and deprivation myopia in rhesus monkeys, but the underlying mechanisms are not known. Also retinal dopamine seems to be involved in the control of eye growth, and in the brain, ARs and dopamine receptors interact widely by heteromerization. We have studied whether 7-MX can inhibit deprivation myopia also in chickens and whether inhibition may involve the retinal dopamine system. METHODS: 7-MX was applied by either tube-feeding (100 µg/g body weight, twice a day) or intravitreal injection (12.5 µg, every other day). Forty-eight 2-week-old chicks wore unilateral diffusers and were randomly assigned to either the tube-feeding group (involving 7-MX, vehicle [xanthan gum], or no feeding, for 13 days) or the intravitreal injection group (involving 7-MX, vehicle, or DMSO, for 8 days). Refractions (REs), ocular biometry (AL, VCD), and scleral and choroidal thickness (ChT) were measured before and after treatment. Dopamine and dihydroxyphenylacetic acid (DOPAC) content were determined in retina and vitreous by HPLC at the end of the experiments. RESULTS: No matter how 7-MX was applied, it did not inhibit deprivation myopia in chicks. No significant differences were observed in RE, VCD, AL, and scleral fibrous layer thickness. Feeding 7-MX produced more choroidal thinning in the open contralateral eye compared to control eyes in the vehicle-fed group (-40 ± 14 vs. -1 ± 7 µm, unpaired t test, p < 0.05). DOPAC and dopamine concentration in vitreous and DOPAC concentration in retina did not change with 7-MX. Vitreal dopamine content was significantly decreased in deprived eyes in the groups fed with the vehicle xanthan gum (paired t test, p < 0.01) but not in 7-MX-treated eyes, perhaps indicating a small effect of 7-MX on dopamine. CONCLUSIONS: In our study, 7-MX had no effect on DM in chicks and only minor effects on ChT and retinal dopamine. It remains unclear whether 7-MX inhibits myopia through a retinal mechanism or whether it acts directly on choroid and sclera. In the latter case, the finding that myopia is suppressed in mammals but not birds might be explained by differences in scleral structure.

Mechanistic actions of oxygen and methylxanthines on respiratory neural control and for the treatment of neonatal apnea.

Apnea remains one of the most concerning and prevalent respiratory disorders spanning all ages from infants (particularly those born preterm) to adults. Although the pathophysiological consequences of apnea are fairly well described, the neural mechanisms underlying the etiology of the different types of apnea (central, obstructive, and mixed) still remain incompletely understood. From a developmental perspective, however, research into the respiratory neural control system of immature animals has shed light on both central and peripheral neural pathways underlying apnea of prematurity (AOP), a highly prevalent respiratory disorder of preterm infants. Animal studies have also been fundamental in furthering our understanding of how clinical interventions (e.g. pharmacological and mechanical) exert their beneficial effects in the clinical treatment of apnea. Although current clinical interventions such as supplemental O2 and positive pressure respiratory support are critically important for the infant in respiratory distress, they are not fully effective and can also come with unfortunate, unintended (and long-term) side-effects. In this review, we have chosen AOP as one of the most common clinical scenarios involving apnea to highlight the mechanistic basis behind how some of the interventions could be both beneficial and also deleterious to the respiratory neural control system. We have included a section on infants with critical congenital heart diseases (CCHD), in whom apnea can be a clinical concern due to treatment with prostaglandin, and who may benefit from some of the treatments used for AOP.

A Randomized, Placebo-Controlled Trial to Assess the Effects of 8 Weeks of Administration of GSK256073, a Selective GPR109A Agonist, on High-Density Lipoprotein Cholesterol in Subjects With Dyslipidemia.

GPR109A (HM74A), a G-protein-coupled receptor, is hypothesized to mediate lipid and lipoprotein changes and dermal flushing associated with niacin administration. GSK256073 (8-chloro-3-pentyl-1H-purine-2,6[3H,7H]-dione) is a selective GPR109A agonist shown to suppress fatty acid levels and produce mild flushing in short-term clinical studies. This study evaluated the effects of GSK256073 on lipids in subjects with low high-density lipoprotein cholesterol (HDLc). Subjects (n = 80) were randomized (1:1:1:1) to receive GSK256073 5, 50, or 150 mg/day or matching placebo for 8 weeks. The primary end point was determining the GSK256073 exposure-response relationship for change from baseline in HDLc. No significant exposure response was observed between GSK256073 and HDLc levels. GSK256073 did not significantly alter HDLc levels versus placebo, but rather revealed a trend at the 150-mg dose for a nonsignificant decrease in HDLc (-6.31%; P = .12) and an increase in triglycerides (median, 24.4%; 95% confidence interval, 7.3%-41.6%). Flushing was reported in 21%, 25%, and 60% of subjects (5, 50, and 150 mg, respectively) versus 24% for placebo. Results indicated that selective activation of the GPR109A receptor with GSK256073 did not produce niacin-like lipid effects. These findings add to the increasing evidence that niacin-mediated lipoprotein changes occur predominantly via GPR109A-independent pathways.

Current and emerging pharmaceutical interventions for myopia.

Myopia is a major cause of visual impairment. Its prevalence is growing steadily, especially in East Asia. Despite the immense disease and economic burden, there are currently no Food and Drug Administration-approved drugs for myopia. This review aims to summarise pharmaceutical interventions of myopia at clinical and preclinical stages in the last decade and discuss challenges for preclinical myopia drugs to progress to clinical trials. Atropine and oral 7-methylxanthine are shown to reduce myopia progression in human studies. The former has been extensively studied and is arguably the most successful medication. However, it has side effects and trials on low-dose atropine are ongoing. Other pharmaceutical agents being investigated at a clinical trial level include ketorolac tromethamine, oral riboflavin and BHVI2 (an experimental drug). Since the pathophysiology of myopia is not fully elucidated, numerous drugs have been tested at the preclinical stage and can be broadly categorised based on the proposed mechanisms of myopisation, namely antimuscarinic, dopaminergic, anti-inflammatory and more. However, several agents were injected intravitreally or subconjunctivally, hindering their progress to human trials. Furthermore, with atropine being the most successful medication available, future preclinical interventions should be studied in combination with atropine to optimise the treatment of myopia.

The role of netrin-1 in the mouse cornea during Aspergillus fumigatus infection.

PURPOSE: To explore the effects of netrin-1 on inflammation in Aspergillus fumigatus-infected mouse corneas and on proliferation and migration in human corneal epithelial cells (HCECs). METHODS: Netrin-1 and the receptor A2BAR were detected in normal and infected corneas from C57BL/6 mice and RAW 264.7 cells. The mice were injected subconjunctivally with recombinant netrin-1. The severity of the disease was determined by clinical scores, photography with a slit lamp, RT-PCR, western blotting, myeloperoxidase (MPO) assays and immunofluorescence staining of polymorphonuclear neutrophilic leukocytes (PMNs). The effects of netrin-1 on RAW 264.7 cells in vitro were determined by RT-PCR. The role of A2BAR was demonstrated in vivo by detecting the expression of IL-1ß, TNF-α, and IL-10 in corneas pretreated subconjunctivally with an A2BAR antagonist (PSB1115). RAW 264.7 cells were stimulated with Aspergillus fumigatus (A. fumigates) and netrin-1 with or without PSB1115 pretreatment. A cell counting kit-8 (CCK-8) assay was used to evaluate cell proliferation ability, and cell migration ability was determined by cell scratch experiments with HCECs. RESULTS: Netrin-1 expression decreased slightly after A. fumigatus infection and then increased to its peak. A2BAR expression increased at 1 day post infection (p.i.), with a subsequent decline. Compared to the PBS control, exogenous netrin-1 attenuated the inflammatory response, PMN infiltration, and expression of the proinflammatory factors IL-1ß and TNF-α, while IL-10 expression was up-regulated. In RAW 264.7 cells, recombinant netrin-1 obviously inhibited the mRNA expression of IL-1ß and TNF-α and promoted the mRNA expression of the anti-inflammatory cytokine IL-10. Pretreatment with PSB1115 resulted in disease aggravation and higher levels of the proinflammatory factors IL-1ß and TNF-α both in vivo and in vitro. And the effect of netrin-1 on inflammatory factors was abolished by PSB1115. Moreover, compared to the control treatment, exogenous netrin-1 significantly facilitated the proliferation and migration of HCECs. CONCLUSIONS: Netrin-1 attenuates inflammation in C57BL/6 mice infected with A. fumigatus, and it may play this role via the receptor A2BAR. Additionally, netrin-1 can promote the proliferation and migration of HCECs.

The safety profile of new antidiabetic xanthine derivatives and their chitosan based formulations.

The safety profile of new antidiabetic xanthine derivatives with thiazolidine­4­one scaffold (6, 7) and their new chitosan based formulations (CS-6, CS-7), administrated to diabetic rats, have been evaluated in terms of biochemical markers of liver and kidney function as well as of hematological markers. The effect on lipid profile and clinic parameters (body weight, food and water intake) has been also evaluated. The treatment of diabetic rats with xanthine derivatives (6, 7) and chitosan based formulations (CS-6, CS-7) was associated with lower liver enzymes (AST, ALT, LDH) and bilirubin (direct, total) values compared to the non-treated diabetic rats, that means the tested derivatives/formulations have improved the liver function injured in diabetes mellitus conditions. Also the kidney biochemical markers (creatinine, uric acid, urea) were significantly decreased in diabetic rats treated with 6, 7 and chitosan microparticles (CS-6, CS-7). The values of biochemical markers of liver and kidney functions were even better than the values recorded for pioglitazone, used as standard antidiabetic drug. The improving effect on kidney function was proved by the histopathological study. Moreover, the xanthine derivatives and their chitosan based formulation were associated with improved hematological markers compared to the non-treated diabetic rats which mean the improving of the hemorheological state. These results support the safety profile of new xanthine derivatives with thiazolidine­4­one scaffold (6, 7) and their new chitosan based formulations (CS-6, CS-7) and their potential applications for the treatment of diabetes mellitus syndrome.

Involvement of adenosine A1 receptor in electroacupuncture-mediated inhibition of astrocyte activation during neuropathic pain.

OBJECTIVE: Neuropathic pain is a chronic pain condition caused by damage or dysfunction of the central or peripheral nervous system. Electroacupuncture (EA) has an antinociceptive effect on neuropathic pain, which is partially due to inhibiting astrocyte activation in the spinal cord. METHODS: We found that an intrathecal injection of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective adenosine A1 receptor antagonist, reversed the antinociceptive effects of EA in a chronic constriction injury-induced neuropathic pain model. RESULTS: The expression of GFAP in L4-L6 spinal cord was significantly upgraded, while DPCPX suppressed the effect of the EA-mediating inhibition of astrocyte activation, as well as wiping out the EA-induced suppression of cytokine content (TNF-α). CONCLUSIONS: These results indicated that the adenosine A1 receptor is involved in EA actions during neuropathic pain through suppressing astrocyte activation as well as TNF-α upregulation of EA, giving enlightenment to the mechanisms of acupuncture analgesia and development of therapeutic targets for neuropathic pain.

Involvement of adenosine A1 receptor in electroacupuncture-mediated inhibition of astrocyte activation during neuropathic pain / Envolvimento do receptor de adenosina A1 na inibição da ativação de astrócitos mediada por eletroacupuntura durante a dor neuropática

ABSTRACT Neuropathic pain is a chronic pain condition caused by damage or dysfunction of the central or peripheral nervous system. Electroacupuncture (EA) has an antinociceptive effect on neuropathic pain, which is partially due to inhibiting astrocyte activation in the spinal cord. We found that an intrathecal injection of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective adenosine A1 receptor antagonist, reversed the antinociceptive effects of EA in a chronic constriction injury-induced neuropathic pain model. The expression of GFAP in L4-L6 spinal cord was significantly upgraded, while DPCPX suppressed the effect of the EA-mediating inhibition of astrocyte activation, as well as wiping out the EA-induced suppression of cytokine content (TNF-α). These results indicated that the adenosine A1 receptor is involved in EA actions during neuropathic pain through suppressing astrocyte activation as well as TNF-α upregulation of EA, giving enlightenment to the mechanisms of acupuncture analgesia and development of therapeutic targets for neuropathic pain.

RESUMO A dor neuropática é uma condição de dor crônica causada por dano ou disfunção do sistema nervoso central ou periférico. A eletroacupuntura (EA) tem um efeito antinociceptivo durante a dor neuropática, que é parcialmente devido à inibição da ativação de astrócitos na medula espinhal. Descobrimos que a injeção intratecal de 8-ciclopentil-1,3-dipropilxantina (DPCPX), um antagonista seletivo do receptor de adenosina A1, reverteu os efeitos antinociceptivos da EA no modelo de dor neuropática induzida por lesão por constrição crônica (CCI). A expressão da GFAP na medula espinal L4-L6 foi significativamente melhorada, enquanto a DPCPX suprimiu o efeito da inibição mediadora da EA na ativação de astrócitos, bem como eliminou a supressão induzida pela EA do conteúdo de citocina (TNF-α). Esses resultados indicam que o receptor de adenosina A1 está envolvido nas ações da EA durante a dor neuropática, suprimindo a ativação astrocitária, bem como o aumento da TNF-α na EA, fornecendo esclarecimentos sobre os mecanismos de analgesia da acupuntura e o desenvolvimento de alvos terapêuticos para dor neuropática.

Propentofylline decreases hypothalamic astrogliosis induced by hypercaloric diet in the rat.

Obesity is associated with a chronic and low-grade inflammatory response in the hypothalamus, where astrogliosis occurs with the upregulation of the astrocyte structural protein GFAP. As propentofylline (PPF) has inhibitory effects on astrocyte and microglial activation during inflammation, this study aimed to investigate if this xanthine derivative could decrease the astrocyte reaction induced by a hypercaloric diet (HD). Male Wistar rats were divided into four groups: NDS - rats receiving a normocaloric diet (ND) and daily saline solution; NDP - rats receiving ND and daily PPF (12.5 mg/kg/day, intraperitoneal route); HDS - rats receiving HD and saline solution, HDP - rats receiving HD and PPF. On the 21st day, rats were anesthetized, and perfused, and brains were collected for GFAP immunohistochemical study in the hypothalamus. Results showed that HD induced increased weight gain and hypothalamic astrogliosis. Propentofylline decreased the expression of GFAP in the HDP group, although it did not affect the weight gain induced by this diet.

Propentofylline decreases hypothalamic astrogliosis induced by hypercaloric diet in the rat / A propentofilina diminui a astrogliose hipotalâmica induzida pela dieta hipercalórica no rato

ABSTRACT Obesity is associated with a chronic and low-grade inflammatory response in the hypothalamus, where astrogliosis occurs with the upregulation of the astrocyte structural protein GFAP. As propentofylline (PPF) has inhibitory effects on astrocyte and microglial activation during inflammation, this study aimed to investigate if this xanthine derivative could decrease the astrocyte reaction induced by a hypercaloric diet (HD). Male Wistar rats were divided into four groups: NDS - rats receiving a normocaloric diet (ND) and daily saline solution; NDP - rats receiving ND and daily PPF (12.5 mg/kg/day, intraperitoneal route); HDS - rats receiving HD and saline solution, HDP - rats receiving HD and PPF. On the 21st day, rats were anesthetized, and perfused, and brains were collected for GFAP immunohistochemical study in the hypothalamus. Results showed that HD induced increased weight gain and hypothalamic astrogliosis. Propentofylline decreased the expression of GFAP in the HDP group, although it did not affect the weight gain induced by this diet.

RESUMO A obesidade está associada com uma resposta inflamatória crônica e de baixo grau no hipotálamo, onde ocorre astrogliose com a superexpressão da proteína astrocitária GFAP. Como a propentofilina (PPF) possui efeitos inibitórios sobre a ativação astrocitária e microglial durante a inflamação, este estudo visou a investigar se esta xantina podia diminuir a reação astrocitária induzida pela dieta hipercalórica (HD). Ratos Wistar machos foram divididos em 4 grupos: NDS- ratos recebendo dieta normocalórica (ND) e solução salina diária; NDP- ratos recebendo ND e PPF diária (12.5 mg/kg/dia, via intraperitoneal); HDS- ratos recebendo HD e solução salina, HDP- ratos recebendo HD e PPF. No 21° dia, os ratos foram perfundidos e os encéfalos, coletados para estudo imuno-histoquímico para a GFAP no hipotálamo. Os resultados mostram que a HD induziu aumento do ganho de peso e astrogliose no hipotálamo. A PPF diminuiu a expressão de GFAP no grupo HD, embora não tenha afetado o ganho de peso induzido por esta dieta.

The Adenosine Receptor Antagonist, 7-Methylxanthine, Alters Emmetropizing Responses in Infant Macaques.

Purpose: Previous studies suggest that the adenosine receptor antagonist, 7-methylxanthine (7-MX), retards myopia progression. Our aim was to determine whether 7-MX alters the compensating refractive changes produced by defocus in rhesus monkeys. Methods: Starting at age 3 weeks, monkeys were reared with -3 diopter (D; n = 10; 7-MX -3D/pl) or +3D (n = 6; 7-MX +3D/pl) spectacles over their treated eyes and zero-powered lenses over their fellow eyes. In addition, they were given 100 mg/kg of 7-MX orally twice daily throughout the lens-rearing period (age 147 ± 4 days). Comparison data were obtained from lens-reared controls (-3D/pl, n = 17; +3D/pl, n = 9) and normal monkeys (n = 37) maintained on a standard diet. Refractive status, corneal power, and axial dimensions were assessed biweekly. Results: The -3D/pl and +3D/pl lens-reared controls developed compensating myopic (-2.10 ± 1.07 D) and hyperopic anisometropias (+1.86 ± 0.54 D), respectively. While the 7-MX +3D/pl monkeys developed hyperopic anisometropias (+1.79 ± 1.11 D) that were similar to those observed in +3D/pl controls, the 7-MX -3D/pl animals did not consistently exhibit compensating myopia in their treated eyes and were on average isometropic (+0.35 ± 1.96 D). The median refractive errors for both eyes of the 7-MX -3D/pl (+5.47 D and +4.38 D) and 7-MX +3D/pl (+5.28 and +3.84 D) monkeys were significantly more hyperopic than that for normal monkeys (+2.47 D). These 7-MX-induced hyperopic ametropias were associated with shorter vitreous chambers and thicker choroids. Conclusions: In primates, 7-MX reduced the axial myopia produced by hyperopic defocus, augmented hyperopic shifts in response to myopic defocus, and induced hyperopia in control eyes. The results suggest that 7-MX has therapeutic potential in efforts to slow myopia progression.

Nucleotide P2Y13-stimulated phosphorylation of CREB is required for ADP-induced proliferation of late developing retinal glial progenitors in culture.

Nucleotides stimulate phosphorylation of CREB to induce cell proliferation and survival in diverse cell types. We report here that ADP induces the phosphorylation of CREB in a time- and concentration-dependent manner in chick embryo retinal progenitors in culture. ADP-induced increase in phospho-CREB is mediated by P2 receptors as it is blocked by PPADS but not by the adenosine antagonists DPCPX or ZM241385. Incubation of the cultures with the CREB inhibitor KG-501 prevents ADP-induced incorporation of [3H]-thymidine, indicating that CREB is involved in retinal cell proliferation. No effect of this compound is observed on the viability of retinal progenitors. While no significant increase in CREB phosphorylation is observed with the P2Y1 receptor agonist MRS2365, ADP-induced phosphorylation of CREB is blocked by the P2Y13 receptor selective antagonist MRS2211, but not by MRS2179 or PSB0739, two antagonists of the P2Y1 and P2Y12 receptors, respectively, suggesting that ADP-induced CREB phosphorylation is mediated by P2Y13 receptors. ADP-induced increase in phospho-CREB is attenuated by the PI3K inhibitor LY294002 and completely prevented by the MEK inhibitor U0126, suggesting that at least ERK is involved in ADP-induced CREB phosphorylation. A pharmacological profile similar to the activation and inhibition of CREB phosphorylation is observed in the phosphorylation of ERK, suggesting that P2Y13 receptors mediate ADP induced ERK/CREB pathway in the cultures. While no increase in [3H]-thymidine incorporation is observed with the P2Y1 receptor agonist MRS2365, both MRS2179 and MRS2211 prevent ADP-mediated increase in [3H]-thymidine incorporation, but not progenitor's survival, suggesting that both P2Y1 and P2Y13 receptor subtypes are involved in ADP-induced cell proliferation. P2Y1 receptor-mediated increase in [Ca2+]i is observed in glial cells only when cultures maintained for 9days are used. In glia from cultures cultivated for only 2days, no increase in [Ca2+]i is detected with MRS2365 and no inhibition of ADP-mediated calcium response is observed with MRS2179. In contrast, MRS2211 attenuates ADP-mediated increase in [Ca2+]i in glial cells from cultures at both stages, suggesting the presence of P2Y13 receptors coupled to calcium mobilization in proliferating retinal glial progenitors in culture.

Inosine attenuates spontaneous activity in the rat neurogenic bladder through an A2B pathway.

Neurogenic detrusor overactivity (NDO) is among the most challenging complications of spinal cord injury (SCI). A recent report by us demonstrated an improvement in NDO in SCI rats following chronic systemic treatment with the purine nucleoside inosine. The objective of this study was to investigate the mechanism of action of inosine underlying improvement of NDO. Male Sprague-Dawley rats underwent complete spinal cord transection at T8. Inosine (1 mM) delivered intravesically to SCI rats during conscious cystometry significantly decreased the frequency of spontaneous non-voiding contractions. In isolated tissue assays, inosine (1 mM) significantly decreased the amplitude of spontaneous activity (SA) in SCI bladder muscle strips. This effect was prevented by a pan-adenosine receptor antagonist CGS15943, but not by A1 or A3 receptor antagonists. The A2A antagonist ZM241385 and A2B antagonist PSB603 prevented the effect of inosine. The effect of inosine was mimicked by the adenosine receptor agonist NECA and the A2B receptor agonist BAY60-6583. The inhibition of SA by inosine was not observed in the presence of the BK antagonist, iberiotoxin, but persisted in the presence of KATP and SK antagonists. These findings demonstrate that inosine acts via an A2B receptor-mediated pathway that impinges on specific potassium channel effectors.

Methylxanthines enhance the effects of cocoa flavanols on cardiovascular function: randomized, double-masked controlled studies.

BACKGROUND: Cocoa flavanol intake, especially that of (-)-epicatechin, has been linked to beneficial effects on human cardiovascular function. However, cocoa also contains the methylxanthines theobromine and caffeine, which may also affect vascular function. OBJECTIVE: We sought to determine whether an interaction between cocoa flavanols and methylxanthines exists that influences cocoa flavanol-dependent vascular effects. DESIGN: Test drinks that contained various amounts of cocoa flavanols (0-820 mg) and methylxanthines (0-220 mg), either together or individually, were consumed by healthy volunteers (n = 47) in 4 different clinical studies-3 with a randomized, double-masked crossover design and 1 with 4 parallel crossover studies. Vascular status was assessed by measuring flow-mediated vasodilation (FMD), brachial pulse wave velocity (bPWV), circulating angiogenic cells (CACs), and blood pressure before and 2 h after the ingestion of test drinks. RESULTS: Although cocoa flavanol intake increased FMD 2 h after intake, the consumption of cocoa flavanols with methylxanthines resulted in a greater enhancement of FMD. Methylxanthine intake alone did not result in statistically significant changes in FMD. Cocoa flavanol ingestion alone decreased bPWV and diastolic blood pressure and increased CACs. Each of these changes was more pronounced when cocoa flavanols and methylxanthines were ingested together. It is important to note that the area under the curve of the plasma concentration of (-)-epicatechin metabolites over time was higher after the co-ingestion of cocoa flavanols and methylxanthines than after the intake of cocoa flavanols alone. Similar results were obtained when pure (-)-epicatechin and the methylxanthines theobromine and caffeine were consumed together. CONCLUSION: A substantial interaction between cocoa flavanols and methylxanthines exists at the level of absorption, in which the methylxanthines mediate an increased plasma concentration of (-)-epicatechin metabolites that coincides with enhanced vascular effects commonly ascribed to cocoa flavanol intake. This trial was registered at clinicaltrials.gov as NCT02149238.

Methotrexate intolerance in the treatment of rheumatoid arthritis (RA): effect of adding caffeine to the management regimen.

The aim of this study was to investigate the effect of caffeine on the symptoms of methotrexate (MTX) intolerance in patients with RA. The follow-up patients with RA seen over a period of 11 months were included in this work. The degree of MTX intolerance, if present, was classified as 'moderate' and 'severe'. Those with intolerance were advised caffeine (coffee or dark chocolate) synchronised with the MTX dose. The effect was assessed as 'very good', 'good' or 'none'. Among 855 patients seen during this period, 313 (36.6 %) did not have any MTX intolerance, 542 (63.4 %) patients had some degree of MTX intolerance, 422 (77.8 %; 49.3 % of the total patients) had 'minimal' intolerance not requiring any intervention. The remaining 120 (22.1 %) of the 542 (14 % of the total 855) patients had 'moderate' or 'severe' MTX intolerance. Among these, 55 % had complete relief of symptoms and were able to continue taking the advised dose of MTX; 13.3 % had partial improvement and continued taking MTX but only with antiemetics; 7.5 % were minimally better but were somehow managing; 10 % were complete caffeine failure without any relief; 14.2 % did not like caffeine (coffee or dark chocolate) and did not want to take it. Caffeine relieved the symptoms of MTX intolerance in 55 % and partial relief in 13 % of the patients. A significant number of patients did not like to take caffeine (coffee or dark chocolate). It is of note that northern part of India is primarily a tea-drinking population where coffee is not a favourite drink.

Xanthines and Phosphodiesterase Inhibitors.

Theophylline is an orally acting xanthine that has been used since 1937 for the treatment of respiratory diseases including asthma and chronic obstructive pulmonary disease (COPD). However, in most treatment guidelines, xanthines have now been consigned to third-line therapy because of their narrow therapeutic window and propensity for drug-drug interactions. However, lower than conventional doses of theophylline considered to be bronchodilator are now known to have anti-inflammatory actions of relevance to the treatment of respiratory disease. The molecular mechanism(s) of action of theophylline are not well understood, but several potential targets have been suggested including non-selective inhibition of phosphodiesterases (PDE), inhibition of phosphoinositide 3-kinase, adenosine receptor antagonism and increased activity of certain histone deacetylases. Although theophylline has a narrow therapeutic window, other xanthines are in clinical use that are claimed to have a better tolerability such as doxofylline and bamifylline. Nonetheless, xanthines still play an important role in the treatment of asthma and COPD as they can show clinical benefit in patients who are refractory to glucocorticosteroid therapy, and withdrawal of xanthines from patients causes worsening of disease, even in patients taking concomitant glucocorticosteroids.More recently the orally active selective PDE4 inhibitor, roflumilast, has been introduced into clinical practice for the treatment of severe COPD on top of gold standard treatment. This drug has been shown to improve lung function in patients with severe COPD and to reduce exacerbations, but is dose limited by a range side effect, particularly gastrointestinal side effects.

Scaffold-hopping from xanthines to tricyclic guanines: A case study of dipeptidyl peptidase 4 (DPP4) inhibitors.

Molecular modeling of unbound tricyclic guanine scaffolds indicated that they can serve as effective bioisosteric replacements of xanthines. This notion was further confirmed by a combination of X-ray crystallography and SAR studies, indicating that tricyclic guanine DPP4 inhibitors mimic the binding mode of xanthine inhibitors, exemplified by linagliptin. Realization of the bioisosteric relationship between these scaffolds potentially will lead to a wider application of cyclic guanines as xanthine replacements in drug discovery programs for a variety of biological targets. Newly designed DPP4 inhibitors achieved sub-nanomolar potency range and demonstrated oral activity in vivo in mouse glucose tolerance test.