Safety Assessment of Methylxanthines as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) assessed the safety of three methylxanthines, Caffeine, Theobromine, and Theophylline, as used in cosmetics. All of these ingredients are reported to function as skin-conditioning agents in cosmetic products. The Panel reviewed the data relevant to the safety of these ingredients and concluded that Caffeine, Theobromine, and Theophylline are safe in cosmetics in the present practices of use and concentration described in this safety assessment.

Pre-clinical and cellular toxicity evaluation of 7-methylxanthine: an investigational drug for the treatment of myopia.

The present study entails the toxicity evaluation of 7-methyl xanthine (7-MX), first of its kind molecule found effective in phase II clinical trials for the treatment of myopia, in comparison to other clinically used xanthines i.e., caffeine and theobromine. For acute toxicity evaluation, 7-MX was administered orally in two rodent species (rat and mice) at the doses of 300 mg/kg and 2000 mg/kg and for repeated dose 28-d oral toxicity, at 250, 500, and 1000 mg/kg in rats. Further, cellular toxicity was evaluated in normal breast epithelial (fR2), rat brain C6 glioma (C6 glioma) and human colorectal (Caco-2) cell lines. Also, the cell uptake assay to determine the intestinal permeability of drug was performed in Caco-2 cells. In acute toxicity, 7-MX treatment showed no mortality and toxicity, whereas 66.6% (mice) and 33.3% (rat) mortality was observed in both caffeine and theobromine treatment groups. In repeated dose 28-d oral toxicity, 7-MX treatment was found to have no-observed-adverse-effect level up to the dose of 1000 mg/kg in the present study conducted as per OECD guidelines 407. Also, very high IC50 value of 305.5 and 721 µg/mL was observed for 7-MX in fR2 and C6 glioma cells, respectively. In Caco-2 cells, linear bioavailability and high % cell viability was observed. Thus, 7-MX may be classified as Globally Harmonized System (GHS) category 5 drug with LD50 >2000-5000 mg/kg. Also, the repeated dose 28-d oral toxicity study demonstrated 7-MX to be nontoxic in nature, with cell line toxicity results further endorsing its nontoxic nature.

Placentation-related processes in a human first-trimester extravillous trophoblast cell line (HTR-8/SVneo cells) are affected by several xenobiotics.

Our aim was to investigate the effect of some xenobiotics on placentation-related processes in an extravillous trophoblastic cell line (HTR-8/SVneo cells). Amphetamine, MDMA, theophylline, and fluoxetine, but not nicotine, cocaine, and caffeine, had a negative effect on cell proliferation rates, culture growth, viability, or migratory capacity. These compounds have a detrimental effect in placentation-related processes of HTR-8/SVneo cells.

Zebrafish Embryo as an In Vivo Model for Behavioral and Pharmacological Characterization of Methylxanthine Drugs.

Zebrafish embryo is emerging as an important tool for behavior analysis as well as toxicity testing. In this study, we compared the effect of nine different methylxanthine drugs using zebrafish embryo as a model. We performed behavioral analysis, biochemical assay and Fish Embryo Toxicity (FET) test in zebrafish embryos after treatment with methylxanthines. Each drug appeared to behave in different ways and showed a distinct pattern of results. Embryos treated with seven out of nine methylxanthines exhibited epileptic-like pattern of movements, the severity of which varied with drugs and doses used. Cyclic AMP measurement showed that, despite of a significant increase in cAMP with some compounds, it was unrelated to the observed movement behavior changes. FET test showed a different pattern of toxicity with different methylxanthines. Each drug could be distinguished from the other based on its effect on mortality, morphological defects and teratogenic effects. In addition, there was a strong positive correlation between the toxic doses (TC50) calculated in zebrafish embryos and lethal doses (LD50) in rodents obtained from TOXNET database. Taken together, all these findings elucidate the potentiality of zebrafish embryos as an in vivo model for behavioral and toxicity testing of methylxanthines and other related compounds.

Synthesis and Analgesic Activity of Annelated Xanthine Derivatives in Experimental Models in Rodents.

A series of annelated derivatives of xanthine were synthesized and assayed as potential analgesic agents. All synthesized xanthine derivatives were tested in the writhing test and hot-plate test. The pharmacological assays demonstrated that all the compounds prepared, without exception, displayed a significant activity in the mouse writhing assay. The analgesic action of the most active compounds, expressed as ED50 was found to be 1.4-4.3 times more potent than that of acetylsalicylic acid used as the reference compound. However, only some of the compounds demonstrated analgesic activity in the hot-plate test. The analgesic effect of some compounds is probably related to their agonistic, antagonistic, or partial agonistic activity at the adenosine receptors.

Efficacy and toxicity of the DPCPX nanoconjugate drug study for the treatment of spinal cord injury in rats.

Effects of the Adenosine A1 blockade using 8-cyclopentyl-1,3-diprophyxanthine (DPCPX) nanoconjugate on inducing recovery of the hemidiaphragm paralyzed by hemisection have been thoroughly examined previously; however, the toxicology of DPCPX nanoconjugate remains unknown. This research study investigates the therapeutic efficacy and toxicology of the nanoconjugate DPCPX in the cervical spinal cord injury (SCI) rat model. We hypothesized that a single injection of nanoconjugate DPCPX in the paralyzed left hemidiaphragm (LDH) of hemisected rats at the 2nd cervical segment (C2Hx) would lead to the long-term recovery of LDH while showing minimal toxicity. Adult male rats underwent left C2Hx surgery and the diaphragms' baseline electromyography (EMG). Subsequently, rats were randomized into a control group and four treated subgroups. Three subgroups received a single intradiaphragmatic dose of either 0.09, 0.15, or 0.27 µg/kg, and one subgroup received 0.1 mg/kg of native DPCPX two times per day intravenously (i.v.) for 3 days (total 0.6 mg/kg). Rats were monitored for a total of 56 days. Compared with control, the treatment with nanoconjugate DPCPX at 0.09 µg/kg, 0.15 µg/kg, and 0.27 µg/kg doses elicited significant recovery of paralyzed LDH (i.e., 67% recovery at 8 wk) (P < 0.05). DPCPX nanoconjugate-treated rats had significant weight loss for first 2 wk but recovered significantly by day 56 (P < 0.05). The levels of gold in the blood and body tissues were below the recommended levels. No sign of weakness, histology of tissue damage, or organ abnormality was observed. A dose of DPCPX nanoconjugate can induce long-term diaphragm recovery after SCI without observed toxicity.NEW & NOTEWORTHY The intradiaphragmatic administration of nanoconjugate is safe and has the promise to significantly reduce the therapeutic dosage for the treatment and achieve long-term and possibly permanent recovery in respiratory muscle dysfunction after SCI. No toxicity of nanoconjugate was found in any of the experimental animals.

Methylxanthines, seizures, and excitotoxicity.

Clinical evidence, in particular the wide use of theophylline as a bronchodilator, suggests that methylxanthines can cause seizures in patients without known underlying epilepsy. Theophylline is also known to be an added risk factor for seizure exacerbation in patients with epilepsy. The proconvulsant activity of methylxanthines can best be explained by their antagonizing the brain's own anticonvulsant adenosine. Recent evidence suggests that adenosine dysfunction is a pathological hallmark of epilepsy contributing to seizure generation and seizure spread. Conversely, adenosine augmentation therapies are effective in seizure suppression and prevention, whereas adenosine receptor antagonists such as methylxanthines generally exacerbate seizures. The impact of the methylxanthines caffeine and theophylline on seizures and excitotoxicity depends on timing, dose, and acute versus chronic use. New findings suggest a role of free radicals in theophylline-induced seizures, and adenosine-independent mechanisms for seizure generation have been proposed.

The effects of prenatal exposure to methylxanthines.

This review discusses epidemiology and laboratory studies on the effects of prenatal methylxanthine administration on some systems developing organisms. They are mainly absorbed from coffee, tea and cocoa products such as cola beverages and chocolate bars. Prenatal methylxanthine exposure can induce several unfavourables changes in the developing organism, which are persistent even in later phases of life. Based on results obtained from animal studies, the effect on embryogenesis is not only poorly understood but also controversial. It is therefore important to study interspecies differences as results may differ depending on animals used and administration methods.

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.

Differential effects of hydrogen peroxide on indices of endothelial cell function.

The responses of pig aortic endothelial cells to sublethal doses of potentially toxic stimuli were investigated by monitoring K+ efflux, prostaglandin production, and the release of cytoplasmic purines. Xanthine plus xanthine oxidase reversibly stimulated these three parameters of endothelial cell function at doses that were not cytotoxic, as measured by chromium release, adenine uptake, and vital dye exclusion. The effects of xanthine plus xanthine oxidase were inhibited by catalase but not by superoxide dismutase, suggesting that H2O2 was responsible. Reagent H2O2 also reversibly stimulated K+ efflux, prostaglandin production, and the release of purines. The threshold concentration of H2O2 for these effects was approximately 10 microM, which was at least 30-fold lower than that which caused cytotoxicity. In addition to the direct effect of H2O2 in stimulating prostaglandin production (PGI2 and PGE2), prior exposure of endothelial cells to lower doses of H2O2 (less than 0.1 microM) at high oxygen tension inhibited the subsequent stimulation of prostaglandin production by ATP, A23187, and H2O2 itself. We conclude that H2O2 has substantial effects on endothelial physiology at doses up to 3,000-fold lower than those which induce cytotoxicity.

Synthesis and biological evaluation of novel xanthine derivatives as potential apoptotic antitumor agents.

A series of novel xanthine/NO donor hybrids containing 1,3,8-trisubstituted or 1,8-disubstituted xanthine derivatives were designed and synthesized. The synthesized compounds were tested in a cell viability assay using human mammary gland epithelial cell line (MCF-10A) where all the compounds exhibited no cytotoxic effects and more than 90% cell viability at a concentration of 50 µM. The oxime containing compounds 7a-b and 17-24 were more active as antiproliferative agents than their non-oxime congeners 6a-b and 9-16. Hydroxyimino-phenethyl scaffold compounds 17-24 were more active than the hydroxyimino-ethyl phenyl acetamide 7a-b derivatives. Compounds 18-20 and 22-24 exhibited inhibition of EGFR with IC50 ranging from 0.32 to 2.88 µM. Compounds 18-20 and 22-24 increased the level of active caspase 3 by 4-8 folds, compared to the control cells in Panc-1 cell lines compared to doxorubicin as a reference drug. Compounds 18, 22 and 23 were the most caspase-3 inducers. Compounds 22 and 23 increased the levels of caspase-8 and 9 indicating activation of both intrinsic and extrinsic pathways and showed potent induction of Bax, down-regulation of Bcl-2 protein levels and over-expression of cytochrome c levels in Panc-1 human pancreas cancer cells. Compound 23 exhibited mainly cell cycle arrest at the Pre-G1 and G2/M phases in the cell cycle analysis of Panc-1 cell line. The drug likeness profiles of compounds 18-20 and 22-24 were predicted to have good to excellent drug likeness profiles specially compounds 18-20 and 23. Finally molecular docking study was performed at the EGFR active site to suggest thier possible binding mode. The hydroxyimino-phenethyl scaffold compounds 17-24 represent an interesting starting point to optimize their pharmacokinetics and pharmacodynamics profiles.

Cardioprotection by ecto-5'-nucleotidase (CD73) and A2B adenosine receptors.

BACKGROUND: Ecto-5'-nucleotidase (CD73)-dependent adenosine generation has been implicated in tissue protection during acute injury. Once generated, adenosine can activate cell-surface adenosine receptors (A1 AR, A2A AR, A2B AR, A3 AR). In the present study, we define the contribution of adenosine to cardioprotection by ischemic preconditioning. METHODS AND RESULTS: On the basis of observations of CD73 induction by ischemic preconditioning, we found that inhibition or targeted gene deletion of cd73 abolished infarct size-limiting effects. Moreover, 5'-nucleotidase treatment reconstituted cd73-/- mice and attenuated infarct sizes in wild-type mice. Transcriptional profiling of adenosine receptors suggested a contribution of A2B AR because it was selectively induced by ischemic preconditioning. Specifically, in situ ischemic preconditioning conferred cardioprotection in A1 AR-/-, A2A AR-/-, or A3 AR-/- mice but not in A2B AR-/- mice or in wild-type mice after inhibition of the A2B AR. Moreover, A2B AR agonist treatment significantly reduced infarct sizes after ischemia. CONCLUSIONS: Taken together, pharmacological and genetic evidence demonstrate the importance of CD73-dependent adenosine generation and signaling through A2B AR for cardioprotection by ischemic preconditioning and suggests 5'-nucleotidase or A2B AR agonists as therapy for myocardial ischemia.

8-(3-phenylpropyl)-1,3,7-triethylxanthine is a synthetic caffeine substitute with stronger metabolic modulator activity.

Caffeine is one of the most worldwide consumed methylxanthines. It is well-known for its thermogenic and cell metabolism modulating effects. Based on methylxanthines' chemical structure, 8-(3-phenylpropyl)-1,3,7-triethylxanthine (PTX) is a novel adenosine antagonist with higher receptor affinity than caffeine. Therefore, we hypothesized that PTX metabolic effects could be stronger than those of caffeine. For that purpose, murine 3T3-L1 cells were cultured in the presence of increasing doses of PTX or caffeine (0.1, 1, 10 and 100 µM) for 24 h. Cytotoxicity was evaluated by reduction of tetrazolium salt (MTT) and lactate dehydrogenase (LDH) release. Cell metabolites released to the culture medium were identified and quantified by proton nuclear magnetic resonance (1H NMR). Cellular oxidative profile was also evaluated. Our results showed that PTX displayed no signs of cytotoxicity at all studied concentrations. When compared with caffeine, PTX increased glucose, pyruvate, and glutamine consumption, as well as lactate, alanine, and acetate production. Additionally, PTX decreased protein oxidation, thus protecting against oxidative stress-induced damage. These results illustrate that PTX is a stronger and less cytotoxic caffeine substitute with potential applications as metabolic modulator and a good candidate for novel drug design.

Correlation between emetic effect of phosphodiesterase 4 inhibitors and their occupation of the high-affinity rolipram binding site in Suncus murinus brain.

We employed an ex vivo [(3)H]rolipram binding experiment to elucidate the mechanism of emetic activity of phosphodiesterase 4 inhibitors. In Suncus murinus (an insectivore used for evaluation of emesis), emetic potential as well as ability to occupy the high-affinity rolipram binding site in brain membrane fraction in vivo were determined for phosphodiesterase 4 inhibitors. In vitro, [(3)H]rolipram bound to the membrane fraction of S. murinus brain with high affinity and its value was comparable to that for rat brain (K(d)=3.6 nM and 3.5 nM, respectively). The test compounds included denbufylline, rolipram, piclamilast, CDP840 and KF19514, each of which possessed similar affinities for the rolipram binding sites in both S. murinus and rat brain. In S. murinus, these compounds induced emesis via intraperitoneal administration. Their ED(50) values were as follows: denbufylline (1.4 mg/kg), rolipram (0.16 mg/kg), piclamilast (1.8 mg/kg), CDP840 (20 mg/kg), and KF19514 (0.030 mg/kg). In addition, these compounds occupied the high-affinity rolipram binding site in vivo as detected by dose-dependent reduction in capacity of ex vivo [(3)H]rolipram binding in brain membrane fractions. A clear correlation was observed between dose required to induce emesis and that to occupy the high-affinity rolipram binding site for individual phosphodiesterase 4 inhibitors. We conclude that the emetic effect of phosphodiesterase 4 inhibitors is caused at least in part via binding to the high-affinity rolipram binding site in brain in vivo.

Selection against expression of the Escherichia coli gene gpt in hprt+ mouse teratocarcinoma and hybrid cells.

Thioxanthine is toxic for mammalian cells transformed by the dominant selectable marker gpt. It allowed us to select, in the presence of the endogenous hypoxanthine-guanine phosphoribosyltransferase gene, mutants that did not express gpt any more and also hybrid cells that had lost the chromosome carrying it. The gpt marker is thus dominant in negative as well as in positive selection, which makes it potentially very useful for genetic studies of mammalian cells.

Methylxanthines induce structural and functional alterations of the cardiac system in zebrafish embryos.

BACKGROUND: Zebrafish embryos are emerging as a model for pharmacological and toxicological studies. We used zebrafish embryos to study the general toxicity and cardiovascular effects of eight methylxanthines: aminophylline, caffeine, diprophylline, doxofylline, etophylline, 3-isobutyl-1-methylxanthine (IBMX), pentoxifylline and theophylline. METHODS: Microinjections of the eight methylxanthines were performed in 1-2 cell stage zebrafish embryos and the general toxicity and cardiovascular effects were analyzed at different time points. Embryotoxicity and teratogenicity were evaluated to understand the general toxicity of these compounds. Structural and functional alterations of the heart were evaluated to assess the cardiovascular effects. RESULTS: Our results showed different activity patterns of the methylxanthines drugs. Caffeine, IBMX, pentoxifylline and theophylline were highly embryotoxic and teratogenic; aminophylline, doxofylline and etophylline were embryotoxic and teratogenic only at higher doses, and diprophylline showed a minimal (<10%) embryotoxicity and teratogenicity. Most of these drugs induced structural alteration of the heart in 20-40% of the injected embryos with the maximum dose. This structural alteration was fatal with the embryos ultimately dying within 120 hpf. All the drugs induced a transient increase in heart rate at 48 hpf which returned to baseline within 96 hpf. This functional effect of methylxanthines showed similarity to the studies done in humans and other vertebrates. CONCLUSION: Our results indicate the potential toxicity and teratogenicity of different methylxanthines in the embryos during embryonic development, the most sensitive period of life. Although interspecies differences need to be considered before drawing any conclusion, our study elucidated that a single exposure of methylxanthines at therapeutic range could induce cardiac dysfunction besides causing embryotoxicity and teratogenicity. Of all the drugs, diprophylline appeared to be safer, with lower degree of embryotoxicity, teratogenicity and cardiac toxicity as compared to other methylxanthines.

Regulation of Expression of Hyperalgesic Priming by Estrogen Receptor α in the Rat.

Hyperalgesic priming, a sexually dimorphic model of transition to chronic pain, is expressed as prolongation of prostaglandin E2-induced hyperalgesia by the activation of an additional pathway including an autocrine mechanism at the plasma membrane. The autocrine mechanism involves the transport of cyclic adenosine monophosphate (AMP) to the extracellular space, and its conversion to AMP and adenosine, by ecto-5'phosphodiesterase and ecto-5'nucleotidase, respectively. The end product, adenosine, activates A1 receptors, producing delayed onset prolongation of prostaglandin E2 hyperalgesia. We tested the hypothesis that the previously reported, estrogen-dependent, sexual dimorphism observed in the induction of priming is present in the mechanisms involved in its expression, as a regulatory effect on ecto-5'nucleotidase by estrogen receptor α (EsRα), in female rats. In the primed paw AMP hyperalgesia was dependent on conversion to adenosine, being prevented by ecto-5'nucleotidase inhibitor α,ß-methyleneadenosine 5'-diphosphate sodium salt and A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. To investigate an interaction between EsRα and ecto-5'nucleotidase, we treated primed female rats with oligodeoxynucleotide antisense or mismatch against EsRα messenger RNA. Whereas in rats treated with antisense AMP-induced hyperalgesia was abolished, the A1 receptor agonist N6-cyclopentiladenosine still produced hyperalgesia. Thus, EsRα interacts with this autocrine pathway at the level of ecto-5'nucleotidase. These results demonstrate a sexually dimorphic mechanism for the expression of priming. PERSPECTIVE: This study presents evidence of an estrogen-dependent mechanism of expression of chronic pain in female rats, supporting the suggestion that differential targets must be considered when establishing protocols for the treatment of painful conditions in men and women.

Pre- and postjunctional effects of angiotensin II in hypertension due to adenosine receptor blockade.

Prejunctional facilitation of [3H]noradrenaline release induced by sympathetic nerve stimulation and postjunctional contractile effects of angiotensin II were studied in the mesenteric artery and vein of 1,3-dipropyl-8-sulfophenylxanthine (DPSPX)-hypertensive rats. Male Wistar rats received infusions of saline or DPSPX (90 microg/kg/h) i.p.. Blood pressure was determined by tail-cuff. The prejunctional effect of angiotensin II was similar in artery and vein preparations of control rats and was increased in DPSPX-hypertensive rats. In contrast, the contractile effect of angiotensin II was much more pronounced in the mesenteric vein than in the mesenteric artery of control rats and was markedly reduced in DPSPX-hypertensive rats. We conclude that (1) the increased prejunctional effect of angiotensin II may contribute to, while (2) the decreased contractile effect of angiotensin II may attenuate DPSPX-induced hypertension. This study also supports the hypothesis that pre- and postjunctional angiotensin II receptors are different.

Increased incidence of mammary tumors in rats after direct or transplacental exposure to 3-hydroxyxanthine.

Rats were exposed directly or transplacentally to the carcinogenic purine N-oxide 3-hydroxyxanthine (3-OH-X). Among female noninbred Wistar rats given 3-OH-X sc, beginning at suckling or weaning ages, there was a significantly greater proportion of mammary tumor bearers and a higher mean number of mammary tumors per rat than among controls. This effect was seen even at low doses producing no other neoplasms such as hepatic carcinomas or fibrosarcomas and fibromas at the site of injection. Noninbred Sprague-Dawley rats, treated transplacentally with 3-OH-X by ip injection into pregnant females, also also had a significantly greater incidence of mammary tumors than did controls.

3-Hydroxyxanthine: transplacental effects and ontogeny of related sulfate metabolism in rats and mice.

The ontogeny of sulfate metabolism related to the metabolic activation of the carcinogenic purine N-oxide 3-hydroxyxanthine (3-OH-X) was studied in noninbred Sprague-Dawley rats. Sulfotransferase activity toward 3-OH-X was detectable in most fetal livers near term at about 25% of adult values and increased slowly after birth. This activity was also present in placentas. Compared to 3-OH-X sulfotransferase, sulfotransferase activity toward p-nitrophenol was lower in fetal livers and was not detected in placentas. Sulfohydrolase activity toward 3'-phosphoadenosine-5'-phosphosulfate was higher in fetal and newborn livers and in placentas than in adult liver. In a parallel transplacential carcinogenicity assay, a low but significant percentage of male rats exposed as fetuses to multiple high doses of 3-OH-X developed single liver carcinomas. After the lowest transplacental dose, the incidence of degenerative kidney disease in old male offspring was significantly higher than that in controls. In an assay with mice, (C57BL/6 X BALB/c)F1 mice exposed transplacentally to 3-OH-X experienced significantly greater perinatal morality and fewer lung adenomas among the surviviors at 20 months of age than did the controls.