Fernanda A. Rosa

Identification of the Plant Steroid α-Spinasterol as a Novel Transient Receptor Potential Vanilloid 1 Antagonist with Antinociceptive Properties

The transient receptor potential vanilloid 1 (TRPV1) receptor is relevant to the perception of noxious information and has been studied as a therapeutic target for the development of new analgesics. The goal of this study was to perform in vivo and in vitro screens to identify novel, efficacious, and safe TRPV1 antagonists isolated from leaves of the medicinal plant Vernonia tweedieana Baker. All of the fractions and the hydroalcoholic extract produced antinociception in mice during the capsaicin test, but the dichloromethane fraction also had antioedematogenic effect. Among the compounds isolated from the dichloromethane fraction, only α-spinasterol reduced the nociception and edema induced by capsaicin injection. Moreover, α-spinasterol demonstrated good oral absorption and high penetration into the brain and spinal cord of mice. α-Spinasterol was able to displace [3H]resiniferatoxin binding and diminish calcium influx mediated by capsaicin. Oral administration of the dichloromethane fraction and α-spinasterol also produced antinociceptive effect in the noxious heat-induced nociception test; however, they did not change the mechanical threshold of naive mice. The treatment with α-spinasterol did not produce antinociceptive effect in mice systemically pretreated with resiniferatoxin. In addition, α-spinasterol and the dichloromethane fraction reduced the edema, mechanical, and heat hyperalgesia elicited by complete Freunds adjuvant paw injection. The dichloromethane fraction and α-spinasterol did not affect body temperature or locomotor activity. In conclusion, α-spinasterol is a novel efficacious and safe antagonist of the TRPV1 receptor with antinociceptive effect.

Ultrasound promoted synthesis of 2-imidazolines in water: a greener approach toward monoamine oxidase inhibitors.

A series of sixteen 2-substituted-2-imidazolines (where the substituent R=Ph, Me-4-Ph; MeO-4-Ph; (MeO)(2)-3,4-Ph; (MeO)(3)-3,4,5-Ph; Ph-4-O-C(O)-Ph; Cl-4-Ph; Cl-2-Ph; Cl(2)-2,4-Ph; NO(2)-4-Ph; NO(2)-3-Ph; Naphth-2-yl; Fur-2-yl; Benzofur-2-yl; Pyridin-2-yl; Quinolin-2-yl) has been synthesized from the reaction of the substituted-aldehydes and ethylenediamine by ultrasound irradiation with NBS in an aqueous medium in high yields (80-99%). The 2-imidazoline ability to inhibit the activity of the A and B isoforms of monoamine oxidase (MAO) was investigated and some of them showed potent and selective MAO inhibitory activity especially for the MAO-B isoform and could become promising candidates for future development.

Spider peptide Phα1β induces analgesic effect in a model of cancer pain

The marine snail peptide ziconotide (ω‐conotoxin MVIIA) is used as an analgesic in cancer patients refractory to opioids, but may induce severe adverse effects. Animal venoms represent a rich source of novel drugs, so we investigated the analgesic effects and the side‐effects of spider peptide Phα1β in a model of cancer pain in mice with or without tolerance to morphine analgesia. Cancer pain was induced by the inoculation of melanoma B16‐F10 cells into the hind paw of C57BL/6 mice. After 14 days, painful hypersensitivity was detected and Phα1β or ω‐conotoxin MVIIA (10–100 pmol/site) was intrathecally injected to evaluate the development of antinociception and side‐effects in control and morphine‐tolerant mice. The treatment with Phα1β or ω‐conotoxin MVIIA fully reversed cancer‐related painful hypersensitivity, with long‐lasting results, at effective doses 50% of 48 (32–72) or 33 (21–53) pmol/site, respectively. Phα1β produced only mild adverse effects, whereas ω‐conotoxin MVIIA induced dose‐related side‐effects in mice at analgesic doses (estimated toxic dose 50% of 30 pmol/site). In addition, we observed that Phα1β was capable of controlling cancer‐related pain even in mice tolerant to morphine antinociception (100% of inhibition) and was able to partially restore morphine analgesia in such animals (56 ± 5% of inhibition). In this study, Phα1β was as efficacious as ω‐conotoxin MVIIA in inducing analgesia in a model of cancer pain without producing severe adverse effects or losing efficacy in opioid‐tolerant mice, indicating that Phα1β has a good profile for the treatment of cancer pain in patients.

Gallic acid functions as a TRPA1 antagonist with relevant antinociceptive and antiedematogenic effects in mice

The transient receptor potential ankyrin 1 (TRPA1) has been identified as a relevant target for the development of novel analgesics. Gallic acid (GA) is a polyphenolic compound commonly found in green tea and various berries and possesses a wide range of biological activities. The goal of this study was to identify GA as a TRPA1 antagonist and observe its antinociceptive effects in different pain models. First, we evaluated the ability of GA to affect cinnamaldehyde-induced calcium influx. Then, we observed the antinociceptive and antiedematogenic effects of GA (3–100 mg/kg) oral administration after the intraplantar (i.pl.) injection of TRPA1 agonists (allyl isothiocyanate, cinnamaldehyde, or hydrogen peroxide—H2O2) in either an inflammatory pain model (carrageenan i.pl. injection) or a neuropathic pain model (chronic constriction injury) in male Swiss mice (25–35 g). GA reduced the calcium influx mediated by TRPA1 activation. Moreover, the oral administration of GA decreased the spontaneous nociception triggered by allyl isothiocyanate, cinnamaldehyde, and H2O2. Carrageenan-induced allodynia and edema were largely reduced by the pretreatment with GA. Moreover, the administration of GA was also capable of decreasing cold and mechanical allodynia in a neuropathic pain model. Finally, GA was absorbed after oral administration and did not produce any detectable side effects. In conclusion, we found that GA is a TRPA1 antagonist with antinociceptive properties in relevant models of clinical pain without detectable side effects, which makes it a good candidate for the treatment of painful conditions.

2-methyl-7-substituted pyrazolo[1,5-a]pyrimidines: highly regioselective synthesis and bromination

The reaction of 3-amino-5-methyl-1H-pyrazole with 1,1,1-trichloro-4-alkoxy-3-alken-2-ones [CCl3C(O)CH=C(R1)OR, where R1/R = H/Me, Me/Et, Et/Me, Pr/Et, Bu/Me, iso-Bu/Me] or β-dimethylaminovinyl ketones [R2C(O)CH=CHNMe2, where R2 = Ph, Ph-4-Me, Ph-4-F, Ph-4-Cl, Ph-4-Br, Ph-4-NO2, fur-2-yl, thien-2-yl, pyrrol-2-yl, pyrid-2-yl], in acetic acid under reflux for 16 hours, furnished highly regioselective the halomethylated pyrazolo[1,5-a]pyrimidines and aryl[heteroaryl]pyrazolo[1,5-a]pyrimidines, respectively. A protocol for the bromination reaction at the 3-position pyrazolo[1,5-a]pyrimidines also was investigated.

A novel, potent, oral active and safe antinociceptive pyrazole targeting kappa opioid receptors

Pyrazole compounds are an intriguing class of compounds with potential analgesic activity; however, their mechanism of action remains unknown. Thus, the goal of this study was to explore the antinociceptive potential, safety and mechanism of action of novel 1-pyrazole methyl ester derivatives, which were designed by molecular simplification, using in vivo and in vitro methods in mice. First, tree 1-pyrazole methyl ester derivatives (DMPE, MPFE, and MPCIE) were tested in the capsaicin test and all presented antinociceptive effect; however the MPClE (methyl 5-trichloromethyl-3-methyl-1H-pyrazole-1-carboxylate) was the most effective. Thus, we selected this compound to assess the effects and mechanisms in subsequent pain models. MPCIE produced antinociception when administered by oral, intraperitoneal, intrathecal and intraplantar routes and was effective in the capsaicin and the acetic acid-induced nociception tests. Moreover, this compound reduced the hyperalgesia in diverse clinically-relevant pain models, including postoperative, inflammatory, and neuropathic nociception in mice. The antinociception produced by orally administered MPClE was mediated by κ-opioid receptors, since these effects were prevented by systemically pre-treatment with naloxone and the κ-opioid receptor antagonist nor-binaltorphimine. Moreover, MPCIE prevented binding of the κ-opioid ligand [(3)H]-CI-977 in vitro (IC₅₀ of 0.68 (0.32-1.4) μM), but not the TRPV1 ([(3)H]-resiniferatoxin) or the α₂-adrenoreceptor ([(3)H]-idazoxan) binding. Regarding the drug-induced side effects, oral administration of MPClE did not produce sedation, constipation or motor impairment at its active dose. In addition, MPCIE was readily absorbed after oral administration. Taken together, these results demonstrate that MPClE is a novel, potent, orally active and safe analgesic drug that targets κ-opioid receptors.

PET depolymerisation in supercritical ethanol catalysed by [Bmim][BF4]

Poly(ethylene terephthalate) (PET) was successfully depolymerised under supercritical ethanol. Robust conversion of 98 wt% from PET to diethylterephthalate (DET) was obtained by adding [Bmim][BF4], as catalyst, accompanied by reduction of depolymerization time from ca. 6 h to 45 min. DET formation in the depolymerization process was characterized by HPLC, 1H NMR, FTIR, TGA, DSC and SEM showing high purity and yield. The yields for different runs were determined by HPLC combined with interpolation from the standard/calibration curve. A 23 factorial design was employed to evaluate the effect of different inputs such as (i) reaction time after supercritical condition, (ii) volume of ionic liquid (VIL) and (iii) amount of PET in the yield of DET. By the analysis of variance (ANOVA), including F-test and P-values, it was found that reaction time and amount of PET inputs correspond, respectively, to 44% and 23% of the evaluated response. Another positive aspect showed by the factorial design is that the amount of catalyst was not significant in the process, and the depolymerization can be conducted successfully since a small amount (this study used VIL ranging 0.15 to 0.35 mL) is present in the reaction media. The method proposed in this paper is advantageous over others, reported in the literature, due to the lower reaction time required for PET depolymerization and the higher DET yield.

Phα1β, a Peptide from the Venom of the Spider phoneutria Nigriventer Shows Antinociceptive Effects after Continuous Infusion in a Neuropathic Pain Model in Rats

BACKGROUND:Neuropathic pain is a severe painful pathology that is difficult to treat. One option for its management is the continuous intrathecal (i.t.) infusion of ziconotide (the Conus magnus peptide &ohgr;-conotoxin MVIIA), which, in addition to being effective, produces serious adverse effects at analgesic doses. Single i.t. administration of Ph&agr;1&bgr;, a peptide purified from the venom of the spider Phoneutria nigriventer, has antinociceptive effects with a greater therapeutic window than ziconotide in rodents. To further evaluate its analgesic potential, we investigated the antinociceptive and toxic effects of Ph&agr;1&bgr; after single or continuous i.t. infusion in a rat model of neuropathic pain. METHODS:Adult male Wistar rats (200–300 g) bred in-house were used. Chronic constriction injury (CCI) of the sciatic nerve was used as the neuropathic pain model. Nociception was assessed by detecting mechanical hyperalgesia, considering a significant reduction in 50% paw withdrawal threshold values after CCI compared with baseline values. First, we assessed the antinociceptive effect of a single i.t. injection of Ph&agr;1&bgr; (10, 30, or 100 pmol/site) in a model of neuropathic pain 8 days after nerve injury. In a different experiment, we delivered Ph&agr;1&bgr; (60 pmol/&mgr;L/h) or vehicle (phosphate-buffered saline, 1.0 &mgr;L/h) through continuous infusion using an osmotic pump by spinal catheterization for 7 days in rats submitted to nerve injury. Behavioral adverse effects were evaluated after single or continuous Ph&agr;1&bgr; i.t. administration, and histopathological analysis of spinal cord, brainstem, and encephalon was performed after continuous Ph&agr;1&bgr; i.t. injection. RESULTS:We observed that CCI of the sciatic nerve but not sham surgery caused intense (reduction of approximately 2.5 times in mechanical withdrawal threshold) and persistent (up to 14 days) nociception in rats. The single i.t. injection of Ph&agr;1&bgr; (30 or 100 pmol/site) reduced neuropathic nociception from 1 to 6 hours after administration, without showing detectable side effects. Similarly, the continuous infusion of Ph&agr;1&bgr; (60 pmol/&mgr;L/h for 7 days) was also able to reverse nerve injury–induced nociception from 1 to 7 days, but did not cause either behavioral side effects or histopathological changes in the central nervous system. CONCLUSIONS:Thus, we have shown for the first time that the continuous i.t. delivery of Ph&agr;1&bgr; produces analgesia disconnected from toxicity in a relevant model of neuropathic pain, indicating that it is an effective and safe drug with a great potential to treat pain.

Theoretical and experimental investigation of the polyeletrophilic β-enamino diketone: straightforward and highly regioselective synthesis of 1,4,5-trisubstituted pyrazoles and pyrazolo[3,4-d]pyridazinones

Obtaining a new precursor enamino diketone with five electrophilic centers is reported, along with theoretical and experimental studies of its reactivity against mono- or dinucleophiles. The Fukui function showed that the β-carbon is the most electrophilic center, followed by the carbonyl ketone and the carbonyl ester, respectively. The reaction of enamino diketone with aniline and hydrazines allowed for the synthesis of a new enamino diketone and 1,4-disubstituted pyrazoles-5-carboxylates, respectively. The regiochemistry and mechanism of syntheses of 1,4-disubstituted pyrazoles-5-carboxylates were determined from reaction monitoring by ESI-MS, NMR analysis and crystallographic data, and fully agreed with the theoretical results. The versatility and efficiency of the enamino diketone was demonstrated by the reaction with hydrazines furnishing multi-functionalized pyrazoles and pyrazolo[3,4-d]pyridazinone derivatives with high regioselectivity.

Antinociceptive and antiedematogenic effect of pecan (Carya illinoensis) nut shell extract in mice: a possible beneficial use for a by-product of the nut industry

Abstract Background: Interest in pecan (Carya illinoensis) nut shells, a by-product of the nut industry, has increased due to its anti-inflammatory and antioxidant activities. The goal of this study was to evaluate the antinociceptive and antiedematogenic activity and the mechanisms of the pecan shell aqueous extract (AE). Methods: First, we performed fingerprinting of C. illinoensis AE. The antinociceptive and antiedematogenic effects of AE intragastric (i.g.) administration in mice (male Swiss mice 20–30 g) were evaluated using the acetic acid test or after subcutaneous (s.c.) paw injection of diverse transient receptor potential ankyrin 1 (TRPA1) agonists, including hydrogen peroxide (H2O2), allyl isothiocyanate, or cinnamaldehyde. We also observed AE antinociceptive and antiedematogenic effects after carrageenan s.c. paw injection and measured H2O2 production. Moreover, we observed the development of adverse effects after AE i.g. treatment. Results: The high-performance liquid chromatography fingerprinting of AE showed the presence of rutin. AE or rutin i.g. treatment produced antinociception in the acetic acid test and reduced the nociception and edema mediated by H2O2 s.c. hind paw injection or nociception induced by other TRPA1 agonists. Moreover, AE or rutin reduced the hyperalgesia, edema, and H2O2 production induced by carrageenan s.c. paw injection. No motor, gastric, or toxicological alterations were observed after AE administration. Conclusions: Collectively, the present results show that AE and its constituent rutin produced antinociceptive and antiedematogenic action in models of acute and persistent inflammatory nociception and it seems to be related to the inhibition of TRPA1 receptor activation.