Fabio C. Amendoeira

Integrin αDβ2 Is Dynamically Expressed by Inflamed Macrophages and Alters the Natural History of Lethal Systemic Infections

The leukocyte integrins have critical roles in host defense and inflammatory tissue injury. We found that integrin αDβ2, a novel but largely uncharacterized member of this family, is restricted to subsets of macrophages and a small population of circulating leukocytes in wild-type mice in the absence of inflammatory challenge and is expressed in regulated fashion during cytokine-induced macrophage differentiation in vitro. αDβ2 is highly displayed on splenic red pulp macrophages and mediates their adhesion to local targets, identifying key functional activity. In response to challenge with Plasmodium berghei, a malarial pathogen that models systemic infection and inflammatory injury, new populations of αD+ macrophages evolved in the spleen and liver. Unexpectedly, targeted deletion of αD conferred a survival advantage in P. berghei infection over a 30-day observation period. Mechanistic studies demonstrated that the increased survival of αD−/− animals at these time points is not attributed to differences in magnitude of anemia or parasitemia or to alterations in splenic microanatomy, each of which is a key variable in the natural history of P. berghei infection, and indicated that an altered pattern of inflammatory cytokines may contribute to the difference in mortality. In contrast to the outcome in malarial challenge, death of αD−/− animals was accelerated in a model of Salmonella sepsis, demonstrating differential rather than stereotyped roles for αDβ2 in systemic infection. These studies identify previously unrecognized and unique activities of αDβ2, and macrophages that express it, in host defense and injury.

Inhibition of advanced glycation end products by aminoguanidine restores mast cell numbers and reactivity in alloxan-diabetic rats

Mast cell number and reactivity have been shown to be down-regulated under diabetic conditions. This study was undertaken in order to investigate the role of the advanced glycation end products in the reduction of mast cell number and reactivity in diabetic rats. The effect of aminoguanidine on mast cell apoptosis was also evaluated. Diabetes was induced by intravenous injection of alloxan into fasted rats and aminoguanidine was administered after 3 days of diabetes induction, once daily for 18 consecutive days. Mast cell apoptosis and levels of Bax, a pro-apoptotic member of Bcl-2 family, were evaluated by TUNEL and western blot, respectively. Diabetes led to increased levels of fructosamine and AGEs in the plasma, an effect prevented by aminoguanidine. Treatment with aminoguanidine restored mast cell numbers in the pleural cavity and in mesenteric tissue of diabetic rats. Aminoguanidine also significantly reversed the diabetes-induced reduction in histamine release, as measured by fluorescence, following activation with substance P or antigen in vitro. Increased apoptosis and levels of Bax in mast cells from diabetic rats were inhibited by aminoguanidine. In conclusion, our findings showed that aminoguanidine restored the number and reactivity of mast cells in diabetic rats, accompanied by suppression of apoptosis, evidencing that advanced glycation end product formation has a critical role in mast cell behavior of diabetic rats.

Analgesic and anti-inflammatory activity of the aqueous extract of Rheedia longifolia Planch & Triana

Rheedia longifolia Planch et Triana belongs to the Clusiaceae family. This plant is widely distributed in Brazil, but its chemical and pharmacological properties have not yet been studied. We report here that leaves aqueous extract of R. longifolia (LAE) shows analgesic and anti-inflammatory effects. Oral or intraperitoneal administration of this extract dose-dependently inhibited the abdominal constrictions induced by acetic acid in mice. The analgesic effect and the duration of action were similar to those observed with sodium diclofenac, a classical non-steroidal analgesic. In addition to the effect seen in the abdominal constriction model, LAE was also able to inhibit the hyperalgesia induced by lipopolysaccharide from gram-negative bacteria (LPS) in rats. We also found that R. longifolia LAE inhibited an inflammatory reaction induced by LPS in the pleural cavity of mice. Acute toxicity was evaluated in mice treated with the extract for seven days with 50 mg/kg/day. Neither death, nor alterations in weight, blood leukocyte counts or hematocrit were noted. Our results suggest that aqueous extract from R. longifolia leaves has analgesic and anti-inflammatory activity with minimal toxicity and are therefore endowed with a potential for pharmacological control of pain and inflammation.

Antinociceptive Activity of Stephanolepis hispidus Skin Aqueous Extract Depends Partly on Opioid System Activation

Stephanolepis hispidus is one of the most common filefish species in Brazil. Its skin is traditionally used as a complementary treatment for inflammatory disorders. However, there are very few studies on chemical and pharmacological properties using the skin of this fish. This study was undertaken in order to investigate the effect of aqueous crude extract of S. hispidus skin (SAE) in different nociception models. Here, we report that intraperitoneal administration of SAE inhibited the abdominal constrictions induced by acetic acid in mice. In addition to the effect seen in the abdominal constriction model, SAE was also able to inhibit the hyperalgesia induced by carrageenan and prostaglandin E2 (PGE2) in mice. This potent antinociceptive effect was observed in the hot plate model too, but not in tail-flick test. Naloxone, an opioid receptor antagonist, was able to block the antinociceptive effect of SAE in the abdominal constriction and hot plate models. In addition, SAE did not present cytotoxic or genotoxic effect in human peripheral blood cells. Our results suggest that aqueous crude extract from S. hispidus skin has antinociceptive activity in close relationship with the partial activation of opioid receptors in the nervous system. Moreover, aqueous crude extract from S. hispidus skin does not present toxicity and is therefore endowed with the potential for pharmacological control of pain.

Chemical Profile and Antinociceptive Efficacy of Rheedia longifolia Leaf Extract

Different species of the family Clusiaceae, including Rheedia longifolia, are used in folk medicine to treat inflammatory diseases. This family is largely distributed in tropical and subtropical areas of Brazil, but their chemical and pharmacological properties have been the subject of a few studies. In previous studies, we found that the aqueous extract from R. longifolia leaves presented important anti-inflammatory and analgesic activity. We investigated the chemical profile of R. longifolia and characterized the pharmacological effect of different chemically identified fractions in pharmacological models of neurogenic and inflammatory nociception. The pharmacological tests showed that oral treatment with aqueous crude extract and fractions of methanol extract of R. longifolia leaf induced a significant antinociceptive effect using von Frey filaments. In addition, the most polar fractions presented antinociceptive activity in a neurogenic model of nociception (capsaicin model). The chromatographic analysis indicated the presence of bisflavonoids in the fractions obtained from the methanol extract. These results suggest that bisflavonoids found in methanol-extracted fractions are involved in the inhibition of inflammatory and neurogenic nociception. It is important that the R. longifolia aqueous extract treatment inhibited ulcer formation induced by indomethacin, suggesting an anti-ulcerogenic activity closely associated with its analgesic effect.

Post-marketing surveillance of generic amoxicillin using a microbiological assay and pharmacokinetic approach in rats

Generic medicines were developed to increase population access to health treatment, to reduce costs and to allow drugs with the same outcomes to be purchased at lower prices. They are therapeutically equivalent to their brand-name counterparts and are interchangeable with them. However, the acceptance of generic medicines by physicians and general consumers is often affected by distrust related to quality and efficacy. In this study three different brands of generic amoxicillin were tested. The results showed that two of them were indistinguishable from the innovator in terms of microbiological potency; however, generic B was unable to reach the Brazilian Pharmacopoeia specifications for potency limits. In contrast, generic B was bioequivalent to the innovator amoxicillin in pharmacokinetic assessment and, surprisingly, generic A, which was approved in the microbiological potency assay, lacked pharmacokinetic equivalence compared with the innovator. Both tests, when used singly, may not be effective at detecting quality deviations in antimicrobial medicines, which indicates that pharmacokinetic tests in rats in association with microbiological potency assays are a valuable tool for post-marketing surveillance of generic antibiotics.

Development of a HPLC/MS/MS methodology for determining 3-O-methyldopa in human plasma and its application in a bioequivalence study

A simple, sensitive and specific HPLC/MS/MS methodology was developed and it was validated for determining 3-O-methyldopa, the major metabolite of dopamine, in human plasma. The separation was achieved on Atlantis T3 C18 analytical column (5 μm; 150 x 4.6 mm i.d.) using a mobile phase consisted of a solution of water and methanol (85:15, v/v) and containing formic acid 0.05%. The extraction from the analyte and the internal standard sample was performed using a simple protein plasma precipitation with perchloric acid. The detection was conducted on a triple quadrupole tandem mass spectrometer with a positive multiple reaction monitoring mode (MRM). The monitored fragmentation transitions were m/z 212.0 - m/z 166.0 for 3-O-methyldopa and m/z 227.10 - m/z 181.0 for carbidopa (internal standard). The calibration curves were linear in the range of 50–4000 ng/mL for 3-O-methyldopa. The methodology presented a good precision and accuracy in accordance to the criteria for biomedical analysis. And it was successfully applied to the bioequivalence study of two formulations levodopa + benserazide (200 + 50 mg) in plasma samples from healthy human volunteers, following the ANVISA guidelines.