Benefits of flavonoid and metronidazole use after excisional hemorrhoidectomy: a randomized double-blind clinical trial.

BACKGROUND: Excisional hemorrhoidectomy remains the most effective treatment for a significant group of patients with hemorrhoids, despite the potential for postoperative pain. The purpose of this study was to evaluate the effects of flavonoid and metronidazole use in the postoperative period on patients undergoing excisional hemorrhoidectomy. METHODS: A double-blind randomized clinical study was performed. Sixty-eight patients underwent excisional hemorrhoidectomy and were randomized into 4 groups of 17 patients each to receive double-placebo (G1), metronidazole plus placebo (G2), flavonoids plus placebo (G3) or metronidazole plus flavonoids (G4) in the postoperative period. A standard analgesic protocol was offered equally for all groups. Postoperative pain, bleeding, edema, pruritus and tenesmus were evaluated during the following three periods: from immediately after the operation until postoperative day (POD)7, from POD 8 to POD 14, and from POD 15 to POD 30. The patients were required to complete symptom questionnaires and to attend postoperative follow-up on PODs 7, 14 and 30. The effect of each drug was assessed for each symptom, and the groups were compared with each other and over time. RESULTS: There was less severe pain in all postoperative periods in the groups using flavonoids (G3 and G4, both p < 0.0001), with an observed synergistic effect of flavonoids combined with metronidazole during the first 14 days after surgery (p < 0.0001). Flavonoid use was also associated with decreased bleeding (G3, p = 0.031 and G4, p = 0.016) between the first and second postoperative weeks CONCLUSIONS: The use of flavonoids alone and in combination with metronidazole resulted in a reduction of most symptoms, particularly pain, after excisional hemorrhoidectomy. TRIAL REGISTRATION: The present study was registered in the SISNEP (document CAAE-0035.0.240.000-11), after approval by the research ethics committee (CEP) of the Hospital Felício Rocho (protocol nº393 / 11).

CD73 as a target to improve temozolomide chemotherapy effect in glioblastoma preclinical model.

Glioblastoma is the most devastating primary brain tumor and effective therapies are not available. Treatment is based on surgery followed by radio and chemotherapy with temozolomide (TMZ), but TMZ increases patient survival only by 2 months. CD73, an enzyme responsible for adenosine production, emerges as a target for glioblastoma treatment. Indeed, adenosine causes tumor-promoting actions and CD73 inhibition increases sensitivity to TMZ in vitro. Here, a cationic nanoemulsion to nasal delivery of siRNA CD73 (NE-siRNA CD73) aiming glioblastoma treatment was employed alone or in combination with TMZ. In vitro, two glioblastoma cell lines (C6 and U138MG) with a chemo-resistant profile were used. Treatment alone with NE-siRNA CD73 reduced C6 and U138MG glioma cell viability by 70% and 25%, respectively. On the other hand, when NE-siRNA + TMZ combined treatment was employed, a reduction of 85% and 33% of cell viability was observed. Notably, treatment with NE-siRNA CD73 of glioma-bearing Wistar rats reduced tumor size by 80%, 60% more than the standard chemotherapy with TMZ, but no synergistic or additive effect was observed in vivo. Additionally, NE-siRNA CD73, TMZ or combined therapy decreased adenosine levels in liquor confirming the importance of this nucleoside on in vivo GB growth. Finally, no hemolytic potential was observed. These results suggest that nasal administration of NE-siRNA CD73 exhibits higher antiglioma effect when compared to TMZ. However, no synergistic or additive in vivo was promoted by the therapeutic regimen employed in this study.

Nasal Administration of Cationic Nanoemulsions as CD73-siRNA Delivery System for Glioblastoma Treatment: a New Therapeutical Approach.

Glioblastoma is the most devastating primary brain tumor. Effective therapies are not available, mainly due to high tumor heterogeneity, chemoresistance, and the difficulties imposed by blood-brain barrier. CD73, an enzyme responsible for adenosine (ADO) production, is overexpressed in cancer cells and emerges as a target for glioblastoma treatment. Indeed, ADO causes a variety of tumor-promoting actions, particularly by inducing tumor immune escape, whereas CD73 inhibition impairs tumor progression. Here, a cationic nanoemulsion to deliver CD73siRNA (NE-siRNA CD73R) via nasal route aiming glioblastoma treatment was developed. NE-siRNA CD73R was uptaken by glioma cells in culture, resulting in a parallel 60-80% decrease in AMPase activity and 30-50% in cell viability. Upon nasal delivery, NE-siRNA CD73R was detected in rat brain and serum. Notably, treatment with CD73siRNA complexes of glioma-bearing Wistar rats reduced tumor growth by 60%. Additionally, NE-siRNA CD73R treatment decreased 95% ADO levels in liquor and tumor CD73 expression, confirming in vivo CD73 silencing. Finally, no toxicity was observed in either primary astrocytes or rats with this cationic nanoemulsion. These results suggest that nasal administration of cationic NE as CD73 siRNA delivery system represents a novel potential treatment for glioblastoma. Graphical Abstract Glioblastoma is the most common and devastating form of primary brain tumor. CD73, a protein involved in cell-cell adhesion and migration processes and also responsible for extracellular adenosine (ADO) production, is overexpressed by glioma cells and emerges as an important target for glioma treatment. Indeed, ADO participates in tumor immune escape, cell proliferation, and angiogenesis, and CD73 inhibition impairs those processes. Here, a cationic nanoemulsion to deliver CD73 siRNA (NE-siRNA CD73R) via nasal route aiming glioblastoma treatment was developed. NE-siRNA CD73R knockdown in vitro and in vivo CD73. Upon nasal delivery of NE-siRNA CD73R, the treatment markedly reduced tumor volume by 60% in a rat preclinical glioblastoma model. The treatment was well tolerated, and did not induce kidney, liver, lung, olfactory, bone marrow, or behavior alterations. These results indicate that the nasal administration of NE as a CD73 siRNA delivery system offered an efficient means of gene knockdown and may represent a potential alternative for glioblastoma treatment.

CD73 Downregulation Decreases In Vitro and In Vivo Glioblastoma Growth.

Glioblastoma is the worst and most common primary brain tumor. Here, we demonstrated the role of CD73, an enzyme responsible for adenosine (ADO) production, in glioblastoma progression. ADO increased glioma cell viability via A1 receptor sensitization. CD73 downregulation decreased glioma cell migration and invasion by reducing metalloproteinase-2 and vimentin expression and reduced cell proliferation by 40%, which was related to necrosis and sub-G1 phase blockage of cell cycle. Those effects also involved the stimulation of Akt/NF-kB pathways. Additionally, CD73 knockdown or enzyme inhibition potentiated temozolomide cytotoxic effect on glioma cells by decreasing the IC50 value and sensitizing cells to a non-cytotoxic drug concentration. CD73 inhibition also decreased in vivo rat glioblastoma progression. Delivery of siRNA-CD73 or APCP reduced tumor size by 45 and 40%, respectively, when compared with control. This effect was followed by a parallel 95% reduction of ADO levels in cerebrospinal fluid, indicating the role of extracellular ADO in in vivo glioma growth. Treatment did not induce systemic damage or mortality. Altogether, we conclude that CD73 is an interesting target for glioblastoma treatment and its inhibition may provide new opportunities to improve the treatment of brain tumors. Graphical Abstract ᅟ.

1,4-Naphthoquinones potently inhibiting P2X7 receptor activity.

P2X7 receptor (P2X7R) is an ATP-gated ion-channel with potential therapeutic applications. In this study, we prepared and searched a series of 1,4-naphthoquinones derivatives to evaluate their antagonistic effect on both human and murine P2X7 receptors. We explored the structure-activity relationship and binding mode of the most active compounds using a molecular modeling approach. Biological analysis of this series (eight analogues and two compounds) revealed significant in vitro inhibition against both human and murine P2X7R. Further characterization revealed that AN-03 and AN-04 had greater potency than BBG and A740003 in inhibiting dye uptake, IL-1ß release, and carrageenan-induced paw edema in vivo. Moreover, we used electrophysiology and molecular docking analysis for characterizing AN-03 and AN-04 action mechanism. These results suggest 1,4-napthoquinones, mainly AN-04, as potential leads to design new P2X7R blockers and anti-inflammatory drugs.

Assessing families' and patients' attitudes toward brain donation for research purposes in a Brazilian population sample.

The neuropathological examination of postmortem human brain tissue is an essential resource for the definitive diagnosis and research on neurodegenerative diseases. Due to the growing need of donated brains to supply the Brain Banks, the understanding of the factors associated with the consent for the donation in our context is an important aspect of the process of brain donation. The verbal answers and the donation consent rate were evaluated in three groups: 30 relatives of patients who underwent verification of the cause of death, 14 patients assisted at a neurology ambulatory outpatient clinic, and 18 patients' relatives. The donation consent rates were of 46.6, 92.8 and 88.8 %, respectively. The main reasons for refusal were the disagreement with the autopsy, philosophical and religious issues, objections from other family members, and the consideration of the wishes of the deceased. The consent was specially motivated by the interest in the advances of scientific knowledge, altruistic reasons and the personal experiences with the disease. Factors as the emotional fragility at the moment of death, the beliefs, family matters, and the lack of knowledge are key elements in the donation process. Future goals include the establishment of a brain donor program with the support of academic institutions, hospitals, scientists, community, patient's associations and autopsy assistants. Approaching patients and relatives in specialized ambulatories clinic during assistance is probably the most efficient mean of obtaining brains for research.

Disruption of redox homeostasis and histopathological alterations caused by in vivo intrastriatal administration of D-2-hydroxyglutaric acid to young rats.

High accumulation of D-2-hydroxyglutaric acid (D-2-HG) is the biochemical hallmark of patients affected by the inherited neurometabolic disorder D-2-hydroxyglutaric aciduria (D-2-HGA). Clinically, patients present neurological symptoms and basal ganglia injury whose pathophysiology is poorly understood. We investigated the ex vivo effects of intrastriatal administration of D-2-HG on important parameters of redox status in the striatum of weaning rats. D-2-HG in vivo administration increased malondialdehyde (MDA) and carbonyl formation (lipid and protein oxidative damage, respectively), as well as the production of reactive nitrogen species (RNS). D-2-HG also compromised the antioxidant defenses by decreasing reduced glutathione (GSH) concentrations, as well as the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Increased amounts of oxidized glutathione (GSSG) with no significant alteration of total glutathione (tGS) were also found. Furthermore, D-2-HG-induced lipid oxidation and reduction of GSH concentrations and GPx activity were prevented by the N-methyl-d-aspartate (NMDA) receptor antagonist dizocilpine maleate (MK-801) and the nitric oxide synthase (NOS) inhibitor N(ω)-nitro-l-arginine methyl ester (l-NAME), suggesting the participation of NMDA receptors and nitric oxide derivatives in these effects. Creatine also impeded D-2-HG-elicited MDA increase, but did not change the D-2-HG-induced diminution of GSH and of the activities of SOD and GPx. We also found that DCFH oxidation and H2O2 production were not altered by D-2-HG, making unlikely an important role for reactive oxygen species (ROS) and reinforcing the participation of RNS in the oxidative damage and the reduction of antioxidant defenses provoked by this organic acid. Vacuolization, lymphocytic infiltrates and macrophages indicating brain damage were also observed in the striatum of rats injected with D-2-HG. The present data provide in vivo solid evidence that D-2-HG disrupts redox homeostasis and causes histological alterations in the rat striatum probably mediated by NMDA overstimulation and RNS production. It is therefore presumed that disturbance of redox status may contribute at least in part to the basal ganglia alterations characteristic of patients affected by D-2-HGA.

Disruption of redox homeostasis and brain damage caused in vivo by methylmalonic acid and ammonia in cerebral cortex and striatum of developing rats.

Hyperammonemia is a common finding in children with methylmalonic acidemia and propionic acidemia, but its contribution to the development of the neurological symptoms in the affected patients is poorly known. Considering that methylmalonic acid (MMA) and propionic acid (PA) predominantly accumulate in these disorders, we investigated the effects of hyperammonemia induced by urease treatment in 30-day-old rats receiving an intracerebroventricular (ICV) injection of MMA or PA on important parameters of redox homeostasis in cerebral cortex and striatum. We evaluated glutathione (GSH) concentrations, sulfhydryl content, nitrate and nitrite concentrations, 2',7'-dichlorofluorescein (DCFH) oxidation, and the activity of antioxidant enzymes. MMA decreased GSH concentrations and sulfhydryl content and increased nitrate and nitrite concentrations in cerebral cortex and striatum from hyperammonemic rats, whereas MMA or ammonia per se did not alter these parameters. MMA plus hyperammonemia also decreased glutathione reductase activity in rat cerebral cortex, but did not affect catalase, superoxide dismutase and glutathione peroxidase activities, neither DCFH oxidation. Furthermore, ICV PA administration alone or combined with hyperammonemia did not alter any of the evaluated parameters. We also found that pre-treatment with antioxidants prevented GSH reduction and sulfhydryl oxidation, whereas N(ω)-nitro-L-arginine methyl ester (L-NAME) prevented the increased nitrate and nitrite concentrations provoked by MMA plus ammonia treatments. Histological alterations, including vacuolization, ischemic neurons, and pericellular edema, were observed in brain of hyperammonemic rats injected with MMA. The data indicate a synergistic effect of MMA and ammonia disturbing redox homeostasis and causing morphological brain abnormalities in rat brain.

p16INK4 expression in precursor lesions of squamous cell cervical cancer related to the presence of HPV-DNA.

The purpose of the present study was to identify the expression of p16INK4 in cervical cancer precursor lesions by immunohistochemistry and to correlate it with lesion grade and presence of human papillomavirus (HPV) infection. Cervical specimens from 144 women seen consecutively at the gynecology outpatient clinic of our institution from December 2003 to May 2005 were analyzed by cytopathology, histopathology, polymerase chain reaction for HPV-DNA, and p16INK4 immunostaining. Histologically normal biopsies, HPV-DNA negative by polymerase chain reaction, were used as control. HPV-DNA prevalence, including the control group, was 68.1% and the prevalence of p16INK4 expression was 55.0%. The percentage of cells stained by p16INK4 ranged from 10 to 100%, both in the group consisting of cervical intraepithelial neoplasia (CIN)1/HPV specimens and in the group of CIN2/CIN3 specimens with P value of 0.0001. p16INK4 expression was 48.3% in the CIN1/HPV group, as opposed to 94.3% in the CIN2/CIN3 group (P = 0.001), showing a statistically significant difference between the two groups. The quantitative method used here is simple and less subjective than the different semiquantitative methods described in the literature. In view of the different definitions of a p16INK4-positive case, it is almost impossible to compare the findings reported by different investigators. This study confirms the association between p16INK4 and CIN2 and CIN3 lesions. Moreover, it shows that some low grade lesions expressed high levels of this protein. This may indicate that such low grade lesions may be predisposed to progress to high grade lesions. This means that p16INK4 may be a strong marker for "neoplastic lesions" induced by HPV and not just an infection marker.

p16INK4 expression in precursor lesions of squamous cell cervical cancer related to the presence of HPV-DNA

The purpose of the present study was to identify the expression of p16INK4 in cervical cancer precursor lesions by immunohistochemistry and to correlate it with lesion grade and presence of human papillomavirus (HPV) infection. Cervical specimens from 144 women seen consecutively at the gynecology outpatient clinic of our institution from December 2003 to May 2005 were analyzed by cytopathology, histopathology, polymerase chain reaction for HPV-DNA, and p16INK4 immunostaining. Histologically normal biopsies, HPV-DNA negative by polymerase chain reaction, were used as control. HPV-DNA prevalence, including the control group, was 68.1 percent and the prevalence of p16INK4 expression was 55.0 percent. The percentage of cells stained by p16INK4 ranged from 10 to 100 percent, both in the group consisting of cervical intraepithelial neoplasia (CIN)1/HPV specimens and in the group of CIN2/CIN3 specimens with P value of 0.0001. p16INK4 expression was 48.3 percent in the CIN1/HPV group, as opposed to 94.3 percent in the CIN2/CIN3 group (P = 0.001), showing a statistically significant difference between the two groups. The quantitative method used here is simple and less subjective than the different semiquantitative methods described in the literature. In view of the different definitions of a p16INK4-positive case, it is almost impossible to compare the findings reported by different investigators. This study confirms the association between p16INK4 and CIN2 and CIN3 lesions. Moreover, it shows that some low grade lesions expressed high levels of this protein. This may indicate that such low grade lesions may be predisposed to progress to high grade lesions. This means that p16INK4 may be a strong marker for "neoplastic lesions" induced by HPV and not just an infection marker.

Extended administration of the association of zidovudine plus ritonavir during rat pregnancy: maternal and fetal effects.

The purpose of the study was to evaluate at term, the effects of the association of zidovudine/ritonavir administered during the entire period of rat pregnancy. Forty pregnant EPM-1 Wistar rats were divided randomly into four groups: one control (drug vehicle control, n=10) and three experimental treated with an oral solution of zidovudine/ritonavir (Exp 1 = 10/20 mg/kg bw, n = 10; Exp 2 = 30/60 mg/kg bw, n=10; Exp 3 = 90/180 mg/kg bw, n=10) from day 0 up to day 20 of pregnancy. Maternal body weights were recorded at the start of the experiment and at the 7th, 14th and the 20th day thereafter. At term (20th day) the rats were anesthetized and, upon laparotomy and hysterotomy, the number of implantations, resorptions, living fetuses, placentae and intrauterine deaths were recorded. The collected fetuses and placentae were weighed, and the concepts were examined under a stereoscopic microscope for external malformations. The maternal body gain and the mean fetal weight at term were both significantly lower (p < 0.01 and p < 0.0001, respectively) in the experimental groups compared to the control. The recorded resorptions were higher in Exp 2 and Exp 3 groups than in the control group. The other parameters were not affected. The exposure of pregnant rats at term to a 1:2 association of zidovudine plus ritonavir resulted in a significant reduction in maternal body weight gain and increased rate of fetal resorption.

Effects of lopinavir-ritonavir combined therapy during the rat pregnancy. Morphological and biochemical aspects.

OBJECTIVE: To evaluate the biochemical and morphological effects in rats subjected to three different dose associations of the protease inhibitors lopinavir and ritonavir administered throughout the entire period of pregnancy. STUDY DESIGN: The animals were treated throughout pregnancy with daily oral doses of lopinavir+ritonavir starting at the day one of pregnancy, and were divided into four groups: E1, 13.3+3.3 mg/kg; E2, 39.9+9.9 mg/kg; E3, 119.7+29.9 mg/kg and C, control (drug vehicle, propyleneglycol). The animals were then sacrificed and maternal blood and fetal and maternal organ samples were taken for morphological and biochemical analysis. RESULTS: No major changes were identified in the group treated with the lowest dose as compared with the control. In the group E2, we found hepatocytes with signs of atrophy, eosinophilic cytoplasm, picnotic nuclei and vasodilatation. The proximal convoluted tubules of maternal kidneys showed eosinophilic areas and hyperchromatic nuclei, as well as signs of vasodilation. In the group treated with the highest dose (group E3), in the maternal kidneys and livers, the morphological changes were similar to those found in E2, although more prominent. Regarding the fetal organs, the single abnormality observed was some liver vasodilation in the group E3 (highest dose). The treatment with lopinavir+ritonavir caused discrete, yet significant, alterations of aspartate aminotransferase activity, blood urea nitrogen and creatinine plasma levels. CONCLUSIONS: Our results showed that the administration of a combination of lopinavir plus ritonavir to pregnant rats can cause morphological as well as functional changes in maternal and fetal liver and kidneys and, in higher than therapeutic doses, might be toxic to those animals.

Morphological and biochemical appraisal of the liver and renal effects of indinavir on rat pregnancy.

Since indinavir is currently used in combination with other antiretroviral agents, there is a scarcity of studies in the literature on its single-drug perinatal safety. Thus, we decided to examine the gross maternal and fetal effects of indinavir administered alone during the entire period of rat pregnancy. Forty pregnant animals were assigned at random to four groups (C = control) treated with the drug vehicle (distilled water); the experimental groups were treated with indinavir as follows: E1 = 40 mg/kg; E2 = 120 mg/kg; E3 = 360 mg/kg from "zero" up to the 20th day of gestation. Drug or vehicle were administered daily by gavage. Each group consisted of ten animals. At term-pregnancy, the rats were deeply anesthetized and blood samples were collected for alanine aminotransferase (ALT) and aspartate aminotransferase (AST), creatinine and urea determinations. Fragments of maternal and fetal livers and kidneys were taken and routinely processed for histopathological study. Serum ALT activity in the E2 group was significantly higher (p < 0.01) than that of the other groups. The concentration of creatinine in blood was lower in the E2 and E3 groups than in group E1 (p < 0.01), whereas blood urea in group E3 was significantly lower than in the other groups (p < 0.01). Morphological (light microscopy) studies revealed that no significant effects of the drug could be detected regarding either maternal or fetal organs of the E1 and E2 groups. However, the maternal hepatocytes in the E3 group showed heterochromatic nuclei. In addition, there was some fatty infiltration, congested sinusoids and portal dilation. Maternal kidneys in the E2 and E3 groups revealed vascular dilation around the convoluted tubules. Regarding the biochemical determinations, the alterations observed were mild, without biological relevance, thus indicating that the treatment with indinavir during the entire gestation was essentially devoid of hepatic or renal effects which could result in altered metabolic parameters. It is concluded that indinavir was well tolerated in therapeutic and even in 9-fold higher doses. Notwithstanding, discrete morphological alterations occurred in the maternal compartment, but with no functional expression that could indicate deleterious effects on mothers and/or fetuses.