Resumo em Inglês:

The process of drug development involves non-clinical and clinical studies. Non-clinical studies are conducted using different protocols including animal studies, which mostly follow the Good Laboratory Practice (GLP) regulations. During the early pre-clinical development process, also known as Go/No-Go decision, a drug candidate needs to pass through several steps, such as determination of drug availability (studies on pharmacokinetics), absorption, distribution, metabolism and elimination (ADME) and preliminary studies that aim to investigate the candidate safety including genotoxicity, mutagenicity, safety pharmacology and general toxicology. These preliminary studies generally do not need to comply with GLP regulations. These studies aim at investigating the drug safety to obtain the first information about its tolerability in different systems that are relevant for further decisions. There are, however, other studies that should be performed according to GLP standards and are mandatory for the safe exposure to humans, such as repeated dose toxicity, genotoxicity and safety pharmacology. These studies must be conducted before the Investigational New Drug (IND) application. The package of non-clinical studies should cover all information needed for the safe transposition of drugs from animals to humans, generally based on the non-observed adverse effect level (NOAEL) obtained from general toxicity studies. After IND approval, other GLP experiments for the evaluation of chronic toxicity, reproductive and developmental toxicity, carcinogenicity and genotoxicity, are carried out during the clinical phase of development. However, the necessity of performing such studies depends on the new drug clinical application purpose.

Resumo em Inglês:

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disorder caused by expansion of the polyglutamine domain of the ataxin-3 (ATX3) protein. MJD/SCA3 is the most frequent autosomal dominant ataxia in many countries. The mechanism underlying MJD/SCA3 is thought to be mainly related to protein misfolding and aggregation leading to neuronal dysfunction followed by cell death. Currently, there are no effective treatments for patients with MJD/SCA3. Here, we report on the potential use of lithium carbonate and coenzyme Q10 to reduce cell death caused by the expanded ATX3 in cell culture. Cell viability and apoptosis were evaluated by MTT assay and by flow cytometry after staining with annexin V-FITC/propidium iodide. Treatment with lithium carbonate and coenzyme Q10 led to a significant increase in viability of cells expressing expanded ATX3 (Q84). In addition, we found that the increase in cell viability resulted from a significant reduction in the proportion of apoptotic cells. Furthermore, there was a significant change in the expanded ATX3 monomer/aggregate ratio after lithium carbonate and coenzyme Q10 treatment, with an increase in the monomer fraction and decrease in aggregates. The safety and tolerance of both drugs are well established; thus, our results indicate that lithium carbonate and coenzyme Q10 are good candidates for further in vivo therapeutic trials.

Resumo em Inglês:

Glucagon-like peptide 1 (GLP-1), a kind of gut hormone, is used in the treatment of type 2 diabetes (T2D). Emerging evidence indicates that GLP-1 has anti-inflammatory activity. Chronic inflammation in the adipose tissue of obese individuals is a cause of insulin resistance and T2D. We hypothesized that GLP-1 analogue therapy in patients with T2D could suppress the inflammatory response of macrophages, and therefore inhibit insulin resistance. Our results showed that GLP-1 agonist (exendin-4) not only attenuated macrophage infiltration, but also inhibited the macrophage secretion of inflammatory cytokines including TNF-β, IL-6, and IL-1β. Furthermore, we observed that lipopolysaccharide (LPS)-induced macrophage conditioned media could impair insulin-stimulated glucose uptake. This effect was compensated by treatment with the conditioned media from macrophages treated with the combination of LPS and exendin-4. It was also observed that exendin-4 directly inhibited the activation of NF-κB in macrophages. In conclusion, our results indicated that GLP-1 improved inflammatory macrophage-derived insulin resistance by inhibiting NF-κB pathway and secretion of inflammatory cytokines in macrophages. Furthermore, our observations suggested that the anti-inflammatory effect of GLP-1 on macrophages can contribute to GLP-1 analogue therapy of T2D.

Resumo em Inglês:

This study aimed to estimate the absorption, distribution, metabolism and excretion (ADME) properties and safety of LDT5, a lead compound for oral treatment of benign prostatic hyperplasia that has previously been characterized as a multi-target antagonist of α1A-, α1D-adrenoceptors and 5-HT1A receptors. The preclinical characterization of this compound comprised the evaluation of its in vitro properties, including plasma, microsomal and hepatocytes stability, cytochrome P450 metabolism and inhibition, plasma protein binding, and permeability using MDCK-MDR1 cells. De-risking and preliminary safety pharmacology assays were performed through screening of 44 off-target receptors and in vivo tests in mice (rota-rod and single dose toxicity). LDT5 is stable in rat and human plasma, human liver microsomes and hepatocytes, but unstable in rat liver microsomes and hepatocytes (half-life of 11 min). LDT5 is highly permeable across the MDCK-MDR1 monolayer (Papp ∼32×10-6 cm/s), indicating good intestinal absorption and putative brain penetration. LDT5 is not extensively protein-bound and is a substrate of human CYP2D6 and CYP2C19 but not of CYP3A4 (half-life >60 min), and did not significantly influence the activities of any of the human cytochrome P450 isoforms screened. LDT5 was considered safe albeit new studies are necessary to rule out putative central adverse effects through D2, 5-HT1A and 5-HT2B receptors, after chronic use. This work highlights the drug-likeness properties of LDT5 and supports its further preclinical development.

Resumo em Inglês:

The presence of the single nucleotide polymorphisms in exon 1 of the mannose-binding lectin 2 (MBL2) gene was evaluated in a sample of 159 patients undergoing coronary artery bypass surgery (71 patients undergoing valve replacement surgery and 300 control subjects) to investigate a possible association between polymorphisms and heart disease with Chlamydia infection. The identification of the alleles B and D was performed using real time polymerase chain reaction (PCR) and of the allele C was accomplished through PCR assays followed by digestion with the restriction enzyme. The comparative analysis of allelic and genotypic frequencies between the three groups did not reveal any significant difference, even when related to previous Chlamydia infection. Variations in the MBL plasma levels were influenced by the presence of polymorphisms, being significantly higher in the group of cardiac patients, but without representing a risk for the disease. The results showed that despite MBL2 gene polymorphisms being associated with the protein plasma levels, the polymorphisms were not enough to predict the development of heart disease, regardless of infection with both species of Chlamydia.

Resumo em Inglês:

Propofol is one of the most commonly used intravenous anesthetic agents during cancer resection surgery. A previous study has found that propofol can inhibit invasion and induce apoptosis of ovarian cancer cells. However, the underlying mechanisms are not known. miR-9 has been reported to be little expressed in ovarian cancer cells, which has been related to a poor prognosis in patients with ovarian cancer. Studies have also demonstrated that propofol could induce microRNAs expression and suppress NF-κB activation in some situations. In the present study, we assessed whether propofol inhibits invasion and induces apoptosis of ovarian cancer cells by miR-9/NF-κB signaling. Ovarian cancer ES-2 cells were transfected with anti-miR-9 or p65 cDNA or p65 siRNA for 24 h, after which the cells were treated with different concentrations of propofol (1, 5, and 10 μg/mL) for 24 h. Cell growth and apoptosis were detected using MTT assay and flow cytometry analysis. Cell migration and invasion were detected using Transwell and Wound-healing assay. Western blot and electrophoretic mobility shift assay were used to detect different protein expression and NF-κB activity. Propofol inhibited cell growth and invasion, and induced cell apoptosis in a dose-dependent manner, which was accompanied by miR-9 activation and NF-κB inactivation. Knockdown of miR-9 abrogated propofol-induced NF-κB activation and MMP-9 expression, reversed propofol-induced cell death and invasion of ES-2 cells. Knockdown of p65 inhibited NF-κB activation rescued the miR-9-induced down-regulation of MMP-9. In addition, overexpression of p65 by p65 cDNA transfection increased propofol-induced NF-κB activation and reversed propofol-induced down-regulation of MMP-9. Propofol upregulates miR-9 expression and inhibits NF-κB activation and its downstream MMP-9 expression, leading to the inhibition of cell growth and invasion of ES-2 cells.

Resumo em Inglês:

The ankle-brachial index (ABI) is a marker of subclinical atherosclerosis related to health-adverse outcomes. ABI is inexpensive compared to other indexes, such as coronary calcium score and determination of carotid artery intima-media thickness (IMT). Our objective was to identify how the ABI can be applied to primary care. Three different methods of calculating the ABI were compared among 13,921 men and women aged 35 to 74 years who were free of cardiovascular diseases and enrolled in the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil). The ABI ratio had the same denominator for the three categories created (the highest value for arm systolic blood pressure), and the numerator was based on the four readings for leg systolic blood pressure: the highest (ABI-HIGH), the mean (ABI-MEAN), and the lowest (ABI-LOW). The cut-off for analysis was ABI<1.0. All determinations of blood pressure were done with an oscillometric device. The prevalence of ABI<1% was 0.5, 0.9, and 2.7 for the categories HIGH, MEAN and LOW, respectively. All methods were associated with a high burden of cardiovascular risk factors. The association with IMT was stronger for ABI-HIGH than for the other categories. The proportion of participants with a 10-year Framingham Risk Score of coronary heart disease >20% without the inclusion of ABI<1.0 was 4.9%. For ABI-HIGH, ABI-MEAN and ABI-LOW, the increase in percentage points was 0.3, 0.7, and 2.3%, respectively, and the relative increment was 6.1, 14.3, and 46.9%. In conclusion, all methods were acceptable, but ABI-LOW was more suitable for prevention purposes.

Resumo em Inglês:

The current study aimed to investigate the effects of perinatal exposure to nonylphenol (NP) on delivery outcome of pregnant rats and subsequent inflammatory hepatic injury in newborn rats. The pregnant rats were divided into 2 groups: control group (corn oil) and NP exposure group. Thirty-four pregnant rats were administered NP or corn oil by gavage from the sixth day of pregnancy to 21 days postpartum, with blood samples collected at 12 and 21 days of pregnancy and 60 days after delivery. The NP concentration was measured by HPLC, with chemiluminescence used for detection of estrogen and progesterone levels. Maternal delivery parameters were also observed. Liver and blood of the newborn rats were collected and subjected to automatic biochemical detection of liver function and blood lipid analyzer (immunoturbidimetry), and ultrastructural observation of the hepatic microstructure, with the TNF-α and IL-1β hepatic tissue levels evaluated by immunohistochemistry. Compared with the control group, the pregnant and postpartum serum NP and estradiol levels of the mother rats in the NP group were significantly increased, together with lowered progesterone level, increased number of threatened abortion and dystocia, and fewer newborn rats and lower litter weight. Serum and hepatic NP levels of the newborn rats measured 60 days after birth were significantly higher than those of the control group, as well as lower testosterone levels and increased estradiol levels. When observed under electron microscope, the hepatocyte nuclei of the control group were large and round, with evenly distributed chromatin. The chromatin of hepatocytes in the NP group presented deep staining of the nuclei, significant lipid decrease in the cytoplasm, and the majority of cells bonded with lysate. The results of immunohistochemistry showed that there was almost no TNF-α or IL-1β expression in the hepatocytes of the control group, while the number of TNF-α-, PCNA-, and IL-1β-positive cells in the NP group was increased, with higher integral optical density than the control group. Compared to the control group, the serum levels of alanine aminotransferase, aspartate aminotransferase, triglyceride and low-density lipoprotein in the newborn rats of the NP group were significantly increased. There was no significant difference in the serum level of high-density lipoprotein or cholesterol between the groups. Perinatal exposure to NP can interfere with the in vivo estrogen and progesterone levels of pregnant rats, resulting in threatened abortion, dystocia and other adverse delivery outcomes. High liver and serum NP levels of the newborn rats led to alteration of liver tissue structure and function. The NP-induced hepatotoxicity is probably mediated by inflammatory cytokines TNF-α and IL-1α.