A Structural In Silico Analysis of the Immunogenicity of L-Asparaginase from Penicillium cerradense.

L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with L-asparaginase have been observed, mainly caused by immunogenicity and allergenicity. Some strategies have been adopted, such as searching for new microorganisms that produce the enzyme and applying protein engineering. Therefore, this work aimed to elucidate the molecular structure and predict the immunogenic profile of L-asparaginase from Penicillium cerradense, recently revealed as a new fungus of the genus Penicillium and producer of the enzyme, as a motivation to search for alternatives to bacterial L-asparaginase. In the evolutionary relationship, L-asparaginase from P. cerradense closely matches Aspergillus species. Using in silico tools, we characterized the enzyme as a protein fragment of 378 amino acids (39 kDa), including a signal peptide containing 17 amino acids, and the isoelectric point at 5.13. The oligomeric state was predicted to be a homotetramer. Also, this L-asparaginase presented a similar immunogenicity response (T- and B-cell epitopes) compared to Escherichia coli and Dickeya chrysanthemi enzymes. These results suggest a potentially useful L-asparaginase, with insights that can drive strategies to improve enzyme production.

Hepatoprotective Effects of Four Brazilian Savanna Species on Acetaminophen-Induced Hepatotoxicity in HepG2 Cells.

We investigated four Cerrado plant species, i.e., Cheiloclinium cognatum (Miers) A.C.Sm, Guazuma ulmifolia Lam., Hancornia speciosa Gomes, and Hymenaea stigonocarpa Mart. ex Hayne, against acetaminophen toxicity using an in vitro assay with HepG2 cells. The activity against acetaminophen toxicity was evaluated using different protocols, i.e., pre-treatment, co-treatment, and post-treatment of the cells with acetaminophen and the plant extracts. HepG2 cell viability after treatment with acetaminophen was 39.61 ± 5.59% of viable cells. In the pre-treatment protocol, the extracts could perform protection with viability ranging from 50.02 ± 15.24% to 78.75 ± 5.61%, approaching the positive control silymarin with 75.83 ± 5.52%. In the post-treatment protocol, all extracts and silymarin failed to reverse the acetaminophen damage. In the co-treatment protocol, the extracts showed protection ranging from 50.92 ± 11.14% to 68.50 ± 9.75%, and silymarin showed 77.87 ± 4.26%, demonstrating that the aqueous extracts of the species also do not increase the toxic effect of acetaminophen. This protection observed in cell viability was accompanied by a decrease in ROS. The extracts' hepatoprotection can be related to antioxidant compounds, such as rutin and mangiferin, identified using HPLC-DAD and UPLC-MS/MS. The extracts were shown to protect HepG2 cells against future APAP toxicity and may be candidates for supplements that could be used to prevent liver damage. In the concomitant treatment using the extracts with APAP, it was demonstrated that the extracts do not present a synergistic toxicity effect, with no occurrence of potentiation of toxicity. The extracts showed considerable cytoprotective effects and important antioxidant characteristics.

l-Asparaginase Type II from Fusarium proliferatum: Heterologous Expression and In Silico Analysis.

The search for new drug-producing microorganisms is one of the most promising situations in current world scientific scenarios. The use of molecular biology as well as the cloning of protein and compound genes is already well established as the gold standard method of increasing productivity. Aiming at this increase in productivity, this work aims at the cloning, purification and in silico analysis of l-asparaginase from Fusarium proliferatum in Komagataella phaffii (Pichia pastoris) protein expression systems. The l-asparaginase gene (NCBI OQ439985) has been cloned into Pichia pastoris strains. Enzyme production was analyzed via the quantification of aspartic B-hydroxamate, followed by purification on a DEAE FF ion exchange column. The in silico analysis was proposed based on the combined use of various technological tools. The enzymatic activity found intracellularly was 2.84 IU/g. A purification factor of 1.18 was observed. The in silico analysis revealed the position of five important amino acid residues for enzymatic activity, and likewise, it was possible to predict a monomeric structure with a C-score of 1.59. The production of the enzyme l-asparaginase from F. proliferatum in P. pastoris was demonstrated in this work, being of great importance for the analysis of new methodologies in search of the production of important drugs in therapy.

A new class of peptides from wasp venom: a pathway to antiepileptic/neuroprotective drugs.

The ability of venom-derived peptides to disrupt physiological processes in mammals provides an exciting source for pharmacological development. Our research group has identified a new class of neuroactive peptides from the venom of a Brazilian social wasp, Polybia occidentalis, with the potential pharmacological profile to treat epilepsies. The study was divided into five phases: Phase 1 concerned the extraction, isolation and purification of Occidentalin-1202(n) from the crude venom, followed by the synthesis of an identical analogue peptide, named Occidentalin-1202(s). In Phase 2, we described the effects of both peptides in two acute models of epilepsy-kainic acid and pentylenetetrazole-induced model of seizures-and measured estimated ED50 and therapeutic index values, electroencephalographic studies and C-fos evaluation. Phase 3 was a compilation of advanced tests performed with Occidentalin-1202(s) only, reporting histopathological features and its performance in the pilocarpine-induced status epilepticus. After the determination of the antiepileptic activity of Occidentalin-1202(s), Phase 4 consisted of evaluating its potential adverse effects, after chronic administration, on motor coordination (Rotarod) and cognitive impairment (Morris water maze) tests. Finally, in Phase 5, we proposed a mechanism of action using computational models with kainate receptors. The new peptide was able to cross the blood-brain barrier and showed potent antiseizure effects in acute (kainic acid and pentylenetetrazole) and chronic (temporal lobe epilepsy model induced by pilocarpine) models. Motor and cognitive behaviour were not adversely affected, and a potential neuroprotective effect was observed. Occidentalin-1202 can be a potent blocker of the kainate receptor, as assessed by computational analysis, preventing glutamate and kainic acid from binding to the receptor's active site. Occidentalin-1202 is a peptide with promising applicability to treat epilepsy and can be considered an interesting drug model for the development of new medicines.

L-Asparaginase from Penicillium sizovae Produced by a Recombinant Komagataella phaffii Strain.

L-asparaginase is an important enzyme in the pharmaceutical field used as treatment for acute lymphoblastic leukemia due to its ability to hydrolyze L-asparagine, an essential amino acid synthesized by normal cells, but not by neoplastic cells. Adverse effects of L-asparaginase formulations are associated with its glutaminase activity and bacterial origin; therefore, it is important to find new sources of L-asparaginase produced by eukaryotic microorganisms with low glutaminase activity. This work aimed to identify the L-asparaginase gene sequence from Penicillium sizovae, a filamentous fungus isolated from the Brazilian Savanna (Cerrado) soil with low glutaminase activity, and to biosynthesize higher yields of this enzyme in the yeast Komagataella phaffii. The L-asparaginase gene sequence of P. sizovae was identified by homology to L-asparaginases from species of Penicillium of the section Citrina: P. citrinum and P. steckii. Partial L-asparaginase from P. sizovae, lacking the periplasmic signaling sequence, was cloned, and expressed intracellularly with highest enzymatic activity achieved by a MUT+ clone cultured in BMM expression medium; a value 5-fold greater than that obtained by native L-asparaginase in P. sizovae cells. To the best of our knowledge, this is the first literature report of the heterologous production of an L-asparaginase from a filamentous fungus by a yeast.

Optimization of aqueous two-phase micellar system for partial purification of L-asparaginase from Penicillium sp. grown in wheat bran as agro-industrial residue.

L-asparaginase has been used in the remission of malignant neoplasms such as acute lymphoblastic leukemia. The search for new sources of this enzyme has become attractive for therapeutics. Traditional methods for biomolecule purification involve several steps. A two-phase system may be a good strategy to anticipate one of these stages. This study aimed to produce and purify a fungal L-asparaginase through an aqueous two-phase micellar system (ATPMS) using Triton X-114. The fungus Penicillium sp.-encoded 2DSST1 was isolated from Cerrado soil. Plackett-Burman design followed by a 24 full factorial design was used to determine the best conditions to produce L-asparaginase. The evaluated variables were L-asparagine, L-proline, wheat bran, potato dextrose broth, ammonium sulfate, yeast extract, sucrose and glucose concentrations, incubation temperature, incubation period, and initial pH of the culture medium. L-asparaginase quantification was valued by the formation of ß-aspartyl hydroxamate. The significant positive variables, L-asparagine, L-proline, potato dextrose broth, and sucrose concentrations, were evaluated at 2 levels (+ 1 and - 1) with triplicate of the central point. After 34 runs, maximum activity (2.33 IU/mL) was achieved at the factorial design central point. A central composite design was performed in ATPMS at two levels (+ 1 and - 1) varying Triton X-114 concentration (w/v), separation phase temperature, and crude extract concentration (w/v). The L-asparaginase partition coefficient (K) was considered the experimental design response. Out of the 16 systems that were examined, the most promising presented a purification factor of 1.4 and a yield of 100%.

Erg6 affects membrane composition and virulence of the human fungal pathogen Cryptococcus neoformans.

Ergosterol is the most important membrane sterol in fungal cells and a component not found in the membranes of human cells. We identified the ERG6 gene in the AIDS-associated fungal pathogen, Cryptococcus neoformans, encoding the sterol C-24 methyltransferase of fungal ergosterol biosynthesis. In this work, we have explored its relationship with high-temperature growth and virulence of C. neoformans by the construction of a loss-of-function mutant. In contrast to other genes involved in ergosterol biosynthesis, C. neoformans ERG6 is not essential for growth under permissive conditions in vitro. However, the erg6 mutant displayed impaired thermotolerance and increased susceptibility to osmotic and oxidative stress, as well as to different antifungal drugs. Total lipid analysis demonstrated a decrease in the erg6Δ strain membrane ergosterol content. In addition, this mutant strain was avirulent in an invertebrate model of C. neoformans infection. C. neoformans Erg6 was cyto-localized in the endoplasmic reticulum and Golgi complex. Our results demonstrate that Erg6 is crucial for growth at high temperature and virulence, likely due to its effects on C. neoformans membrane integrity and dynamics. These pathogen-focused investigations into ergosterol biosynthetic pathway components reinforce the multiple roles of ergosterol in the response of diverse fungal species to alterations in the environment, especially that of the infected host. These studies open perspectives to understand the participation of ergosterol in mechanism of resistance to azole and polyene drugs. Observed synergistic growth defects with co-inhibition of Erg6 and other components of the ergosterol biosynthesis pathway suggests novel approaches to treatment in human fungal infections.

Predictive Power of In Silico Approach to Evaluate Chemicals against M. tuberculosis: A Systematic Review.

Mycobacterium tuberculosis (Mtb) is an endemic bacterium worldwide that causes tuberculosis (TB) and involves long-term treatment that is not always effective. In this context, several studies are trying to develop and evaluate new substances active against Mtb. In silico techniques are often used to predict the effects on some known target. We used a systematic approach to find and evaluate manuscripts that applied an in silico technique to find antimycobacterial molecules and tried to prove its predictive potential by testing them in vitro or in vivo. After searching three different databases and applying exclusion criteria, we were able to retrieve 46 documents. We found that they all follow a similar screening procedure, but few studies exploited equal targets, exploring the interaction of multiple ligands to 29 distinct enzymes. The following in vitro/vivo analysis showed that, although the virtual assays were able to decrease the number of molecules tested, saving time and money, virtual screening procedures still need to develop the correlation to more favorable in vitro outcomes. We find that the in silico approach has a good predictive power for in vitro results, but call for more studies to evaluate its clinical predictive possibilities.

Optimization and partial purification of beta-galactosidase production by Aspergillus niger isolated from Brazilian soils using soybean residue.

ß-Galactosidases are widely used for industrial applications. These enzymes could be used in reactions of lactose hydrolysis and transgalactosylation. The objective of this study was the production, purification, and characterization of an extracellular ß-galactosidase from a filamentous fungus, Aspergillus niger. The enzyme production was optimized by a factorial design. Maximal ß-galactosidase activity (24.64 U/mL) was found in the system containing 2% of a soybean residue (w/v) at initial pH 7.0, 28 °C, 120 rpm in 7 days. ANOVA of the optimization study indicated that the response data on temperature and pH were significant (p < 0.05). The regression equation indicated that the R2 is 0.973. Ultrafiltration at a 100 and 30 kDa cutoff followed by gel filtration and anion exchange chromatography were carried out to purify the fungal ß-galactosidase. SDS-PAGE revealed a protein with molecular weight of approximately 76 kDa. The partially purified enzyme showed an optimum temperature of 50 °C and optimum pH of 5.0, being stable under these conditions for 15 h. The enzyme was exposed to conditions approaching gastric pH and in pepsin's presence, 80% of activity was preserved after 2 h. These results reveal a A. niger ß-galactosidase obtained from residue with favorable characteristics for food industries.

Optimization of Xylanase Production from Aspergillus foetidus in Soybean Residue.

Enzymatic hydrolysis is an important but expensive step in the process to obtain enzyme derived products. Thus, the production of efficient enzymes is of great interest for this biotechnological application. The production of xylanase by Aspergillus foetidus in soybean residues was optimized using 2 × 23 factorial designs. The experimental data was fitted into a polynomial model for xylanase activity. Statistical analyses of the results showed that variables pH and the interaction of pH and temperature had influenced the production of xylanase, with the best xylanase production level (13.98 U/mL) occurring at fermentation for 168 hours, pH 7.0, 28°C, and 120 rpm.

Biological activity of dihydropyrimidinone (DHPM) derivatives: A systematic review.

Dihydropyrimidinones are heterocycles with a pyrimidine moiety in the ring nucleus, which, in recent decades, have aroused interest in medicinal chemistry due to alleged versatile biological activity. In this systematic review, we describe the currently published activities of dihydropyrimidinone derivatives. Between 1990 and December 31st, 2016, 115 articles outlined biological activities or toxicity of DHPM derivatives, 12 of those involved in vivo experiments. The main activities associated with this class of compounds are antitumoral (43 articles), anti-inflammatory (12 articles), antibacterial (20 articles) and calcium channel antagonism/inhibition (14 articles). Antitumoral activity is the main biological property evaluated, since the main representative compound of this class (monastrol) is a known Eg5 kinesin inhibitor. This review depicts a variety of other pharmacological activities associated with DHPM derivatives, but the main findings are essentially in vitro characteristics of the substances. This review presents the current state of the art of DHPM biological activities and demonstrates that there is still a need for further in vivo studies to better delineate the pharmacological potential of this class of substances.

Extracts of Morus nigra L. Leaves Standardized in Chlorogenic Acid, Rutin and Isoquercitrin: Tyrosinase Inhibition and Cytotoxicity.

Melanogenesis is a process responsible for melanin production, which is stored in melanocytes containing tyrosinase. Inhibition of this enzyme is a target in the cosmetics industry, since it controls undesirable skin conditions such as hyperpigmentation due to the overproduction of melanin. Species of the Morus genus are known for the beneficial uses offered in different parts of its plants, including tyrosinase inhibition. Thus, this project aimed to study the inhibitory activity of tyrosinase by extracts from Morus nigra leaves as well as the characterization of its chromatographic profile and cytotoxicity in order to become a new therapeutic option from a natural source. M. nigra leaves were collected, pulverized, equally divided into five batches and the standardized extract was obtained by passive maceration. There was no significant difference between batches for total solids content, yield and moisture content, which shows good reproducibility of the extraction process. Tyrosinase enzymatic activity was determined for each batch, providing the percentage of enzyme inhibition and IC50 values obtained by constructing dose-response curves and compared to kojic acid, a well-known tyrosinase inhibitor. High inhibition of tyrosinase activity was observed (above 90% at 15.625 µg/mL). The obtained IC50 values ranged from 5.00 µg/mL ± 0.23 to 8.49 µg/mL ± 0.59 and were compared to kojic acid (3.37 µg/mL ± 0.65). High Performance Liquid Chromatography analysis revealed the presence of chlorogenic acid, rutin and, its major compound, isoquercitrin. The chromatographic method employed was validated according to ICH guidelines and the extract was standardized using these polyphenols as markers. Cytotoxicity, assessed by MTT assay, was not observed on murine melanomas, human keratinocytes and mouse fibroblasts in tyrosinase IC50 values. This study demonstrated the potential of M. nigra leaf extract as a promising whitening agent of natural source against skin hyperpigmentation.

Access to medicines in Brazil based on monetary and non-monetary acquisition data obtained from the 2008/2009 Household Budget Survey.

OBJECTIVE: To investigate the access to medicines by Brazilian families by monetary and non-monetary acquisition data. METHODS: This is a cross-sectional study based on data obtained from the 2008/2009 Brazilian Household Budget Survey. The units of assessment were households that participated in the survey and the data on the acquisition of medicines over the 30 days prior to the interviews. The medicines were classified according to the Anatomical Therapeutic Chemical classification system. RESULTS: Acquisition of medicines was reported by 82.9% of Brazilian households, with 2.38 medicines/household, and 0.72 medicine/individual. In the South and Southeast regions, the average acquisition was slightly greater than the national average (2.53 and 2.49, respectively). In 22.3% of Brazilian households, it was reported that a medicine was not acquired due to lack of financial resources, mainly in the North and Northeastern regions, and in rural areas. Approximately 15.0% of medicines were obtained with no costs, 90.1% of them by the Brazilian Unified Health System. The medicines most acquired were those acting on the nervous system (28.8% of Brazilian households), on the cardiovascular system (15.7%), on the digestive tract and metabolism (14.3%), and on the respiratory system (12.1%). Overall, the quantity of medicines acquired was greater in higher socioeconomic classes of the population, with the exception of antiparasitic products, most likely because of the precarious sanitary conditions faced by less privileged social classes. CONCLUSIONS: The acquisition of medicines is a common practice in Brazil, being reported by over 80.0% of the Brazilian households in 2008/2009. Although the data obtained from the Brazilian Household Budget Survey have some limitations, the information obtained in this study can help health authorities to design national and regional policies to guarantee access to these products while promoting their rational use.

Access to medicines in Brazil based on monetary and non-monetary acquisition data obtained from the 2008/2009 Household Budget Survey

ABSTRACT OBJECTIVE To investigate the access to medicines by Brazilian families by monetary and non-monetary acquisition data. METHODS This is a cross-sectional study based on data obtained from the 2008/2009 Brazilian Household Budget Survey. The units of assessment were households that participated in the survey and the data on the acquisition of medicines over the 30 days prior to the interviews. The medicines were classified according to the Anatomical Therapeutic Chemical classification system. RESULTS Acquisition of medicines was reported by 82.9% of Brazilian households, with 2.38 medicines/household, and 0.72 medicine/individual. In the South and Southeast regions, the average acquisition was slightly greater than the national average (2.53 and 2.49, respectively). In 22.3% of Brazilian households, it was reported that a medicine was not acquired due to lack of financial resources, mainly in the North and Northeastern regions, and in rural areas. Approximately 15.0% of medicines were obtained with no costs, 90.1% of them by the Brazilian Unified Health System. The medicines most acquired were those acting on the nervous system (28.8% of Brazilian households), on the cardiovascular system (15.7%), on the digestive tract and metabolism (14.3%), and on the respiratory system (12.1%). Overall, the quantity of medicines acquired was greater in higher socioeconomic classes of the population, with the exception of antiparasitic products, most likely because of the precarious sanitary conditions faced by less privileged social classes. CONCLUSIONS The acquisition of medicines is a common practice in Brazil, being reported by over 80.0% of the Brazilian households in 2008/2009. Although the data obtained from the Brazilian Household Budget Survey have some limitations, the information obtained in this study can help health authorities to design national and regional policies to guarantee access to these products while promoting their rational use.

Tablet splitting of psychotropic drugs for patients with dementia: a pharmacoepidemiologic study in a Brazilian sample.

PURPOSE: The objective of this study was to assess the frequency of tablet splitting of psychotropic drugs in a population of older adults with a diagnosis of dementia. METHODS: This retrospective, cross-sectional study examined a sample of geriatric outpatients seen at a public center specializing in the care of elderly patients, a referral center for management of dementias in general, especially Alzheimer dementia to identify the frequency of tablet splitting of psychotropic drugs and the factors that may be involved in this practice. Comparison of the presence or absence of tablet splitting in relation to several parameters was assessed by means of P values; between-group differences with an α < 5% (P < 0.05) were deemed significant. FINDINGS: The presence of dementia was significantly associated with prescriptions implying to split tablets, which was found in 88 patients with dementia (34.9%) versus 90 patients without dementia (23.7%) (P = 0.002). Among the 88 patients with dementia who split tablets, 64 (72.7%) split tablets of psychotropic drugs. IMPLICATIONS: These results indicate the importance of identifying the practice of tablet splitting, particularly when it involves psychotropic drugs, because it entails several factors that can reduce the efficacy of the drug therapy.

Advice on Degradation Products in Pharmaceuticals: A Toxicological Evaluation.

Degradation products are unwanted chemicals that can develop during the manufacturing, transportation, and storage of drug products and can affect the efficacy of pharmaceutical products. Moreover, even small amounts of degradation products can affect pharmaceutical safety because of the potential to cause adverse effects in patients. Consequently, it is crucial to focus on mechanistic understanding, formulation, storage conditions, and packaging to prevent the formation of degradation products that can negatively affect the quality and safety of the drug product. In this sense, databases and software that help predict the reactions involving the pharmaceutically active substance in the presence of degradation conditions can be used to obtain information on major degradation routes and the main degradation products formed during pharmaceutical product storage. In some cases, when the presence of a genotoxic degradation product is verified, it is necessary to conduct more thorough assessments. It is important to consider the chemical structure to distinguish between compounds with toxicologically alerting structures with associated toxic/genotoxic risks and compounds without active structures that can be treated as ordinary impurities. Evaluating the levels of degradation products based on a risk/benefit analysis is mandatory. Controlling critical variables during early development of drug products and conducting a follow-up study of these impurities can prevent degradation impurities present at concentrations greater than threshold values to ensure product quality. The definition of the impurity profile has become essential per various regulatory requirements. Therefore, this review includes the international regulatory perspective on impurity documents and the toxicological evaluation of degradation products. Additionally, some techniquesused in the investigation of degradation products and stability-indicating assay methods are highlighted. LAY ABSTRACT: Degradation products are impurities resulting from chemical changes that occur during drug manufacturing. They can also form during storage and transportation in response to changes in light, temperature, pH, and humidity, or due to inherent characteristics of the active pharmaceutical substance, such as their reaction with excipients or on contact with the packaging. The presence of these chemicals can affect product safety and quality. Therefore, it is necessary to know and follow the guidelines and standards regarding degradation products and existing regulatory environments to assess the toxicity and risk related to their presence in pharmaceutical products.

Olanzapine, weight change and metabolic effects: a naturalistic 12-month follow up.

OBJECTIVE: Olanzapine is an atypical antipsychotic drug used to treat schizophrenia. Some of the adverse effects related to its use are obesity, hyperlipidemia, type 2 diabetes and hypertension, which may result in development of metabolic syndrome. This study aimed to investigate a possible increase in some anthropometric and biochemical parameters, and the existence of any correlation between them in Brazilian patients with schizophrenia treated with olanzapine in the mid term. METHODS: Thirty subjects with schizophrenia were evaluated, 16 women and 14 men, aged between 18 and 47 years. All patients underwent blood collection and anthropometric measurements at four different times during 12 months of follow up; thus each patient was his or her own control. RESULTS: Evaluation of some anthropometric measurements showed significant differences when comparing the mean values obtained in each of the different data collection times (p < 0.05). However, the biochemical indicators of development of metabolic syndrome measured in our study did not show the same rate of increment, with only the total cholesterol and glucose levels presenting statistically significant changes (p < 0.05), but without the same magnitude of weight change. CONCLUSION: We conclude that medium-term treatment with olanzapine promoted a substantial weight gain and increased visceral fat, while the metabolic profile did not show the same magnitude of change, suggesting a dissociation between weight gain and blood parameters, despite the severe weight gain observed among subjects evaluated.