Preparation and characterization of a microencapsulated polyethylene glycol cross-linked polyhemoglobin.

Many complications are associated to the therapeutic use of blood, among which are not only transfusion adverse events but also other issues such as lack of donors and high costs for collecting, testing, preserving, and distributing blood packages. Therefore, a clinically viable "blood substitute" is considered the holy grail of traumatology and may greatly benefit medicine. One of the most successful approaches to date is conjugating hemoglobin with polyethylene glycol (PEG). This conjugation aims mainly at overcoming free cell hemoglobin toxicity, which makes its use as oxygen carrier in pure form unfeasible. To improve PEG-hemoglobin conjugates feasibility, we propose applying dual functional PEG cross-linking hemoglobin molecules encapsulated by a protein carrier. The new oxygen carrier showed mean values of the hydrodynamic diameter, dispersity, and zeta potential of 1370 nm, 0.029 and -36 mV, respectively, evidencing the successful synthesis of PEG bis(N-succinimidyl succinate) and polyhemoglobin as well as the structuring of protein carrier.

Cytotoxicity of doxycycline effluent generated by the Fenton process.

This study aims at determining the Minimum Inhibitory Concentration with Escherichia coli ATCC 25922 and cytotoxicity to L929 cells (ATCC CCL-1) of the waste generated by doxycycline degradation by the Fenton process. This process has shown promise in this treatment thanks mainly to the fact that the waste did not show any relevant inhibitory effect on the test organism and no cytotoxicity to L-929 cells, thus demonstrating that the antibiotic properties were inactivated.

Development of Ac2-26 Mesoporous Microparticle System as a Potential Therapeutic Agent for Inflammatory Bowel Diseases.

Introduction: Inflammatory bowel diseases (IBDs) disrupt the intestinal epithelium, leading to severe chronic inflammation. Current therapies cause adverse effects and are expensive, invasive, and ineffective for most patients. Annexin A1 (AnxA1) is a pivotal endogenous anti-inflammatory and tissue repair protein in IBD. Nanostructured compounds loading AnxA1 or its active N-terminal mimetic peptides improve IBD symptomatology. Methods: To further explore their potential as a therapeutic candidate, the AnxA1 N-terminal mimetic peptide Ac2-26 was incorporated into SBA-15 ordered mesoporous silica and covered with EL30D-55 to deliver it by oral treatment into the inflamed gut. Results: The systems SBA-Ac2-26 developed measurements revealed self-assembled rod-shaped particles, likely on the external surface of SBA-15, and 88% of peptide incorporation. SBA-15 carried the peptide Ac2-26 into cultured Raw 264.7 macrophages and Caco-2 epithelial cells. Moreover, oral administration of Eudragit-SBA-15-Ac2-26 (200 µg; once a day; for 4 days) reduced colitis clinical symptoms, inflammation, and improved epithelium recovery in mice under dextran-sodium sulfate-induced colitis. Discussion: The absorption of SBA-15 in gut epithelial cells is typically low; however, the permeable inflamed barrier can enable microparticles to cross, being phagocyted by macrophages. These findings suggest that Ac2-26 is successfully delivered and binds to its receptors in both epithelial and immune cells, aligning with the clinical results. Conclusion: Our findings demonstrate a simple and cost-effective approach to delivering Ac2-26 orally into the inflamed gut, highlighting its potential as non-invasive IBD therapy.

CD14 and IL6 polymorphisms are associated with a pro-atherogenic profile in young adults with acute myocardial infarction.

This study investigated the relationship of polymorphisms in genes encoding CD14, IL-6 and TLR4 with metabolic, inflammatory and endothelial markers in young adults with acute myocardial infarction (AMI). Glucose, lipids, nitrate and inflammatory markers, flow mediated vasodilatation (FMV) and flow mediated by nitrate (FMN) were evaluated in 102 AMI and 108 non-AMI (control group) young individuals (<45 years). CD14 -260C>T (rs2569190), IL6 -174G>C (rs1800795) and TLR4 c.896A>G (rs4986790) and TLR4 c.1196C>T (rs4986791) polymorphisms were analyzed by PCR-RFLP. Minor allele frequencies of CD14, IL6 and TLR4 polymorphisms were similar between AMI and control groups (p > 0.05). In AMI group, individuals carrying IL6 -174CC genotype had higher serum triglycerides, VLDL cholesterol and glucose compared to the IL6 -174GG/GC genotype carriers (p < 0.05). Multiple logistic analysis showed that IL6 -174CC genotype carriers had increased risk for hyperglycemia (>5.77 mmol/l) [OR: 6.75, 95 % CI: 1.80-24.40, p = 0.004] and hypertriglyceridemia (>2.68 mmol/l) [OR: 3.00, 95 % CI: 1.00-9.00, p = 0.043]. Moreover, CD14 -260TT genotype was associated with reduced serum HDL cholesterol [OR: 3.10, 95 % CI: 1.00-9.01, p = 0.044] and apolipoprotein AI [OR: 3.20, 95 % CI: 1.00-9.70, p = 0.038] in AMI group. Relationship between CD14 and IL6 variants and altered inflammatory and endothelial (nitrate, FMV and FMN) markers was not found in both AMI and control groups. The IL6 -174G>C and CD14 -260C>T polymorphisms are likely to be associated with a pro-atherogenic profile but not with increased inflammatory markers and endothelial dysfunction in young AMI patients.

Cell-free protein synthesis: advances on production process for biopharmaceuticals and immunobiological products.

Biopharmaceutical products are of great importance in the treatment or prevention of many diseases and represent a growing share of the global pharmaceutical market. The usual technology for protein synthesis (cell-based expression) faces certain obstacles, especially with 'difficult-to-express' proteins. Cell-free protein synthesis (CFPS) can overcome the main bottlenecks of cell-based expression. This review aims to present recent advances in the production process of biologic products by CFPS. First, key aspects of CFPS systems are summarized. A description of several biologic products that have been successfully produced using the CFPS system is provided. Finally, the CFPS system's ability to scale up and scale down, its main limitations and its application for biologics production are discussed.

Protein drug delivery: current dosage form profile and formulation strategies.

Protein drugs present specific challenges to the maintenance of long-term stability, which can be accomplished by altering parameters of obtention, purification, molecule structure and formulation. As we believe, commercial formulations are undervalued; therefore, this review focuses on screening, categorising and discussing all formulations of protein drugs approved and not withdrawn by regulatory agencies from United States, Canada and Europe until mid-2018. Peptides (<50 amino acids) were not included to allow a more precise evaluation of choices for larger molecules. We extracted data from the DrugBank database, cross-checked it with the FDA purple book and supplemented it with patient information leaflets and papers. We further classified and discussed the entries according to protein function, drug delivery, route of administration and types of excipient (freeze-dried forms). In addition, alternative choices of excipients were discussed. Experimental work included here relates to targeting strategies with verified pharmacokinetics or in vivo effectiveness to identify physiologically relevant options. Although no single rule can be set for efficient protein formulation, our data help to better understand and optimise the choice for excipients and pharmaceutical dosage forms. For more information, see the Supplemental Data.

Biodegradable nanocarriers coated with polymyxin B: Evaluation of leishmanicidal and antibacterial potential.

Most treatments of leishmaniasis require hospitalization and present side effects or parasite resistance; innovations in drug formulation/reposition can overcome these barriers and must be pursued to increase therapeutic alternatives. Therefore, we tested polymyxin B (polB) potential to kill Leishmania amazonensis, adsorbed or not in PBCA nanoparticles (PBCAnp), which could augment polB internalization in infected macrophages. PBCAnps were fabricated by anionic polymerization and analyzed by Dynamic Light Scattering (size, ζ potential), Nanoparticle Tracking Analysis (size/concentration), vertical diffusion cell (release rate), drug incorporation (indirect method, protein determination) and in vitro cell viability. Nanoparticles coated with polB (PBCAnp-polB) presented an adequate size of 261.5 ± 25.9 nm, low PDI and ζ of 1.79 ± 0.17 mV (stable for 45 days, at least). The 50% drug release from PBCAnp-polB was 6-7 times slower than the free polB, which favors a prolonged and desired release profile. Concerning in vitro evaluations, polB alone reduced in vitro amastigote infection of macrophages (10 µg/mL) without complete parasite elimination, even at higher concentrations. This behavior limits its future application to adjuvant leishmanicidal therapy or antimicrobial coating of carriers. The nanocarrier PBCAnp also presented leishmanicidal effect and surpassed polB activity; however, no antimicrobial activity was detected. PolB maintained its activity against E. coli, Pseudomonas and Klebsiella, adding antimicrobial properties to the nanoparticles. Thus, this coated drug delivery system, described for the first time, demonstrated antileishmanial and antimicrobial properties. The bactericidal feature helps with concomitant prevention/treatment of secondary infections that worst ulcers induced by cutaneous L. amazonensis, ultimately ending in disfiguring or disabling lesions.

Small cell lung cancer: an overview of the targets

Abstract Lung cancer is the leading cause of cancer deaths worldwide. Small cell lung cancer (SCLC) accounts for approximately 15% of all lung cancer cases. Despite a frequently good response to first-line treatment with chemotherapy and/or radiotherapy, early relapse occurs in the majority of patients and 5-year survival is only about 5%. This histological subtype of lung cancer is strongly associated with tobacco smoking. The behavior of SCLC is unique within solid tumors. Initially, it positively responds to chemotherapy or radiotherapy. However, at relapse, which occurs early in the majority of cases, the tumor is resistant to available therapy and eventually will cause the death of the patient. These results in an overall 5-year survival of approximately 5% for the entire population of patients diagnosed with SCLC. This dismal prognosis has not significantly changed in past years. There is an urgent need for discovery targets to select patients more prone to having a proper response to the treatment, avoiding to reduce their resistance and resulting the increase of overall and progression-free survivals.

Action of bromelain and ficin on horse anti Bothrops sp venom antibodies

The treatment with hyperimmune sera constitute the only specific and effective therapy available against snakebite envenomation, most common in developing countries. Serum quality is an important factor on patient recovery time and in the incidence of death and permanent disability. To date, most sera consist of pepsin digested IgG antibodies harvested from hyperimmune animals. The use of animal derived enzymes, such as pepsin, to digest IgG, constitute a source of adventitious agents and contaminants, such as porcine circovirus. The present study aims to evaluate the use of the plant derived enzymes bromelain and ficin, as an alternative to pepsin. To this purpose, horse serum immunized against Bothrops venoms was purified with caprylic acid and digested with bromelain or ficin. SDS-PAGE results evidence the formation of F(ab)’2 fragments and suggest that a digestion time superior to 8 hours may be required to completely digest the antibodies with bromelain or ficin. F(ab)’2 fragments obtained by digestion with either bromelain or ficin digestion preserved the ability to recognize Bothrops sp. venom in western blotting assays. Therefore, both enzymes are suitable for use in large-scale production, minimizing contamination risks and increasing safety and efficiency of serotherapy treatments.

Action of Bromelain and Ficin on horse anti Bothrops sp venom Antibodies

Abstract The treatment with hyperimmune sera constitute the only specific and effective therapy available against snakebite envenomation, most common in developing countries. Serum quality is an important factor on patient recovery time and in the incidence of death and permanent disability. To date, most sera consist of pepsin digested IgG antibodies harvested from hyperimmune animals. The use of animal derived enzymes, such as pepsin, to digest IgG, constitute a source of adventitious agents and contaminants, such as porcine circovirus. The present study aims to evaluate the use of the plant derived enzymes bromelain and ficin, as an alternative to pepsin. To this purpose, horse serum immunized against Bothrops venoms was purified with caprylic acid and digested with bromelain or ficin. SDS-PAGE results evidence the formation of F(ab)'2 fragments and suggest that a digestion time superior to 8 hours may be required to completely digest the antibodies with bromelain or ficin. F(ab)'2 fragments obtained by digestion with either bromelain or ficin digestion preserved the ability to recognize Bothrops sp. venom in western blotting assays. Therefore, both enzymes are suitable for use in large-scale production, minimizing contamination risks and increasing safety and efficiency of serotherapy treatments.

Production of the first effective hyperimmune equine serum antivenom against Africanized bees.

Victims of massive bee attacks become extremely ill, presenting symptoms ranging from dizziness and headache to acute renal failure and multiple organ failure that can lead to death. Previous attempts to develop specific antivenom to treat these victims have been unsuccessful. We herein report a F(ab)(´)(2)-based antivenom raised in horse as a potential new treatment for victims of multiple bee stings. The final product contains high specific IgG titers and is effective in neutralizing toxic effects, such as hemolysis, cytotoxicity and myotoxicity. The assessment of neutralization was revised and hemolysis, the primary toxic effect of these stings, was fully neutralized in vivo for the first time.

Immunogenicity of a whole-cell pertussis vaccine with low lipopolysaccharide content in infants.

The lack of a clear correlation between the levels of antibody to pertussis antigens and protection against disease lends credence to the possibility that cell-mediated immunity provides primary protection against disease. This phase I comparative trial had the aim of comparing the in vitro cellular immune response and anti-pertussis toxin (anti-PT) immunoglobulin G (IgG) titers induced by a cellular pertussis vaccine with low lipopolysaccharide (LPS) content (wP(low) vaccine) with those induced by the conventional whole-cell pertussis (wP) vaccine. A total of 234 infants were vaccinated at 2, 4, and 6 months with the conventional wP vaccine or the wP(low) vaccine. Proliferation of CD3(+) T cells was evaluated by flow cytometry after 6 days of peripheral blood mononuclear cell culture with stimulation with heat-killed Bordetella pertussis or phytohemagglutinin (PHA). CD3(+), CD4(+), CD8(+), and T-cell receptor gammadelta-positive (gammadelta(+)) cells were identified in the gate of blast lymphocytes. Gamma interferon, tumor necrosis factor alpha, interleukin-4 (IL-4), and IL-10 levels in supernatants and serum anti-PT IgG levels were determined using enzyme-linked immunosorbent assay (ELISA). The net percentage of CD3(+) blasts in cultures with B. pertussis in the group vaccinated with wP was higher than that in the group vaccinated with the wP(low) vaccine (medians of 6.2% for the wP vaccine and 3.9% for the wP(low) vaccine; P = 0.029). The frequencies of proliferating CD4(+), CD8(+), and gammadelta(+) cells, cytokine concentrations in supernatants, and the geometric mean titers of anti-PT IgG were similar for the two vaccination groups. There was a significant difference between the T-cell subpopulations for B. pertussis and PHA cultures, with a higher percentage of gammadelta(+) cells in the B. pertussis cultures (P < 0.001). The overall data did suggest that wP vaccination resulted in modestly better specific CD3(+) cell proliferation, and gammadelta(+) cell expansions were similar with the two vaccines.

Biological and physicochemical stability of ceftazidime and aminophylline on glucose parenteral solution

Ceftazidime is a broad spectrum antibiotic administered mainly by the parenteral route, and it is especially effective against Pseudomonas aeruginosa. The period of time in which serum levels exceed the Minimum Inhibitory Concentration (MIC) is an important pharmacodynamic parameter for its efficacy. One of the forms to extend this period is to administer the antibiotic by continuous infusion, after prior dilution in a Parenteral Solution (PS). The present work assessed the stability of ceftazidime in 5% glucose PS for 24 hours, combined or not with aminophylline, through High Performance Liquid Chromatography (HPLC). The physicochemical evaluation was accompanied by in vitro antimicrobial activity compared MIC test in the 24-hour period. Escherichia coli and Pseudomonas aeruginosa were the microorganisms chosen for the MIC comparison. The HPLC analysis confirmed ceftazidime and aminophylline individual stability on PS, while the MIC values were slightly higher than the mean described in the literature. When both drugs were associated in the same PS, the ceftazidime concentration by HPLC decreased 25% after 24 hours. Not only did the MIC values show high loss of antibiotic activity within the same period, but also altered MIC values immediately after the preparation, which was not detected by HPLC. Our results indicate that this drug combination is not compatible, even if used right away, and that PS might not be the best vehicle for ceftazidime, emphasizing the importance of the MIC evaluation for drug interactions.

Ceftazidima é um antimicrobiano administrado por via parenteral, que apresenta amplo espectro de ação, principalmente contra Pseudomonas aeruginosa. O tempo em que a concentração sérica de ceftazidima permanece acima da concentração mínima inibitória (MIC) é um importante parâmetro farmacodinâmico para a determinação da eficácia antimicrobiana e pode ser potencializado através da utilização de infusão contínua em soluções parenterais (PS). Este artigo visa a avaliar a estabilidade da ceftazidima em solução de glicose 5%, na presença e na ausência do fármaco aminofilina, através de cromatografia líquida de alta eficiência HPLC e MIC durante o período de 24 horas. Os microorganismos selecionados para a determinação do MIC foram Escherichia coli e Pseudomonas aeruginosa. Os ensaios em cromatógrafo líquido confirmaram a estabilidade dos fármacos ceftazidima e aminofilina quando são individualmente associados em PS, enquanto os valores de MIC ficaram maiores que os valores encontrados na literatura. Quando ambos os fármacos foram associados na mesma solução parenteral a concentração de ceftazidima obtida por HPLC diminuiu 25% depois de 24 horas. Os valores de MIC mostraram maior decaimento da atividade antimicrobiana neste mesmo período e também valores de MIC alterados nas soluções preparadas no tempo zero, decaimento este que não foi detectado em HPLC. Os resultados indicaram incompatibilidade na associação dos fármacos em PS, enfatizando a importância dos resultados de MIC para interações de fármacos.

Assessment of the viral safety of antivenoms fractionated from equine plasma.

Antivenoms are preparations of intact or fragmented (F(ab')2 or Fab) immunoglobulin G (IgG) used in human medicine to treat the severe envenomings resulting from the bites and stings of various animals, such as snakes, spiders, scorpions, or marine animals, or from the contact with poisonous plants. They are obtained by fractionating plasma collected from immunized horses or, less frequently, sheep. Manufacturing processes usually include pepsin digestion at acid pH, papain digestion, ammonium sulphate precipitation, caprylic acid precipitation, heat coagulation and/or chromatography. Most production processes do not have deliberately introduced viral inactivation or removal treatments, but antivenoms have never been found to transmit viruses to humans. Nevertheless, the recent examples of zoonotic diseases highlight the need to perform a careful assessment of the viral safety of antivenoms. This paper reviews the characteristics of equine viruses of antivenoms and discusses the potential of some manufacturing steps to avoid risks of viral contamination. Analysis of production parameters indicate that acid pH treatments and caprylic acid precipitations, which have been validated for the manufacture of some human IgG products, appear to provide the best potential for viral inactivation of antivenoms. As many manufacturers of antivenoms located in developing countries lack the resources to conduct formal viral validation studies, it is hoped that this review will help in the scientific understanding of the viral safety factors of antivenoms, in the controlled implementation of the manufacturing steps with expected impact on viral safety, and in the overall reinforcement of good manufacturing practices of these essential therapeutic products.

Immunogenicity of a Whole-Cell Pertussis Vaccine with Low Lipopolysaccharide Content in Infants

The lack of a clear correlation between the levels of antibody to pertussis antigens and protection against disease lends credence to the possibility that cell-mediated immunity provides primary protection against disease...

CD14 and IL6 polymorphisms are associated with a pro-atherogenic profile in young adults with acute myocardial infarction

Abstract This study investigated the relationship of polymorphismsin genes encoding CD14, IL-6 and TLR4 withmetabolic, inflammatory and endothelial markers in youngadults with acute myocardial infarction (AMI). Glucose,lipids, nitrate and inflammatory markers, flow mediatedvasodilatation (FMV) and flow mediated by nitrate (FMN)were evaluated in 102 AMI and 108 non-AMI (controlgroup) young individuals (% years). CD14 -260C[T(rs2569190), IL6 -174G[C (rs1800795) and TLR4c.896A[G (rs4986790) and TLR4 c.1196C[T (rs4986791)polymorphisms were analyzed by PCR–RFLP. Minor allelefrequencies of CD14, IL6 and TLR4 polymorphisms weresimilar between AMI and control groups (p[0.05). In AMIgroup, individuals carrying IL6 -174CC genotype hadhigher serum triglycerides, VLDL cholesterol and glucosecompared to the IL6 -174GG/GC genotype carriers(p84 2013-11-12

Effect of beta-propiolactone treatment on the complement activation mediated by equine antisera

A reducao da ativacao do complemento atraves de uma alteracao do fragmento Fc das imunoglobulinas pela beta-propiolactona foi obtida em soros hiperimunes equinos antivirus rabico, venenos Bothrops e toxina difterica. Os resultados foram avaliados por teste de anafilaxia em cobaias, e comparados com aqueles obtidos com os mesmos soros purificados por precipitacao salina (sulfato de amonio), seguidos ou nao por digestao enzimatica com pepsina. Os niveis de pureza proteica foram para o soro antibotropico de 184,5 mg/g e 488,5 mg/g tratado pela beta-propiolactona e digeridos pela pepsina, respectivamente...(au)

Produçäo de anticorpos antiveneno total de Crotalus durissus terrificus em cavalos por fosfolipase A2 / Antigens antivenom's total production of Crotalus durissus terrificus in horses by phospholipase A2

Fosfolipases A2 purificada de veneno de Crotalus durissus terrificus ou veneno integral foram usados, como antígenos, para imunizar cavalos e burros. A imunizaçäo de base foi feita injetando os animais, pela via subcutânea, com 5mg do antígeno emulsionado em adjuvante de Freund completo. Quatro meses depois, os animais foram reimunizados injetando-se os antígenos correspondentes, dissolvidos em salina, ao redor dos granulomas resultantes da imunizaçäo primária. Esta injeçäo foi repetida por mais cinco vezes a intervalos de uma semana..

Cross - reactivity of horse monovalent antivenoms to venoms of ten bothrops species

Horses were immunized with B. alternatus, B. atrox, B. cotiara, B. erythromelas, B. insularis, B. jararaca, B. jararacussu, B, moojeni, B. neuwiedi and B. pradoi venoms. Antibodies recognizing the venom antigenic components were either immunochemically detected by the precipattion methods or biologically by the assays measuring the venomns indirect hemolytic and lethal toxic activities . Specific and cross - reacting antivenoms.Modifications in the serum electrophoretic patterns characterized by a reduction of the albumin peak and by a correspondent increase of the y-globulins with a pattent or no modification of the α or β globulins were found in these sera.

Cavalos foram imunizados com veneno de B. alternatus, B. atrox, B. cotiara, B. erythromelas, B. insularis, B. jararaca, B. jararacussu, B, moojeni, B. neuwiedi and B. pradoi. Anticorpos específicos para componentes antigênicos dos venenos foram detectados pelos métodos imuno- enzimático, dupla-difusão e precipitação quantitativa enquanto que os anticorpos neutralizantes foram analisados pelos métodos da hemólise indireta em placas e pela neutralização de seus efeitos letais. Anticorpos, tanto específicos como dando reações cruzadas com venenos botrópicos foram encontrados em todos os dez soros monovalentes. Modificações nos padrões eletroforéticos, caracterizadas por uma redução no pico da albumina e por um correspondente aumento das γ. globulinas com modificações ora acentuadas ora pouco perceptíveis nas frações das α e β globolinas, foram detectadas todos esses soros.

Veneno de Bothrops jararaca modificado com PEG (monometoxipolietileno glicol) para elaboração de um adjuvante complexado à partícula antigênica / Bothrops jararaca venon modified for elaboration of complexed adjuvant to the antigenic particule with (monomethoxypolyethylene glycol (mPEG)

Enzimas, biofármacos e produtos de origem biológica de modo geral sempre foram obstáculos para o uso terapêutico. Isto em função dos riscos de reação adversa que poderiam ocasionar bem como a indução de uma resposta imunológica desejável ou não. A utilização do monometoxipolietileno glicol (mPEG) conjugado a proteínas e enzimas trouxe novas perspectivas de utilização de substâncias naturais como substâncias farmacológicas ativas, pois o fato de estarem conjugadas a um polímero inerte atribui uma nova estrutura físico-química a essas substâncias, diminuindo consideravelmente os riscos de anafilaxia e a biodegradação por formação de complexos antígeno-anticorpo...