RESUMO
This study aimed to assess the formation of nevirapine (NVP) co-amorphs systems (CAM) with different co-formers (lamivudine-3TC, citric acid-CAc, and urea) through combined screening techniques as computational and thermal studies, solubility studies; in addition to develop and characterize suitable NVP-CAM. NVP-CAM were obtained using the quench-cooling method, and characterized by differential scanning calorimetry (DSC), X-ray diffractometry (XRD), Fourier Transform Infrared Spectroscopy (FTIR), and polarized light microscopy (PLM), in addition to in vitro dissolution in pH 6.8. The screening results indicated intermolecular interactions occurring between NVP and 3TC; NVP and CAc, where shifts in the melting temperature of NVP were verified. The presence of CAc impacted the NVP equilibrium solubility, due to hydrogen bonds. DSC thermograms evidenced the reduction and shifting of the endothermic peaks of NVP in the presence of its co-formers, suggesting partial miscibility of the compounds. Amorphization was proven by XRD and PLM assays. In vitro dissolution study exhibited a significant increase in solubility and dissolution efficiency of NVP-CAM compared to free NVP. Combined use of screening studies was useful for the development of stable and amorphous NVP-CAM, with increased NVP solubility, making CAM promising systems for combined antiretroviral therapy.
Assuntos
Varredura Diferencial de Calorimetria , Química Farmacêutica , Nevirapina , Solubilidade , Difração de Raios X , Nevirapina/química , Varredura Diferencial de Calorimetria/métodos , Difração de Raios X/métodos , Química Farmacêutica/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Composição de Medicamentos/métodos , Lamivudina/química , Ligação de Hidrogênio , Fármacos Anti-HIV/químicaRESUMO
INTRODUCTION: Human Immunodeficiency Virus (HIV) infection is still a major global problem, whose drug treatment consists of prophylactic prevention and antiretroviral combination therapy for better pharmacological efficacy and control of the circulating virus. However, there are still pharmacological problems that need to be overcome, such as low aqueous solubility of drugs, toxicity, and low patient adherence. Drug delivery technologies can be used to overcome these barriers. OBJECTIVE: This review summarized the latest drug delivery systems for HIV treatment. Initially, an overview of the current therapy was presented, along with the problems it presents. Then, the latest drug delivery systems used to overcome the challenges imposed in conventional HIV therapy were discussed. CONCLUSION: This review examines innovative approaches for HIV treatment, where various drug delivery systems have shown significant advantages, such as high drug encapsulation, improved solubility, and enhanced bioavailability both in vitro and in vivo. Strategies like cyclodextrins, solid dispersions, microneedles, and nanoparticles are explored to address challenges in drug solubility, bioavailability, and administration routes. Despite progress, obstacles like limited clinical trials and industrial scalability hinder the widespread adoption of these formulations, emphasizing the need for further research and collaboration to optimize and ensure accessibility of innovative HIV therapies, mainly in regions where access to HIV treatment is scarce and remains a challenge.
RESUMO
Asparagine is a non-essential amino acid crucial for protein biosynthesis and function, and therefore cell maintenance and growth. Furthermore, this amino acid has an important role in regulating several metabolic pathways, such as tricarboxylic acid cycle and the urea cycle. When compared to normal cells, tumor cells typically present a higher demand for asparagine, making it a compelling target for therapy. In this review article, we investigate different facets of asparagine bioavailability intricate role in malignant tumors raised from solid organs. We take a comprehensive look at asparagine synthetase expression and regulation in cancer, including the impact on tumor growth and metastasis. Moreover, we explore asparagine depletion through L-asparaginase as a potential therapeutic method for aggressive solid tumors, approaching different formulations of the enzyme and combinatory therapies. In summary, here we delve into studies about endogenous and exogenous asparagine availability in solid cancers, analyzing therapeutic implications and future challenges.
Assuntos
Asparagina , Aspartato-Amônia Ligase , Neoplasias , Humanos , Asparagina/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/tratamento farmacológico , Aspartato-Amônia Ligase/metabolismo , Aspartato-Amônia Ligase/genética , Asparaginase/uso terapêutico , AnimaisRESUMO
In the current biopharmaceutical scenario, constant bioprocess monitoring is crucial for the quality and integrity of final products. Thus, process analytical techniques, such as those based on Raman spectroscopy, have been used as multiparameter tracking methods in pharma bioprocesses, which can be combined with chemometric tools, like Partial Least Squares (PLS) and Artificial Neural Networks (ANN). In some cases, applying spectra pre-processing techniques before modeling can improve the accuracy of chemometric model fittings to observed values. One of the biological applications of these techniques could have as a target the virus-like particles (VLP), a vaccine production platform for viral diseases. A disease that has drawn attention in recent years is Zika, with large-scale production sometimes challenging without an appropriate monitoring approach. This work aimed to define global models for Zika VLP upstream production monitoring with Raman considering different laser intensities (200 mW and 495 mW), sample clarification (with or without cells), spectra pre-processing approaches, and PLS and ANN modeling techniques. Six experiments were performed in a benchtop bioreactor to collect the Raman spectral and biochemical datasets for modeling calibration. The best models generated presented a mean absolute error and mean relative error respectively of 3.46 × 105 cell/mL and 35 % for viable cell density (Xv); 4.1 % and 5 % for cell viability (CV); 0.245 g/L and 3 % for glucose (Glc); 0.006 g/L and 18 % for lactate (Lac); 0.115 g/L and 26 % for glutamine (Gln); 0.132 g/L and 18 % for glutamate (Glu); 0.0029 g/L and 3 % for ammonium (NH4+); and 0.0103 g/L and 2 % for potassium (K+). Sample without conditioning (with cells) improved the models' adequacy, except for Glutamine. ANN better predicted CV, Gln, Glu, and K+, while Xv, Glc, Lac, and NH4+ presented no statistical difference between the chemometric tools. For most of the assessed experimental parameters, there was no statistical need for spectra pre-filtering, for which the models based on the raw spectra were selected as the best ones. Laser intensity impacts quality model predictions in some parameters, Xv, Gln, and K+ had a better performance with 200 mW of intensity (for PLS, ANN, and ANN, respectively), for CV the 495 mW laser intensity was better (for PLS), and for the other biochemical variables, the use of 200 or 495 mW did not impact model fitting adequacy.
RESUMO
Viral infections have been the cause of high mortality rates throughout different periods in history. Over the last two decades, outbreaks caused by zoonotic diseases and transmitted by arboviruses have had a significant impact on human health. The emergence of viral infections in different parts of the world encourages the search for new inputs to fight pathologies of viral origin. Antibodies represent the predominant class of new drugs developed in recent years and approved for the treatment of various human diseases, including cancer, autoimmune and infectious diseases. A promising group of antibodies are single-domain antibodies derived from camelid heavy chain immunoglobulins, or VHHs, are biomolecules with nanometric dimensions and unique pharmaceutical and biophysical properties that can be used in the diagnosis and immunotherapy of viral infections. For viral neutralization to occur, VHHs can act in different stages of the viral cycle, including the actual inhibition of infection, to hindering viral replication or assembly. This review article addresses advances involving the use of VHHs in therapeutic propositions aimed to battle different viruses that affect human health.
Assuntos
Antivirais , Anticorpos de Domínio Único , Viroses , Anticorpos de Domínio Único/uso terapêutico , Animais , Camelidae/metabolismo , Antivirais/uso terapêutico , Terapia de Alvo Molecular , Viroses/tratamento farmacológico , Viroses/virologia , Humanos , Vírus/classificaçãoRESUMO
L-asparaginase (ASNase) is an efficient inhibitor of tumor development, used in chemotherapy sessions against acute lymphoblastic leukemia (ALL) tumor cells; its use results in 80% complete remission of the disease in treated patients. Saccharomyces cerevisiae's L-asparaginase II (ScASNaseII) has a high potential to substitute bacteria ASNase in patients that developed hypersensitivity, but the endogenous production of it results in hypermannosylated immunogenic enzyme. Here we describe the genetic process to acquire the ScASNaseII expressed in the extracellular medium. Our strategy involved a fusion of mature sequence of protein codified by ASP3 (amino acids 26-362) with the secretion signal sequence of Pichia pastoris acid phosphatase enzyme; in addition, this DNA construction was integrated in P. pastoris Glycoswitch® strain genome, which has the cellular machinery to express and secrete high quantity of enzymes with humanized glycosylation. Our data show that the DNA construction and strain employed can express extracellular asparaginase with specific activity of 218.2 IU mg-1. The resultant enzyme is 40% more stable than commercially available Escherichia coli's ASNase (EcASNaseII) when incubated with human serum. In addition, ScASNaseII presents 50% lower cross-reaction with anti-ASNase antibody produced against EcASNaseII when compared with ASNase from Dickeya chrysanthemi.
Assuntos
Antineoplásicos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Saccharomyces , Humanos , Asparaginase/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Antineoplásicos/farmacologiaRESUMO
The oral route is the preferred way of drug administration for most drugs, whose treatment success is directly related to the compound intestinal absorption. This absorption process, in its turn, is influenced by several factors impacting the drug bioavailability, which is extremely dependent on the maximum solubility and permeability. However, optimizing these last two factors, without chemical structural modification, is challenging. Although poly(amidoamine) dendrimers (PAMAM) are an innovative and promising strategy as drug delivery compounds, there are few studies that determine the permeability and solubility of PAMAM-drugs derivatives. Considering this scenario, this paper aimed to carry out a literature review of the last five years concerning biopharmaceutical characterizations of dendrimer delivery systems. In vitro methodologies, such as the Parallel artificial membrane permeability assay (PAMPA) (non-cellular based model) and Caco-2 cells (cellular based model), used for the permeability evaluation in the early stages of drug discovery proved to be the most promising methodologies. As a result, we discussed, for instance, that through the usage of PAMPA it was possible to evaluate the higher capacity for transdermal delivery of DNA of TAT-conjugated PAMAM, when in comparison with unmodified PAMAM dendrimer with a P<0.05. We also presented the importance of choosing the best methods of biopharmaceutical characterization, which will be essential to guarantee the efficacy and safety of the drug candidate.
Assuntos
Produtos Biológicos , Dendrímeros , Células CACO-2 , Dendrímeros/química , Humanos , Membranas ArtificiaisRESUMO
The depth and versatility of siRNA technologies enable their use in disease targets that are undruggable by small molecules or that seek to achieve a refined turn-off of the genes for any therapeutic area. Major extracellular barriers are enzymatic degradation of siRNAs by serum endonucleases and RNAases, renal clearance of the siRNA delivery system, the impermeability of biological membranes for siRNA, activation of the immune system, plasma protein sequestration, and capillary endothelium crossing. To overcome the intrinsic difficulties of the use of siRNA molecules, therapeutic applications require nanometric delivery carriers aiming to protect double-strands and deliver molecules to target cells. This review discusses the history of siRNAs, siRNA design, and delivery strategies, with a focus on progress made regarding siRNA molecules in clinical trials and how siRNA has become a valuable asset for biopharmaceutical companies.
RESUMO
For a drug to excerpt pharmacological action after oral intake, it first needs to be released from the formulation, get into solution (dissolve), be absorbed, and reach the systemic circulation. Since only solubilized drugs can be absorbed, and thus have therapeutic effect, the understanding of the dissolution and drug release processes of a drug product is of primary importance. Such understanding allows a robust formulation development with an ideal in vivo performance. In order to meet set standards, the performance assessment of oral drug products, such as dissolution testing, often applies conditions that are not reflective of the in vivo environment. The use of non-physiologically relevant dissolution method during the drug product development phase can be misleading and give poor mechanistic understanding of the in vivo dissolution process. Hence, we hypothesized that applying physiologically relevant conditions to the dissolution test would result in more accurate in vivo predictability for a robust and precise development process. Since the buffering system in the intestinal lumen operates at low molarity values, phosphate buffer at low buffer capacity was used as a first approach to an in vivo relevant parameter. Furthermore, a biphasic system was used, that is, the low buffer capacity medium was paired with an organic layer (n-octanol) to mimic the concurrent drug absorption that happens with the in vivo dissolution. Both poorly and highly soluble drugs in immediate release formulations (ibuprofen and metronidazole, respectively) were tested in this set-up to assess the dissolution in the aqueous medium and the partitioning to the organic phase. Additionally, enteric coated formulations were tested in bicarbonate buffer at the in vivo reported molarities values to assess the impact of buffer species on drug dissolution. The evaluated parameters were the buffer system (bicarbonate buffer vs. phosphate buffer), buffer capacity and medium pH. In all approaches, dissolution was also carried out in compendial buffer for comparison purposes. Our results demonstrate that the USP-recommended dissolution method greatly lacked discriminatory power, whereas low buffer capacity media discriminated between manufacturing methods. The use of an absorptive phase in the biphasic dissolution test assisted in controlling the medium pH due to the drug removal from the aqueous medium. Hence, the applied noncompendial methods were more discriminative to drug formulation differences and manufacturing methods than conventional dissolution conditions. In this study, it was demonstrated how biphasic dissolution and a low buffer capacity can be used to assess drug product performance differences. This can be a valuable approach during the early stages of drug product development for investigating drug release with improved physiological relevance. Similarly, all the enteric coated formulations displayed a fast release in phosphate buffer and complied with the compendial performance specifications. On the other hand, they all had a much slower drug release in bicarbonate buffer and failed the USP acceptance criteria. Also, the nature of the drug (acid vs base) impacted the dissolution behavior in bicarbonate buffer. This study indicates that compendial dissolution test for enteric coated tablets lacks physiological relevance and it needs to be reevaluated. Thus, an in vivo relevant performance method for EC products is needed. Overall, the findings of this thesis comprehensively demonstrates that meaningful differences in performance and accordance to clinical reports were only obtained when physiological relevant conditions were applied. Hence, our results indicate that the central hypothesis was answered positively
Para que um medicamento exerça a ação farmacológica após a ingestão oral, ele primeiro precisa ser liberado da formulação, dissolver, ser absorvido e atingir a circulação sistêmica. Uma vez que apenas medicamentos solubilizados podem ser absorvidos e, assim, ter efeito terapêutico, a compreensão dos processos de dissolução e liberação de um medicamento é de extrema importância. Tal compreensão permite o desenvolvimento de uma formulação robusta com o desempenho in vivo ideal. Para atender aos padrões regulatórios previamente estabelecidos, a avaliação da performance de formulações orais, como por exemplo, o teste de dissolução, frequentemente aplica condições que não refletem o ambiente fisiológico. O uso de métodos de dissolução não fisiologicamente relevante durante a fase de desenvolvimento do medicamento pode gerar resultados equivocados sem uma compreensão mecanistica do processo de dissolução in vivo. Portanto, a hipótese desse trabalho é que a aplicação de condições fisiologicamente relevantes no teste de dissolução resultaria em uma predição mais precisa da dissolução in vivo para um processo de desenvolvimento robusto e preciso. Uma vez que o sistema tampão no lúmen intestinal possui baixa molaridade, o tampão fosfato com baixa capacidade tamponante foi usado como uma primeira abordagem como um meio de dissolução fisiologicamente relevante. Além disso, foi utilizado um sistema bifásico, ou seja, o meio de baixa capacidade tamponante combinado a uma fase orgânica (n-octanol) para imitar a absorção in vivo. Formulações de liberação imediata contendo fármacos de baixa e de alta solubilidade (ibuprofeno e metronidazol, respectivamente) foram testadas no sistema bifásico para avaliar a dissolução no meio aquoso e a partição para a fase orgânica. Ademais, formulações com revestimento entérico foram testadas em tampão bicarbonato nos valores de molaridades fisiológicos para avaliar o impacto da espécie tamponante na dissolução do fármaco. Os parâmetros avaliados foram o sistema tampão (tampão bicarbonato vs. tampão fosfato), capacidade tamponante e pH médio. Em todas as abordagens, a dissolução também foi realizada em tampão farmacopeico para fins de comparação. Nossos resultados demonstraram que o método de dissolução farmacopeico não foi discriminativo, enquanto o meio com menor capacidade tamponante diferenciou entre as formulações obtidas via granulação úmida ou compressão direta. Ademais, a utilização da fase orgânica no teste de dissolução bifásica auxiliou no controle do pH do meio aquoso. Portanto, os métodos não compendiais aplicados foram mais discriminativos do que as condições de dissolução convencionais. Neste estudo, foi demonstrado como a dissolução bifásica e uma baixa capacidade tamponante podem ser usadas para avaliar as diferenças na performance de formulações. Esta pode ser uma abordagem valiosa durante os estágios iniciais do desenvolvimento de medicamentos para investigar a liberação destes sob condições fisiologicamente relevantes. Da mesma forma, todas as formulações com revestimento entérico exibiram uma liberação rápida em tampão de fosfato e atenderam às especificações farmacopeicas. Entretanto, a liberação do fármaco foi muito mais lenta em tampão de bicarbonato e consequentemente não cumpriram com as especificações farmacopeicas. Além disso, a natureza do fármaco (ácido vs. base) impactou o comportamento de dissolução no tampão de bicarbonato. Este estudo indica que o teste de dissolução convencional para comprimidos de liberação retardada não possui relevância fisiológica e precisa ser reavaliado. Portanto, os resultados desta tese demonstram de forma abrangente que diferenças significativas na performance condizentes com relatórios clínicos foram obtidas apenas quando as condições fisiológicas relevantes foram aplicadas. Esses resultados indicam que a hipótese central foi respondida positivamente
Assuntos
Preparações Farmacêuticas/análise , Ações Farmacológicas , Otimização de Processos , Dissolução , Desenvolvimento de Medicamentos/instrumentação , Química Farmacêutica/instrumentação , Composição de Medicamentos , Eficiência , Liberação Controlada de Fármacos , Necessidades e Demandas de Serviços de Saúde/classificação , Concentração de Íons de Hidrogênio , Metronidazol/efeitos adversosRESUMO
Os parâmetros de permeabilidade e solubilidade são fundamentais à absorção oral de fármacos e a partir dessas características, foi criado o Sistema de Classificação Biofarmacêutica, através do qual os fármacos são divididos em quatro classes. Atualmente, para a determinação da solubilidade de um fármaco, existem diversos métodos padronizados por agências regulatórias, no entanto, para a determinação da permeabilidade, os ensaios são passíveis de diversas variações em sua execução, diminuindo a confiabilidade dos resultados obtidos e impossibilitando a comparação dos mesmos quando realizados com técnicas diferentes umas das outras. O objetivo do presente trabalho é avaliar as variáveis experimentais do modelo do saco intestinal que podem influenciar nos resultados de permeabilidade aparente de fármacos e na viabilidade do tecido. O presente estudo foi aprovado pelo Comitê de Ética no Uso de Animais da FCF-USP (109.2018-P574). Foram utilizados 33 Rattus norvegicus da linhagem Wistar, machos, jovens adultos, com peso entre 200 g e 300 g. Para realização do procedimento, cada animal permaneceu em jejum por cerca de quatro horas e após adequada anestesia a porção do jejuno do intestino delgado foi retirada e dividida em seis segmentos de aproximadamente 8,5cm cada. Foram realizados experimentos com e sem inversão do saco intestinal, submetidos a diferentes tempos de banho de gelo após sua ressecção, na presença ou ausência de inibidor da glicoproteína-P (verapamil). Os fármacos naproxeno e famotidina foram empregados como marcadores de alta e baixa permeabilidade, respectivamente. A losartana foi utilizada como substrato da glicoproteína P. Cada um dos sacos intestinais foi colocado em um tubo de ensaio contendo tampão Krebs, a 37°C, saturado com gás carbogênio. Para avaliação da integridade e viabilidade dos segmentos intestinais, observou-se a presença de movimentos peristálticos e coletaram-se amostras do meio de incubação nos tempos 0, 30, 45, 60, 90 e 120 minutos para quantificação dos fármacos e de glicose, uma vez que esta é ativamente transportada para a serosa do intestino delgado. Determinou-se a permeabilidade aparente de cada fármaco e as concentrações de glicose nas diferentes condições experimentais, realizou-se planejamento fatorial multinível e os resultados foram analisados por análise variância (ANOVA), seguida de pós-teste de Tukey. Observou-se que as variáveis experimentais interferiram de forma significativa na viabilidade tecidual e na permeabilidade aparente dos fármacos. Não foram observadas diferenças significativas da permeabilidade de fármacos nos diferentes segmentos do jejuno. A glicose mostrou-se um bom marcador de viabilidade tecidual e foi constatado que a presença ou ausência de movimentos peristálticos não está relacionada diretamente com a viabilidade do tecido. Uma vez que foram constatadas tantas interferências nos resultados, é imprescindível que os procedimentos experimentais sejam padronizados, para que os resultados apresentem menor variabilidade e possam ser comparados entre si
The permeability and solubility parameters are fundamental to the oral absorption of drugs and from these characteristics, the Biopharmaceutical Classification System was created, through which drugs are divided into four classes. Currently, for the determination of the solubility of a drug, there are several methods standardized by regulatory agencies, however, for the determination of permeability, the tests are subject to several variations in their execution, reducing the reliability of the results obtained and making it impossible to compare the results obtained. same when performed with different techniques. The aim of this study is to evaluate if different experimental conditions can influence the results of apparent drug permeability and tissue viability on gut sac model. The present study was approved by the Ethics Committee for the Use of Animals of FCF-USP (109.2018-P574). Thirty-three male, young adult Rattus norvegicus were used, weighing between 200 g and 300 g. To perform the procedure, each animal fasted for about four hours and after adequate anesthesia, the portion of the jejunum of the small intestine was removed and divided into six segments of approximately 8.5 cm each. Experiments were performed with and without inversion of the gut sac, submitted to different times of ice bath after its resection, in the presence or absence of a P-glycoprotein inhibitor (verapamil). The drugs naproxen and famotidine were used as markers of high and low permeability, respectively. Losartan was used as a substrate for P-glycoprotein. Each of the gut sacs was placed in a test tube containing Krebs buffer, at 37°C, saturated with carbogen gas. To evaluate the integrity and viability of the intestinal segments, the presence of peristaltic movements was observed and samples of the incubation medium were collected at 0, 30, 45, 60, 90 and 120 minutes for quantification of drugs and glucose, as it is actively transported to the serosa of the small intestine. The apparent permeability of each drug and the glucose concentrations were determined under different experimental conditions, multilevel factorial design was performed and the results were analyzed by analysis of variance (ANOVA), followed by Tukey's post-test. It was observed that the experimental variables significantly interfered in the tissue viability and in the apparent permeability of the drugs. No significant differences in drug permeability were observed in the different segments of the jejunum. Glucose proved to be a good marker of tissue viability and it was found that the presence or absence of peristaltic movements is not directly related to tissue viability. Since so many interferences were found in the results, it is essential that the experimental procedures be standardized, so that the results show less variability and can be compared between different authors
Assuntos
Animais , Masculino , Ratos , Permeabilidade , Solubilidade , Biofarmácia/instrumentação , Preparações Farmacêuticas/análise , Intestino Delgado/metabolismo , Métodos , Padrões de Referência , Análise de Variância , Jejum/efeitos adversos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos adversos , Absorção , Jejuno/anormalidadesRESUMO
Biopharmaceutical production is currently a multibillion-dollar industry with high growth perspectives. The research and development of biologically sourced pharmaceuticals are extremely important and a reality in our current healthcare system. Interferon alpha consensus (cIFN) is a non-natural synthetic antiviral molecule that comprises all the most prevalent amino acids of IFN-α into one consensus protein sequence. For clinical use, cIFN is produced in E. coli in the form of inclusion bodies. Here, we describe the use of two solubility tags (Fh8 and DsbC) to improve soluble cIFN production. Furthermore, we analyzed cIFN production in different culture media and temperatures in order to improve biopharmaceutical production. Our results demonstrate that Fh8-cIFN yield was improved when bacteria were cultivated in autoinduction culture medium at 30 °C. After hydrolysis, the recovery of soluble untagged cIFN was 58% from purified Fh8-cIFN molecule, fourfold higher when compared to cIFN recovered from the DsbC-cIFN, which achieved 14% recovery. The biological activity of cIFN was tested on in vitro model of antiviral effect against Zika, Mayaro, Chikungunya and SARS-CoV-2 virus infection in susceptible VERO cells. We show, for the first time, that cIFN has a potent activity against these viruses, being very low amounts of the molecule sufficient to inhibit virus multiplication. Thus, this molecule could be used in a clinical approach to treat Arboviruses and SARS-CoV-2.
RESUMO
O acesso a medicamentos pode ser facilitado por programas globais de desenvolvimento farmacêutico, mas há necessidade de que as agencias regulatórias e as indústrias farmoquímicas e farmacêuticas interajam e haja um consenso quanto as exigências para o registro de medicamentos. Este artigo examinou a legislação especifica sobre bi isenção com base no Sistema de Classificação Biofarmacêutica, comparando os cenários do Brasil e do mundo. A partir dessa análise, identificou os entraves a aplicação dos critérios internacionais na realidade regulatória nacional, identificando algumas fragilidades da legislação, como no caso de pro-fármacos. Analisaram-se os critérios de cinco organismos regulatórios (Agência Europeia de Medicamentos, Food and Drug Administration, Health Canada, Conselho Internacional para Harmonização de Requisitos Técnicos para Medicamentos de Uso Humano e Organização Mundial da Saúde) frente aos requisitos da Agência Nacional de Vigilância Sanitária, pontuando as diferenças e o que já se encontra pacificado no tocante a classe do Sistema de Classificação Biofarmacêutica aceita, a comparabilidade entre formulação teste e de referência, solubilidade, permeabilidade intestinal e perfil de dissolução in vitro. Concluiu--se que a Agência Nacional de Vigilância Sanitária deve internalizar os preceitos e critérios da bioisenção com base no Sistema de Classificação Biofarmacêutica por meio de um novo marco regulatório. Além disso, para que esse marco regulatório seja bem-sucedido e produza resultados palpáveis, em especial na área de saúde publica e vigilância sanitária, a agência brasileira deve estar aberta ao diálogo com o setor regulado e as inovações e orientações da academia, sem desviar o foco de sua missão institucional.
Access to medicines can be facilitated by global pharmaceutical development programs, but there is a need for regulatory agencies and the pharmochemical and pharmaceutical industries to interact and to have a consensus on the requirements for drug registration. This article examined the specific legislation concerning to biowaiver based on the Biopharmaceutical Classification System, comparing the scenario in Brazil and worldwide. Based on this analysis, it identified the obstacles to the application of international criteria in the national regulatory reality, identifying some weaknesses of the legislation, as in the case of prodrugs. The criteria of five regulatory bodies (European Medicines Agency, Food and Drug Administration, Health Canada, International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use and World Health Organization) were analyzed in relation to the requirements of the Brazilian Health Regulatory Agency (Anvisa), pointing out the differences and what has already been settled regarding the accepted class of the Biopharmaceutical Classification System, the comparability between test and reference formulations, solubility, intestinal permeability and in vitro dissolution profile. It was concluded that Anvisa should internalize the percepts and criteria of the biowaiver based on Biopharmaceutical Classification System, through a new regulatory framework. Moreover, for this regulatory framework to be successful and produce tangible results, especially in the area of public health and health surveillance, Anvisa must be open to dialogue with the regulated sector and to innovations and guidance from academia, without losing focus of its institutional mission.
RESUMO
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.
Assuntos
Produtos Biológicos , Sistema Livre de Células , Biossíntese de Proteínas , ProteínasRESUMO
Biopharmaceutical production is currently a multibillion-dollar industry with high growth perspectives. The research and development of biologically sourced pharmaceuticals are extremely important and a reality in our current healthcare system. Interferon alpha consensus (cIFN) is a non-natural synthetic antiviral molecule that comprises all the most prevalent amino acids of IFN-α into one consensus protein sequence. For clinical use, cIFN is produced in E. coli in the form of inclusion bodies. Here, we describe the use of two solubility tags (Fh8 and DsbC) to improve soluble cIFN production. Furthermore, we analyzed cIFN production in different culture media and temperatures in order to improve biopharmaceutical production. Our results demonstrate that Fh8-cIFN yield was improved when bacteria were cultivated in autoinduction culture medium at 30 °C. After hydrolysis, the recovery of soluble untagged cIFN was 58% from purified Fh8-cIFN molecule, fourfold higher when compared to cIFN recovered from the DsbC-cIFN, which achieved 14% recovery. The biological activity of cIFN was tested on in vitro model of antiviral effect against Zika, Mayaro, Chikungunya and SARS-CoV-2 virus infection in susceptible VERO cells. We show, for the first time, that cIFN has a potent activity against these viruses, being very low amounts of the molecule sufficient to inhibit virus multiplication. Thus, this molecule could be used in a clinical approach to treat Arboviruses and SARS-CoV-2.
RESUMO
L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.
Assuntos
Antineoplásicos/farmacologia , Asparaginase/farmacologia , Produtos Biológicos/farmacologia , Fenômenos Imunogenéticos/efeitos dos fármacos , Lisossomos/imunologia , Peptídeo Hidrolases/farmacologia , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Asparaginase/química , Asparaginase/uso terapêutico , Produtos Biológicos/química , Produtos Biológicos/uso terapêutico , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Galinhas , Relação Dose-Resposta a Droga , Escherichia coli , Feminino , Cavalos , Humanos , Fenômenos Imunogenéticos/fisiologia , Células Jurkat , Lisossomos/química , Camundongos , Camundongos Endogâmicos BALB C , Peptídeo Hidrolases/química , Peptídeo Hidrolases/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Estrutura Secundária de ProteínaRESUMO
INTRODUCTION: Most drugs used in therapy have low water-solubility, a factor that could reduce their dissolution rate and oral bioavailability, representing a challenge in pharmaceutical development. Nanonization of drugs is the reduction of particles to nanoscale, increasing the surface area and consequently the saturation solubility and dissolution rate and resulting in higher bioavailability. AREAS COVERED: This review provides an overview of the consequences of the poor water-solubility and the main strategies applied to increase the solubility of poorly water-soluble drugs. The relationship between the biopharmaceutical classification system and the solubilization process of the drug is also considered. Finally, it includes how drug nanoparticles and nanocarriers, especially lipid-based nanosystems, can overcome these challenges and which of these approaches are already available on the market. EXPERT OPINION: Due to the growing importance of nanomedicines, especially for applications in poorly water-soluble drugs, it is important to clearly establish the specifications and quality criteria for nanonized drugs to ensure the quality and safety of nanoparticles.
Assuntos
Nanopartículas , Preparações Farmacêuticas/administração & dosagem , Administração Oral , Disponibilidade Biológica , Química Farmacêutica , Portadores de Fármacos/química , Desenvolvimento de Medicamentos , Humanos , Lipídeos/química , Preparações Farmacêuticas/química , Preparações Farmacêuticas/classificação , Solubilidade , Água/químicaRESUMO
Introducción: La evaluación de los factores claves de éxito de la innovación en la industria biotecnológica aplicada a la salud constituye un problema de investigación. Objetivo: Evaluar la innovación en un centro cubano de la biotecnología aplicada a la salud. Métodos: Se aplicó en dos momentos la encuesta de innovación en el Centro de Inmunología Molecular. Los resultados se procesaron con el diagrama de caja, la estadística básica y el análisis de la correlación. Se analizaron artículos científicos que muestran los resultados de la innovación en el centro. Resultados: La encuesta se aplicó en el 2015 y 2018 a 33 miembros del consejo de dirección y a especialistas, identificándose como estrategias genéricas las de innovación, calidad y liderazgo y como objetivos de innovación los de calidad y el mercado. Las fuentes de innovación más importantes se refieren a las actividades de investigación y desarrollo, la producción, la alta dirección y los centros de investigaciones. Los factores que limitan están relacionados con el período largo de rentabilidad de la innovación, el financiamiento y la adquisición de los insumos. Conclusiones: La evaluación de la innovación en el centro de biotecnología corroboró la significación que tiene para la empresa la estrategia de innovación como factor clave, que lo ubica en una posición de excelencia en la industria biofarmacéutica cubana e internacional(AU)
Introduction: The assessment of the key success factors of innovation in the biotechnology industry applied to health constitutes a research problem. Objective: To assess innovation process in a Cuban center of biotechnology applied to health. Methods: It was applied in two moments the survey of innovation in the Molecular Immunology Center. The results were processed by the box plot, basic statistics and analysis of the correlation. Scientific articles that show the results of innovation in the Center were analyzed. Results: The survey was applied in 2015 and 2018 to 33 members of the board of directors and to specialists, and being identified innovation, quality and leadership as generic strategies, and quality and the market as objectives of innovation. The most important sources of innovation referred to the research and development activities, production, top management and research centers. The limiting factors are related to the long term return of innovation, financing, and the acquisition of supplies. Conclusions: The assessment of innovation in the Biotechnology Center corroborated the significance of the innovation strategy for the company as a key factor, which places it in a position of excellence in the Cuban and international biopharmaceutical industry(AU)
Assuntos
Biofarmácia , Biotecnologia , InvençõesRESUMO
Introducción: La evaluación de los factores claves de éxito de la innovación en la industria biotecnológica aplicada a la salud constituye un problema de investigación. Objetivo: Evaluar la innovación en un centro cubano de la biotecnología aplicada a la salud. Métodos: Se aplicó en dos momentos la encuesta de innovación en el Centro de Inmunología Molecular. Los resultados se procesaron con el diagrama de caja, la estadística básica y el análisis de la correlación. Se analizaron artículos científicos que muestran los resultados de la innovación en el centro. Resultados: La encuesta se aplicó en el 2015 y 2018 a 33 miembros del consejo de dirección y a especialistas, identificándose como estrategias genéricas las de innovación, calidad y liderazgo y como objetivos de innovación los de calidad y el mercado. Las fuentes de innovación más importantes se refieren a las actividades de investigación y desarrollo, la producción, la alta dirección y los centros de investigaciones. Los factores que limitan están relacionados con el período largo de rentabilidad de la innovación, el financiamiento y la adquisición de los insumos. Conclusiones: La evaluación de la innovación en el centro de biotecnología corroboró la significación que tiene para la empresa la estrategia de innovación como factor clave, que lo ubica en una posición de excelencia en la industria biofarmacéutica cubana e internacional(AU)
Introduction: The assessment of the key success factors of innovation in the biotechnology industry applied to health constitutes a research problem. Objective: To assess innovation process in a Cuban center of biotechnology applied to health. Methods: It was applied in two moments the survey of innovation in the Molecular Immunology Center. The results were processed by the box plot, basic statistics and analysis of the correlation. Scientific articles that show the results of innovation in the Center were analyzed. Results: The survey was applied in 2015 and 2018 to 33 members of the board of directors and to specialists, and being identified innovation, quality and leadership as generic strategies, and quality and the market as objectives of innovation. The most important sources of innovation referred to the research and development activities, production, top management and research centers. The limiting factors are related to the long term return of innovation, financing, and the acquisition of supplies. Conclusions: The assessment of innovation in the Biotechnology Center corroborated the significance of the innovation strategy for the company as a key factor, which places it in a position of excellence in the Cuban and international biopharmaceutical industry(AU)
Assuntos
Humanos , Produtos Biológicos , Inquéritos e Questionários , Estatística , Invenções/normasRESUMO
A L-Asparaginase (L-ASNase) de Erwinia chrysathemi (ErA) é uma enzima amplamente utilizada para o tratamento da leucemia linfoblástica aguda (LLA). Embora o seu uso como segunda linha de tratamento para a LLA tenha proporcionado consideráveis benefícios clínicos, reações de hipersensibilidade e rápida depuração plasmática ainda são problemas recorrentes. Ademais, extensivos e custosos processos de produção da ErA são necessários para a obtenção da enzima pura. Com base nesses problemas, o presente trabalho propõe (1) o estudo de viabilidade de expressão da ErA em um sistema de síntese proteica livre de células (SPLC) e (2) a conjugação da proteína em bacteriófagos como ferramenta alternativa para o isolamento e monitoramento da depuração plasmática da ErA. Foram utilizados extratos celulares de Escherichia coli suplementados com solução energética contendo creatina fosfato (CP) como fonte de energia para síntese in vitro de ErA. Para conjugação da ErA a bacteriófagos, o sistema SpyTag/SpyCatcher foi implementado: SpyCatcher foi fusionado à porção N-terminal da ErA e bacteriófagos filamentosos da linhagem M13 e fd foram modificados de modo a expressar SpyTag nas proteínas de capsídeo pIII e pVIII, respectivamente. Em relação ao primeiro objetivo, o sistema de SPLC foi capaz de expressar a ErA com atividade. A proteína foi expressa na fração solúvel e apresentou atividade enzimática significativamente superior em relação à reação controle (7,07 ± 0,68 U/mL vs. 1,83 ± 0,14 U/mL). Tempo necessário para obtenção do extrato celular foi reduzido de 45 para 26 hrs, e sete componentes da solução energética foram removidos da composição original sem implicações negativas na eficiência de expressão da ErA, simplificando desta forma o processo de SPLC. Em relação ao segundo objetivo, ErA fusionada à SpyCatcher (SpyCatcher_ErA) foi conjugada com êxito em bacteriófagos capazes de expressar SpyTag fusionadas na porção N-terminal das proteínas pIII (SpyTag_pIII) e pVIII (SpyTag_pVIII). A porcentagem de formação dos conjugados entre SpyCatcher_ErA e SpyTag_pIII ((ErA)5-pIII) foi de 6% enquanto formação dos conjugados entre SpyCatcher_ErA e SpyTag_pVIII ((ErA)50-pVIII) foi de 46%, valores estes confirmados por atividade enzimática. Solução contendo conjugados foram injetados em camundongos e sequenciados/titulados com êxito. Não houve diferença de depuração plasmática entre (ErA)5-pIII e bacteriófago controle, mas houve maior taxa de eliminação de (ErA)50-pVIII em relação ao mesmo bacteriófago não conjugado à SpyCatcher_ErA. Os resultados aqui apresentados confirmam ser possível expressar ErA com atividade biológica em sistemas de SPLC. Além disso, o sistema de conjugação da ErA a bacteriófagos aqui desenvolvido foi capaz de monitorar a concentração de ErA presente na circulação em função do tempo, tornando-se uma potencial plataforma de desenvolvimento de novas proteoformas da ErA com características clínicas melhoradas
L-Asparaginase (L-ASNase) from Erwinia chrysanthemi (ErA) is a widely used enzyme for treatment of acute lymphoblastic leukemia (ALL). Although its use as a second-line treatment has provided significant clinical benefits, hypersensitivity reactions and a fast clearance rate are recurring L-ASNase-related problems. In addition, extensive and costly production processes are required for the manufacturing of pure ErA. Based on these drawbacks, this current work proposes (1) the study of the use of a cell-free protein synthesis (CFPS) system as a viable platform for the synthesis of ErA and (2) the conjugation of the protein on bacteriophages as an alternative tool for the isolation and monitoring of ErA clearance. Escherichia coli-derived cell extracts supplemented with a creatine phosphate-based energy solution were used to synthesize ErA in vitro. To conjugate ErA on bacteriophages, the SpyTag/SpyCatcher system was implemented: SpyCatcher was fused to the N-terminus of the ErA while filamentous phage strains M13 and fd were engineered in order to display SpyTag on their pIII and pVIII capsid proteins, respectively. Regarding the first goal, the CFPS system was able to express an active ErA. The protein was expressed in the soluble fraction and there presented a significant higher enzymatic activity compared to the control reaction (7.07 ± 0.68 U/mL vs. 1.83 ± 0.14 U/mL). Time required to obtain the cell extract was reduced from 45 to 26 hours, and seven energy solution reagents were removed from the original solution without compromising the efficiency of ErA expression, thus simplifying the CFPS process. With respect to the second goal, ErA fused to SpyCatcher (SpyCatcher_ErA) was sucessfully conjugated on bacteriophages capable of displaying SpyTag fused to the Nterminus of the pIII (SpyTag_pIII) or pVIII (SpyTag_pVIII) proteins. Percentage of conjugate formation between SpyCatcher_ErA and SpyTag_pIII (ErA)5-pIII was 6% whereas conjugate formation between SpyCatcher_ErA and SpyTag_pVIII (ErA)50-pVIII was 46%, values that were confirmed by enzymatic activity. Sample containing conjugates were injected into mice and sucessfully sequenced/titrated. No clearance differences were observed between (ErA)5- pIII and a control bacteriophage, but a higher clearance rate was observed for (ErA)50-pVIII compared to SpyTag_VIII non conjugated to SpyCatcher_ErA. The results here presented confirm the expression of a biologically active ErA from a CFPS system. Besides, the development of a conjugation system capable of linking ErA to bacteriophages could be used as a means to monitor the ErA concentration in the blood as a function of time and also as a potential platform to be used in the development of novel ErA proteoforms with improved clinical properties