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
Over the past few years, there has been a surge in the industrial production of recombinant enzymes from microorganisms due to their catalytic characteristics being highly efficient, selective, and biocompatible. L-asparaginase (L-ASNase) is an enzyme belonging to the class of amidohydrolases that catalyzes the hydrolysis of L-asparagine into L-aspartic acid and ammonia. It has been widely investigated as a biologic agent for its antineoplastic properties in treating acute lymphoblastic leukemia. The demand for L-ASNase is mainly met by the production of recombinant type II L-ASNase from Escherichia coli and Erwinia chrysanthemi. However, the presence of immunogenic proteins in L-ASNase sourced from prokaryotes has been known to result in adverse reactions in patients undergoing treatment. As a result, efforts are being made to explore strategies that can help mitigate the immunogenicity of the drug. This review gives an overview of recent biotechnological breakthroughs in enzyme engineering techniques and technologies used to improve anti-leukemic L-ASNase, taking into account the pharmacological importance of L-ASNase.
Assuntos
Antineoplásicos , Asparaginase , Produtos Biológicos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Antineoplásicos/uso terapêutico , Asparaginase/uso terapêutico , Fatores Biológicos , Produtos Biológicos/uso terapêutico , Escherichia coli/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Engenharia de Proteínas/métodosRESUMO
Acute lymphoblastic leukemia (ALL) is the most common cancer among children worldwide, characterized by an overproduction of undifferentiated lymphoblasts in the bone marrow. The treatment of choice for this disease is the enzyme L-asparaginase (ASNase) from bacterial sources. ASNase hydrolyzes circulating L-asparagine in plasma, leading to starvation of leukemic cells. The ASNase formulations of E. coli and E. chrysanthemi present notorious adverse effects, especially the immunogenicity they generate, which undermine both their effectiveness as drugs and patient safety. In this study, we developed a humanized chimeric enzyme from E. coli L-asparaginase which would reduce the immunological problems associated with current L-asparaginase therapy. For these, the immunogenic epitopes of E. coli L-asparaginase (PDB: 3ECA) were determined and replaced with those of the less immunogenic Homo sapiens asparaginase (PDB:4O0H). The structures were modeled using the Pymol software and the chimeric enzyme was modeled using the SWISS-MODEL service. A humanized chimeric enzyme with four subunits similar to the template structure was obtained, and the presence of asparaginase enzymatic activity was predicted by protein-ligand docking.
Assuntos
Antineoplásicos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Criança , Humanos , Asparaginase/genética , Asparaginase/uso terapêutico , Escherichia coli/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Asparagina , Proteínas Recombinantes de Fusão/uso terapêutico , Antineoplásicos/uso terapêuticoRESUMO
L-Asparaginase is an antileukemic drug long approved for clinical use to treat childhood acute lymphoblastic leukemia, the most common cancer in this population worldwide. However, the efficacy and its use as a drug have been subject to debate due to the variety of adverse effects that patients treated with it present, as well as the prompt elimination in plasma, the need for multiple administrations, and high rates of allergic reactions. For this reason, the search for new, less immunogenic variants has long been the subject of study. This review presents the main aspects of the L-asparaginase enzyme from a structural, pharmacological, and clinical point of view, from the perspective of its use in chemotherapy protocols in conjunction with other drugs in the different treatment phases.
Assuntos
Antineoplásicos , Hipersensibilidade a Drogas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Criança , Asparaginase/uso terapêutico , Asparaginase/efeitos adversos , Antineoplásicos/efeitos adversos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológicoRESUMO
OBJECTIVE: The aim of this study was to analyze and identify documented infections and possible risk factors for Clostridioides difficile infections in children with cancer. METHODS: This is a retrospective case-control study, carried out in a pediatric cancer hospital, covering the years 2016-2019. Matching was performed by age and underlying disease, and for each case, the number of controls varied from 1 to 3. Logistic regression models were used to assess risk factors. RESULTS: We analyzed 63 cases of documented infection by C. difficile and 125 controls. Diarrhea was present in all cases, accompanied by fever higher than 38°C in 52.4% of the patients. Mortality was similar among cases (n=4; 6.3%) and controls (n=6; 4.8%; p=0.7). In all, 71% of patients in the case group and 53% in the control group received broad-spectrum antibiotics prior to the infection. For previous use of vancomycin, the Odds Ratio for C. difficile infection was 5.4 (95% confidence interval [95%CI] 2.3-12.5); for meropenem, 4.41 (95%CI 2.1-9.2); and for cefepime, 2.6 (95%CI 1.3-5.1). For the antineoplastic agents, the Odds Ratio for carboplatin was 2.7 (95%CI 1.2-6.2), melphalan 9.04 (95%CI 1.9-42.3), busulfan 16.7 (95%CI 2.1-134.9), and asparaginase 8.97 (95%CI 1.9-42.9). CONCLUSIONS: C. difficile symptomatic infection in children with cancer was associated with previous hospitalization and the use of common antibiotics in cancer patients, such as vancomycin, meropenem, and cefepime, in the last 3 months. Chemotherapy drugs, such as carboplatin, melphalan, busulfan, and asparaginase, were also risk factors.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Infecção Hospitalar , Neoplasias , Humanos , Criança , Estudos de Casos e Controles , Estudos Retrospectivos , Vancomicina , Cefepima/uso terapêutico , Meropeném , Bussulfano/uso terapêutico , Melfalan/uso terapêutico , Asparaginase/uso terapêutico , Institutos de Câncer , Carboplatina/uso terapêutico , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/induzido quimicamente , Infecção Hospitalar/tratamento farmacológico , Antibacterianos/uso terapêutico , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/induzido quimicamente , Infecções por Clostridium/tratamento farmacológico , Neoplasias/complicações , Fatores de RiscoRESUMO
BACKGROUND: L-asparaginase (L-ASNase) is an essential component of chemotherapy strategies due to its differential action between normal and leukemic cells. Recently, concerns about the efficiency of commercial formulations administered in developing countries have been reported, and available methods have limitations for directly determining the quality of the formulation of the medications. PROCEDURE: We developed a cell-based protocol to analyze the activity of different L-ASNase formulations used in Colombia to induce apoptosis of the NALM-6 cell line after 24, 48, and 72 hours, using flow cytometry. Then we compared results and determined the statistically significant differences. RESULTS: Three statistically different groups, ranging from full to no activity against leukemic cells, using 0.05, 0.5, and 5.0 IU/ml concentrations, were identified. Group 1 (asparaginase codified [ASA]2-4) exhibited very low to no activity against B-cell acute lymphoblastic leukemia (B-ALL) cells. Group 2 (ASA6) exhibited intermediate-level activity, and group 3 (ASA1 and ASA5) exhibited high activity. CONCLUSIONS: Differences found between the therapeutic formulations of L-ASNase distributed in Colombia raise concerns about the quality of the treatment administered to patients in low- and middle-income countries. Therefore, we recommend a preclinical evaluation of formulations of L-ASNase in order to prevent therapeutical impacts on the outcome of ALL patients.
Assuntos
Antineoplásicos , Asparaginase , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Antineoplásicos/uso terapêutico , Asparaginase/uso terapêutico , Linhagem Celular , Colômbia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológicoAssuntos
Apendicite , Leucemia-Linfoma Linfoblástico de Células Precursoras , Criança , Humanos , Asparaginase/uso terapêutico , Metotrexato/uso terapêutico , Estado Terminal , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Protocolos de Quimioterapia Combinada AntineoplásicaRESUMO
The l-asparaginase (l-ASNase) enzyme catalyzes the conversion of the non-essential amino acid l-asparagine into l-aspartic acid and ammonia. Importantly, the l-ASNases are used as a key part of the treatment of acute lymphoblastic leukemia (ALL); however, despite their benefits, they trigger severe side effects because they have their origin in bacterial species (Escherichia coli and Erwinia chrysanthemi). Therefore, one way to solve these side effects is the use of l-ASNases with characteristics similar to those of bacterial types, but from different sources. In this sense, Cavia porcellus l-ASNase (CpA) of mammalian origin is a promising enzyme because it possesses similarities with bacterial species. In this work, the hydrolysis reaction for C. porcellus l-asparaginase was studied from an atomistic point of view. The QM/MM methodology was employed to describe the reaction, from which it was found that the conversion mechanism of l-asparagine into l-aspartic acid occurs in four steps. It was identified that the nucleophilic attack and release of the ammonia group is the rate-limiting step of the reaction. In this step, the nucleophile (Thr19) attacks the substrate (ASN) leading to the formation of a covalent intermediate and release of the leaving group (ammonia). The calculated energy barrier is 18.9 kcal mol-1, at the M06-2X+D3(0)/6-311+G(2d,2p)//CHARMM36 level of theory, which is in agreement with the kinetic data available in the literature, 15.9 kcal mol-1 (derived from the kcat value of 38.6 s-1). These catalytic aspects will hopefully pave the way toward enhanced forms of CpA. Finally, our work emphasizes that computational calculations may enhance the rational design of mutations to improve the catalytic properties of the CpA enzyme.
Assuntos
Asparaginase , Asparagina , Animais , Cobaias/metabolismo , Amônia/química , Asparaginase/genética , Asparaginase/metabolismo , Asparaginase/uso terapêutico , Asparagina/química , Asparagina/genética , Asparagina/metabolismo , Ácido Aspártico , Mamíferos/metabolismo , MutaçãoRESUMO
L-Asparaginase (L-ASNase) is an enzyme applied in the treatment of lymphoid malignancies. However, an innovative L-ASNase with high yield and lower side effects than the commercially available preparations are still a market requirement. Here, a new-engineered Bacillus subtilis strain was evaluated for Aliivibrio fischeri L-ASNase II production, being the bioprocess development and the enzyme characterization studied. The pBS0E plasmid replicative in Bacillus sp and containing PxylA promoter inducible by xylose and its repressive molecule sequence (XylR) was used for the genetic modification. Initially, cultivations were carried out in orbital shaker, and then the process was scaled up to stirred tank bioreactor (STB). After the bioprocess, the cells were recovered and submitted to ultrasound sonication for cells disruption and intracellular enzyme recovery. The enzymatic extract was characterized to assess its biochemical, kinetic and thermal properties using L-Asparagine and L-Glutamine as substrates. The results indicated the potential enzyme production in STB achieving L-ASNase activity up to 1.539 U mL-1. The enzymatic extract showed an optimum pH of 7.5, high L-Asparagine affinity (Km = 1.2275 mmol L-1) and low L-Glutaminase activity (0.568-0.738 U mL-1). In addition, thermal inactivation was analyzed by two different Kinect models to elucidate inactivation mechanisms, low kinetic thermal inactivation constants for 25 ºC and 37 ºC (0.128 and 0.148 h-1, respectively) indicate an elevated stability. The findings herein show that the produced recombinant L-ASNase has potential to be applied for pharmaceutical purposes.
Assuntos
Antineoplásicos , Produtos Biológicos , Aliivibrio fischeri , Antineoplásicos/química , Asparaginase/química , Asparaginase/genética , Asparaginase/uso terapêutico , Asparagina , Bacillus subtilis/genética , Glutaminase , Glutamina , Preparações Farmacêuticas , XiloseRESUMO
ANTECECENTES: Como parte de las funciones de la UFETS, se ha elaborado el presente informe sobre el uso de L-asparaginasa pegilada en el tratamiento de pacientes con leucemia linfoblástica aguda (LLA) y linfoma linfoblástico (LL) que presentan hipersensibilidad a L-asparaginasa nativa. En el Instituto Nacional de Enfermedades Neoplásicas se registran más de 500 casos de linfoma no Hodgkin (LNH) y 90 casos de LL anualmente. La población esperada en el INEN para ambas patologías que requieren asparaginasa pegilada es de 12 pacientes al año. El uso de algún tipo de asparaginasa se encuentra dentro del esquema de inducción ECOG. TECNOLOGÍA: En el presente documento se reporta la evaluación del uso de asparaginasa pegilada en pacientes que presentan hipersensibilidad a asparaginasa nativa. En el INEN se estima una población objetivo de 12 pacientes al año. METODOLOGÍA; Se hizo una búsqueda sistemática de la literatura hasta marzo del 2022, en la cual se encontró un ECA que respondía la pregunta PICO. En este estudio realizado en 76 pacientes con LLA, se observó una menor tasa de respuesta al tratamiento en aquellos con hipersensibilidad a L-asparaginasa nativa que recibieron L-asparaginasa pegilada (38%), en comparación con aquellos sin hipersensibilidad que recibieron L-Asparaginasa pegilada (41%) o Lasparaginasa nativa (47%). Además, los pacientes con hipersensibilidad que recibieron L-asparaginasa pegilada no mostraron mayores niveles de toxicidad que aquellos que recibieron L-asparaginasa nativa. Estos resultados resaltan la potencial utilidad de L-asparaginasa pegilada en el tratamiento antineoplásico de pacientes con hipersensibilidad a L-asparaginasa nativa. Sin embargo, el estudio mostró una baja calidad de evidencia. DISCUSIÓN: Tomando los criterios para un marco de valor de la Health Technology Assessment International (2018) para la toma de decisiones y formulación de la recomendación, se describe: a calidad de la evidencia encontrada es baja. En el ECA se halló que el uso de asparaginasa pegilada en pacientes que presentaron hipersensibilidad por asparaginasa nativa tuvo resultados similares en respuesta completa que en aquellos pacientes que no presentaron hipersensibilidad y continuaron con asparaginasa nativa o asparaginasa pegilada. La calidad de evidencia fue baja, a pesar de que el estudio para el grupo intervención principal (asparaginasa pegilada en pacientes que presentaron hipersensibilidad a la asparaginasa nativa) no recibió aleatorización basal, este presentó características similares a la comparación. El ensayo clínico presentó sesgos, por enmascaramiento de la intervención y solo se encontró un estudio clínico para responder esta pregunta PICO. No se cuenta con estudios de evaluación económica en la región. Sin embargo, en el Reino Unido se realizó una estimación de costos del uso de asparaginasa pegilada. Aquí se proyectó que su uso puede conllevar a ahorro de costos en relación con el uso de asparaginasa nativa. No obstante, se basa en evidencia local del Reino Unido. Se tiene aceptación por parte de los médicos tratantes para continuar el uso de asparaginasa pegilada en pacientes que presentaron hipersensibilidad a la asparaginasa nativa en leucemia linfoblástica aguda y linfoma linfoblástico. Luego de la discusión, el panel concluye que la asparaginasa pegilada es una opción de apoyo en el tratamiento de inducción en pacientes con LLA o LNH que presentan hipersensibilidad a asparaginasa nativa. CONCLUSIONES: La presente evaluación evaluó el uso de asparaginasa pegilada en pacientes que presentaron hipersensibilidad a asparaginasa nativa. La leucemia linfoblástica aguda y el linfoma linfoblástico son el grupo más frecuente de neoplasias hematológicas malignas en población pediátrica. Dentro del esquema de inducción, se encuentra el uso de algún tipo de asparaginasa. Se realizó una búsqueda sistemática hasta marzo del 2022, encontrando 1 ECA que respondía la pregunta PICO. El ECA encontró que el uso de asparaginasa pegilada en pacientes que presentaron hipersensibilidad por asparaginasa nativa, tuvo resultados similares en respuesta completa de aquellos pacientes que no presentaron hipersensibilidad y continuaron con asparaginasa nativa o asparaginasa pegilada. La calidad de evidencia fue baja. La asparaginasa pegilada se encuentra disponible en el Perú, y el costo de cada ampolla (750 UI/ml) es de S/ 5 500.00. Revisamos un estudio de costo-efectividad realizado en Reino Unido, donde compararon el uso de asparaginasa pegilada versus asparaginasa nativa como primera línea. Se evidenció que el uso de asparaginasa pegilada aumentaba costos del medicamento, pero reducía costos de estancia hospitalaria y de otros gastos sanitarios comparado con asparaginasa nativa, llevando a un ahorro de £ 4741 libras (Colocar conversión en dólares) (para el sistema sanitario de Reino Unido). En pacientes con leucemia linfoblástica aguda y linfoma linfoblástico se sugiere el uso de asparaginasa pegilada en pacientes que presentan hipersensibilidad al uso de asparaginasa nativa.