RESUMO
PURPOSE: The aim of this study was to determine the potential of magnetic resonance imaging to evaluate the biodistribution of exogenous iron within 24 h after one single injection of Venofer® (iron sucrose). METHODS: Venofer® was evaluated in vitro for its ability to generate contrast in MR images. Subsequently, iron disposition was assessed in rats with MRI, in vivo up to 3 h and post mortem at 24 h after injection of Venofer®, at doses of 10- and 40 mg/kg body weight (n = 2 × 4), or saline (n = 4). RESULTS: Within 10-20 min after injection of Venofer®, transverse relaxation rates (R2) clearly increased, representative of a local increase in iron concentration, in liver, spleen and kidney, including the kidney medulla and cortex. In liver and spleen R2 values remained elevated up to 3 h post injection, while the initial R2 increase in the kidney was followed by gradual decrease towards baseline levels. Bone marrow and muscle tissue did not show significant increases in R2 values. Whole-body post mortem MRI showed most prominent iron accumulation in the liver and spleen at 24 h post injection, which corroborated the in vivo results. CONCLUSIONS: MR imaging is a powerful imaging modality for non-invasive assessment of iron distribution in organs. It is recommended to use this whole-body imaging approach complementary to other techniques that allow quantification of iron disposition at a (sub)cellular level.
Assuntos
Óxido de Ferro Sacarado/farmacocinética , Hematínicos/farmacocinética , Imageamento por Ressonância Magnética , Imagem Corporal Total , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Óxido de Ferro Sacarado/administração & dosagem , Meia-Vida , Hematínicos/administração & dosagem , Injeções Intravenosas , Rim/diagnóstico por imagem , Rim/metabolismo , Fígado/diagnóstico por imagem , Fígado/metabolismo , Masculino , Modelos Animais , Ratos , Ratos Sprague-Dawley , Baço/diagnóstico por imagem , Baço/metabolismo , Distribuição TecidualRESUMO
PURPOSE: The high prevalence of pure red cell aplasia in Thailand has been associated with the sharp increase in number of recombinant human erythropoietin (rhEPO) copy products, based on a classical generic regulatory pathway, which have entered the market. This study aims to assess the quality of rhEPO copy products being used in Thailand. METHODS: Twelve rhEPO copy products were purchased from pharmacies in Thailand, shipped under controlled cold chain conditions to the Netherlands and characterized using (1) high performance size-exclusion chromatography, (2) asymmetrical flow field-flow fractionation, (3) sodium dodecyl sulfate polyacrylamide gel electrophoresis in combination with (4) Western blotting and additionally tested for (5) host cell protein impurities as well as (6) endotoxin contamination. RESULTS: Some of the tested rhEPO copy products showed high aggregate levels and contained a substantial amount of protein fragments. Also, one of rhEPO copy products had a high endotoxin level, exceeding the FDA limit. CONCLUSIONS: Our observations show that some of the tested copy products on the Thai market differ significantly from the originator rhEPO product, Epogen®. This comparison study supports a link between the quality attributes of copy rhEPO products and their immunogenicity.
Assuntos
Eritropoetina/farmacologia , Western Blotting , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Eritropoetina/isolamento & purificação , Fracionamento por Campo e Fluxo , Humanos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , TailândiaRESUMO
PURPOSE: The immunological processes underlying immunogenicity of recombinant human therapeutics are poorly understood. Using an immune tolerant mouse model we previously demonstrated that aggregates are a major trigger of the antidrug antibody (ADA) response against recombinant human interferon beta (rhIFNß) products including Betaferon®, and that immunological memory seems to be lacking after a rechallenge with non-aggregated rhIFNß. The apparent absence of immunological memory indicates a CD4+ T-cell independent (Tind) immune response underlying ADA formation against Betaferon®. This hypothesis was tested. METHODS: Using the immune tolerant mouse model we first validated that rechallenge with highly aggregated rhIFNß (Betaferon®) does not lead to a subsequent fast increase in ADA titers, suggesting a lack of immunological memory. Next we assessed whether Betaferon® could act as Tind antigen by inactivation of marginal zone (MZ) B-cells during treatment. MZ B-cells are major effector cells involved in a Tind immune response. In a following experiment we depleted the mice from CD4+ T-cells to test their involvement in the ADA response against Betaferon®. RESULTS: Inactivation of MZ B-cells at the start of Betaferon® treatment drastically lowered ADA levels, suggesting a Tind immune response. However, persistent depletion of CD4+ T-cells before and during Betaferon® treatment abolished the ADA response in almost all mice. CONCLUSION: The immune response against rhIFNß in immune tolerant mice is neither a T-cell independent nor a classical T-cell dependent immune response. Further studies are needed to confirm absence of immunological memory (cells).
Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Tolerância Imunológica/efeitos dos fármacos , Imunoglobulina G/biossíntese , Memória Imunológica/efeitos dos fármacos , Interferon beta/antagonistas & inibidores , Interferon beta/imunologia , Tecido Linfoide/imunologia , Animais , Subpopulações de Linfócitos B/classificação , Subpopulações de Linfócitos B/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Feminino , Interferon beta-1b , Interferon beta/administração & dosagem , Cooperação Linfocítica/efeitos dos fármacos , Cooperação Linfocítica/imunologia , Depleção Linfocítica , Tecido Linfoide/citologia , Tecido Linfoide/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/imunologiaRESUMO
Oxidation via Cu(2+)/ascorbate of recombinant human interferon beta-1a (IFNß1a) leads to highly immunogenic aggregates, however it is unknown which amino acids are modified and how covalent aggregates are formed. In the present work we mapped oxidized and cross-linked amino acid residues in aggregated IFNß1a, formed via Cu(2+)/ascorbate catalyzed oxidation. Size exclusion chromatography (SEC) was used to confirm extensive aggregation of oxidized IFNß1a. Circular dichroism and intrinsic fluorescence spectroscopy indicated substantial loss of secondary and tertiary structure, respectively. Derivatization with 4-(aminomethyl)benzenesulfonic acid was used to demonstrate, by fluorescence in combination with SEC, the presence of tyrosine (Tyr) oxidation products. High performance liquid chromatography coupled to electrospray ionization mass spectrometry of reduced, alkylated, and digested protein was employed to localize chemical degradation products. Oxidation products of methionine, histidine, phenylalanine (Phe), tryptophan, and Tyr residues were identified throughout the primary sequence. Covalent cross-links via 1,4- or 1,6-type addition between primary amines and DOCH (2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl)propanoic acid, an oxidation product of Phe and Tyr) were detected. There was no evidence of disulfide bridge, Schiff base, or dityrosine formation. The chemical cross-links identified in this work are most likely responsible for the formation of covalent aggregates of IFNß1a induced by oxidation, which have previously been shown to be highly immunogenic.
Assuntos
Interferon beta/química , Ácido Ascórbico/química , Derivados de Benzeno/química , Cromatografia em Gel , Dicroísmo Circular , Cobre/química , Humanos , Interferon beta/imunologia , Metilaminas/química , Estrutura Molecular , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
PURPOSE: Interferon beta is commonly used as therapeutic in the first line of therapy for multiple sclerosis. However, depending on the product, it induces an antibody response in up to 60% of patients. This study evaluated the impact of therapy related factors like dose, route of administration and administration frequency on the immunogenicity of one of the originator interferon beta drugs (Betaferon®) in an immune tolerant transgenic mouse model. METHODS: Immune tolerant transgenic mice received injections with Betaferon® via different routes, doses and injection frequencies. Anti-drug antibody (ADA) production was measured by ELISA to assess immunogenicity. RESULTS: A single injection of Betaferon® was found to be sufficient for the induction of ADAs. The antibody titer was enhanced with increasing dose and treatment frequency. Among the tested administration routes, the intravenous route was the most immunogenic one, which is in contradiction with one of the dogma in immunogenicity research according to which subcutaneous administration is the most immunogenic route. Intramuscular, intraperitoneal and subcutaneous injections resulted in comparable immunogenicity. CONCLUSION: This study shows that treatment related factors affect significantly immunogenicity of Betaseron® and therefore substantiate the need for further studies on these factors in patients.
Assuntos
Anticorpos/imunologia , Interferon beta/administração & dosagem , Interferon beta/imunologia , Animais , Formação de Anticorpos , Vias de Administração de Medicamentos , Humanos , Tolerância Imunológica , Camundongos Transgênicos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologiaRESUMO
An increasing number of pegylated therapeutic proteins and drug targeting compounds are being introduced in the clinic. Pegylation is intended to increase circulation time and to reduce an immunogenic response. Recently however a number of publications have appeared claiming that the polyethylene glycol (PEG) moiety of these products in itself may be immunogenic and that the induced anti-PEG antibodies are linked to enhanced blood clearance and reduced efficacy of the products. A critical review of the literature shows that most, if not all assays for anti-PEG antibodies are flawed and lack specificity. Also the biological effects induced by anti-PEG antibodies lack the characteristics of a bona fide antibody reaction. Standardization of the anti-PEG assays and the development of reference sera are urgently needed.
Assuntos
Anticorpos/imunologia , Polietilenoglicóis/metabolismo , Proteínas/imunologia , Proteínas/uso terapêutico , Animais , Formação de Anticorpos , Sistemas de Liberação de Medicamentos/métodos , Humanos , Lipossomos/química , Lipossomos/imunologia , Polietilenoglicóis/química , Proteínas/químicaRESUMO
All therapeutic proteins are potentially immunogenic. Antibodies formed against these drugs can decrease efficacy, leading to drastically increased therapeutic costs and in rare cases to serious and sometimes life threatening side-effects. Many efforts are therefore undertaken to develop therapeutic proteins with minimal immunogenicity. For this, immunogenicity prediction of candidate drugs during early drug development is essential. Several in silico, in vitro and in vivo models are used to predict immunogenicity of drug leads, to modify potentially immunogenic properties and to continue development of drug candidates with expected low immunogenicity. Despite the extensive use of these predictive models, their actual predictive value varies. Important reasons for this uncertainty are the limited/insufficient knowledge on the immune mechanisms underlying immunogenicity of therapeutic proteins, the fact that different predictive models explore different components of the immune system and the lack of an integrated clinical validation. In this review, we discuss the predictive models in use, summarize aspects of immunogenicity that these models predict and explore the merits and the limitations of each of the models.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas/imunologia , Proteínas/uso terapêutico , Animais , Simulação por Computador , Humanos , Sistema Imunitário/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Modelos BiológicosRESUMO
Immunogenicity of therapeutic proteins lowers patient well-being and drastically increases therapeutic costs. Preventing immunogenicity is an important issue to consider when developing novel therapeutic proteins and applying them in the clinic. Animal models are increasingly used to study immunogenicity of therapeutic proteins. They are employed as predictive tools to assess different aspects of immunogenicity during drug development and have become vital in studying the mechanisms underlying immunogenicity of therapeutic proteins. However, the use of animal models needs critical evaluation. Because of species differences, predictive value of such models is limited, and mechanistic studies can be restricted. This review addresses the suitability of animal models for immunogenicity prediction and summarizes the insights in immunogenicity that they have given so far.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Modelos Animais , Proteínas/imunologia , Proteínas/farmacologia , Animais , Produtos Biológicos/imunologia , Produtos Biológicos/farmacologia , Humanos , Valor Preditivo dos TestesRESUMO
PURPOSE: To study the effect of oxidation on the structure of recombinant human interferon beta-1a (rhIFNß-1a) and its immunogenicity in wild-type and immune-tolerant transgenic mice. METHODS: Untreated rhIFNß-1a was degraded by metal-catalyzed oxidation, H(2)O(2)-mediated oxidation, and guanidine-mediated unfolding/refolding. Four rhIFNß-1a preparations with different levels of oxidation and aggregation were injected intraperitoneally in mice 15× during 3 weeks. Both binding and neutralizing antibodies were measured. RESULTS: All rhIFNß-1a preparations contained substantial amounts of aggregates. Metal-catalyzed oxidized rhIFNß-1a contained high levels of covalent aggregates as compared with untreated rhIFNß-1a. H(2)O(2)-treated rhIFNß-1a showed an increase in oligomer and unrecovered protein content by HP-SEC; RP-HPLC revealed protein oxidation. Guanidine-treated rhIFNß-1a mostly consisted of dimers and oligomers and some non-covalent aggregates smaller in size than those in untreated rhIFNß-1a. All degraded samples showed alterations in tertiary protein structure. Wild-type mice showed equally high antibody responses against all preparations. Transgenic mice were discriminative, showing elevated antibody responses against both metal-catalyzed oxidized and H(2)O(2)-treated rhIFNß-1a as compared to untreated and guanidine-treated rhIFNß-1a. CONCLUSIONS: Oxidation-mediated aggregation increased the immunogenicity of rhIFNß-1a in transgenic mice, whereas aggregated preparations devoid of measurable oxidation levels were hardly immunogenic.
Assuntos
Interferon beta/química , Interferon beta/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos/imunologia , Cromatografia Líquida de Alta Pressão/métodos , Guanidina/química , Humanos , Peróxido de Hidrogênio/química , Tolerância Imunológica/imunologia , Interferon beta-1a , Interferon beta/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxirredução , Ligação Proteica/imunologia , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologiaRESUMO
PURPOSE: To compare the quality of therapeutic erythropoietin (EPO) products, including two biosimilars, with respect to content, aggregation, isoform profile and potency. METHODS: Two original products, Eprex (epoetin alpha) and Dynepo (epoetin delta), and two biosimilar products, Binocrit (epoetin alpha) and Retacrit (epoetin zeta), were compared using (1) high performance size exclusion chromatography, (2) ELISA, (3) SDS-PAGE, (4) capillary zone electrophoresis and (5) in-vivo potency. RESULTS: Tested EPO products differed in content, isoform composition, and potency. CONCLUSION: Of the tested products, the biosimilars have the same or even better quality as the originals. Especially, the potency of originals may significantly differ from the value on the label.
Assuntos
Produtos Biológicos/química , Eritropoetina/química , Equivalência Terapêutica , Animais , Produtos Biológicos/uso terapêutico , Cromatografia em Gel , Eletroforese Capilar , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Eritropoetina/imunologia , Eritropoetina/uso terapêutico , Camundongos , Isoformas de Proteínas/química , Proteínas RecombinantesRESUMO
PURPOSE: To study the influence of protein aggregation on the immunogenicity of recombinant human interferon beta (rhIFNbeta) in wild-type mice and transgenic, immune-tolerant mice, and to evaluate the induction of immunological memory. METHODS: RhIFNbeta-1b and three rhIFNbeta-1a preparations with different aggregate levels were injected intraperitoneally in mice 15x during 3 weeks, and the mice were rechallenged with rhIFNbeta-1a. The formation of binding (BABs) and neutralizing antibodies (NABs) was monitored. RESULTS: Bulk rhIFNbeta-1a contained large, mainly non-covalent aggregates and stressed rhIFNbeta-1a mainly covalent, homogeneous (ca. 100 nm) aggregates. Reformulated rhIFNbeta-1a was essentially aggregate-free. All products induced BABs and NABs in wild-type mice. Immunogenicity in the transgenic mice was product dependent. RhIFNbeta-1b showed the highest and reformulated rhIFNbeta-1a the lowest immunogenicity. In contrast with wild-type mice, transgenic mice did not show NABs, nor did they respond to the rechallenge. CONCLUSIONS: The immunogenicity of the products in transgenic mice, unlike in wild-type mice, varied. In the transgenic mice, neither NABs nor immunological memory developed. The immunogenicity of rhIFNbeta in a model reflecting the human immune system depends on the presence and the characteristics of aggregates.
Assuntos
Anticorpos Neutralizantes/sangue , Tolerância Imunológica , Memória Imunológica , Interferon Tipo I/efeitos adversos , Interferon Tipo I/imunologia , Animais , Sítios de Ligação de Anticorpos , Western Blotting , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Tolerância Imunológica/genética , Memória Imunológica/genética , Interferon Tipo I/genética , Luz , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes , Espalhamento de Radiação , Espectrometria de FluorescênciaRESUMO
Background: Eluforsen (previously known as QR-010) is a 33-mer antisense oligonucleotide under development for oral inhalation in cystic fibrosis (CF) patients with the delta F508 mutation. Previous work has shown that eluforsen restores CF transmembrane conductance regulator (CFTR) function in vitro and in vivo. To be effective, eluforsen has first to reach its primary target, the lung epithelial cells. Therefore, it has to diffuse through the CF airway surface layer (ASL), which in CF is characterized by the presence of thick and viscous mucus, impaired mucociliary clearance, and persistent infections. The goal of this study was to assess delivery of eluforsen through CF-like ASL. Methods and Results: First, air-liquid interface studies with cultured primary airway epithelial cells revealed that eluforsen rapidly diffuses through CF-like mucus at clinically relevant doses when nebulized once or repeatedly, over a range of testing doses. Furthermore, eluforsen concentrations remained stable in CF patient sputum for at least 48 hours, and eluforsen remained intact in the presence of various inhaled CF medications for at least 24 hours. When testing biodistribution of eluforsen after orotracheal administration in vivo, no differences in lung, liver, trachea, and kidney eluforsen concentration were observed between mice with a CF-like lung phenotype (ENaC-overexpressing mice) and control wild-type (WT) littermates. Also, eluforsen was visualized in the airway epithelial cell layer of CF-like muco-obstructed mice and WT littermates. Finally, studies of eluforsen uptake and binding to bacteria prevalent in CF lungs, and diffusion through bacterial biofilms showed that eluforsen was stable and not absorbed by, or bound to bacteria. In addition, eluforsen was found to be able to penetrate Pseudomonas aeruginosa biofilms. Conclusions: The thickened and concentrated CF ASL does not constitute a significant barrier for delivery of eluforsen, and feasibility of oral inhalation of eluforsen is supported by these data.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/terapia , Pulmão/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos/farmacologia , Administração por Inalação , Animais , Biofilmes , Células Cultivadas , Fibrose Cística/genética , Células Epiteliais/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/farmacocinética , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/farmacocinética , Pseudomonas aeruginosa/fisiologia , Fatores de Tempo , Distribuição TecidualRESUMO
Most drug delivery procedures induce stress, which might interfere with the pharmacological action of the drug and behaviour. Stress is deduced from high and long-lasting elevations of the hormone corticosterone. We set out to develop a non-invasive, stress-free method of drug delivery in mice. Validation consisted of delivery of glucocorticoid ligands via oats to male C57BL/6J mice. Oat consumption induced a small increase in corticosterone concentrations after 15 min (<50 ng/ml) that returned to low resting levels at t=30 (<10 ng/ml). Gavage and intraperitoneal (i.p.) vehicle injections resulted in long-lasting corticosterone elevations (>100 ng/ml at t=30 and approximately 50 ng/ml at t=60 min after delivery). Adding corticosterone to oats resulted in threefold higher plasma corticosterone in the 15.0-mg/kg group (+/-250 ng/ml) compared to the 4.5-mg/kg group at t=30 and 90. Application of the glucocorticoid receptor antagonist RU38486 (200 mg/kg) elevated plasma corticosterone for at least 8h. Additional swimming increased corticosterone even further. Presumably, already the small oat-consumption-induced increase of corticosterone requires negative feedback via glucocorticoid receptors. In conclusion, the context-dependent and dose-controlled application of drugs via oats avoids confounding strong stress system activation and makes it suitable for studies on learning and memory processes.
Assuntos
Glucocorticoides/farmacologia , Adrenalectomia , Animais , Avena , Glicemia/metabolismo , Relação Dose-Resposta a Droga , Glucocorticoides/agonistas , Glucocorticoides/antagonistas & inibidores , Antagonistas de Hormônios/administração & dosagem , Antagonistas de Hormônios/farmacologia , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Injeções Intraperitoneais , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Mifepristona/administração & dosagem , Mifepristona/farmacologia , Veículos Farmacêuticos , Receptores de Glucocorticoides/antagonistas & inibidores , Estresse Psicológico/fisiopatologiaRESUMO
The aim of the study was to examine the reproducibility of a rat model to assess the preclinical similarity in safety profiles and tissue accumulation of iron products. Accordingly, the effect of several doses of intravenously administered Venofer® and of Ferrlecit® on blood parameters, and on kidney and particularly liver toxicity were examined in non-anemic Sprague Dawley rats. The different analysis showed neither a clear treatment nor a dose effect after multiple injections. The parameters measured in this rat strain showed some iron induced adverse effects, but these could not be correlated to treatment specific differences. The findings presented in this paper indicate the difficulty to define a useful preclinical model to evaluate iron-based nano-colloidal preparations.
Assuntos
Hematínicos/toxicidade , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Modelos Animais , Ratos , Animais , Coloides/administração & dosagem , Coloides/toxicidade , Compostos Férricos/administração & dosagem , Compostos Férricos/toxicidade , Óxido de Ferro Sacarado , Ácido Glucárico/administração & dosagem , Ácido Glucárico/toxicidade , Hematínicos/administração & dosagem , Infusões Intravenosas , Injeções Intravenosas , Masculino , Nanopartículas/administração & dosagem , Nanopartículas/toxicidade , Ratos Sprague-Dawley , Reprodutibilidade dos TestesRESUMO
Corticosteroids regulate stress response and influence emotion, learning, and memory via two receptors in the brain, the high-affinity mineralocorticoid (MR) and low-affinity glucocorticoid receptor (GR). We test the hypothesis that MR- and GR-mediated effects interact in emotion and cognition when a novel situation is encountered that is relevant for a learning process. By adrenalectomy and additional constant corticosterone supplement we obtained four groups of male C57BL/6J mice with differential chronic MR and GR activations. Using a hole board task, we found that mice with continuous predominant MR and moderate GR activations were fast learners that displayed low anxiety and arousal together with high directed explorative behavior. Progressive corticosterone concentrations with predominant action via GR induced strong emotional arousal at the expense of cognitive performance. These findings underline the importance of a balanced MR/GR system for emotional and cognitive functioning that is critical for mental health.
Assuntos
Transtornos Cognitivos/metabolismo , Cognição/fisiologia , Emoções/fisiologia , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Corticosteroides/administração & dosagem , Adrenalectomia , Animais , Ansiedade/sangue , Ansiedade/metabolismo , Transtornos Cognitivos/sangue , Corticosterona/sangue , Emoções/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Glucocorticoides/agonistas , Receptores de Mineralocorticoides/agonistasRESUMO
Comprehensive physicochemical characterization and biological assays are essential parts in assessing quality attributes of biologicals. Here, we compared the quality of different marketed recombinant human erythropoietin (epoetin) products: originators, Eprex and NeoRecormon as well as 2 biosimilars, Retacrit and Binocrit. In addition, assessment of batch-to-batch variability was included by collecting 2 or more batches of each product. Common assays which included sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-performance size-exclusion chromatography, asymmetrical flow field-flow fractionation, capillary zone electrophoresis, and potency testing were used. Of the tested products and among batches of single products, variations in epoetin content, isoform profiles, and potency were found. Ultimately, this study demonstrated the high quality of epoetin products with some degree of variation among products and batches, confirming the "similar but not identical" paradigm of biologicals.
Assuntos
Medicamentos Biossimilares/normas , Química Farmacêutica/métodos , Epoetina alfa/normas , Eritropoetina/normas , Animais , Medicamentos Biossimilares/análise , Medicamentos Biossimilares/química , Epoetina alfa/análise , Epoetina alfa/química , Eritropoetina/análise , Eritropoetina/química , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/normas , Equivalência TerapêuticaRESUMO
Anemia resulting from iron deficiency is one of the most prevalent diseases in the world. As iron has important roles in several biological processes such as oxygen transport, DNA synthesis and cell growth, there is a high need for iron therapies that result in high iron bioavailability with minimal toxic effects to treat patients suffering from anemia. This study aims to develop a novel oral iron-complex formulation based on hemin-loaded polymeric micelles composed of the biodegradable and thermosensitive polymer methoxy-poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl)methacrylamide-dilactate], abbreviated as mPEG-b-p(HPMAm-Lac2). Hemin-loaded micelles were prepared by addition of hemin dissolved in DMSO:DMF (1:9, one volume) to an aqueous polymer solution (nine volumes) of mPEG-b-p(HPMAm-Lac2) followed by rapidly heating the mixture at 50°C to form hemin-loaded micelles that remain intact at room and physiological temperature. The highest loading capacity for hemin in mPEG-b-p(HPMAm-Lac2) micelles was 3.9%. The average particle diameter of the hemin-micelles ranged from 75 to 140nm, depending on the concentration of hemin solution that was used to prepare the micelles. The hemin-loaded micelles were stable at pH 2 for at least 3 h which covers the residence time of the formulation in the stomach after oral administration and up to 17 h at pH 7.4 which is sufficient time for uptake of the micelles by the enterocytes. Importantly, incubation of Caco-2 cells with hemin-micelles for 24 h at 37°C resulted in ferritin levels of 2500ng/mg protein which is about 10-fold higher than levels observed in cells incubated with iron sulfate under the same conditions. The hemin formulation also demonstrated superior cell viability compared to iron sulfate with and without ascorbic acid. The study presented here demonstrates the development of a promising novel iron complex for oral delivery.
Assuntos
Administração Oral , Portadores de Fármacos/química , Hemina/química , Polímeros/química , Acrilamidas/química , Anemia/sangue , Ácido Ascórbico/química , Células CACO-2 , Sobrevivência Celular , Sistemas de Liberação de Medicamentos , Compostos Férricos/química , Ferritinas/química , Heme/química , Humanos , Concentração de Íons de Hidrogênio , Ferro/química , Micelas , Microscopia Confocal , Peso Molecular , Tamanho da Partícula , Polietilenoglicóis/química , Sulfatos/química , Temperatura , Raios UltravioletaRESUMO
Immunological processes leading to formation of antidrug antibodies (Abs) against recombinant human proteins remain poorly understood. Animal and clinical studies revealed that immunogenicity shares both T-cell-dependent (requirement of CD4(+) T cells, isotype switching) and T-cell-independent (involvement of Marginal Zone B cells, apparent lack of memory) characteristics. We used immune tolerant mice to study the mechanism underlying immunogenicity in more detail. We found that CD4(+) T cells were crucial at early stages of Ab responses against rhIFNß. In addition, we found a similar number of germinal centers (GCs) in spleen after rhIFNß treatment as after treatment with a foreign protein. However, neither Ab titers nor the number of GCs was increased by adsorption of rhIFNß on aluminum hydroxide. Therefore, we tested the effect of several immune adjuvants in a follow-up study. We found that only conjugation of rhIFNß to a carrier protein (cholera toxin subunit B) was effective in boosting Ab titers. However, these conjugates failed to trigger rhIFNß specific memory formation. Our findings show that early events of the immunogenicity reaction to self-proteins are CD4(+) T-cell dependent. Nevertheless, despite those similarities, immunogenicity of human proteins is clearly not a classical CD4(+) T-cell-dependent response.
Assuntos
Adjuvantes Imunológicos , Anticorpos/imunologia , Linfócitos T CD4-Positivos/imunologia , Centro Germinativo/imunologia , Tolerância Imunológica , Interferon beta/imunologia , Animais , Anticorpos/administração & dosagem , Linfócitos T CD4-Positivos/citologia , Centro Germinativo/citologia , Humanos , Interferon beta/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologiaRESUMO
Protein aggregates are a major risk factor for immunogenicity. Until now most studies on aggregate-driven immunogenicity have focused on linking physicochemical features of the aggregates to the formation of anti-drug antibodies. Lacking is however, basic knowledge on the effect of aggregation on the biodistribution and clearance of therapeutic proteins in vivo. The aim of current study was to get insight into the effect of aggregation on biodistribution in mice using different routes of administration. Fluorescently labeled stressed and unstressed mouse serum albumin was injected via different routes in mice and detected via in vivo fluorescence imaging up to 48 hrs post-injection. We found that biodistribution of stressed MSA significantly differed from its unstressed counterpart. Subcutaneous and intramuscular administration resulted in accumulation of protein at the site of injection, from which clearance of stressed MSA was considerably slower than clearance of unstressed MSA. Upon intravenous and intraperitoneal injection of stressed MSA, fluorescent "hotspots" were observed in the spleens, livers and lungs. Further and more detailed examination of biodistribution after intraperitoneal injection showed higher fluorescence in most of tested organs suggesting more efficient diffusion and/or lymphatic uptake from peritoneum of unstressed MSA than the stressed formulation.
Assuntos
Fígado/metabolismo , Pulmão/metabolismo , Albumina Sérica/administração & dosagem , Albumina Sérica/farmacocinética , Baço/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Feminino , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Injeções Intramusculares , Injeções Intraperitoneais , Injeções Intravenosas , Injeções Subcutâneas , Medições Luminescentes/métodos , Camundongos , Albumina Sérica/química , Distribuição TecidualRESUMO
Interferon beta may induce antibodies in multiple sclerosis patients and the incidence of immunogenicity depends on the type of product. These antibodies can reduce the efficacy of interferon beta. Two transgenic immune tolerant mouse models for human interferon beta (hIFNß) (C57Bl/6, and C57Bl/6×FVB/N F1 hybrid mice) have been developed previously for studying immunogenicity. These models, however, may not be used for every interferon beta product because of the lack of immunogenicity in the wildtype genetic background. We therefore developed a modified transgenic mouse model by backcrossing the F1 hybrid C57Bl/6×FVB/N transgenic mice with wildtype FVB/N for 10 generations. These F10 offspring (referred to hear as FVB/N) have a genetic background consisting of mostly FVB/N (99.9%) and very little C57Bl/6 (0.1%), and are expected to have the more sensitive antibody producing phenotype of the parental FVB/N strain. The newly generated "FVB/N" strain was assessed for antibody formation against different rhIFNß formulations compared to the C57Bl/6, and C57Bl/6×FVB/N transgenic mouse models. The new FVB/N transgenic mouse model was more sensitive for all tested rhIFNß products, and the difference in antibody titers between the transgenic and non-transgenic mice of the FVB/N strain was much bigger compared to the antibody levels of the C57Bl/6, and C57Bl/6×FVB/N strains.