RESUMEN
Achieving therapeutic efficacy in protein replacement therapies requires sustaining pharmacokinetic (PK) profiles, while maintaining the bioactivity of circulating proteins. This is often achieved via PEGylation in protein-based therapies, but it remains challenging for proteins produced in vivo in mRNA-based therapies due to the lack of a suitable post-translational modification method. To address this issue, we integrated a genetically encoded zwitterionic polypeptide, EKP, into mRNA constructs to enhance the PK properties of product proteins. Composed of alternating glutamic acid (E), lysine (K), and proline (P), EKP exhibits unique superhydrophilic properties and low immunogenicity. Our results demonstrate that EKP fusion significantly extends the circulation half-life of proteins expressed from mRNA while preserving their bioactivity using human interferon alpha and Neoleukin-2/15 as examples. This EKP fusion technology offers a new approach to overcoming the current limitations in mRNA therapeutics and has the potential to significantly advance the development of mRNA-based protein replacement therapy.
Asunto(s)
Péptidos , ARN Mensajero , Humanos , ARN Mensajero/genética , ARN Mensajero/química , Péptidos/química , Péptidos/farmacocinética , Animales , Interferón-alfa/farmacocinética , Interferón-alfa/química , Interferón-alfa/genética , RatonesRESUMEN
BACKGROUND: Virologic breakthrough (VBT) may occur in chronic hepatitis B (CHB) patients after switching from nucleos(t)ide analogues (NAs) to pegylated interferon alpha (Peg-IFN-É). This study aimed to characterize the clinical and immunological features of VBT. METHODS: In NAs-treated patients switching to Peg-IFN-É, innate and adaptive immune cell proportions were examined in peripheral blood and liver biopsy specimens. In vitro effect of IFN-É on the expressions of toll-like receptors 2 (TLR2) and programmed cell death ligand 1 (PDL1) on monocytes, programmed cell death 1 (PD1) on CD8+T cells was examined. Peripheral blood mononuclear cells (PBMCs) were treated with TLR2 agonist and/or PDL1 blockade to evaluate their effect on HBV replication. RESULTS: 33 of 166 patients switching to Peg-IFN-É experienced VBT after NA cessation, with majority being hepatitis B e antigen (HBeAg) positive or having higher hepatitis B core-related antigen (HBcrAg) levels. Patients with VBT exhibited lower proportions of TLR2+monocyte and increased PD1+HBV-specific CD8+T cell during the early phase of Peg-IFN-É therapy after NA cessation in peripheral blood, as well as fewer TLR2+CD68+macrophages but more PDL1+CD68+macrophages and PD1+CD8+T cells in liver tissues. Simultaneous use of TLR2 agonist and PDL1 blockage ex vivo suppressed HBV replication by promoting cytokines production and CD8+T cells cytotoxicity. Upon in vitro IFN-É stimulation, PDL1+monocytes and PD1+CD8+T cells were upregulated, whereas TLR2+monocytes were not increased in PBMC isolated from HBeAg-positive patients, or those with high HBcrAg titers. CONCLUSIONS: In NAs-treated patients, lower TLR2+monocyte and increased PD1+HBV-specific CD8+T cell proportions potentially contribute to VBT after switching to Peg-IFN-É therapy. This insufficient immunity may be associated with the HBeAg status and HBcrAg levels.
Asunto(s)
Antivirales/uso terapéutico , Sustitución de Medicamentos/efectos adversos , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/inmunología , Interferón-alfa/uso terapéutico , Nucleósidos/uso terapéutico , Adolescente , Adulto , Anciano , Antivirales/farmacología , Femenino , Hepatitis B Crónica/virología , Humanos , Inmunidad/efectos de los fármacos , Interferón-alfa/farmacocinética , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/virología , Masculino , Persona de Mediana Edad , Nucleósidos/efectos adversos , Nucleósidos/análogos & derivados , Adulto JovenRESUMEN
A sensitive method for determination of PEG-IFN-α-2b in human serum was developed using ultra performance liquid chromatography aligned with tandem mass spectrometric detection. A two-treatment, two-period, cross over study was conducted to establish bioequivalence between a test and reference formulation and the method was successfully applied to the quantification of PEG-IFN-α-2b in serum samples of this clinical study. The sample concentrations obtained from LC-MS/MS technique were compared with the concentrations obtained from ELISA technique. PEG-IFN-α-2b was isolated from serum using protein precipitation technique with isopropyl alcohol followed by overnight tryptic digestion. The signature peptide formed as result of tryptic digestion was separated on a chromatograph and detected using a mass detector. The mass transition ion-pair of m/z 741.3â¯ââ¯1047.1 for PEG-IFN-α-2b and m/z 387.4â¯ââ¯205.2 for internal standard were used for MS/MS detection. The sample extraction involves a simple protein precipitation method followed by tryptic digestion of the supernatant and further sample cleanup was not needed. The method has been validated over a linear range of 1.028-3200â¯ng/mL with a correlation coefficientâ¯≥â¯0.99. The precision (%RSD) was 5.52 to 7.90 and accuracy (%RE) was within -1.80 to 1.68. The total run time was 22.0â¯min. The sensitivity of LC-MS/MS method was 1.0â¯ng/ml which was found to be more sensitive than ELISA and resulted in improving the overall study data by being able to quantify all the samples without any below LOQ results helping to further improve the pharmacokinetic modeling. This improved method is a promising anti-body free LC-MS/MS based methodology for estimation of PEG-IFN-α-2b in human serum and may be applied for other such pegylated molecules.
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Cromatografía Líquida de Alta Presión/métodos , Interferón alfa-2/sangre , Interferón-alfa/sangre , Fragmentos de Péptidos/sangre , Espectrometría de Masas en Tándem/métodos , Estudios Cruzados , Humanos , Interferón alfa-2/farmacocinética , Interferón-alfa/farmacocinética , Límite de Detección , Modelos Lineales , Fragmentos de Péptidos/metabolismo , Polietilenglicoles/farmacocinética , Proteínas Recombinantes/sangre , Proteínas Recombinantes/farmacocinética , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/métodos , Equivalencia Terapéutica , Tripsina/metabolismoRESUMEN
PURPOSE: IFN4N is a glycoengineered version of recombinant human interferon alpha 2 (rhIFN-α2) that was modified to exhibit four N-glycosylation sites. It shows reduced in vitro specific biological activity (SBA) mainly due to R23 mutation by N23. However, it has improved pharmacokinetics and led to a high in vivo antitumor activity in mice. In order to prepare a new IFN-based biobetter, this work compares the influence of glycosylation (affecting pharmacokinetics) with the in vitro antiproliferative SBA on the in vivo efficacy. METHODS: Based on IFN4N, three groups of muteins were designed, produced, and characterized. Group A: variants with the same glycosylation degree (4N) but higher in vitro antiproliferative SBA (R23 restored); group B: muteins with higher glycosylation degree (5N) but similar in vitro antiproliferative activity; and group C: variants with improved glycosylation (5N and 6N) and in vitro antiproliferative bioactivity. RESULTS: Glycoengineering was successful for improving pharmacokinetics, and R23 restoration considerably increased in vitro antiproliferative activity of new muteins compared to IFN4N. Hyperglycosylation was able to improve the in vivo efficacy similarly to or even better than R23 restoration. Additionally, the highest glycosylated mutein exhibited the lowest immunogenicity. CONCLUSIONS: Hyperglycosylation constitutes a successful strategy to prepare a novel IFN biobetter.
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Antineoplásicos/farmacocinética , Interferón-alfa/farmacocinética , Adulto , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/aislamiento & purificación , Células CHO , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cricetulus , Glicosilación , Células HEK293 , Semivida , Voluntarios Sanos , Humanos , Interferón-alfa/administración & dosificación , Interferón-alfa/genética , Interferón-alfa/aislamiento & purificación , Leucocitos Mononucleares , Ratones , Persona de Mediana Edad , Cultivo Primario de Células , Ingeniería de Proteínas , Ratas , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacocinética , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto JovenRESUMEN
Polymer-protein conjugates are a class of biohybrids with unique properties that are highly useful in biomedicine ranging from protein therapeutics to biomedical imaging; however, it remains a considerable challenge to conjugate polymers to proteins in a site-specific, mild, and efficient way to form polymer-protein conjugates with uniform structures and properties and optimal functions. Herein we report pyridine-2,6-dicarboxaldehyde (PDA)-enabled N-terminal modification of proteins with polymerization initiators for in situ growth of poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA) conjugates uniquely at the N-termini of a range of natural and recombinant proteins in a mild and efficient fashion. The formed POEGMA-protein conjugates showed highly retained in vitro bioactivity as compared with free proteins. Notably, the in vitro bioactivity of a POEGMA-interferon α (IFN) conjugate synthesized by this new chemistry is 8.1-fold higher than that of PEGASYS that is a commercially available and Food and Drug Administration (FDA) approved PEGylated IFN. The circulation half-life of the conjugate is similar to that of PEGASYS but is 46.2 times longer than that of free IFN. Consequently, the conjugate exhibits considerably improved antiviral bioactivity over free IFN and even PEGASYS in a mouse model. These results indicate that the PDA-enabled N-terminal grafting-from method is applicable to a number of proteins whose active sites are far away from the N-terminus for the synthesis of N-terminal polymer-protein conjugates with high yield, well-retained activity, and considerably improved pharmacology for biomedical applications.
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Aldehídos/química , Antivirales/farmacología , Indicadores y Reactivos/química , Interferón-alfa/farmacología , Polietilenglicoles/farmacología , Piridinas/química , 2',5'-Oligoadenilato Sintetasa/metabolismo , Secuencia de Aminoácidos , Animales , Antivirales/química , Antivirales/farmacocinética , Semivida , Interferón-alfa/química , Interferón-alfa/farmacocinética , Ratones , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , PolimerizacionRESUMEN
INTRODUCTION: Polycythemia vera (PV) is a Philadelphia chromosome-negative chronic myeloproliferative neoplasm (MPN). A newly developed PV treatment option, ropeginterferon alfa-2b, contains recombinant human alfa monoisomer as an active ingredient, resulting in a novel pharmacologic profile and improved tolerability. Efficacy studies conclude remarkable long-term hematological response and sustained JAK2V617F allele burden reduction. Ropeginterferon alfa-2b compound has been approved for the treatment of polycythemia vera without symptomatic splenomegaly. AREAS COVERED: Current clinical trials are investigating the role of ropeginterferon alfa-2b in the first-line setting of treatment for PV. The safety and efficacy results of completed trials are summarized in this review. Metabolic, pharmacokinetic issues are also discussed of ropeginterferon alfa-2b. EXPERT OPINION: Ropeginterferon alfa-2b is a targeted therapeutic option in the treatment of PV, representing a significant improvement compared to conventional cytoreductive therapies. The single isomer entity of the recombinant human interferon alfa-2b and the mono-pegylation method imparts favorable properties to the compound. The use of ropeginterferon alfa-2b allows extended dosing interval, reduces side effects, and may increase the overall survival of PV patients by reducing the risk of progression to myelofibrosis or acute leukemia. Clinical data suggests that the compound may provide a disease-modifying option for PV patients with asymptomatic splenomegaly.
Asunto(s)
Interferón alfa-2/farmacocinética , Interferón-alfa/farmacocinética , Policitemia Vera/tratamiento farmacológico , Polietilenglicoles/farmacocinética , Alelos , Animales , Progresión de la Enfermedad , Humanos , Interferón alfa-2/administración & dosificación , Interferón alfa-2/efectos adversos , Interferón-alfa/administración & dosificación , Interferón-alfa/efectos adversos , Janus Quinasa 2/genética , Policitemia Vera/genética , Polietilenglicoles/administración & dosificación , Polietilenglicoles/efectos adversos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/farmacocinética , Tasa de SupervivenciaRESUMEN
PEGylation had been used successfully to improve the circulation half-lives and some physicochemical properties of protein therapeutics. However, anti-polyethylene glycol (anti-PEG) antibodies, either pre-existing or treatment-induced, can negatively affect the pharmacokinetics and pharmacological efficacy of PEGylated proteins. We have examined anti-PEG immune responses in mice for peginterferon alfa-2a (Pegasys), a clinically approved PEGylated protein therapeutic, at both the recommended dose (equivalent to 3 µg/kg in mice) and at higher doses (150 µg/kg) for single or repeated subcutaneous (s.c.) administrations. The effect of treatment-induced anti-PEG IgM on serum concentrations of Pegasys, following repeated administrations, was evaluated. In addition, the effect of pre-existing anti-PEG IgM elicited by a different PEGylated protein, PEG-OVA, on the systemic clearance of Pegasys, was investigated. At a s.c. dose of 3 µg/kg, single injections of Pegasys barely elicited anti-PEG immune responses. Four repeated doses of 150 µg/kg Pegasys elicited anti-PEG IgM production, depending on dose frequency, and triggered the rapid clearance of subsequent doses. In addition, anti-PEG-IgM produced in response to prior administration of PEG-OVA caused a rapid blood clearance of Pegasys. Our results, therefore, underscore the importance of screening for both pre-existing and treatment-induced anti-PEG antibodies in patients prior to and during treatment with PEGylated protein drugs.
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Anticuerpos Antiidiotipos/inmunología , Inmunoglobulina M/inmunología , Interferón-alfa/farmacocinética , Polietilenglicoles/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos BALB C , Polietilenglicoles/farmacocinética , Proteínas Recombinantes/farmacocinéticaRESUMEN
PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.
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Antivirales , Interferón-alfa , Polietilenglicoles , Transglutaminasas , Animales , Humanos , Interferón alfa-2/metabolismo , Interferón-alfa/biosíntesis , Interferón-alfa/farmacocinética , Polietilenglicoles/farmacocinética , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Reproducibilidad de los Resultados , Transglutaminasas/metabolismoRESUMEN
Sustained release of active interferon-α (IFN-α) has been achieved from core-shell nanoparticles (NPs) prepared by aqueous precipitation of IFN-α-enriched human serum albumin (HSA-IFN-α) and layer-by-layer (L-b-L) by coating of the IFN-α NPs with poly(sodium-4-styrene) sulphonate (PSS) and chitosan (Chit). The concentration and the pH of HSA solution were optimized during the development of this method. Dynamic light scattering (DLS), zeta-potential, thermal analysis (differential scanning calorimetry (DSC) and termogravimetry (TG)), X-ray diffraction (XRD), IFN-α activity and morphology (transmission electron microscope (TEM)) studies were used to control the preparation and analyse the products. The dissolution kinetics of NPs was measured in vitro over 7â¯days in Hanson dissolution tester with Millex membrane. In vivo studies in Pannon white rabbit detected steady IFN-α plasma level for 10â¯days after subcutaneous injection administration of the HSA-IFN-α NPs. The IFN-α plasma concentration was detected by using the enzyme-linked immunosorbent assay (ELISA) method. In the present paper we discuss the preparation method, the optimization steps and the results of in vitro and in vivo release studies. It was established that 76.13% HSA-IFN-α are encapsulated in the core-shell NPs.
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Portadores de Fármacos/química , Composición de Medicamentos/métodos , Interferón-alfa/administración & dosificación , Nanopartículas/química , Animales , Quitosano/química , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/farmacocinética , Liberación de Fármacos , Interferón-alfa/química , Interferón-alfa/farmacocinética , Modelos Animales , Tamaño de la Partícula , Poliestirenos/química , Conejos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Albúmina Sérica Humana/administración & dosificación , Albúmina Sérica Humana/química , Albúmina Sérica Humana/farmacocinética , SolubilidadRESUMEN
Introduction: Interferon (IFN) had both antiviral and immunomodulatory effects, and was one of the approved treatments for hepatitis B virus (HBV). Herein, we reviewed the pharmacokinetics and pharmacodynamics of pegylated IFN-α (PegIFN-α) for the treatment of HBV. Areas covered: The steady-state serum levels of PegIFN-α were reached within 5 to 8 weeks, and the week 48 mean trough concentrations were approximately 2-fold higher than week 1. There was also no difference of the pharmacokinetics in male or female, healthy volunteers or patients with hepatitis B or C infection. PegIFN-α did not affect the metabolism of the cytochrome P450 (CYP) isozymes, except inhibition of CYP1A2. There was also no pharmacokinetic interaction between PegIFN-α and HBV nucleot(s)ide analogues (NUCs). Forty-eight weeks of PegIFN-α achieved 32% of HBeAg seroconversion, 32-43% of HBV DNA suppression, 41-59% of ALT normalization, and 3% of HBsAg seroconversion rate with a post-treatment durable response up to 80% in the initial responders. Expert opinion: On-treatment HBsAg titer guided the treatment of HBV with PegIFN-α. The recommendation of PegIFN-α and NUC combination or switch remained controversial. New immunotherapeutic agents are now in development. Although, PegIFN-α should continue to play a role in the treatment of HBV.
Asunto(s)
Hepatitis B Crónica/tratamiento farmacológico , Interferón alfa-2/administración & dosificación , Interferón-alfa/administración & dosificación , Polietilenglicoles/administración & dosificación , Animales , Antivirales/administración & dosificación , Antivirales/farmacocinética , Antivirales/farmacología , ADN Viral/efectos de los fármacos , Interacciones Farmacológicas , Femenino , Antígenos e de la Hepatitis B/sangre , Humanos , Interferón alfa-2/farmacocinética , Interferón alfa-2/farmacología , Interferón-alfa/farmacocinética , Interferón-alfa/farmacología , Masculino , Polietilenglicoles/farmacocinética , Polietilenglicoles/farmacología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologíaRESUMEN
Different strategies have been developed and successfully applied to biotherapeutics in order to improve their in vivo efficacy. The genetic fusion to natural or synthetic glycosylated peptides constitutes a promising strategy since it conserves the protein sequence and results in the improvement of the pharmacokinetic properties. The ANITVNITV peptide described by Perlmann and coworkers presents 9 amino acids and 2 potential N-glycosylation sites. Its fusion to FSH resulted in the increase of the molecular mass and negative charge of the protein. Consequently, the pharmacokinetics was considerably improved. The aim of the present study was to compare the influence of ANITVNITV peptide fusion on the physicochemical, biological and pharmacokinetic properties of native hIFN-α2b (IFNwt), which contains a single O-glycosylation site, and a hyperglycosylated variant (IFN4N), that bears, in addition, 4 N-linked glycans. The resulting molecules, IFNwtNter and IFN4NNter, evidenced a higher molecular mass and negative charge compared to IFNwt and IFN4N, respectively. Therefore, the pharmacokinetic properties of the new molecules were significantly improved. The molecules obtained by the synthetic peptide fusion strategy evidenced a decrease in their in vitro antiviral specific biological activities (SBA). However, in vitro antiproliferative SBA was differentially modified for IFNwtNter and IFN4NNter in comparison with the parental molecules. For IFNwtNter, a reduction in the antiproliferative SBA was also observed. Remarkably, the addition of the ANITVNITV peptide to the N-terminus of IFN4N had a positive impact on its growth-inhibitory activity. This feature together with its improved pharmacokinetics encourages the development of IFN4NNter as an IFN-α based biobetter.
Asunto(s)
Interferón-alfa/genética , Interferón-alfa/farmacocinética , Péptidos/química , Secuencia de Aminoácidos , Animales , Células CHO , Línea Celular , Proliferación Celular/efectos de los fármacos , Cricetulus , Perros , Variación Genética , Glicosilación , Humanos , Interferón alfa-2 , Interferón-alfa/química , Células de Riñón Canino Madin Darby , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacocinéticaRESUMEN
The therapeutic effect of a drug is governed by its pharmacokinetics which determine the downstream pharmacodynamic response within the cellular network. A complete understanding of the drug-effect relationship therefore requires multi-scale models which integrate the properties of the different physiological scales. Computational modelling of these individual scales has been successfully established in the past. However, coupling of the scales remains challenging, although it will provide a unique possibility of mechanistic and holistic analyses of therapeutic outcomes for varied treatment scenarios. We present a methodology to combine whole-body physiologically-based pharmacokinetic (PBPK) models with mechanistic intracellular models of signal transduction in the liver for therapeutic proteins. To this end, we developed a whole-body distribution model of IFN-α in human and a detailed intracellular model of the JAK/STAT signalling cascade in hepatocytes and coupled them at the liver of the whole-body human model. This integrated model infers the time-resolved concentration of IFN-α arriving at the liver after intravenous injection while simultaneously estimates the effect of this dose on the intracellular signalling behaviour in the liver. In our multi-scale physiologically-based pharmacokinetic/pharmacodynamic (PBPK/PD) model, receptor saturation is seen at low doses, thus giving mechanistic insights into the pharmacodynamic (PD) response. This model suggests a fourfold lower intracellular response after administration of a typical IFN-α dose to an individual as compared to the experimentally observed responses in in vitro setups. In conclusion, this work highlights clear differences between the observed in vitro and in vivo drug effects and provides important suggestions for future model-based study design.
Asunto(s)
Factores Inmunológicos/farmacología , Interferón-alfa/farmacología , Células Cultivadas , Simulación por Computador , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Factores Inmunológicos/farmacocinética , Interferón-alfa/farmacocinética , Quinasas Janus/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Modelos Biológicos , Factores de Transcripción STAT/metabolismo , Transducción de SeñalRESUMEN
Recombinant interferon-α (rIFN-α) has been widely used for treating viral infections. However, the clinical efficacy of unmodified rIFN-α is limited due to small molecular size and rapid clearance from circulation. In this study we developed a novel strategy for half-life extension of porcine IFN-α (PoIFN-α) by fusion to the immunoglobulin (Ig)-binding C2 domain of streptococcal protein G (SPG). The coding sequences for PoIFN-α6 and SPG C2 domain, with a tobacco etch virus (TEV) protease recognition sequence introduced at the 5-end, were cloned into an elastin-like polypeptide (ELP) fusion expression vector and expressed as an ELP-PoIFNα-C2 fusion protein. After optimization of the conditions for soluble protein expression and purification, the fusion protein was purified to more than 90% purity by two rounds of inverse transition cycling (ITC) in the presence of 0.5% Triton X-100. After cleavage with self-aggregating peptide ELK-16-tagged tobacco etch virus protease, the protease was removed by quick centrifugation and PoIFNα-C2 protein was recovered by an additional round of ITC with 98% purity. Western blotting analysis showed that PoIFNα-C2 protein had the specific affinity for pig IgG binding. The antiviral assay showed that PoIFNα-C2 protein had potent antiviral activities against vesicular stomatitis virus and porcine pseudorabies virus. After single intravenous or subcutaneous injection into rats, PoIFNα-C2 protein showed 16- or 4-fold increase in serum half-life with significantly improved bioavailability.
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Proteínas Bacterianas/farmacocinética , Herpesvirus Suido 1/efectos de los fármacos , Interferón-alfa/farmacocinética , Proteínas Recombinantes de Fusión/farmacocinética , Vesiculovirus/efectos de los fármacos , Secuencias de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Bioensayo , Disponibilidad Biológica , Línea Celular , Clonación Molecular , Elastina/genética , Elastina/metabolismo , Endopeptidasas/química , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Semivida , Herpesvirus Suido 1/crecimiento & desarrollo , Herpesvirus Suido 1/inmunología , Humanos , Interferón-alfa/genética , Interferón-alfa/inmunología , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Porcinos , Vesiculovirus/crecimiento & desarrollo , Vesiculovirus/inmunologíaRESUMEN
Polycythemia vera (PV) is the most common Philadelphia chromosome-negative myeloproliferative neoplasm. Whereas low-risk patients are treated with aspirin and phlebotomy, high-risk patients receive cytoreductive therapy, which most commonly consists of hydroxyurea in the United States. Concerns about the long-term safety of hydroxyurea, as well as a desire for more efficacious and targeted therapy, have led to the development of novel therapies for high-risk patients with PV. Pegylated interferon (IFN) has shown promise in phase 2 studies of PV, and preliminary data from ongoing phase 3 studies suggest noninferiority as a frontline therapy. Efficient count control, tolerability, and even molecular responses as a salvage therapy have been demonstrated. Ropeginterferon-α-2b, a monopegylated IFN with a longer half-life and less frequent dose interval compared with recombinant or pegylated IFN, is an impressive agent in development. Ruxolitinib has a proven role as second-line therapy for PV, but an ongoing trial combining ruxolitinib and IFN as salvage therapy is under way. Early-phase clinical trials have also suggested that MDM2 inhibitors such as idasanutlin and histone deacetylase inhibitors should continue in their development. If these novel agents are able to modify the natural history of PV, the treatment paradigm in newly diagnosed patients will evolve from risk-adapted or reactive treatment toward early interventions.
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Interferón alfa-2/uso terapéutico , Interferón-alfa/uso terapéutico , Policitemia Vera/tratamiento farmacológico , Polietilenglicoles/uso terapéutico , Pirazoles/uso terapéutico , Pirrolidinas/uso terapéutico , para-Aminobenzoatos/uso terapéutico , Ensayos Clínicos Fase II como Asunto , Ensayos Clínicos Fase III como Asunto , Semivida , Humanos , Hidroxiurea/farmacocinética , Hidroxiurea/uso terapéutico , Interferón alfa-2/farmacocinética , Interferón-alfa/farmacocinética , Nitrilos , Policitemia Vera/metabolismo , Policitemia Vera/patología , Polietilenglicoles/farmacocinética , Pirazoles/farmacocinética , Pirimidinas , Pirrolidinas/farmacocinética , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapéutico , Terapia Recuperativa/métodos , para-Aminobenzoatos/farmacocinéticaRESUMEN
Most therapeutic proteins except antibodies necessitate frequent dosing at high concentrations due to their short circulation half-lives, leading to limited therapeutic efficacy, serious adverse side effects and poor patient compliance. Herein we report a strategy of thermoresponsive polypeptide fusion to genetically engineer a super-long-acting interferon alpha fused with a body-temperature-responsive polypeptide. After a single subcutaneous injection in a mouse model, this interferon alpha can in situ form a depot to show a one-month zero-order sustained release, which would enable a once-trimonthly dosing in humans. As a result, it exhibits greatly enhanced tumor accumulation and tumor eradication as well as substantially improved tolerability and biosafety. This strategy provides a promising solution to dramatically enhance the pharmacological performance of therapeutic proteins with short circulation half-lives while reducing the side effects.
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Preparaciones de Acción Retardada/uso terapéutico , Interferón-alfa/uso terapéutico , Neoplasias/tratamiento farmacológico , Péptidos/uso terapéutico , Animales , Temperatura Corporal , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/farmacocinética , Femenino , Inyecciones Subcutáneas , Interferón-alfa/administración & dosificación , Interferón-alfa/química , Interferón-alfa/farmacocinética , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/patología , Péptidos/administración & dosificación , Péptidos/química , Péptidos/farmacocinética , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/uso terapéuticoRESUMEN
The rational combination of recombinant IFN-α2b and IFN-γ resulted in a new formulation of interferons (HeberFERON) with improved pharmacodynamics. In basal cell carcinomas HeberFERON produces a more rapid antitumor effect and results in a larger number of complete responses. In patients with glioblastoma multiforme, the administration of HeberFERON after surgery and radiotherapy results in an estimated overall survival of 19 months. Patients with stage III or IV renal cell carcinoma also appear to benefit from the intravenous administration of HeberFERON, with prolongation of survival and good quality of live. HeberFERON offers a promising alternative formulation of interferons for the treatment of cancer with a very favorable safety profile.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Interferón-alfa/uso terapéutico , Interferón gamma/uso terapéutico , Neoplasias/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica , Humanos , Interferón alfa-2 , Interferón-alfa/administración & dosificación , Interferón-alfa/farmacocinética , Interferón gamma/administración & dosificación , Interferón gamma/farmacocinética , Neoplasias/genética , Neoplasias/metabolismo , Proteoma/metabolismo , Calidad de Vida , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
Pegylated interferon alfa-2a (PEG-IFN alfa-2a), which was developed to overcome the disadvantages of conventional formulations, is widely prescribed for hepatitis B or C virus infection. It is characterized by pharmacokinetic (PK) and pharmacodynamic (PD) properties much different from those of conventional forms. The present study developed a population PK-PD model of subcutaneous PEG-IFN alfa-2a in a Korean population. For PK, IFN alfa-2a concentrations were described by a 1-compartment model with first-order absorption, preceded by skin-to-depot first-order input. For PD, serum 2'-5' oligoadenylsynthetase activity was described by an effect compartment model incorporating a tolerance compartment. The baseline serum 2'-5' oligoadenylsynthetase level was found to have an inverse relationship with sensitivity to tolerance, leading to high tolerance at low baseline. The model revealed that subjects with low baselines experienced time delay, while those with high baselines showed tolerance in their concentration-effect relationships. The developed models matched well with data and simulation results, and the model showed that the optimal dose decreases with the baseline, with no dose effective for a baseline >250 pmol/dL. Our results can serve as a basis for improving dosing regimens of PEG-IFN alfa-2a in adult patients with chronic hepatitis B or C infection.
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Antivirales/farmacología , Antivirales/farmacocinética , Interferón-alfa/farmacología , Interferón-alfa/farmacocinética , Polietilenglicoles/farmacología , Polietilenglicoles/farmacocinética , Administración Cutánea , Adulto , Algoritmos , Antivirales/administración & dosificación , Antivirales/sangre , Simulación por Computador , Tolerancia a Medicamentos , Humanos , Interferón-alfa/administración & dosificación , Interferón-alfa/sangre , Masculino , Modelos Biológicos , Polietilenglicoles/administración & dosificación , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/sangre , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Adulto JovenRESUMEN
Neutropenia is a hematologic disorder commonly reported in patients with chronic hepatitis C viral (HCV) infection. The objective of the present analysis is to describe the change in neutrophil count resulting from peglated interferon alpha 2-a (PEG-IFN α-2a) therapy in HCV-infected patients. A population pharmacodynamic model will be developed. We also plan to identify patient characteristics that contribute to the development of PEG-IFN α-2a-induced neutropenia in hepatitis C patients. A population pharmacodynamic modeling approach was applied to a cohort of patients (n = 292) with chronic HCV infection. Modeling was performed using NONMEM 6. Data was obtained from two phases III studies sponsored by Hoffmann-La Roche. Covariate screening was applied to evaluate various demographic and clinical characteristics as possible predictors of pharmacodynamic parameter during model development. A total of 4517 neutrophil counts from 292 subjects were analyzed by the proposed population pharmacodynamic model. A constant residual error model was used to the log-transformed neutrophil count. Platelet baseline count and uric acid level were identified as predictors of neutrophil pharmacodynamic model. Increased baseline platelet count is expected to result in higher neutrophil baseline. A higher neutrophil baseline is also expected in patients with increased uric acid level. In conclusion, a mechanistic pharmacodynamic model was developed. The effect of various covariates was included in the model. This allows the prediction of neutrophil count following antiviral therapy in patients with hepatitis C infection. Clinical studies: NV15942 and NV15801.
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Antivirales/efectos adversos , Hepatitis C Crónica/tratamiento farmacológico , Interferón-alfa/efectos adversos , Modelos Biológicos , Neutropenia/inducido químicamente , Polietilenglicoles/efectos adversos , Adulto , Anciano , Femenino , Hepatitis C Crónica/inmunología , Humanos , Interferón-alfa/farmacocinética , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Neutropenia/inmunología , Neutrófilos/efectos de los fármacos , Polietilenglicoles/farmacocinética , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/farmacocinética , Ribavirina/farmacologíaRESUMEN
BACKGROUND: Several countries have used pegylation technology to improve the pharmacokinetic properties of essential drugs. Recently, a novel interferon alfa-2b protein conjugated to four-branched 12 kDa polyethylene glycol molecules was developed jointly between Cuba and Brazil. The aim of this study was to compare the pharmacokinetic properties of BIP48 (pegylated interferon alfa-2b from Bio-Manguinhos/Fiocruz, Brazil) to those of PEGASYS® (commercially available pegylated interferon alfa-2a from Roche Pharmaceutical). METHODS: This phase I, single-centre, randomized, double-blind crossover trial enrolled 31 healthy male volunteers aged 19 to 35 who were allocated to two stages, either side of a 5-week wash-out period, with each arm lasting 14 consecutive days after subcutaneous administration of 180 µg of one formulation or the other (study or comparator). The main outcome variable was serum pegylated interferon concentrations in 15 samples collected during the course of the study and tested using an enzyme immunoassay. RESULTS: There were no differences between formulations in terms of magnitude or absorption parameters. Analysis of time parameters revealed that BIP48 remained in the body significantly longer than PEGASYS® (Tmax: 73 vs. 54 h [p = 0.0010]; MRT: 133 vs. 115 h [p = 0.0324]; ke: 0.011 vs. 0.013 h(-1) [p = 0.0153]; t1/2: 192 vs. 108 h [p = 0.0218]). CONCLUSION: BIP48 showed the expected pharmacokinetic profile for a pegylated product with a branched molecular structure. Compared to PEGASYS®, the magnitude absorption was similar, but time parameters were consistent with slower elimination. Further studies should be conducted to evaluate the clinical implications of these findings. A phase II-III repeated-dose clinical trial is ongoing to study these findings in patients with chronic hepatitis C virus infection. TRIAL REGISTRATION: This study is registered on the ClinicalTrials.gov platform (accession number NCT01889849 ). This trial was retrospectively registered in June 2013.
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Interferón alfa-2/farmacocinética , Interferón-alfa/farmacocinética , Polietilenglicoles/farmacocinética , Adulto , Estudios Cruzados , Método Doble Ciego , Voluntarios Sanos , Humanos , Interferón alfa-2/sangre , Interferón-alfa/sangre , Masculino , Proteínas Recombinantes/sangre , Proteínas Recombinantes/farmacocinética , Adulto JovenRESUMEN
Systemically administered interferons are rapidly cleared from the circulation thus requiring frequent dosing to maintain the therapeutic levels of circulating interferon. This is particularly problematic for their use in the treatment of chronic diseases. The purpose of this study was to evaluate the potential of proprietary calcium phosphate (CaP) particles to deliver biologically active interferon alpha (IFNα) via the lungs into systemic circulation. Recombinant human IFNα-2a was formulated with proprietary CaP particles. In vitro biological activity of IFNα was assessed for its potential to activate IFN-induced cellular pathways in HEK-Blu-IFN α/ß cell cultures. Antiviral activity was evaluated against vesicular stomatitis virus (VSV) infection of HeLa cells. Male BALB/c mice were used to evaluate the absorption of IFNα from CaP-IFNα across the lungs following intratracheal (IT) instillation. Serum IFNα concentrations up to 9 h post-treatment were determined. Data were analyzed to obtain pharmacokinetic (PK) parameters. Data from these studies indicated that IFNα formulated with CaP retains its biological activity, and it is transported into circulation in a dose-dependent manner. PK analysis showed larger than two-fold area under the serum concentration-time curve (AUC) for CaP-IFNα compared to non-formulated IFNα administered IT. The IFNα formulated with CaP had two-fold longer half-life (t1/2) and mean residence time (MRT) relative to IFNα alone administered by injection. Clearance of CaP-IFNα was slower than IFNα administered IM or IT. Relative bioavailability of CaP-IFNα was 1.3-fold of IFNα injection and twofold of IFNα administered IT. Furthermore, inhalation of aerosolized CaP did not indicate any lung toxicity in animals.