RESUMO
The pleiotropic biologic effects of interferon (IFN) are mediated through regulation of the expression of numerous IFN-sensitive genes. Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were analyzed to study the immunoregulatory and antiviral messenger RNAs (mRNAs) and proteins regulated by pegylated IFN-alpha2b (PEG-IFN-alpha2b) and IFN-alpha2b. A dose-dependent and time-dependent response for multiple IFN-regulated genes was observed. IFN-dependent protein production and secretion were correlated with IFN-regulated mRNA induction. Overall regulation of gene expression patterns for PEG-IFN-alpha2b and IFN-alpha2b was comparable, even though the antiviral activity of PEG-IFN-alpha2b demonstrated a longer biologic halflife in vitro compared with IFN-alpha2b. To study the heterogeneity of responses, PBMCs obtained from over 25 healthy donors were analyzed. Within a particular donor dataset, gene-specific and dose-dependent responses to PEG-IFN-alpha2b treatment, demonstrated in both the amplitude of transcriptional upregulation and the duration of sustained mRNA upregulation, were observed. However because of donor heterogeneity, the amplitude of a given transcriptional response could not be predicted for a specific dose of PEG-IFN-alpha2b. Notably, mRNA levels of oligoadenylate synthetase (OAS), double-stranded RNA (dsRNA)-activated protein kinase (PKR), IP-10, IFN-stimulated gene 54 (ISG54), and ISG15 were upregulated after 120 h of continuous PEG-IFN-alpha2b treatment. These results suggest that the use of antiviral and immunoregulatory protein mRNA levels as markers to assess the therapeutic efficacy of IFN-alpha2b and PEG-IFN-alpha2b against viral and neoplastic diseases in clinical trials is promising but will require further analysis using clinical patient samples.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Interferon-alfa/farmacologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Antivirais/farmacologia , Células Cultivadas , Quimiocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Humanos , Interferon alfa-2 , Interferon-alfa/metabolismo , Cinética , Leucócitos/imunologia , Leucócitos/virologia , Análise de Sequência com Séries de Oligonucleotídeos , Polietilenoglicóis , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas RecombinantesAssuntos
Interferon-alfa/química , Interferon-alfa/farmacologia , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Interferon alfa-2 , Interferon-alfa/imunologia , Interferon-alfa/toxicidade , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Polietilenoglicóis/toxicidade , Transporte Proteico , Proteínas Recombinantes , Fator de Transcrição STAT1 , Transativadores/metabolismoRESUMO
Therapeutic pegylated interferon-alphas (IFN-alpha) are mixtures of positional isomers that have been monopegylated at specific sites on the core IFN-alpha molecule. The pegylation results in lower in vitro specific activity associated with the core IFN-alpha molecule that is related to the site of pegylation and size of polyethylene glycol (PEG) attached. We prepared purified, homogeneous, positional pegylation isomers of IFN-alpha2b that were monopegylated using 5-30-kDa linear PEG molecules attached at 7 primary reactive amino acid residues: Cys(1), His(34), Lys(31), Lys(83), Lys(121), Lys(131), and Lys(134). The isomers were evaluated for STAT translocation and antiviral and antiproliferative activity. The site of pegylation strongly influenced activity relative to an IFN-alpha2b control. The highest residual activity was observed with the His(34) positional isomers, and the lowest was observed with the Cys(1) positional isomers. The Lys positional isomers demonstrated intermediate activity, with a general order of Lys(134) > Lys(83) approximately Lys(131) approximately Lys(121) > Lys(31). The progressive relationship between decreased activity and increased PEG size suggests that pegylation may interfere with interaction and binding of IFN-alpha to the IFNAR1-IFNAR2 heterodimeric receptor. The higher specific activity associated with the His(34) positional isomer suggests that this site may be favorable for pegylating IFN-alpha2b molecules.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Portadores de Fármacos/síntese química , Interferon-alfa/química , Polietilenoglicóis/síntese química , Polietilenoglicóis/farmacologia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Antivirais/química , Antivirais/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Humanos , Interferon alfa-2 , Interferon-alfa/farmacologia , Janus Quinase 1 , Peso Molecular , Polietilenoglicóis/química , Proteínas Recombinantes , Fator de Transcrição STAT1 , Relação Estrutura-AtividadeRESUMO
The relative activities of interferon-alpha2b (IFN-alpha2b) and polyethylene glycol(12000)-IFN-alpha2b (PEG-IFN-alpha2b) were assessed in cell culture studies using WM9 melanoma or ACHN renal cell carcinoma cell lines. Interferon-alpha2b and PEG-IFN-alpha2b had identical antiproliferative activities when tested in cell proliferation studies conducted with equivalent antiviral units of each IFN preparation. Neither IFN formulation was effective in inducing apoptosis in WM9 melanoma cells, but both increased slightly the percentage of ACHN cells undergoing apoptosis as assessed by Annexin V staining. Interferon-alpha2b and PEG-IFN-alpha2b both activated signal transducer and activator of transcription complexes, and the duration of complex activation was similar for both IFN formulations. Induction of different IFN-stimulated genes was assessed by Northern blotting and the quantitative real-time reverse transcription-coupled polymerase chain reaction (RT-PCR) in WM9 melanoma, ACHN renal cell carcinoma, U937 lymphoma, and MOLT-4 and Mono Mac 6 leukemia cell lines. Interferon-alpha2b and PEG-IFN-alpha2b had equivalent gene-modulatory activities within each of these tumor cell lines, although cell line-specific induction patterns were observed. When compared with the antiviral 50% inhibitory concentration (IC(50)) values, the dose-dependent gene expression data correlated with cell sensitivity to IFN treatment. Together, the drug comparability and cell sensitivity data suggest a predictive relation between dose, time, antiviral activity, and gene transcription effects. Therefore, although the specific activity of IFN-alpha2b is approximately three times greater than PEG-IFN-alpha2b, the two preparations have identical in vitro biologic activities when applied to cells at equivalent antiviral units.