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Improved Pharmacokinetic Properties of a Polyethylene Glycol-Modified Form of Interferon-??-1a with Preserved in Vitro Bioactivity
Abstract
Interferon therapies suffer from a relatively short half-life of the products in circulation. To address this issue we investigated the effects of polyethylene glycol modification (PEGylation) on the pharmacokinetic properties of human interferon (IFN)-beta-1a. PEGylation with a linear 20-kDa PEG targeted at a single site on the N-terminal amine had no deleterious effect on its specific activity in an in vitro antiviral assay. In monkeys, PEG IFN-beta-1a treatment induced neopterin and beta2-microglobulin expression (pharmacodynamic markers of activity). Systemic clearance values in monkeys, rats, and mice decreased, respectively, from 232, 261, and 247 ml/h/kg for the unmodified IFN-beta-1a to 30.5, 19.2, and 18.7 ml/h/kg for the PEGylated form, while volume of distribution values decreased from 427, 280, and 328 ml/kg to 284, 173, and 150 ml/kg. The decreased clearance and volume of distribution resulted in higher serum antiviral activity in the PEG IFN-beta-1a-treated animals. In the rat, a more extensive set of dosing routes was investigated, including intraperitoneal, intratracheal, and oral administration. Bioavailability for the PEG IFN-beta-1a was similar to the unmodified protein for each of the extravascular routes examined. For the intraperitoneal route, bioavailability was almost 100%, whereas for the oral and intratracheal routes absorption was low (<5%). In rats, subcutaneous bioavailability was moderate (28%), whereas in monkeys it was approximately 100%. In all instances an improved pharmacokinetic profile for the PEGylated IFN-beta-1a was observed. These findings demonstrate that PEGylation greatly alters the pharmacokinetic properties of IFN-beta-1a, resulting in an increase in systemic exposure following diverse routes of administration.
... Site directed mutagenesis revealed that the N terminal region of IFN beta is the least critical for its activity [10,11]. In animal experiments, it has been shown that IFN beta having an N terminus modified with PEG demonstrated improved phar macokinetic parameters [12]. Despite a decrease in the specific activity in an in vitro assay, the biological activity of the protein was higher due to a significant increase in its half life. ...
... Despite a decrease in the specific activity in an in vitro assay, the biological activity of the protein was higher due to a significant increase in its half life. The antiviral activity of the obtained PEG IFN beta conjugates depended on the molecular weight (5-20 kDa) and structure of PEG; it was 15-50% of the specific activity of the unmod ified protein [12,13]. These findings led to the assumption that the use of 20 kDa PEG for the mod ification could result in the preparation of PEG IFN beta with a high therapeutic efficacy in the treatment of multiple sclerosis. ...
... The specific activity of the IFN beta 1a conju gate modified at its N terminus with 20 kDa PEG was 50% [19]. Similar results were published previously for IFN beta 1b [12]. Studies of the specific antiviral activity of the IFN beta 1a conjugate with 30 kDa PEG showed that the specific antiviral activity of the modified protein in vitro was (5.04 ± 0.3) × 10 7 IU/mg. ...
The recombinant human interferon (IFN) beta-1a was modified with polyethylene glycol (PEG). The reaction was performed with the use of activated linear butyraldehyde PEG derivative with a molecular weight of 30 kDa. As a result of the multifactorial experiment, the correlation between the reaction conditions and the yield of the monoPEGylated protein was demonstrated and the optimal PEGylation conditions were established. We developed a one-step chromatographic purification scheme that makes it possible to obtain monoPEG-IFN beta-1a conjugate a purity above 98%. Mass-spectrometric studies showed that the purified conjugate is the PEGylated IFN beta-1a, in which the N-terminal methionine is bound with a PEG molecule. The molecular weight of the conjugate is 54130 Da. The obtained PEG-IFN beta-1a has a specific antiviral activity; it can be considered as a promising candidate in the design of prolonged-release medicine for the treatment of multiple sclerosis.
... Previous studies had shown that the half-life of IFNβ in the bloodstream was less than 5 min, and 1 h after venous injection of 6 × 10 6 U IFNβ, the serum concentration declined to 8 U/ml which was much less than the required amount for preventing tumor cell reproduction and activating natural killer cells (Pepinsky et al. 2001). On the other hand, such a short half-life made its pharmaceutical production very expensive. ...
... Production of IFNβ with enhanced solubility has also been reported so far (Otto et al. 2009;Carr 2009;Shin et al. 2012). A polyethylene glycol-modified form of IFNβ1 was furthermore designed to improve its solubility and half-life (Pepinsky et al. 2001). In other studies, substitution of asparagine to aspartic acid at residue 25 and modification of its polypeptides was used in order to improve its biological activity and to reduce its immunogenicity, respectively (Carr 2000). ...
Interferon beta (IFNβ) is transiently expressed in response to viral infections and widely used to treat relapsing-remitting multiple sclerosis (MS). We introduced mutations in the IFNβ gene (in the 27th and 101st codons and in the Kozak sequence, and also deletion of 3′ and 5′ unstable, untranslated region, UTR) with the aim of increasing the expression of IFNβ. Computational analyses of mutant and wild-type RNAs and proteins of IFNβ by RNAfold, ASAView, HOPE and Ramachandran plot, and iStable web servers showed that the mutations could decrease RNA stability, protein solvent accessibility, and protein stability but could not change correct folding. Two recombinant IFNβ101 and IFNβ101+27 constructs were designed by site-directed mutagenesis. The wild-type IFNβ gene also was used as a control. In vitro experiments by quantitative real-time PCR, dot blot, SDS-PAGE, and Western blot assays showed an increased expression level of recombinant IFNβs. 79.9-fold, 99.7-fold, and 190-fold elevations in the mRNA expression of IFNβw, IFNβ101, and IFNβ101+27 were seen, respectively, in comparison with the endogenous IFNβ mRNA in untransfected HEK293T cells. The IFNβ mRNA expression was increased 2.38-fold and 1.25-fold for 101+27 and 101 mutated forms, respectively, in comparison with the IFNβ wild-type construct. An elevation in IFNβ protein production was also clearly detected in the transfected HEK293T cell containing recombinant IFNβ101 and IFNβ101+27 constructs. Finally, these directed mutations in the IFNβ gene successfully elevated protein and mRNA production but in silico analyses of mutant mRNAs showed decreased mRNA stability, unlike previous studies, in comparison with the wild-type mRNA.
... Previously, another PEG-IFN-b, called PEG-IFN-b-1a, was generated that contains 20-kDa PEG at the N-terminal region. (27) However, this form of PEG-IFN-b totally loses its activity when PEG with 40 kDa is conjugated. (27) Another IFN-b-1b bound with 40-kDa PEG also exhibited attenuated anti-viral activity to 19-35%, compared with non-pegylated IFN-b-1b. ...
... (27) However, this form of PEG-IFN-b totally loses its activity when PEG with 40 kDa is conjugated. (27) Another IFN-b-1b bound with 40-kDa PEG also exhibited attenuated anti-viral activity to 19-35%, compared with non-pegylated IFN-b-1b. (28) Furthermore, the IFN-b-1b molecule itself has reduced anti-viral activity compared to natural IFN-b. ...
Malignant ascites manifests as an end-stage event during the progression of a number of cancers and lacks a generally accepted standard therapy. Interferon-β (IFN-β) has been used to treat several cancer indications; however, little is known about the efficacy of IFN-β on malignant ascites. In the present study, we report on the development of a novel, engineered form of human and murine IFN-β, each conjugated with a polyethylene glycol molecule (PEG-hIFN-β and PEG-mIFN-β, respectively). We provide evidence that these IFN-β molecules retain anti-viral potency comparable to unmodified IFN-β in vitro and manifested improved pharmacokinetics in vivo. Interestingly, PEG-mIFN-β significantly inhibited the accumulation of ascites fluid and vascular permeability of the peritoneal membrane in models of ovarian cancer and gastric cancer cell xenograft mice. We further show that PEG-hIFN-β directly suppresses VEGF165 -induced hyperpermeability in a monolayer of human vascular endothelial cells and that PEG-mIFN-β enhanced gene expression for a number of cell adhesion related molecules in mouse vascular endothelial cells. Taken together, these findings unveil a hitherto unrecognized potential of IFN-β in maintaining vascular integrity, and provide proof-of-mechanism for a novel and long-acting pegylated hIFN-β for the therapeutic treatment of malignant ascites. This article is protected by copyright. All rights reserved.
... As expected, MX2-LUC mice that do not express the HyBNAR transgene demonstrate minimal IFN-I induced luminescence (Fig. 9B). These data support that in contrast to humans, there is a very limited timeframe of PD response to single injection of IFN-Is in the mouse, and is consistent with published PK studies suggesting a serum half-life of injected IFN-Is in mice of less than one hour [30,31]. ...
... It has been published that the serum half-life of IFN-I in mice ranges from as little as 0.5 hours to as much as 4 hours [30,31,33,34]. The PD data presented in this paper indirectly supports that the PK half-life of IFN-Is in mouse is indeed very short. ...
We have generated transgenic mice that harbor humanized type I interferon receptors (IFNARs) enabling the study of type I human interferons (Hu-IFN-Is) in mice. These "HyBNAR" (Hybrid IFNAR) mice encode transgenic variants of IFNAR1 and IFNAR2 with the human extracellular domains being fused to transmembrane and cytoplasmic segments of mouse sequence. B16F1 mouse melanoma cells harboring the HyBNAR construct specifically bound Hu-IFN-Is and were rendered sensitive to Hu-IFN-I stimulated anti-proliferation, STAT1 activation and activation of a prototypical IFN-I response gene (MX2). HyBNAR mice were crossed with a transgenic strain expressing the luciferase reporter gene under the control of the IFN-responsive MX2 promoter (MX2-Luciferase). Both the HyBNAR and HyBNAR/MX2-Luciferase mice were responsive to all Hu-IFN-Is tested, inclusive of IFNα2A, IFNβ, and a human superagonist termed YNSα8. The mice displayed dose-dependent pharmacodynamic responses to Hu-IFN-I injection, as assessed by measuring the expression of IFN-responsive genes. Our studies also demonstrated a weak activation of endogenous mouse interferon response, especially after high dose administration of Hu-IFNs. In sharp contrast to data published for humans, our pharmacodynamic readouts demonstrate a very short-lived IFN-I response in mice, which is not enhanced by sub-cutaneous (SC) injections in comparison to other administration routes. With algometric differences between humans and mice taken into account, the HyBNAR mice provides a convenient non-primate pre-clinical model to advance the study of human IFN-Is.
... Additionally, PEGylated interferon α-2b has shown effectiveness in treating hepatitis C virus (HCV) among children with end-stage renal disease (ESRD) (Mogahed et al., 2016). Moreover, PEGylation of interferon β-1a has been found to improve its pharmacokinetic and pharmacodynamic properties (Pepinsky et al., 2001;Cocco and Marrosu, 2015). These findings collectively demonstrate that optimal PEG modification enhances the bioavailability of interferons. ...
With the rapid advancement of genetic and protein engineering, proteins and peptides have emerged as promising drug molecules for therapeutic applications. Consequently, there has been a growing interest in the field of chemical modification technology to address challenges associated with their clinical use, including rapid clearance from circulation, immunogenicity, physical and chemical instabilities (such as aggregation, adsorption, deamination, clipping, oxidation, etc.), and enzymatic degradation. Polyethylene glycol (PEG) modification offers an effective solution to these issues due to its favorable properties. This review presents recent progress in the development and application of PEGylated therapeutic proteins and peptides (TPPs). For this purpose, firstly, the physical and chemical properties as well as classification of PEG and its derivatives are described. Subsequently, a detailed summary is provided on the main sites of PEGylated TPPs and the factors that influence their PEGylation. Furthermore, notable instances of PEG-modified TPPs (including antimicrobial peptides (AMPs), interferon, asparaginase and antibodies) are highlighted. Finally, we propose the chemical modification of TPPs with PEG, followed by an analysis of the current development status and future prospects of PEGylated TPPs. This work provides a comprehensive literature review in this promising field while facilitating researchers in utilizing PEG polymers to modify TPPs for disease treatment.
... In addition, PEGylation could also lower the immunogenicity of peptides through steric masking of potential antigenic sites in the peptides to prevent recognition by the body's immune system [1,80]. FDA-approved PEGylated protein therapeutics which can be found in the market include Krystexxa, PEGASYS (peginterferon alpha 2b), Adagen (pegademase), Oncaspar (pegaspargase), Somavert (pegvisomant), Neulasta (pegfilgrastim), Mircera (CERA; PEG-EPO), Cimzia (certolizumab), Macugen (pegaptanib), and Plegridy (peginterferon beta-1a) [45,[82][83][84][85][86][87][88][89][90][91]. However, there is a potential safety concern involving PEG because it is non-biodegradable. ...
Peptides are a rapid-growing class of therapeutics with unique and desirable physicochemical properties. Due to disadvantages such as low membrane permeability and susceptibility to proteolytic degradation, peptide-based drugs have limited bioavailability, a short half-life, and rapid in vivo elimination. Various strategies can be applied to improve the physicochemical properties of peptide-based drugs to overcome limitations such as limited tissue residence time, metabolic instability, and low permeability. Applied strategies including backbone modifications, side chain modifications, conjugation with polymers, modification of peptide termini, fusion to albumin, conjugation with the Fc portion of antibodies, cyclization, stapled peptides, pseudopeptides, cell-penetrating peptide conjugates, conjugation with lipids, and encapsulation in nanocarriers are discussed.
... Moreover, the relatively short half-life time of IFN-β may limit sufficient circulating IFN-β concentrations. Strategies to increase the half-life time of IFN-β, such as IFN-based conjugates or PEGylated form of IFN-β, have demonstrated promising results to achieve higher serum concentration, requiring lower and less frequent doses compared with the conventional IFNs (36,37). While recombinant IFN therapies are generally given as exogenous pharmaceuticals, it is suggested that the autocrine and paracrine actions of endogenous type I IFNs on tumor growth control (both the direct and indirect effects) are much stronger. ...
Resistance to gemcitabine is common and critically limits its therapeutic efficacy in patients with pancreatic cancer. Interferon‑beta (IFN‑β) induces numerous antitumor effects and synergizes with gemcitabine treatment. The immunomodulatory effects of this treatment regimen have not yet been described. In the present study, the antitumor effect of IFN‑β combined with gemcitabine was investigated in immune competent mice. Mouse KPC3 cells were used in all experiments. Treatment effects were determined with cell proliferation assay. Reverse transcription‑quantitative PCR was used to measure gene expression. For in vivo experiments, cells were subcutaneously injected in immune competent mice. For immune profiling, NanoString analysis was performed on tumor samples of treated and untreated mice. Baseline expression of Ifnar‑1 and Ifnar‑2c in KPC3 cells was 1.42±0.16 and 1.50±0.17, respectively. IC50 value of IFN‑β on cell growth was high (>1,000 IU/ml). IFN‑β pre‑treatment increased the in vitro response to gemcitabine (1.3‑fold decrease in EC50; P<0.001). In vivo, tumor size was not statistically significant smaller in mice treated with IFN‑β plus gemcitabine (707±92 mm3 vs. 1,239±338 mm3 in vehicle‑treated mice; P=0.16). IFN‑β alone upregulated expression of numerous immune‑related genes. This effect was less pronounced when combined with gemcitabine. For the first time, to the best of our knowledge, the immunomodulatory effects of IFN‑β, alone and combined with gemcitabine, in pancreatic cancer were reported. Prognostic markers for predicting effective responses to IFN‑β therapy are urgently needed.
... For Plegridy ® (a 20 kDa-PEG interferon beta-1a indicated for multiple sclerosis), the purification process after PEGylation consists of two sequential chromatography steps: SEC followed by CEX. In the SEC step, the PEGylation reaction mixture is loaded into a Superose 6 resin and results in the separation of the mono-PEGylated conjugate from the native protein (Pepinsky et al., 2001;Pepinsky et al., 2005). The nominal molecular weight of the PEG reactant (20 kDa) and the molecular weight of interferon beta-1a (∼23 kDa) are basically equal, resulting in a conjugate with doubled molecular weight compared to that of the native protein. ...
Proteins, which have inherent biorecognition properties, have long been used as therapeutic agents for the treatment of a wide variety of clinical indications. Protein modification through covalent attachment to different moieties improves the therapeutic’s pharmacokinetic properties, affinity, stability, confers protection against proteolytic degradation, and increases circulation half-life. Nowadays, several modified therapeutic proteins, including PEGylated, Fc-fused, lipidated, albumin-fused, and glycosylated proteins have obtained regulatory approval for commercialization. During its manufacturing, the purification steps of the therapeutic agent are decisive to ensure the quality, effectiveness, potency, and safety of the final product. Due to the robustness, selectivity, and high resolution of chromatographic methods, these are recognized as the gold standard in the downstream processing of therapeutic proteins. Moreover, depending on the modification strategy, the protein will suffer different physicochemical changes, which must be considered to define a purification approach. This review aims to deeply analyze the purification methods employed for modified therapeutic proteins that are currently available on the market, to understand why the selected strategies were successful. Emphasis is placed on chromatographic methods since they govern the purification processes within the pharmaceutical industry. Furthermore, to discuss how the modification type strongly influences the purification strategy, the purification processes of three different modified versions of coagulation factor IX are contrasted.
... To date, numerous drugs with PEGylated compounds have been approved. For example, PEGylated interferons have been used for therapies of multiple sclerosis [111] and hepatitis C [112]. Pegademase bovine has been applied in enzyme replacement therapy for severe combined immunodeficiency [113], while PEGylated peptides have been used for acromegaly [114,115]. ...
Adenovirus-based vectors are playing an important role as efficacious genetic vaccines to fight the current COVID-19 pandemic. Furthermore, they have an enormous potential as oncolytic vectors for virotherapy and as vectors for classic gene therapy. However, numerous vector–host interactions on a cellular and noncellular level, including specific components of the immune system, must be modulated in order to generate safe and efficacious vectors for virotherapy or classic gene therapy. Importantly, the current widespread use of Ad vectors as vaccines against COVID-19 will induce antivector immunity in many humans. This requires the development of strategies and techniques to enable Ad-based vectors to evade pre-existing immunity. In this review article, we discuss the current status of genetic and chemical capsid modifications as means to modulate the vector–host interactions of Ad-based vectors.
... One example is the use of IFN based conjugates, which increase the half-life time of IFN and potentially results into higher concentrations at the tumor site [44]. In addition, the PEGylated form of IFN resulted in a higher serum concentration, requiring lower and less frequent doses compared to the conventional IFNs [45]. The PEGylated form of IFN-α has already proven to be effective in the treatment of melanoma and metastatic renal cell carcinoma patients [46,47]. ...
Background:
Adjuvant gemcitabine for pancreatic cancer has limited efficacy in the clinical setting. Impaired drug metabolism is associated with treatment resistance. We aimed to evaluate the chemosensitising effect of interferon-beta (IFN-β).
Methods:
BxPC-3, CFPAC-1, and Panc-1 cells were pre-treated with IFN-β followed by gemcitabine monotherapy. The effect on cell growth, colony formation, and cell cycle was determined. RT-qPCR was used to measure gene expression. BxPC-3 cells were used in a heterotopic subcutaneous mouse model.
Results:
IFN-β increased sensitivity to gemcitabine (4-, 7.7-, and 1.7-fold EC50 decrease in BxPC-3, CFPAC-1, and Panc-1, respectively; all P < 0.001). Findings were confirmed when assessing colony formation. The percentage of cells in the S-phase was significantly increased after IFN-β treatment only in BxPC-3 and CFPAC-1 by 12 and 7%, respectively (p < 0.001 and p < 0.05, respectively). Thereby, IFN-β upregulated expression of the drug transporters SLC28A1 in BxPC-3 (252%) and SLC28A3 in BxPC-3 (127%) and CFPAC-1 (223%) (all p < 0.001). In vivo, combination therapy reduced tumor volume with 45% (P = 0.01). Both ex vivo and in vivo data demonstrate a significant reduction in the number of proliferating cells, whereas apoptosis was increased.
Conclusions:
For the first time, we validated the chemosensitising effects of IFN-β when combined with gemcitabine in vitro, ex vivo, and in vivo. This was driven by cell cycle modulation and associated with an upregulation of genes involving intracellular uptake of gemcitabine. The use of IFN-β in combination with gemcitabine seems promising in patients with pancreatic cancer and needs to be further explored.
... (44) In addition, the PEGylated form of IFN resulted in a higher serum concentration, requiring lower and less frequent doses compared to the conventional IFNs. (45) The PEGylated form of IFN-α has already proven to be effective in the treatment of melanoma and metastatic renal cell carcinoma patients. (46,47) Currently, the PEGylated form IFN-β is being tested in a phase III clinical trial (ADVANCE) in patients with multiple sclerosis. ...
Background: Adjuvant gemcitabine for pancreatic cancer has limited efficacy in the clinical setting. Impaired drug metabolism is associated with treatment resistance. We aimed to evaluate the chemosensitising effect of interferon-beta (IFN-β).
Methods: BxPC-3, CFPAC-1, and Panc-1 cells were pre-treated with IFN-β followed by gemcitabine monotherapy. The effect on cell growth, colony formation, and cell cycle was determined. RT-qPCR was used to measure gene expression. BxPC-3 cells were used in a heterotopic subcutaneous mouse model.
Results: IFN-β increased sensitivity to gemcitabine (4-, 7.7-, and 1.7-fold EC50 decrease in BxPC-3, CFPAC-1, and Panc-1, respectively; all P<0.001). Findings were confirmed when assessing colony formation. The percentage of cells in the S-phase was significantly increased after IFN-β treatment only in BxPC-3 and CFPAC-1 by 12% and 7%, respectively (p<0.001 and p<0.05, respectively). Thereby, IFN-β upregulated expression of the drug transporters SLC28A1 in BxPC-3 (252%) and SLC28A3 in BxPC-3 (127%) and CFPAC-1 (223%) (all p<0.001). In vivo, combination therapy reduced tumor volume with 45% (P=0.01). Both ex vivo and in vivo data demonstrate a significant reduction in the number of proliferating cells, whereas apoptosis was increased.
Conclusions: For the first time, we validated the chemosensitising effects of IFN-β when combined with gemcitabine in vitro, ex vivo, and in vivo. This was driven by cell cycle modulation and associated with an upregulation of genes involving intracellular uptake of gemcitabine. The use of IFN-β in combination with gemcitabine seems promising in patients with pancreatic cancer and needs to be further explored.
... They reported that the PEG propionaldéhyde derivative 4 is specific for the N-terminal a-amine (pKa -7.8) at pH 5.0 due to its lower pKa compared to other nucleophilic reactive groups. The conjugation reaction occurs by reductive alkylation via formation of a Schiff s base 5 which requires reduction in situ with sodium cyanoborohydride to yield a stable secondary amine (Guerra et al. 1998), IFN (3-la (Pepinsky et al. 2001;Baker et al. 2006); rhEGF (Lee et al. 2003) and octreotide which demonstrate that there is sitespecific PEGylation. A product available for clinical use based on this technology is Neulasta® (PEG-G-CSF, Amgen Inc.). ...
Disulfide bonds are ubiquitous in proteins and are broadly involved with protein structure, function and stability. Most therapeutic proteins which act extracellularly have disulfides. Frequently at least one disulfide is accessible to protein disulfide reductants. Disulfide bridging-poly (ethylene glycol) (DB-PEG) reacts with the two sulfurs derived by reduction of such an accessible disulfide and inserts a 3-carbon bridge between the thiols thus attaching the PEG to the protein. This thesis investigates the site-specificity and application of the bis-functional DB-PEG for the monoPEGylation of therapeutic proteins with accessible disulfides. The hypotheses of the project were i) some protein disulfides can be modified with DB-PEG with the protein retaining its structural features and biological activity, ii) upon reaction, the 3-carbon bridge forms between the sustain thiols, iii) DB-PEGylation can be efficient with high yields of the PEGylated protein and iv) since many therapeutic proteins possess accessible disulfides that mostly maintain protein stability, this conjugation strategy will have a broad applicability. DB-PEG was prepared by a simple synthetic route and their reactivity and bis-thiol specificity was studied using reduced glutathione. The characteristics of this PEGylation approach are i) a requirement to reduce the protein disulfides and ii) reaction efficiency depended on the disulfide bond position within the protein. Molecular modelling was done to study the 3-carbon disulfide bridged protein structure. The use of the DB-PEG to conjugate PEG to proteins was studied with L-asparaginase, interferon α-2b, leptin and somatostatin. Evaluation of the biological activities of the conjugates indicated that the observed activities were predominantly due to the steric shielding of PEG. The DB-PEG was found to be stoichiometrically efficient. Disulfide reduction without loss of protein structure was possible and the DB-PEG was specific to the thiols generated from a reduced disulfide bond.
... Indicated for the treatment of patients with relapsing forms of multiple sclerosis. (Pepinsky et al. 2001) efficacious by masking undesirable properties, such as surface cationic charge, or by enhancing water solubility. The high stability and low immunogenicity of PEGylated proteins result in sustained clinical response to drugs and allows for minimal dose and less frequent administration. ...
PEGylation is a biochemical modification process of bioactive molecules with polyethylene glycol (PEG), which lends several desirable properties to proteins/peptides, antibodies, and vesicles considered to be used for therapy or genetic modification of cells. However, PEGylation of proteins is a complex process and can be carried out using more than one strategy that depends on the nature of the protein and the desired application. Proteins of interest are covalently conjugated or non-covalently complexed with inert PEG strings. Purification of PEGylated protein is another critical step, which is mainly carried out based on electrostatic interactions or molecular sizes using chromatography. Several PEGylated drugs are being used for diseases like anemia, kidney disease, multiple sclerosis, hemophilia and cancers. With the advancement and increased specificity of the PEGylation process, the world of drug therapy, and specifically cancer therapy could benefit by utilizing this technique to create more stable and non-immunogenic therapies. In this article we describe the structure and functions of PEGylation and how this chemistry helps in drug discovery. Moreover, special emphasis has been given to CCN-family proteins that can be targeted or used as therapy to prevent or block cancer progression through PEGylation technology.
... Treatments were begun at 30 dpi, with a single weekly subcutaneous injection of 100 μL, and were continued in each animal until necropsy. PegIFNβtreated animals were thus given 1 × 10 4 units of pegIFNβ per injection, a dose deemed optimal from pharmacokinetic studies in the mouse of the murine pegIFNβ and close to the per kilogram dosing for human pegIFNβ (Pepinsky et al., 2001). Importantly, experimenters were masked as to whether the treatment was pegIFNβ or vehicle, as Biogen provided Dartmouth with drug aliquots in tubes labeled as A and B only. ...
We evaluated the effects of pegylated-interferonβ-1a (pegIFNβ) therapy on intrathecal antibody responses, disability progression, and viral load in the CNS in mice infected with the Theiler's virus (TMEV), an animal model of progressive disability in Multiple Sclerosis (MS). The lack of a direct antiviral activity in the CNS, the absence of any effect upon the intrathecal immune response, and the failure to treat disease progression, indicate that the immunomodulatory effects of pegIFNβ-1a likely occur in the systemic circulation rather than within the CNS. These results may be relevant to the relative lack of effect of IFNβ in progressive MS relative to relapsing MS.
... Conjugating PEG to IFN-β-1a resulted in growth of systemic exposure during different routes of administration. Compar- ing to the non-PEGylated form, PEG-IFN-β-1a has longer terminal half-life and elevated PD markers for receptor activation by IFNs type 1. Subcutaneous administration of 125 µg of PEG-IFN-β-1a every two weeks during 48 weeks can significantly reduce relapses and annualized relapse rate, risk of disability progression, T2 lesions counts, and tertiary magnetic resonance imaging measures [34,35]. Dosing every 2 weeks was demonstrated to be more efficient than every 4 weeks. ...
Therapeutic proteins can be pharmacologically improved by conjugative post-translational
modifications through the rational design of their structure and production process. N- and O- linked
glycosylation can confer advantages to proteins and can be controlled by the process conditions, producing cell lines and enzymatic expression or activity. The resulting glycan profile influences their
pharmacological features such as in the biopharmaceutical velaglucerase alfa. Conjugation of polyethylene glycol onto therapeutic proteins has been used to overcome pharmacological limitations. They
can be site-specifically or randomly linked through the reaction between a terminal group of the polyethylene glycol molecule and an amino acid residue group of the protein. Both components (protein
and polyethylene glycol) participate in pharmacological mechanisms, enabling a longer half-life for
peginterferon beta-1a, for example. Fatty acylation of proteins occurs in cells via some known reactions that may involve different linkages, fatty acids, modified residues, and enzymes. Insulin degludec
is a fatty acylated biopharmaceutical formulated to solve the problem of variability in insulin exposure
associated with other products. Conjugative post-translational modifications can be used to pharmacologically improve even therapeutic proteins produced by prokaryotic cell lines while exploring them
may result in the development of other potential uses as well as new generations of biopharmaceuticals.
... jp/pdf/newPINS/00050436.pdf). Pepinsky et al. also reported that following intravenous administration of PEG-IFN to rhesus monkeys, 10 3 -10 4 U/mL PEG-IFN was observed by 24 hr post-administration. 29 In this study, the knockdown efficiencies of the shRNAs were reduced when cells were treated with IFN-a at more than 10 3 U/mL ( Figure 2C). We should exercise care when applying shRNAexpressing vectors to patients undergoing an IFN therapy or other therapies associated with the production of type I IFNs. ...
... Various pre-clinical studies using shRNA-expressing vectors have been reported. 29 In this study, the knockdown efficiencies of the shRNAs were reduced when cells were treated with IFNα at more than 10 3 U/ml (Fig. 2c). We should exercise care when applying shRNA-expressing vectors to patients undergoing an IFN therapy or other therapies associated with the production of type I IFNs. ...
RNA interference (RNAi) by short-hairpin RNA (shRNA) is a powerful tool not only for studying gene functions in various organisms, including mammals, but also for treatment of severe disorders. However, shRNA-expressing vectors can induce type I interferon (IFN) expression by activation of innate immune responses, leading to off-target effects and unexpected side effects. Several strategies have been developed to prevent type I IFN induction. On the other hand, it has remained unclear whether type I IFNs have effects on shRNA-mediated RNAi. Here we show that the type I IFNs significantly inhibit shRNA-mediated RNAi. Treatment with recombinant human IFNα significantly inhibited shRNA-mediated knockdown of target genes, while it did not inhibit small interfering RNA (siRNA)-mediated knockdown. Following treatment with IFNα, increased and decreased copy numbers of shRNA and its processed form, respectively, were found in the cells transfected with shRNA-expressing plasmids. Dicer protein levels were not altered by IFNα. These results indicate that type I IFNs inhibit shRNA-mediated RNAi via inhibition of dicer-mediated processing of shRNA to siRNA. Our findings should provide important clues for efficient RNAi-mediated knockdown of target genes in both basic researches and clinical gene therapy.
... A process development sample of recombinant interferon β-1a conjugated with a 20 kDa PEG chain via the N-terminus (PEG-IFNβ) was prepared as described previously. 22,23 All other chemicals and solvents used in this work were of analytical grade or higher. PEG-IFNβ samples for the on-line IXC/MS analysis were prepared by diluting the drug substance sample in 100 mM ammonium acetate, pH 5.5 to the specified concentration. ...
Detailed profiling of both enzymatic (e.g., glycosylation) and non-enzymatic (e.g., oxidation and deamidation) post-translational modifications (PTMs) is frequently required for the quality assessment of protein-based drugs. Challenging as it is, this task is further complicated for the so-called second-generation biopharmaceuticals, which also contain "designer PTMs" introduced to either enhance their pharmacokinetic profiles (e.g., PEGylated proteins) or endow them with therapeutic activity (e.g., protein-drug conjugates). Such modifications of protein covalent structure can dramatically increase structural heterogeneity, making the very notion of "molecular mass" meaningless, as ions representing different glycoforms of a PEGylated protein may have nearly identical distributions of ionic current as a function of m/z, making their contributions to the mass spectrum impossible to distinguish. In this work we demonstrate that a combination of ion exchange chromatography (IXC) with on-line detection by electrospray ionization mass spectrometry (ESI MS) and methods of ion manipulation in the gas phase (limited charge reduction and collision-induced dissociation) allows meaningful structural information to be obtained on a structurally heterogeneous sample of PEGylated interferon β-1a. IXC profiling of the protein sample gives rise to a convoluted chromatogram with several partially resolved peaks which can represent both deamidation and different glycosylation patterns within the protein, as well as varying extent of PEGylation. Thus, profiling the protein with on-line IXC/ESI/MS/MS allows it to be characterized by providing information on three different types of PTMs (designer, enzymatic and non-enzymatic) within a single protein therapeutic.
... Sam proces pegylacji został wprowadzony do produkcji już w latach 70. ubiegłego wieku i był między innymi stosowany do poprawy właściwości farmakokinetycznych IFNb-1a [26]. Wspomniana modyfikacja strukturalna pozwala na wydłużenie czasu działania IFNb w mechanizmie opartym m.in. ...
Stwardnienie rozsiane (³ac. sclerosis multiplex – SM) jest przewlek³± demielinizacyjn± chorob± o¶rodkowego uk³adu nerwowego (OUN), u której podstaw le¿± najprawdopodobniej z³o¿one zaburzenia odpowiedzi immunologicznej oraz procesy neurodegeneracyjne. Intensywne badania nad terapi± w SM umo¿liwi³y w ostatnich latach wprowadzenie na rynek szeregu leków o zró¿nicowanym mechanizmie dzia³ania, odmiennej skuteczno¶ci klinicznej i profilu dzia³añ niepo¿±danych, a tak¿e ró¿nej drodze i schemacie podawania pacjentom. Wczesne rozpoczêcie terapii immunomodulacyjnej oraz mo¿liwo¶æ jej dostosowania do przebiegu choroby skutkuj±, u du¿ej grupy pacjentów, dobr± kontrol± rzutów, a nawet spowolnieniem progresji niepe³nosprawno¶ci. Pozwoli³o to na wprowadzenie pojêcia „brak oznak aktywno¶ci choroby” (ang. no evidence of disease activity – NEDA) do standardu oceny skuteczno¶ci leczenia w SM. Niestety, sytuacja terapeutyczna u pacjentów z SM o wczesnym pocz±tku jest znacznie trudniejsza. W dalszym ci±gu brak jest danych na temat leczenia immunomodulacyjnego uzyskanych w wieloo¶rodkowych badaniach klinicznych przeprowadzonych w populacjach pediatrycznych. W Polsce dodatkowym ograniczeniem s± ramy programów refundacyjnych, które nie przewiduj± ¿adnej opcji terapeutycznej dla pediatrycznych pacjentów z SM niereaguj±cych na leki pierwszego rzutu (interferony i octan glatirameru). Dlatego du¿e nadzieje wi±zane s± z wynikami prowadzonych obecnie u dzieci i m³odzie¿y z SM badañ III fazy leków immunomodulacyjnych o du¿ej skuteczno¶ci dzia³ania wykazanej u osób doros³ych i dogodnej – doustnej formie podawania.Multiple sclerosis (MS) is a chronic demyelinative disease of the central nervous system (CNS) caused, most probably, by complex immune disturbances and neurodegenerative processes. Recent intensive research on the therapy of MS enabled the registration of several pharmaceuticals characterized by various mechanisms of action, different clinical effectiveness and side-effects profile as well as different ways and schemes of administration. Early introduction of the immunomodulatory treatment and the possibility to adjust the therapy to the disease course allow for an efficient control of relapses and even the inhibition of disability progression in a significant group of patients. Accordingly, the term of No Evidence of Disease Activity (NEDA) was included in the standards of assessment of MS therapy effectiveness. Unfortunately, the situation in early onset MS is less positive. There are still no data on the immunomodulatory treatment obtained in multicenter clinical trials performed in pediatric populations. In Poland additional constrictions arise from the governmental therapy programs lacking any options for the pediatric MS patients with no response to first line treatments (interferons or galtiramer acetate). Therefore, there are high expectations regarding the results of currently ongoing in pediatric MS patients Phase 3 clinical trials of immunomodulatory therapies, characterized by significant effectiveness in adult population and convenient, oral way of administration.
... PEGylation technology is effective in reducing glomerular filtration rate and increases the systemic circulation time [73]. Nevertheless, the therapy is characterized by relatively high plasma concentrations of the active drug substance because of its administration as an aqueous solution [74]. Therefore, further optimization of IFN-β therapy is needed to ameliorate adverse effects as well as to prolong the dosing intervals. ...
As a rapidly growing class of therapeutics, biopharmaceuticals have conquered the global market. Despite of the great potential from a therapeutic perspective, such formulations often require frequent injections due to their short half-life. Aiming to establish a parenteral dosage form with prolonged release properties, a biodegradable implant was developed, based on a combination of nanoencapsulation of protein-heparin complexes, creation of a slow release matrix by freeze-drying, and compression using hyaluronan and methylcellulose. In order to investigate this novel delivery system, formulations containing IFN-β-1a and trypsinogen as model proteins were developed. No degradation of the proteins was observed at any stage of the formulation processing. The potential of the delivery system was evaluated in vivo and in vitro after fluorescence-labeling of the biopharmaceuticals. An optimized agarose gel was utilized as in vitro release medium to simulate the subcutaneous environment in a biorelevant manner. In addition, the formulations were administered to female SJL mice and release was innovatively tracked by fluorescence imaging, setting up an in vitro-in vivo correlation. A prolonged time of residence of approximately 12 days was observed for the selected formulation design.
... Initial progress on PEG-IFN-β-1a has already been reported (9,20,21), but a general strategy for creating tailored PEGylated IFN-β-1a has not been developed since 1990 (22). Similar success was recently achieved by conjugating a branched PEG structure to IFN-ß via an amide linkage, as mentioned above. ...
Interferon-beta-1a (IFN-β-1a) is used clinically in the treatment of multiple sclerosis. Similar to other biological molecules, IFN-β-1a has a relatively short serum half-life and is rapidly detected by the host's immune system. PEGylation is a common approach to increase the blood circulation time of therapeutic proteins. In the present study, IFN-β-1a was PEGylated using linear methoxy polyethylene glycols (mPEGs) with molecular weights of 5 and 20 kDa and also 40 kDa branched mPEG-SPA. Prior to PEGylation, the mPEGs were activated by succinimidyl propionic acid (SPA). PEGylation was evaluated by size-exclusion HPLC (SEHPLC) and Ninhydrin method. In the designed experiments, the factors of mPEG molecular weight, pH, and the molecular ratio of protein to mPEG fractions were studied. The results were analyzed using Design-Expert statistical software and the significant factors were determined. Then, in order to find the optimum conditions, Taguchi method (L 9 array) was used by considering the significant factors. Consequently, the optimum conditions for PEGylation, using 20 kDa linear mPEG-SPA was found to be at pH 8 and the protein to mPEG molar ratio of 1/40. The extents of protein modification were obtained 45.5% and 46.8% by HPLC and Ninhydrin methods, respectively. Optimum PEGylation with 40 kDa branched mPEG-SPA was obtained at pH 8 and protein/mPEG of 1/40. In this case, the extents of protein modification were obtained 46.5% and 47.7% by HPLC and Ninhydrin methods, respectively. The biological activity test showed that the PEGylated protein retained about 80% of its activity, were compared to that of the unmodified protein.
... According to their beneficial effects on the clinical stage, genetic engineering methods are used in order to improve IFNb quality and quantity. A polyethylene-glycol modified form of Interferon-b-1 was designed to not only improve its solubility but also its half-life [21]. In another study, a modified IFNb was created by substitution of asparagine to aspartic acid at residue 25. ...
Interferon β (IFNβ) is the most prescribed drug that has been used frequently for the treatment of multiple sclerosis (MS) patients. The aim of this study is to improve the production of IFNβ by induction of site directed mutagenesis. Accordingly, recombinant constructs were designed in order to enhance the expression of IFNβ mRNA and protein. The recombinant plasmids were transfected to the CHO cell line, following RNA extractions and cDNA synthesis. The effects of recombinant constructs were analyzed by real time PCR, ELISA and MTT assay. Transfected samples with either IFNβ101 or IFNβ101+27 have shown 11.55 and 2.26 fold elevation and over-expression compare to the wild type construct respectively. Our data also indicated that the IFNβ101 and IFNβ101+27 constructs increase IFNβ protein expression more than 2.2 and 4.5 fold, respectively compared to the control group. It could be concluded that the substitution of Phe in the codon 101 position, which may increase the binding activity of IFNβ with its receptors and introduction of an additional N glycosylation site (Asn-X-Thr) in the position 27 of IFNβ protein may cause such an effect. The proliferative activity of transfected cells by a recombinant IFNβ101 decreases in comparison to the wild type, although it was not statistically significant. Over-expression of IFNβ in such a level is promising not only for the patients but also for the pharmaceutical industries.
... According to the fast growth of MS in the world, scientists have tried to improve the IFNb quality and quantity by using protein and genetic engineering methods. Different studies have tried to improve IFNb solubility, halflife or reduce its immunogenicity (in order to diminish the drug side effects) [21][22][23]. A modified IFNb with less immunogenicity was provided by inducing a few substitutions at some residues of human IFNb [24]. ...
Interferon β (IFNβ) is the most important drug that has been used frequently for multiple sclerosis treatment. This study has tried to improve the IFNβ production by introducing mutations in the coding region of IFNβ, while its amino acid sequence is intact. Two recombinant vectors IFNβK and IFNβK+CRID were designed by site-directed mutagenesis. The IFNβK and IFNβK+CRID have two substitutions in Kozak sequence and four substitutions in CRID sequence, respectively. The Chinese hamster ovary (CHO) cell codon usage optimization was also performed for both of them. They were transiently transfected to CHO-dhfr(-) cell line using Lipofectamine kit (Invitrogen, Grand Island, NY). The amount of mRNA and protein was determined by real time PCR and ELISA. The results of this study indicate that the amount of IFNβ protein produced by CHO cells containing IFNβK has been elevated up to 3.5-fold. On the other hand, enormous amounts of IFNβ mRNA and protein were produced by cells containing IFNβK+CRID construct; more than 4.6-fold and 6-fold, respectively. It could be concluded that disruption of AT pattern in CRID element increase RNA and protein production, improve IFNβ mRNA stability and, may also enhance mRNA half-life. In a similar way, more proteins are produced by modification of Kozak sequence.
... However, there is limited reported work on the PEG modification of GHRP-2, and it is difficult to evaluate whether PEGylation is beneficial since the PEGylation of the proteins or peptides varied from substrate to substrate. In addition, the PEGylation efficiency is determined by many factors including the site of PEGylation and the physical properties of the PEG, etc. (10)(11)(12). ...
The purpose of this study was to investigate an efficient synthetic route to the mono-PEGylated growth hormone releasing peptide-2 (GHRP-2) and its biological activity in vivo. The commercially available key PEGylating reagent, mPEG-NHS ester, was successfully utilized to the synthesis of mono-PEGylated GHRP-2, during which the PEGylation profiles of GHRP-2 were monitored by high-performance liquid chromatography (HPLC). The product was purified by cation exchange chromatography, and its biological activity was conducted in rats. The desired mono-PEGylated GHRP-2 as the major product was readily obtained in anhydrous aprotic solvent, such as dimethyl formamide (DMF) and dimethylsulfoxide (DMSO), when the molar ratio of mPEG-NHS ester to GHRP-2 was fixed to be 0.8:1. The products were characterized by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The evaluation of the biological activity for the products showed that the mono-PEGylated GHRP-2 gave a more stable activity than GHRP-2, suggesting that PEGylation led to the increase in the half-life of GHRP-2 in plasma without greatly impairing the biological activity. PEGylation of the GHRP-2 is a good choice for the development of the GHRP-2 applications.
... These PEGylated derivatives of insulin showed low degradation against elastase and pepsin in comparison to unmodified derivatives of insulin and significantly reduced the glucose level when formulated with thiolated polymer poly (acrylic acid)-cysteine [51]. On the other hand, Pepinsky et al., reported that PEGylated derivative of interferon-β-1a demonstrated low oral bioavailability in rat in comparison to intraperitoneal and intratracheal routes [52]. ...
Delivery of proteins/peptides in gastrointestinal (GI) tract via peroral/oral route involves tremendous challenges due to unfavorable environmental conditions like harsh pH, presence of proteolytic enzymes and absorption barriers. Detailed research is being conducted at academic and industrial level to diminish these troubles and various products are under clinical trials. Several approaches have been established to optimize oral delivery of proteins and peptides and can be broadly categorized into chemical and physical strategies. Chemical strategies include site specific mutagenesis, proteinylation, glycosylation, PEGylation and prodrug approaches, whereas physical strategies comprise of formulation based approaches including application of absorption enhancers and metabolism modifiers along with delivering them via colloidal carrier systems such as nanoparticles, liposomes, microparticles, micro- and nano-emulsions. This review stands to accomplish the diverse aspects of oral delivery of proteins/peptides and summarizes the key concepts involved in targeting the biodrugs to specific sites of GI tract such as, intestine and colon. Furthermore some light has also been shed on the current industrial practices followed in developing oral formulations of such bioactives.
... We therefore compared the pharmacokinetic properties of R27T, R27TDGlyc and Rebif. After IV administration, the pharmacokinetic parameters (CL, V ss , and t 1/2 ) of Rebif (1 MIU/kg) were consistent with those obtained in a previous study on the pharmacokinetics of native rhIFN-b, administered at a dose of 21 MIU/kg [39]. The AUC and CL values of R27T were significantly higher and lower, respectively, than those of Rebif and R27TDGlyc. ...
The purpose of this study was to develop a biobetter version of recombinant human interferon-β 1a (rhIFN-β 1a) to improve its biophysical properties, such as aggregation, production and stability, and pharmacokinetic properties without jeopardizing its activity. To achieve this, we introduced additional glycosylation into rhIFN-β 1a via site-directed mutagenesis. Glycoengineering of rhIFN-β 1a resulted in a new molecular entity, termed R27T, which was defined as a rhIFN-β mutein with two N-glycosylation sites at 80th (original site) and at an additional 25th amino acid due to a mutation of Thr for Arg at position 27th of rhIFN-β 1a. Glycoengineering had no effect on rhIFN-β ligand-receptor binding, as no loss of specific activity was observed. R27T showed improved stability and had a reduced propensity for aggregation and an increased half-life. Therefore, hyperglycosylated rhIFN-β could be a biobetter version of rhIFN-β 1a with a potential for use as a drug against multiple sclerosis.
... Translation of these findings into a clinical application is, however, not easy, considering the short half-life of IFN-h. But new promising strategies (PEGylated form, carrier proteins like IFNAR-2a, or gene therapy) can improve pharmacokinetic and pharmacodynamic properties of IFN-h, bringing its real clinical application in cancer closer (36,49,50). ...
The acute kidney injury (AKI) and dose-limiting nephrotoxicity, which occurs in 20–60% of patients following systemic administration of colistin, represents a challenge in the effective treatment of multi-drug resistant Gram-negative infections. To reduce clinical toxicity of colistin and improve targeting to infected/inflamed tissues, we previously developed dextrin–colistin conjugates, whereby colistin is designed to be released by amylase-triggered degradation of dextrin in infected and inflamed tissues, after passive targeting by the enhanced permeability and retention effect. Whilst it was evident in vitro that polymer conjugation can reduce toxicity and prolong plasma half-life, without significant reduction in antimicrobial activity of colistin, it was unclear how dextrin conjugation would alter cellular uptake and localisation of colistin in renal tubular cells in vivo. We discovered that dextrin conjugation effectively reduced colistin's toxicity towards human kidney proximal tubular epithelial cells (HK-2) in vitro, which was mirrored by significantly less cellular uptake of Oregon Green (OG)-labelled dextrin–colistin conjugate, when compared to colistin. Using live-cell confocal imaging, we revealed localisation of both, free and dextrin-bound colistin in endolysosome compartments of HK-2 and NRK-52E cells. Using a murine AKI model, we demonstrated dextrin–colistin conjugation dramatically diminishes both proximal tubular injury and renal accumulation of colistin. These findings reveal new insight into the mechanism by which dextrin conjugation can overcome colistin's renal toxicity and show the potential of polymer conjugation to improve the side effect profile of nephrotoxic drugs.
Interferons (IFN) are immunomodulating, antiviral and antiproliferative cytokines for treatment of multiple indications, including cancer, hepatitis, and autoimmune disease. The first IFNs were discovered in 1957, first approved in 1986, and are nowadays listed in the WHO model list of essential Medicines. Three classes of IFNs are known; IFN–α2a and IFN-β belonging to type-I IFNs, IFN-γ a type-II IFN approved for some hereditary diseases and IFN-λs, which form the newest class of type-III IFNs. IFN-λs were discovered in the last decade with fascinating yet under discovered pharmaceutical potential. This article reviews available IFN drugs, their field and route of application, while also outlining available and future strategies for bioconjugation to further optimize pharmaceutical and clinical performances of all three available IFN classes.
Background: Adjuvant gemcitabine for pancreatic cancer has limited efficacy in the clinical setting. Impaired drug metabolism is associated with treatment resistance. We aimed to evaluate the chemosensitising effect of interferon-beta (IFN-β).
Methods: BxPC-3, CFPAC-1, and Panc-1 cells were pre-treated with IFN-β followed by gemcitabine monotherapy. The effect on cell growth, colony formation, and cell cycle was determined. RT-qPCR was used to measure gene expression. BxPC-3 cells were used in a heterotopic subcutaneous mouse model.
Results: IFN-β increased sensitivity to gemcitabine (4-, 7.7-, and 1.7-fold EC50 decrease in BxPC-3, CFPAC-1, and Panc-1, respectively; all P<0.001). Findings were confirmed when assessing colony formation. The percentage of cells in the S-phase was significantly increased after IFN-β treatment only in BxPC-3 and CFPAC-1 by 12% and 7%, respectively (p<0.001 and p<0.05, respectively). Thereby, IFN-β upregulated expression of the drug transporters SLC28A1 in BxPC-3 (252%) and SLC28A3 in BxPC-3 (127%) and CFPAC-1 (223%) (all p<0.001). In vivo, combination therapy reduced tumor volume with 45% (P=0.01). Both ex vivo and in vivo data demonstrate a significant reduction in the number of proliferating cells, whereas apoptosis was increased.
Conclusions: For the first time, we validated the chemosensitising effects of IFN-β when combined with gemcitabine in vitro, ex vivo, and in vivo. This was driven by cell cycle modulation and associated with an upregulation of genes involving intracellular uptake of gemcitabine. The use of IFN-β in combination with gemcitabine seems promising in patients with pancreatic cancer and needs to be further explored.
Background
Adjuvant gemcitabine for pancreatic cancer has limited efficacy. Impaired drug metabolism is associated with treatment resistance. We aimed to evaluate the chemosensitising effect of interferon-bèta (IFN-β).
Methods
BxPC-3, CFPAC-1, and Panc-1 cells were pre-treated with IFN-β followed by gemcitabine monotherapy. The effect on cell growth, colony formation, and cell cycle was determined. RT-qPCR was used to measure gene expression. BxPC-3 cells were used in a heterotopic subcutaneous mouse model.
Results
IFN-β increased sensitivity to gemcitabine (4-, 7.7-, and 1.7-fold EC50 decrease in BxPC-3, CFPAC-1, and Panc-1, respectively; all P < 0.001). Findings were confirmed when assessing colony formation. The percentage of cells in the S-phase was significantly increased after IFN-β treatment only in BxPC-3 and CFPAC-1 by 12% and 7%, respectively (p < 0.001 and p < 0.05, respectively). Thereby, IFN-β upregulated expression of the drug transporters SLC28A1 in BxPC-3 (252%) and SLC28A3 in BxPC-3 (127%) and CFPAC-1 (223%) (all p < 0.001). In vivo, combination therapy reduced tumor volume with 45% (P = 0.01). Both ex vivo and in vivo data demonstrate a significant reduction in the number of proliferating cells, whereas apoptosis was increased.
Conclusions
For the first time, we validated the chemosensitizing effects of IFN-β when combined with gemcitabine in vitro, ex vivo, and in vivo. Thereby, we show the interaction between IFN-β and genes involving intracellular uptake of gemcitabine. The use of IFN-β as adjuvant treatment seems promising and need to be further explored.
The concept of biostimulant has gained an increasing interest in plant science. Biostimulants are not providing nutrients as their primary function, as opposed to fertilizers, and they do not target pests and pathogens, as is the case for pesticides. They provide tools to act on and within the networks, and to bridge the gap between targeted effects in the lab and potential benefits in the field. With the bioactive ingredients encapsulated by nanotechnology, it will boost the efficiency of foliar application of conventional agrochemicals and most likely plant biostimulants (PBs) as well. For agrochemical products, understanding the mode of action is key for quality control and certification that the product will deliver the expected results, and for marketing under a regulatory framework. The use of PBs is usually defined by claims such as enhanced nutrient use efficiency, tolerance to abiotic stress and improved crop quality.
In this work, a novel carrier based-on modified graphene oxide was designed for co-delivery of hydrophobic and hydrophilic anticancer drugs (curcumin (Cur) and doxorubicin (DOX) as the model of drugs). The hydroxyl groups at the edges of graphene oxide (GO) sheets were used as the initiation sites for growing poly(epichlorohydrin) (PCH) chains. Then, hyperbranched polyglycerol (HPG) was grafted on the hydroxyl end groups of PCH (PCH-g-HPG). Pendant chlorines in the main chain of GO-PCH-g-HPG were replaced with hydrazine. The modification of GO sheets with oxygen-rich polymers increased water solubility of graphene oxide. Doxorubicin was loaded onto the nanocarrier by covalent bonding with hydrazine pendant group and curcumin was loaded through π−π stacking with graphene oxide sheets which were partially reduced to graphene sheets during the carrier preparation. The MTT assay revealed that the carrier had low toxicity because of the presence of oxygen-rich polymers. The drug release of the carrier was investigated and a pH-sensitive behavior was observed for the release of drugs from the carrier. The cellular uptake images proved that co-encapsulation of both DOX and Cur drugs showed higher drug internalization in comparison with the single drug loaded carrier. According to the results, a combination of both drugs exhibited a synergistic behavior for chemotherapy.
Recombinant human interferon beta (rIFN-β) has long been used as a first-line treatment for multiple sclerosis (MS), and any attempt to develop a long-acting rIFN-β is desirable since only one pegylated version of long-acting rIFN-β-1a (Plegridy) is currently available in clinics. Previously, we reported that SL335, a human Fab molecule specific to serum albumin, exhibits an extended serum half-life via utilizing the FcRn recycling mechanism. With the ultimate goal of developing a long-acting rIFN-® we generated a fusion construct by linking human IFN-β cDNA to the C-terminus of the SL335 H chain at the DNA level followed by expression of the fusion protein, referred to as SL335-IFN-β-1a, in Chinese hamster ovary-S (CHO-S) cells. In its N-linked glycosylated form, the resulting fusion protein was easily purified from the culture supernatant via a three-step chromatography process. In vitro functional assays revealed that the fusion protein retained its intrinsic binding capabilities to human serum albumin (HSA) and interferon α/β receptor (IFNAR) that were almost identical to those of parental SL335 and rIFN-β-1a (Rebif). In addition, the fusion protein possessed an antiviral potency and anti-proliferation activity comparable to those of Rebif. In pharmacokinetic (PK) analyses using Lewis rats and cynomolgus monkeys, SL335-IFN-β-1a exhibited at least a two-fold longer serum half-life and a significantly reduced renal clearance rate compared to those of Rebif. Finally, a four-week repeated dose toxicity study revealed no abnormal toxicological signs. In conclusion, our results clearly demonstrated that SL335-IFN-β-1a is worthy of further development as an alternative long-acting IFN-β therapeutic.
Synthetic biology is an emerging interdisciplinary field covering the subjects of biology, chemistry and engineering, which promotes the progress from understanding life to creating living systems. As one of important forefront directions of synthetic biology, in vitro synthetic biology (IVSB) focuses on studying the phenomenon and rules of life activities in testing tubes, not relying on living organisms. Cell-free protein synthesis (CFPS) is an effective research platform for IVSB, which utilizes exogenous DNA or mRNA as genetic information for synthesizing protein in vitro, instead of traditional protein expression in cells. CFPS possesses superior advantages over cell-based systems including convenience, simplicity, rapidness, excellent controllability and green economy. Moreover, CFPS is a robust research model for exploration of life evolution and creation of artificial living system. Since 1958, it has been demonstrated for the first time that proteins could be synthesized from cell extract; and afterwards, CFPS has emerged to not only a promising platform for proteomics and pharmaceutics, but also a significant tool for revelation of chemical basis of cells and exploration of origins of life. Cell extracts contained basic transcription and translation machineries, energy regeneration substrates, amino acids, nucleotides and cofactors. A variety of cell extracts have been created from different cell sources such as Escherichia coli (E.coli), wheat germ, yeast and rabbit reticulocyte. E.coli was the earliest prokaryotic system and most widely used due to its high production yield, short production cycle and low cost. The protein yields of E. coli system could reach up to several milligrams per milliliter of reaction. Eukaryotic systems such as wheat germ and rabbit reticulocyte although less productive, provided more robust platform for studying protein functions, in particular for post-translationally modified proteins. Different production formats of CFPS have been developed and classified according to how the reactions were fed, including batch, continuous-flow, continuous-exchange, bilayer and hydrogel. Batch was the simplest format, but the key limitation was the short lifetimes and the consequent low yield. This was primar-
ily because of the rapid depletion of the high-energy phosphate pool, leading to accumulation of free phosphates and further inhibition of protein synthesis. Continuous-flow overcame the problem in batch format because of the continuous supply of energy and substrates and the continuous removal of the by-products. As a result, the reaction time could be extended to 20 hours, and the product yield was increased by two orders of magnitude in comparison with batch format. In continuous-exchange format, passive exchange of substrates and by-products further extended the reaction lifetime. However, continuous formats were not easily applicable to high-throughput processes, in which miniaturization and automation were required. Bilayer format was simple, efficient and high-throughput-friendly. Very recently, hydrogel formats have been developed because of the gel niche resembling the physicochemical nature of cells, subsequently leading to high yield. One of the promising applications of CFPS was to synthesize biologically active proteins. CFPS could produce proteins that were difficult or impossible to express with cell-based systems, such as membrane proteins, cell-toxic proteins, isotope-labeling proteins and proteins with unnatural amino acids incorporated. The open nature and high versatility of the CFPS platform enabled high-throughput tools for genomics and proteomics, allowing for biopharmaceutical applications. Through the highthroughput screening of proteins in CFPS the efficacy of protein drugs could be significantly improved by addition of unnatural amino acids, which provided excellent pharmacological properties and prolonged the half-life of drugs in plasma. In this review, we therefore summarize the development history, compositions, categories and production formats of CFPS, as well as applications in the synthesis of functional proteins, high throughput synthesis and screening of protein, and
production of protein drugs.
Objectives:
Protein-based therapeutics garner significant attention because of exquisite specificity and limited side effects and are now being used to accomplish targeted delivery of small-molecule drugs. This review identifies and highlights individual chemical attributes and categorizes how site-specific changes affect protein stability based on published high-resolution molecular analyses.
Key findings:
Because it is challenging to determine the mechanisms by which the stability of large, complex molecules is altered and data are sparse, smaller, therapeutic proteins (insulin, erythropoietin, interferons) are examined alongside antibody data. Integrating this large pool of information with the limited available studies on antibodies reveals common mechanisms by which specific alterations affect protein structure and stability.
Summary:
Physical and chemical stability of therapeutic proteins and antibody drug conjugates (ADCs) is of critical importance because insufficient stability prevents molecules from making it to market. Individual moieties on/near the surface of proteins have substantial influence on structure and stability. Seemingly small, superficial modification may have far-reaching consequences on structure, conformational dynamics, and solubility of the protein, and hence physical stability of the molecule. Chemical modifications, whether spontaneous (e.g. oxidation, deamidation) or intentional, as with ADCs, may adversely impact stability by disrupting local surface properties or higher order protein structure.
Francesco M Veronese has a degree in Pharmacy from the University of Padova, Italy. In 1962–1970 he was a researcher for the Italian National Council of Researches, 1971–1972 an Associated Research Professor at the Department of Biochemistry, University of California-Los Angeles, CA, 1973–1981 an Assistant Professor of ‘Pharmaceutical Industry,’ University of Ferrara, and of ‘Applied Biochemistry,’ University of Padova, Italy, and from 1982 Full Professor of ‘Applied Pharmaceutical Chemistry,’ School of Pharmacy, University of Padova. His past research activities have included: specific chemical modification of peptides and proteins for structure–activity relationships; induction, purification, sequence, enzymatic, and structural properties of glutamate dehydrogenases; structural and enzyme activity investigation of thermostable enzymes; drug–protein interaction: covalent and noncovalent linkage between furocoumarins and proteins; covalent modification by amphiphylic polymers of polypeptides and proteins to improve their therapeutic potential and their use in biocatalysis; entrapment of bioactive substances by nonbiodegradable matrices (acrylate polymers obtained by γ-ray induced polymerization) or polyvinyl alcohol, and by biodegradable polymers (polyphosphazenes) for controlled release; and polymeric prodrugs with targeting properties. Currently, Prof Veronese is carrying out research in the following areas: covalent binding of polymers to low-molecular-weight drugs, peptides, and proteins for improved delivery and increased therapeutic index; modification of enzymes with amphiphylic polymers for biocatalysis in organic solvents; and noncovalent entrapment of drugs by insoluble nonbiodegradable or biodegradable polymers for slow and controlled release of bioactive compounds. He was chairman of the PhD program in Pharmaceutical Sciences at the University of Padova 1983–2003; Director of the ‘Seminar of Pharmaceutical Sciences’ 1982–2000; co-organizer of the ‘Advanced School of Pharmaceutical Chemistry’ 1980–1992; co-organizer of the ‘National Seminars in Pharmaceutical Sciences’ 1980–1992; and Past President of the ‘Controlled Release Society,’ Italian Chapter, since 2004. Author of over 190 peer research papers, 15 reviews, 11 book chapters, and 19 US or European patents.
Detailed Description of the Concept Technological Aspects Typical Applications and Indications Alternatives and Variants of This Approach Conclusions and Future Perspectives References
Biomolecules are rarely orally bioavailable and parenteral injections are the industry standard. Development of long acting delivery systems will continue to help biopharmaceuticals reach their full therapeutic potential. Long acting injections have manifested into a range of products designed to target optimal therapeutic dosing requirements (prefilled syringes, long acting injections, sustained release depots, controlled release implants, targeted delivery, and larger payloads). Sustained release implants usually provide a longer term delivery duration than long acting injections and can be categorized into bioerodible systems (rods and cylinders) and implantable devices. This chapter reviews biomolecules currently marketed or in clinical trials, utilizing long acting parenteral technology or sustained/controlled release implants.
Introduction:
Multiple sclerosis (MS) is a chronic immune-mediated disease of the CNS characterized in most cases by a relapsing and remitting disease course, often followed by a progressive course. There are many available injectable therapies for the relapsing-remitting form of MS (RRMS); however, efficacy can be affected by poor adherence and compliance, and an increased frequency of side effects as a result of frequent injections is seen.
Areas covered:
This review focuses on the use of subcutaneous PEGylated IFNβ-1a (PEGIFN β-1a) in RRMS. The pharmacological data in addition to clinical safety and tolerability are analyzed. The clinical efficacy is assessed by evaluating results of various end points used in the ADVANCE Phase III study and the differences between treatment groups over 2 years.
Expert opinion:
We discuss the significance of the ADVANCE trial results and how the results compare to other products on the market. Due to a lack of head-to-head comparison of PEGIFN β-1a with other types of drugs for RRMS, it is difficult to draw conclusion about the superiority of the drug. We also discuss whether the results can be applied to patients with more severe forms of the disease. Overall, PEGIFN β-1a is a promising addition to the repertoire of emerging drugs for the treatment of RRMS.
Type 1 interferons (IFNs) have been shown to be efficacious against hepatitis C virus (HCV), hepatitis B virus (HBV), and some cancers with a significant drawback of short drug exposure. We have significantly improved the pharmacokinetic (PK) of consensus interferon (CIFN) by glycoengineering. We generated AL-624 by introducing 4 glycosylation sites. AL-624 was expressed, purified, and fractionated to yield 2-Gly, 3-Gly, and 4-Gly. In a rat PK study, AL-624 4-Gly exhibited a 6-fold increase of area under curve (AUC) and more than an 11-fold increase in time to half life (T1/2) over CIFN, suggesting the potential for weekly dosing (QW). In Yellow fever virus hamster model, QW of 4-Gly achieved similar efficacy to daily dosing (QD) CIFN and QW Peg-IFN-α-2a in overall survival rate and reduction of alanine aminotransferase (ALT) level. Further refinement resulted in development of AL-683 by addition of external glycosylation sites and its mouse homologue. AL-683 maintains undiminished biological potency in HCV replicon. In mouse PK/pharmacodynamic (PD) studies, AL-683 homologue has a ∼37-fold improvement in T1/2 and a ∼33-fold improvement in AUC when compared with the unglycosylated mouse IFN-α-1. Significantly improved PD responses were also observed. The significant improvement of AL-683 PK over AL-624 suggests a bimonthly dosing regimen for AL-683. The possibility for once-a-month dosing could be realized by further optimization of manufacturing conditions.
Pegylation of pharmacological substances was developed in the 1970s as a way of improving their efficacy and elimination and hence reducing the dosage frequency. A prominent example is pegylation of IFNα, which revolutionized the treatment of virus hepatitis in the late 1990s. Efforts have now succeeded in producing a pegylated interferon beta (PEG-IFN-β1a) to treat multiple sclerosis (MS) and the efficacy and safety have been investigated in a phase III trial called the ADVANCE study. The 1-year results of this randomized, double blind, multicenter, placebo-controlled study in more than 1500 MS patients show that administration of subcutaneous PEG-IFN-β1a significantly reduces the annual relapse rate and disability progression. The safety and tolerability profile of PEG-IFN-β1a was found to be similar to that of conventional IFN-β drugs. The most common adverse events were flu-like symptoms and redness at the injection site. The results of this study underscore that PEG-IFN-β1a is an interesting new therapeutic option in the treatment of relapsing-remitting MS that combines highly effective interferon with the established tolerability and safety profile of IFN-β at a reduced dosage frequency.
Introduction:
The use of cytokines as therapeutic agents is important, given their potent biological effects. However, this very potency, coupled with the pleiotropic nature and short half-life of these molecules, has limited their therapeutic use. Strategies to increase the half-life and to decrease toxicity are necessary to allow effective treatment with these molecules.
Areas covered:
A number of strategies are used to overcome the natural limitations of cytokines, including PEGylation, encapsulation in liposomes, fusion to targeting peptides or antibodies and latent cytokines. Latent cytokines are engineered using the latency-associated peptide of transforming growth factor-β to produce therapeutic cytokines/peptides that are released only at the site of disease by cleavage with disease-induced matrix metalloproteinases. The principles underlying the latent cytokine technology are described and are compared to other methods of cytokine delivery. The potential of this technology for developing novel therapeutic strategies for the treatment of diseases with an inflammatory-mediated component is discussed.
Expert opinion:
Methods of therapeutic cytokine delivery are addressed. The latent cytokine technology holds significant advantages over other methods of drug delivery by providing simultaneously increased half-life and localised drug delivery without systemic effects. Cytokines that failed clinical trials should be reassessed using this delivery system.
Die Formulierung und orale Applikation von therapeutischen Peptiden und Proteinen stellt aufgrund des proteolytischen Abbaus im Gastrointestinaltrakt und der geringen Resorption im Darmlumen eine Herausforderung dar. Für eine mögliche orale Verabreichung wurde eine chitosan-basierte partikuläre Formulierung von Interferon-β-1b unter Einsatz eines Sprühtrockungsverfahrens entwickelt. Die Quantifizierung der biologischen Aktivität erfolgte mittels eines Antiviralen-Assays und eines ELISA-Assays. Der Aktivitätstest zeigte den Erhalt der biologischen Aktivität des Interferons nach dem Herstellungsprozess.
The formulation and oral application of therapeutic petides and proteins is still a challenge due to the proteolytic gastritic degradation and the poor adsorption mechanism out of the gut lumen. A particulate chitosan-based formulation of Interferon-β-1b was prepared for the future oral administration using a spray drying process. The quantification of the biological activity of Interferon-β-1b was carried out by means of an Anti Viral Assay (AVA) and an ELISA. The activity test showed no loss of the biological activity of Interferon after the manufacturing process.
The effect on a range of biologic responses of interferon-beta serine (IFN-βser), administered by either the sc or the iv route, was examined in 16 patients. Despite the absence of IFN in the serum of
13 of 16 patients after sc administration, biologic changes associated with IFN administration occurred. Significant increases
in peripheral mononu-clear cell surface proteins were evident. Monocyte human leukocyte antigen-DR (HLA-DR) showed a 23% increase
in mean fluorescent intensity (P =.04) and a 9% increase in percentage of positive cells (P =.02); lymphocyte OKT10 had an 11% increase in percentage of positive cells (P <.0001) and a 26% increase in mean fluorescent intensity (P =.002). Natural killer cell activity against the Chang target increased by 125% (P=.004). Intracellular activity of 2‘, 5’-oligoadenylate synthetase increased 297% at 24 hours and 226% at 48 hours (P <.0001). Significant increases in serum concentrations of β2 microglobulin (24% at 24 hr and 27% at 48 hr, P <.0001) and neopterin (85%, P =.0001 and 165%, P =.00001) were observed. These alterations after sc administration were similar quantitatively to those resulting from the
same dose of IFN-βser. given iv. Thus, serum IFN concentrations did not have to be measurable for IFN-βser to exert biologic activity. The different effects of two dose levels, 45 × 106 IU and 180 × 106 IU, also were compared independent of route. The higher dose resulted in greater increases over baseline of 2′, 5′-oligoadenylate
synthetase activity (344% vs. 145% at 24 hr; 231% vs. 83% at 48 hr) and serum neopterin concentrations (185% vs. 99% at 24
hr; 271% vs. 153% at 48 hr). For all the other parameters, there was no significant difference between the two doses. [J Natl
Cancer Inst 81: 1061–1068, 1989]