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1.
Drug Metab Dispos ; 50(7): 898-908, 2022 07.
Article in English | MEDLINE | ID: mdl-35545256

ABSTRACT

Fragment crystallizable (Fc) fusion is commonly used for extending the half-life of biotherapeutics such as cytokines. In this work, we studied the pharmacokinetics of Fc-fused interleukin-10 (IL-10) proteins that exhibited potent antitumor activity in mouse syngeneic tumor models. At pharmacologically active doses of ≥0.1 mg/kg, both mouse Fc-mouse IL-10 and human Fc-human IL-10, constructed as the C terminus of the Fc domain fused with IL-10 via a glycine-serine polypeptide linker, exhibited nonlinear pharmacokinetics after intravenous administration to mice at the doses of 0.05, 0.5, and 5 mg/kg. With a nominal dose ratio of 1:10:100; the ratio of the area under the curve for mouse Fc-mouse IL-10 and human Fc-human IL-10 was 1:181:1830 and 1:75:633, respectively. In contrast, recombinant mouse or human IL-10 proteins exhibited linear pharmacokinetics in mice. Compartmental analysis, using the Michaelis-Menten equation with the in vitro IL-10 receptor alpha binding affinity inputted as the Km, unified the pharmacokinetic data across the dose range. Additionally, nontarget-mediated clearance estimated for fusion proteins was ∼200-fold slower than that for cytokines, causing the manifestation of target-mediated drug disposition (TMDD) in the fusion protein pharmacokinetics. The experimental data generated with a mouse IL-10 receptor alpha-blocking antibody and a human Fc-human IL-10 mutant with a reduced receptor binding affinity showed significant improvements in pharmacokinetics, supporting TMDD as the cause of nonlinearity. Target expression and its effect on pharmacokinetics must be determined when considering using Fc as a half-life extension strategy, and pharmacokinetic evaluations need to be performed at a range of doses covering pharmacological activity. SIGNIFICANCE STATEMENT: Target-mediated drug disposition can manifest to affect the pharmacokinetics of a fragment crystallizable (Fc)-fused cytokine when the nontarget-mediated clearance of the cytokine is decreased due to neonatal Fc receptor-mediated recycling and molecular weight increases that reduce the renal clearance. The phenomenon was demonstrated with interleukin-10 Fc-fusion proteins in mice at pharmacologically active doses. Future drug designs using Fc as a half-life extension approach for cytokines need to consider target expression and its effect on pharmacokinetics at relevant doses.


Subject(s)
Interleukin-10 , Animals , Half-Life , Humans , Interleukin-10/pharmacokinetics , Mice , Receptors, Interleukin-10 , Recombinant Fusion Proteins/pharmacokinetics
2.
Lancet Oncol ; 20(11): 1544-1555, 2019 11.
Article in English | MEDLINE | ID: mdl-31563517

ABSTRACT

BACKGROUND: IL-10 has anti-inflammatory and CD8+ T-cell stimulating activities. Pegilodecakin (pegylated IL-10) is a first-in-class, long-acting IL-10 receptor agonist that induces oligoclonal T-cell expansion and has single-agent activity in advanced solid tumours. We assessed the safety and activity of pegilodecakin with anti-PD-1 monoclonal antibody inhibitors in patients with advanced solid tumours. METHODS: We did a multicentre, multicohort, open-label, phase 1b trial (IVY) at 12 cancer research centres in the USA. Patients were assigned sequentially into cohorts. Here, we report on all enrolled patients from two cohorts treated with pegilodecakin combined with anti-PD-1 inhibitors. Eligible patients were aged at least 18 years with histologically or cytologically confirmed advanced malignant solid tumours refractory to previous therapies, and an Eastern Cooperative Oncology Group performance status of 0 or 1. Patients with uncontrolled infectious diseases were excluded. Pegilodecakin was provided in single-use 3 mL vials and was self-administered subcutaneously by injection at home at 10 µg/kg or 20 µg/kg once per day in combination with pembrolizumab (2 mg/kg every 3 weeks or 200 mg every 3 weeks) or nivolumab (3 mg/kg every 2 weeks or 240 mg every 2 weeks or 480 mg every 4 weeks at the approved dosing), both of which were given intravenously at the study site. Patients received pembrolizumab or nivolumab with pegilodecakin until disease progression, toxicity necessitating treatment discontinuation, patient withdrawal of consent, or study end. The primary endpoints were safety and tolerability, assessed in all patients enrolled in the study who received any amount of study medication including at least one dose of pegilodecakin, and pharmacokinetics (previously published). Secondary endpoints included objective response by immune-related response criteria in all patients who were treated and had evaluable measurements. The study is active but no longer recruiting, and is registered with ClinicalTrials.gov, NCT02009449. FINDINGS: Between Feb 13, 2015, and Sept 12, 2017, 111 patients were enrolled in the two cohorts. 53 received pegilodecakin plus pembrolizumab, and 58 received pegilodecakin plus nivolumab. 34 (31%) of 111 patients had non-small-cell lung cancer, 37 (33%) had melanoma, and 38 (34%) had renal cell carcinoma; one (<1%) patient had triple-negative breast cancer and one (<1%) had bladder cancer. Data cutoff was July 1, 2018. Median follow-up was 26·9 months (IQR 22·3-31·5) for patients with non-small-cell lung cancer, 33·0 months (29·2-35·1) for those with melanoma, and 22·7 months (20·9-27·0) for those with renal cell carcinoma. At least one treatment-related adverse event occurred in 103 (93%) of 111 patients. Grade 3 or 4 events occurred in 73 (66%) of 111 patients (35 [66%] of 53 in the pembrolizumab group and 38 [66%] of 58 in the nivolumab group), the most common of which were anaemia (12 [23%] in the pembrolizumab group and 16 [28%] in the nivolumab group), thrombocytopenia (14 [26%] in the pembrolizumab group and 12 [21%] in the nivolumab group), fatigue (11 [21%] in the pembrolizumab group and 6 [10%] in the nivolumab group) and hypertriglyceridaemia (three [6%] in the pembrolizumab group and eight [14%] in the nivolumab group). There were no fatal adverse events determined to be related to the study treatments. Of the patients evaluable for response, objective responses were 12 (43%) of 28 (non-small-cell lung cancer), three (10%) of 31 (melanoma), and 14 (40%) of 35 (renal cell carcinoma). INTERPRETATION: In this patient population, pegilodecakin with anti-PD-1 monoclonal antibodies had a manageable toxicity profile and preliminary antitumour activity. Pegilodecakin with pembrolizumab or nivolumab could provide a new therapeutic opportunity for previously treated patients with renal cell carcinoma and non-small-cell carcinoma. FUNDING: ARMO BioSciences, a wholly owned subsidiary of Eli Lilly and Company.


Subject(s)
Antibodies, Monoclonal, Humanized/administration & dosage , Antineoplastic Agents, Immunological/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Interleukin-10/administration & dosage , Neoplasms/drug therapy , Nivolumab/administration & dosage , Polyethylene Glycols/administration & dosage , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Aged , Antibodies, Monoclonal, Humanized/adverse effects , Antineoplastic Agents, Immunological/adverse effects , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Female , Humans , Interleukin-10/adverse effects , Interleukin-10/pharmacokinetics , Male , Middle Aged , Neoplasms/immunology , Neoplasms/pathology , Nivolumab/adverse effects , Polyethylene Glycols/adverse effects , Polyethylene Glycols/pharmacokinetics , Programmed Cell Death 1 Receptor/immunology , United States
3.
Cancer Cell ; 35(6): 901-915.e4, 2019 06 10.
Article in English | MEDLINE | ID: mdl-31185213

ABSTRACT

Increasing evidence demonstrates that interleukin-10 (IL-10), known as an immunosuppressive cytokine, induces antitumor effects depending on CD8+ T cells. However, it remains elusive how immunosuppressive effects of IL-10 contribute to CD8+ T cell-mediated antitumor immunity. We generated Cetuximab-based IL-10 fusion protein (CmAb-(IL10)2) to prolong its half-life and allow tumor-targeted delivery of IL-10. Our results demonstrated potent antitumor effects of CmAb-(IL10)2 with reduced toxicity. Moreover, we revealed a mechanism of CmAb-(IL10)2 preventing dendritic cell (DC)-mediated CD8+ tumor-infiltrating lymphocyte apoptosis through regulating IFN-γ production. When combined with immune checkpoint blockade, CmAb-(IL10)2 significantly improves antitumor effects in mice with advanced tumors. Our findings reveal a DC-regulating role of IL-10 to potentiate CD8+ T cell-mediated antitumor immunity and provide a potential strategy to improve cancer immunotherapy.


Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Apoptosis/drug effects , CD8-Positive T-Lymphocytes/drug effects , Cetuximab/pharmacology , Dendritic Cells/drug effects , Interleukin-10/pharmacology , Lymphocytes, Tumor-Infiltrating/drug effects , Neoplasms/drug therapy , Animals , Antineoplastic Agents, Immunological/pharmacokinetics , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/pathology , Cell Communication , Cell Line, Tumor , Cell Proliferation/drug effects , Cetuximab/pharmacokinetics , Coculture Techniques , Dendritic Cells/immunology , Dendritic Cells/metabolism , Dendritic Cells/pathology , Drug Resistance, Neoplasm , Female , Humans , Interleukin-10/pharmacokinetics , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/pathology , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Molecular Targeted Therapy , Neoplasms/immunology , Neoplasms/metabolism , Neoplasms/pathology , Recombinant Fusion Proteins/pharmacology , Signal Transduction , Tumor Burden/drug effects , Xenograft Model Antitumor Assays
4.
Acta Biomater ; 90: 179-191, 2019 05.
Article in English | MEDLINE | ID: mdl-30936036

ABSTRACT

Although there are numerous medical applications to recover damaged skin tissue, scarless wound healing is being extensively investigated to provide a better therapeutic outcome. The exogenous delivery of therapeutic growth factors (GFs) is one of the engineering strategies for skin regeneration. This study presents an exogenous GF delivery platform developed using coacervates (Coa), a tertiary complex of poly(ethylene argininyl aspartate diglyceride) (PEAD) polycation, heparin, and cargo GFs (i.e., transforming growth factor beta 3 (TGF-ß3) and interleukin 10 (IL-10)). Coa encompasses the advantage of high biocompatibility, facile preparation, protection of cargo GFs, and sustained GF release. We therefore speculated that coacervate-mediated dual delivery of TGF-ß3/IL-10 would exhibit synergistic effects for the reduction of scar formation during physiological wound healing. Our results indicate that the exogenous administration of dual GF via Coa enhances the proliferation and migration of skin-related cells. Gene expression profiles using RT-PCR revealed up-regulation of ECM formation at early stage of wound healing and down-regulation of scar-related genes at later stages. Furthermore, direct injection of the dual GF Coa into the edges of damaged skin in a rat skin wound defect model demonstrated accelerated wound closure and skin regeneration after 3 weeks. Histological evaluation and immunohistochemical staining also revealed enhanced formation of the epidermal layer along with facilitated angiogenesis following dual GF Coa delivery. Based on these results, we conclude that polycation-mediated Coa fabrication and exogenous dual GF delivery via the Coa platform effectively augments both the quantity and quality of regenerated skin tissues without scar formation. STATEMENT OF SIGNIFICANCE: This study was conducted to develop a simple administration platform for scarless skin regeneration using polycation-based coacervates with dual GFs. Both in vitro and in vivo studies were performed to confirm the therapeutic efficacy of this platform toward scarless wound healing. Our results demonstrate that the platform developed by us enhances the proliferation and migration of skin-related cells. Sequential modulation in various gene expression profiles suggests a balanced collagen-remodeling process by dual GFs. Furthermore, in vivo histological evaluation demonstrates that our technique enhances clear epidermis formation with less scab and thicker woven structure of collagen bundle, similar to that of a normal tissue. We propose that simple administration of dual GFs with Coa has the potential to be applied as a clinical approach for fundamental scarless skin regeneration.


Subject(s)
Cicatrix/prevention & control , Dermis/metabolism , Drug Delivery Systems , Fibroblasts/metabolism , Interleukin-10 , Transforming Growth Factor beta3 , Wound Healing/drug effects , Cicatrix/metabolism , Cicatrix/pathology , Dermis/pathology , Fibroblasts/pathology , Humans , Interleukin-10/chemistry , Interleukin-10/pharmacokinetics , Interleukin-10/pharmacology , Transforming Growth Factor beta3/chemistry , Transforming Growth Factor beta3/pharmacokinetics , Transforming Growth Factor beta3/pharmacology
5.
Macromol Biosci ; 19(1): e1800248, 2019 01.
Article in English | MEDLINE | ID: mdl-30259658

ABSTRACT

Shear-thinning hydrogels are useful for biomedical applications, from 3D bioprinting to injectable biomaterials. Although they have the appropriate properties for injection, it may be advantageous to decouple injectability from the controlled release of encapsulated therapeutics. Toward this, composites of hydrogels and encapsulated microgels are introduced with microgels that are fabricated via microfluidics. The microgel cross-linker controls degradation and entrapped molecule release, and the concentration of microgels alters composite hydrogel rheological properties. For the treatment of myocardial infarction (MI), interleukin-10 (IL-10) is encapsulated in microgels and released from composites. In a rat model of MI, composites with IL-10 reduce macrophage density after 1 week and improve scar thickness, ejection fraction, cardiac output, and the size of vascular structures after 4 weeks when compared to saline injection. Improvements are also observed with the composite without IL-10 over saline, emphasizing the role of injectable hydrogels alone on tissue repair.


Subject(s)
Biocompatible Materials , Hydrogels , Interleukin-10 , Animals , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacokinetics , Biocompatible Materials/pharmacology , Delayed-Action Preparations/chemistry , Delayed-Action Preparations/pharmacokinetics , Delayed-Action Preparations/pharmacology , Disease Models, Animal , Hydrogels/chemistry , Hydrogels/pharmacokinetics , Hydrogels/pharmacology , Interleukin-10/chemistry , Interleukin-10/pharmacokinetics , Interleukin-10/pharmacology , Myocardial Infarction/metabolism , Rats
6.
J Clin Oncol ; 34(29): 3562-3569, 2016 10 10.
Article in English | MEDLINE | ID: mdl-27528724

ABSTRACT

Purpose Interleukin-10 (IL-10) stimulates the expansion and cytotoxicity of tumor-infiltrating CD8+ T cells and inhibits inflammatory CD4+ T cells. Pegylation prolongs the serum concentration of IL-10 without changing the immunologic profile. This phase I study sought to determine the safety and antitumor activity of AM0010. Patients and Methods Patients with selected advanced solid tumors were treated with AM0010 in a dose-escalation study, which was followed by a renal cell cancer (RCC) dose-expansion cohort. AM0010 was self-administered subcutaneously at doses of 1 to 40 µg/kg once per day. Primary end points were safety and tolerability; clinical activity and immune activation were secondary end points. Results In the dose-escalation and -expansion cohorts, 33 and 18 patients, respectively, were treated with daily subcutaneous injection of AM0010. AM0010 was tolerated in a heavily pretreated patient population. Treatment-related adverse events (AEs) included anemia, fatigue, thrombocytopenia, fever, and injection site reactions. Grade 3 to 4 nonhematopoietic treatment-related AEs, including rash (n = 2) and transaminitis (n = 1), were observed in five of 33 patients. Grade 3 to 4 anemia or thrombocytopenia was observed in five patients. Most treatment-related AEs were transient or reversible. AM0010 led to systemic immune activation with elevated immune-stimulatory cytokines and reduced transforming growth factor beta in the serum. Partial responses were observed in one patient with uveal melanoma and four of 15 evaluable patients with RCC treated at 20 µg/kg (overall response rate, 27%). Prolonged stable disease of at least 4 months was observed in four patients, including one with colorectal cancer with disease stabilization for 20 months. Conclusion AM0010 has an acceptable toxicity profile with early evidence of antitumor activity, particularly in RCC. These data support the further evaluation of AM0010 both alone and in combination with other immune therapies and chemotherapies.


Subject(s)
Cytokines/blood , Interleukin-10/adverse effects , Neoplasms/drug therapy , Polyethylene Glycols/adverse effects , Adult , Aged , Aged, 80 and over , Anemia/chemically induced , Carcinoma, Renal Cell/drug therapy , Drug Eruptions/etiology , Exanthema/chemically induced , Fatigue/chemically induced , Female , Fever/chemically induced , Humans , Injections, Subcutaneous/adverse effects , Interferon-gamma/blood , Interleukin-10/immunology , Interleukin-10/pharmacokinetics , Interleukin-10/therapeutic use , Interleukin-4/blood , Interleukin-8/blood , Kidney Neoplasms/drug therapy , Male , Melanoma/drug therapy , Middle Aged , Neoplasms/pathology , Polyethylene Glycols/pharmacokinetics , Polyethylene Glycols/therapeutic use , Recombinant Proteins/adverse effects , Recombinant Proteins/immunology , Recombinant Proteins/pharmacokinetics , Recombinant Proteins/therapeutic use , Thrombocytopenia/chemically induced , Transforming Growth Factor beta/blood , Uveal Neoplasms/drug therapy , Young Adult
7.
Nano Lett ; 14(11): 6449-55, 2014 Nov 12.
Article in English | MEDLINE | ID: mdl-25333768

ABSTRACT

Protein therapeutics have gained attention recently for treatment of a myriad of human diseases due to their high potency and unique mechanisms of action. We present the development of a novel polymeric thermosponge nanoparticle for efficient delivery of labile proteins using a solvent-free polymer thermo-expansion mechanism with clinical potential, capable of effectively delivering a range of therapeutic proteins in a sustained manner with no loss of bioactivity, with improved biological half-lives and efficacy in vivo.


Subject(s)
Anti-Inflammatory Agents/administration & dosage , Delayed-Action Preparations/chemistry , Hypoglycemic Agents/administration & dosage , Insulin/administration & dosage , Interleukin-10/administration & dosage , Nanoparticles/chemistry , Polymers/chemistry , Animals , Anti-Inflammatory Agents/pharmacokinetics , Anti-Inflammatory Agents/pharmacology , Cell Line , Drug Delivery Systems , Humans , Hypoglycemic Agents/pharmacokinetics , Hypoglycemic Agents/pharmacology , Insulin/pharmacokinetics , Insulin/pharmacology , Interleukin-10/pharmacokinetics , Interleukin-10/pharmacology , Mice , Nanoparticles/ultrastructure , Temperature
8.
J Control Release ; 162(1): 84-91, 2012 Aug 20.
Article in English | MEDLINE | ID: mdl-22659050

ABSTRACT

Liver fibrosis represents a scar formation process as a response to chronic injury and a major cause of death worldwide. To date, no drug is available for this condition. Interleukin-10 (IL-10) has potent anti-inflammatory and antifibrotic properties but its short half-life in the circulation hampers its clinical use. Our aim was therefore to modify IL-10 with polyethylene glycol (PEG) to prolong its circulation time and enhance its effectivity. IL-10 was modified with 5 or 20 kDa PEG. The biological activity was preserved after PEGylation as assessed by inhibition of TNF-α production by macrophages. In vivo, during CCl(4)-induced fibrogenesis in mice, both 5PEG-IL-10 and 20PEG-IL-10 showed a longer circulation time compared to IL-10, which was associated with a significant increased liver accumulation. Immunohistochemical analysis of fibrotic livers of mice receiving treatment with IL-10 or its PEGylated forms, revealed a decrease in markers reflecting HSC and KC activation induced by 5PEG-IL10. Transcription levels of IL-6 were decreased upon treatment with IL-10 and both PEGylated forms, whereas IL-1ß levels were only down-regulated by 5PEGIL-10 and 20PEGIL-10. We conclude that PEGylation of IL-10 is a good strategy to attenuate liver fibrosis and that 5PEGIL-10 is the most effective conjugate.


Subject(s)
Interleukin-10/chemistry , Interleukin-10/therapeutic use , Liver Cirrhosis/drug therapy , Liver/drug effects , Polyethylene Glycols/chemistry , Animals , Carbon Tetrachloride , Cell Line , Collagen/metabolism , Half-Life , Humans , Interleukin-10/pharmacokinetics , Interleukin-10/pharmacology , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Interleukin-6/genetics , Interleukin-6/immunology , Liver/immunology , Liver/metabolism , Liver/pathology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/immunology , Liver Cirrhosis/pathology , Macrophages/drug effects , Macrophages/immunology , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , Transcriptional Activation/drug effects , Tumor Necrosis Factor-alpha/immunology
9.
Protein Expr Purif ; 83(2): 152-6, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22484198

ABSTRACT

Interleukin (IL)-10 is an anti-inflammatory cytokine that could be potentially applied for clinical therapy. However, its short circulating half-life in the serum limits its clinical applications. In this study, we designed a fusion protein containing human IL-10 and an IgG Fc fragment (hIL-10/Fc), and expressed it in Pichia pastoris. This hIL-10/Fc fusion protein was purified from the culture supernatant using MabSelect affinity chromatography and size-exclusion chromatography. The hIL-10/Fc yield was about 5mg/L in shake flasks, with purity exceeding 95%. In addition, the hIL-10/Fc fusion protein suppressed the phytohemagglutinin-induced IFN-γ production in human peripheral blood mononuclear cells. Pharmacokinetic study also revealed that hIL-10/Fc has a prolonged circulating half-life of about 30h in rats. More importantly, the hIL-10/Fc fusion protein displayed highly specific biological activity, which was slightly higher than that of the commercial recombinant human IL-10 (rhIL-10). Therefore, P. pastoris is useful in the large-scale production of hIL-10/Fc fusion protein for both research and therapeutic applications.


Subject(s)
Immunoglobulin Fc Fragments/isolation & purification , Interleukin-10/isolation & purification , Recombinant Fusion Proteins/isolation & purification , Animals , Humans , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/genetics , Interferon-gamma/analysis , Interferon-gamma/metabolism , Interleukin-10/genetics , Interleukin-10/metabolism , Interleukin-10/pharmacokinetics , Leukocytes, Mononuclear/metabolism , Male , Rats , Rats, Sprague-Dawley , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacokinetics
10.
Drug Metab Dispos ; 40(2): 360-73, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22083830

ABSTRACT

Interleukin 10 (IL-10) is a potent cytokine homodimer with multiple immunoregulatory functions. Here, we have characterized the effects of PEGylation and formation of human IL-10 (hIL-10)/humanized anti-human IL-10 (hαhIL-10) immune complexes in the pharmacokinetics, biodistribution, and biotransformation of IL-10 in mice. To assess the fate of native, PEGylated, and antibody-bound IL-10; we implemented an analytical set of fluorescence emission-linked assays. Plasma size exclusion chromatography analysis indicated that fluoro-labeled native and PEGylated murine IL-10 (PEG-mIL-10) are stable in the circulation. PEGylation of IL-10 resulted in a 21-fold increased exposure, 2.7-fold increase in half-life, and 20-fold reduction in clearance. Kidney is the major organ of disposition for both native and PEGylated mIL-10 with renal uptake directly related to systemic clearance. The fluorescence signal in the kidneys reached tissue/blood ratios up to 150 and 20 for native and PEG-mIL-10, respectively. hIL-10/hαhIL-10 immune complexes are detectable in the circulation without evidence of unbound or degraded hIL-10. The exposure of hIL-10 present in immune complexes versus that of hIL-10 alone increased from 0.53 to 11.28 µg · day/ml, with a half-life of 1.16 days and a 23-fold reduction in clearance. Unlike hIL-10 alone, antibody-bound hIL-10 was targeted mainly to the liver with minimal renal distribution. In addition, we found an 11-fold reduction (from 9.9 to 113 nM) in binding to the neonatal Fc receptor (FcRn) when the hαhIL10 antibody is conjugated to hIL-10. The potential changes in FcRn binding in vivo and increased liver uptake may explain the unique pharmacokinetic properties of hIL-10/hαhIL-10 immune complexes.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Antigen-Antibody Reactions , Interleukin-10/chemistry , Interleukin-10/pharmacokinetics , Polyethylene Glycols/chemistry , Animals , Anti-Inflammatory Agents, Non-Steroidal/antagonists & inhibitors , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Antigen-Antibody Complex/analysis , Female , Half-Life , Histocompatibility Antigens Class I/metabolism , Humans , Injections, Intravenous , Injections, Subcutaneous , Interleukin-10/antagonists & inhibitors , Interleukin-10/metabolism , Kidney/cytology , Kidney/immunology , Kidney/metabolism , Liver/cytology , Liver/immunology , Liver/metabolism , Metabolic Clearance Rate , Mice , Mice, Inbred Strains , Receptors, Fc/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacokinetics , Tissue Distribution
11.
Biotechnol Bioeng ; 108(8): 1977-86, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21391205

ABSTRACT

Interleukin-10 (IL-10) is an anti-inflammatory cytokine, which active form is a non-covalent homodimer. Given the potential of IL-10 for application in various medical conditions, it is essential to develop systems for its effective delivery. In previous work, it has been shown that a dextrin nanogel effectively incorporated and stabilized rIL-10, enabling its release over time. In this work, the delivery system based on dextrin nanogels was further analyzed. The biocompatibility of the nanogel was comprehensively analyzed, through cytotoxicity (lactate dehydrogenase (LDH) release, MTS, Live, and Dead) and genotoxicity (comet) assays. The release profile of rIL-10 and its biological activity were evaluated in vivo, using C57BL/6 mice. Although able to maintain a stable concentration of IL-10 for at least 4 h in mice serum, the amount of protein released was rather low. Despite this, the amount of rIL-10 released from the complex was biologically active inhibiting TNF-α production, in vivo, by LPS-challenged mice. In spite of the significant stabilization achieved using the nanogel, rIL-10 still denatures rather quickly. An additional effort is thus necessary to develop an effective delivery system for this cytokine, able to release active protein over longer periods of time. Nevertheless, the good biocompatibility, the protein stabilization effect and the ability to perform as a carrier with controlled release suggest that self-assembled dextrin nanogels may be useful protein delivery systems.


Subject(s)
Dextrins/administration & dosage , Drug Carriers/administration & dosage , Immunologic Factors/pharmacology , Immunologic Factors/pharmacokinetics , Interleukin-10/pharmacology , Interleukin-10/pharmacokinetics , Polyethylene Glycols/administration & dosage , Polyethyleneimine/administration & dosage , Animals , Dextrins/adverse effects , Drug Carriers/adverse effects , Mice , Mice, Inbred C57BL , Nanogels , Polyethylene Glycols/adverse effects , Polyethyleneimine/adverse effects , Protein Denaturation , Serum/chemistry
12.
Cytokine ; 40(3): 183-92, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17980615

ABSTRACT

The powerful anti-inflammatory and immunosuppressive activities of IL-10 make it attractive for supplemental therapy in translational tolerance induction protocols. This is bolstered by reports of IL-10-mediated inhibition of innate immunity, association of human stem cell and nonhuman primate (NHP) islet allograft tolerance with elevated serum IL-10, and evidence that systemic IL-10 therapy enhanced pig islets survival in mice. IL-10 has not been examined as adjunctive immunosuppression in NHP. To enable such studies, we cloned and expressed rhesus macaque (RM) IL-10 fused to a mutated hinge region of human IgG1 Fc to generate IL-10/Fc(ala-ala). RM IL-10/Fc(ala-ala) was purified to approximately 98% homogeneity by affinity chromatography and shown to be endotoxin-free (<0.008 EU/microg protein). The biological activity of IL-10/Fc(ala-ala) was demonstrated by (1) costimulation of the mouse mast cell line, MC/9 proliferation in a dose-dependent fashion, (2) suppression of LPS-induced septic shock in mice and (3) abrogation of LPS-induced secretion of proinflammatory cytokines/chemokines in vitro and in vivo in NHP. Notably, RM IL-10/Fc(ala-ala) had significantly greater potency than human IL-10/Fc(ala-ala) and exhibited a circulating half-life of approximately 14 days. The availability of this reagent will facilitate definitive studies to determine whether supplemental therapy with RM IL-10/Fc(ala-ala) can influence tolerance outcomes in NHP.


Subject(s)
Cell Proliferation/drug effects , Immunoglobulin Fc Fragments/pharmacology , Interleukin-10/pharmacology , Mast Cells/immunology , Recombinant Fusion Proteins/pharmacology , Shock, Septic/drug therapy , Transplantation Tolerance/drug effects , Animals , Cell Line , Cloning, Molecular , Dose-Response Relationship, Drug , Graft Survival/drug effects , Graft Survival/immunology , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Immunoglobulin G/pharmacology , Insulin-Secreting Cells/immunology , Insulin-Secreting Cells/transplantation , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-10/pharmacokinetics , Lipopolysaccharides/toxicity , Macaca mulatta , Mice , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacokinetics , Shock, Septic/chemically induced , Shock, Septic/immunology , Swine , Transplantation, Heterologous
13.
Drug Metab Dispos ; 35(5): 814-21, 2007 May.
Article in English | MEDLINE | ID: mdl-17312017

ABSTRACT

Cytokines are considered a promising immunotherapy for chronic diseases, because of their potency and fundamental roles in pathological processes. However, their therapeutic use is limited because of their poor pharmacokinetics and pleiotropic effects in various organs. These problems may be overcome by cell-specific delivery of the cytokine. This approach involves chemical modification of the protein with homing devices that recognize receptors on target cells. The cytokine interleukin-10 (IL10) may be valuable as a therapeutic cytokine for patients with liver cirrhosis. However, its rapid renal elimination and general immunosuppressive activities limit therapeutic use. We therefore aim to target this cytokine in the liver, in particular to fibrogenic hepatic stellate cells (HSCs). We show that IL10 is successfully modified with mannose 6-phosphate (M6P), which is a homing device for the mannose 6-phosphate/insulin-like growth factor II (M6P/IGFII) receptor expressed on activated HSCs. Chemical modification did not diminish IL10 efficacy with regard to in vitro anti-inflammatory (lipopolysaccharide-stimulated tumor necrosis factor alpha release) and antifibrotic (collagen deposition and degradation) activities. Biodistribution studies with radiolabeled M6P-IL10 and IL10 in rats with liver fibrosis showed that modification with M6P groups induced a shift in the distribution from the kidneys (IL10) to the liver (M6P-IL10). Hepatocellular binding of M6P-IL10 occurred via M6P/IGFII receptors and scavenger receptors, indicating that not only HSCs but also Kupffer and endothelial cells are target cells. IL10 did not bind to these receptors. We conclude that we prepared an active and liver-specific form of the cytokine IL10 that can be evaluated for its efficacy to treat liver diseases.


Subject(s)
Interleukin-10/pharmacokinetics , Liver/metabolism , Mannosephosphates/chemistry , Animals , Blotting, Western , Cell Line , Dose-Response Relationship, Drug , Immunohistochemistry/methods , Injections, Intravenous , Interleukin-10/administration & dosage , Interleukin-10/chemistry , Iodine Radioisotopes , Kidney/metabolism , Lipopolysaccharides/pharmacology , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/metabolism , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Male , Molecular Structure , Rats , Rats, Wistar , Tissue Distribution , Tumor Necrosis Factor-alpha/metabolism
14.
Pharm Res ; 21(11): 2072-8, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15587930

ABSTRACT

PURPOSE: Because interleukin-10 (IL-10) seems a promising new antifibrotic drug, we investigated the pharmacokinetic and biodistribution profile of this potent therapeutic cytokine in rats with extensive liver fibrosis (BDL-3). IL-10 receptor expression was also determined in relation to these aspects. METHODS: To study the pharmacokinetic and biodistribution of IL-10, rhIL-10 was labeled with 125-iodine. Plasma samples of 125IrhIL-10 were obtained over a 30-min time period after administration of radiolabeled-cytokine to BDL-3 and normal rats. The tissue distribution was assessed 10 and 30 min after i.v. administration of 125IrhlL-10. IL-10 receptor expression was determined by immurohistochemical staining and RT-PCR technique. RESULTS: . The 125IrhIL-10 plasma curves followed two-compartment kinetics with a lower AUC in BDL-3 rats as compared to control. Plasma clearance and distribution volume at steady state were larger in BDL-3 rats. Tissue distribution analysis in normal rats showed that 125IrhIL-10 highly accumulated in kidneys. In BDL-3 rats, the liver content of 125IrhIL-10 increased by a factor of 2, whereas kidney accumulation did not significantly change. Immunohistochemical staining and RT-PCR analysis showed that IL-10 receptor was clearly upregulated in BDL-3 rat livers. CONCLUSIONS: . In normal rats, 125IrhIL-10 rapidly disappears from the circulation, and the kidney is predominantly responsible for this. In BDL-3 rats, the liver largely contributes to this rapid plasma disappearance, probably due to an increase in IL-10 receptor expression. The extensive renal clearance of IL-10 in vivo may limit a clinical application of this cytokine for the treatment of chronic liver diseases. To optimize the therapeutic effects of IL-10 in hepatic diseases, alternative approaches that either decrease renal disposition or that further enhance hepatic delivery should be considered.


Subject(s)
Interleukin-10/pharmacokinetics , Liver Cirrhosis/metabolism , Animals , Area Under Curve , Dose-Response Relationship, Drug , Humans , Injections, Intravenous , Interleukin-10/metabolism , Iodine Radioisotopes , Isotope Labeling , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Receptors, Interleukin/biosynthesis , Receptors, Interleukin/metabolism , Recombinant Proteins/pharmacokinetics , Reverse Transcriptase Polymerase Chain Reaction , Tissue Distribution
15.
Brain Res Mol Brain Res ; 114(2): 168-71, 2003 Jun 10.
Article in English | MEDLINE | ID: mdl-12829328

ABSTRACT

Interleukin (IL)-10 exerts beneficial effects on the central nervous system (CNS) after peripheral administration, but its penetration across the blood-brain barrier (BBB) has not been quantified. We show that 125I-IL-10 is stable in circulating blood but does not cross the intact BBB after intravenous delivery. Thus, peripheral IL-10 probably can serve as a CNS therapeutic only when the BBB is disrupted.


Subject(s)
Blood-Brain Barrier/drug effects , Brain/drug effects , Interleukin-10/pharmacokinetics , Albumins/pharmacokinetics , Animals , Blood-Brain Barrier/physiology , Brain/metabolism , Brain Diseases/drug therapy , Cell Membrane/drug effects , Cell Membrane/metabolism , Diffusion/drug effects , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Interleukin-10/therapeutic use , Iodine Radioisotopes , Male , Membrane Lipids/metabolism , Metabolic Clearance Rate/drug effects , Mice , Spinal Cord/drug effects , Spinal Cord/metabolism , Technetium
16.
J Pharmacol Exp Ther ; 301(1): 59-65, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11907157

ABSTRACT

Interleukin (IL)-10 is an anti-inflammatory cytokine that suppresses the T helper 1 immune response and down-regulates macrophages and monocytes. The therapeutic effect of systemic administration of IL-10 for patients with inflammatory bowel disease, however, has not been satisfactory. We examined whether rectal administration of gelatin microspheres (GM) containing IL-10 (GM-IL-10) prevents colitis in IL-10-deficient (IL-10(-/-)) mice. GM-IL-10 and IL-10 alone were administered rectally. The colon was examined macroscopically and microscopically. IL-12 mRNA expression and CD40 expression in Mac-1-positive cells were also examined. Macroscopic and microscopic examination revealed marked improvement of colitis in IL-10(-/-) mice treated with GM-IL-10. mRNA expression of IL-12 in Mac-1-positive cells in GM-IL-10-treated mice was significantly decreased compared with that in the mice treated with IL-10 alone. Additionally, CD40 expression in Mac-1-positive cells in GM-IL-10-treated mice was decreased more prominently than in mice treated with IL-10 alone. The therapeutic effects of GM-IL-10 were associated with decreased expression of IL-12 mRNA and down-regulation of CD40 expression in Mac-1-positive cells. GM-IL-10 might be useful for treatment of patients with inflammatory bowel disease.


Subject(s)
Cytokines/administration & dosage , Cytokines/toxicity , Inflammatory Bowel Diseases/chemically induced , Interleukin-10/administration & dosage , Interleukin-10/toxicity , Animals , CD40 Antigens/biosynthesis , Cells, Cultured , Colon/metabolism , Colon/pathology , Disease Models, Animal , Drug Delivery Systems , Excipients , Female , Flow Cytometry , Gelatin , Inflammatory Bowel Diseases/pathology , Interleukin-10/pharmacokinetics , Isotope Labeling , Mice , Mice, Inbred C57BL , Mice, Knockout , Microspheres , RNA, Messenger/biosynthesis , Spleen/metabolism
17.
Regul Toxicol Pharmacol ; 35(1): 56-71, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11846636

ABSTRACT

Escherichia coli-derived recombinant human interleukin-10 (rhuIL-10) has been evaluated in an extensive series of in vivo and in vitro nonclinical safety studies, including genetic toxicology, single- and repeat-dose systemic toxicity and toxicokinetics, reproductive toxicity, and specialized irritation studies. The primary test species in the toxicology studies were the mouse and monkey based on rhuIL-10 activity in receptor binding and ex vivo cytokine assays. Supported by a detailed preclinical program of therapeutic and prophylactic animal models in autoimmune diseases, the initial clinical development program has focused on investigating the therapeutic potential of rhuIL-10 (Tenovil) in Crohn's disease and rheumatoid arthritis. The results of the subcutaneous toxicity studies, up to 3 months dosing duration in mice and 6 months dosing duration in monkeys, support the development of rhuIL-10 for present and future clinical indications by the subcutaneous route of administration.


Subject(s)
Interleukin-10/toxicity , Recombinant Proteins/toxicity , Toxicity Tests/methods , Animals , Carcinogenicity Tests/methods , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Haplorhini , Humans , Injections, Intravenous , Injections, Subcutaneous , Interleukin-10/pharmacokinetics , Interleukin-10/standards , Mice , Recombinant Proteins/pharmacokinetics , Recombinant Proteins/standards , Safety
18.
Proc Natl Acad Sci U S A ; 98(1): 277-82, 2001 Jan 02.
Article in English | MEDLINE | ID: mdl-11134537

ABSTRACT

IL-10 is a pleiotropic cytokine that acts as an important regulator of macrophage, T cell, and natural killer cell functions. Human IL-10 (hIL-10) has both stimulatory and inhibitory effects on a wide variety of cell types. Viral IL-10 (vIL-10) possesses only a subset of hIL-10's activities, predominantly its suppression of cytokine synthesis by T helper type 1 clones. In the present report, we evaluated tissue accumulation and biological activity of hIL-10 and vIL-10 in vivo in individual organs by using a first-generation adenoviral (Ad) vector administered intratracheally and intravenously. We report the observation that Ad vectors delivering vIL-10, but not hIL-10, are associated with prolonged expression in the lung (>42 days) when delivered intratracheally. In contrast, there was no prolongation in vIL-10 expression when Ad vectors were intravenously administered, although vIL-10 levels in the tissue, but not serum, were markedly increased relative to hIL-10. Moreover, we report an augmented capacity of expressed vIL-10 versus hIL-10 to suppress the acute inflammatory responses in the lung to intratracheal administration of Ad. These findings confirm fundamental differences in Ad-induced expression of vIL-10 and hIL-10 when administered to the lungs. The results further suggest that Ad vectors expressing vIL-10 may have a role as anti-inflammatory agents in the treatment of acute and chronic lung inflammation.


Subject(s)
Adenoviridae/genetics , Genetic Therapy , Interleukin-10/metabolism , Lung/metabolism , Viral Proteins/metabolism , Adenoviridae/immunology , Animals , Anti-Inflammatory Agents/therapeutic use , Antibodies, Viral/blood , Antibodies, Viral/immunology , Cell Line , Female , Gene Expression Regulation, Viral , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Genetic Vectors/immunology , Humans , Inflammation/immunology , Inflammation/pathology , Inflammation/therapy , Injections, Intravenous , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-10/pharmacokinetics , Intubation, Intratracheal , Liver/metabolism , Liver/virology , Lung/immunology , Lung/pathology , Lung/virology , Mice , Mice, Inbred C57BL , Neutralization Tests , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacokinetics , Time Factors , Transduction, Genetic , Viral Proteins/genetics , Viral Proteins/immunology , Viral Proteins/pharmacokinetics
19.
Gastroenterology ; 119(6): 1473-82, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11113068

ABSTRACT

BACKGROUND & AIMS: Interleukin 10 (IL-10) is an anti-inflammatory, immunomodulatory cytokine that regulates mucosal inflammation. This study evaluated the safety, tolerance, and efficacy of recombinant human IL-10 (rhuIL-10) for mild to moderately active Crohn's disease. METHODS: We conducted a 24-week multicenter, prospective, randomized, double-blind, placebo-controlled, and sequential-escalating-dose study. Ninety-five patients with Crohn's Disease Activity Index of 200-350, not presently undergoing corticosteroid, mesalamine, or immunosuppressive therapy, were treated with subcutaneous rhuIL-10 (1, 5, 10, or 20 microg/kg) or placebo once daily for 28 consecutive days. Patients were followed up for 20 weeks after treatment. Evaluation of safety and tolerance was the first objective, and efficacy was the second objective. RESULTS: Adverse effects were dose-related, mild-to-moderate in severity, and reversible. Asymptomatic and reversible anemia and thrombocytopenia were observed at higher doses. No withdrawal or delayed adverse effects were evident during 20 weeks of follow-up. At the end of treatment (day 29), intent-to-treat analysis showed that 23.5% (confidence interval [CI], 6.8%-49.9%) of patients receiving 5 micro/kg rhuIL-10 experienced clinical remission and endoscopic improvement; 0% (CI, 0%-14.8%) of patients in the placebo group did. Higher doses of recombinant human IL-10 were less effective than 5 microg/kg. No rhuIL-10 serum accumulation and no antibody against IL-10 were detected after 4 weeks. CONCLUSIONS: Subcutaneous rhuIL-10 administered daily for 28 days to patients with mild to moderately active Crohn's disease is safe, well-tolerated, and shows clinical and endoscopic improvement.


Subject(s)
Crohn Disease/drug therapy , Crohn Disease/physiopathology , Interleukin-10/therapeutic use , Adult , Aged , Crohn Disease/pathology , Dose-Response Relationship, Drug , Endoscopy, Digestive System , Female , Humans , Injections, Subcutaneous , Interleukin-10/administration & dosage , Interleukin-10/adverse effects , Interleukin-10/pharmacokinetics , Male , Middle Aged , Prospective Studies , Recombinant Proteins/administration & dosage , Recombinant Proteins/adverse effects , Recombinant Proteins/pharmacokinetics , Recombinant Proteins/therapeutic use , Recurrence , Severity of Illness Index , Treatment Outcome
20.
Clin Cancer Res ; 6(7): 2661-9, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10914707

ABSTRACT

A pilot dose-escalation study of recombinant human interleukin 12 (rhIL-12) was conducted in Japanese patients with advanced malignancies. Cohorts of three patients received escalating doses of rhIL-12 that increased from 50 to 300 ng/kg/day s.c. three times a week for 2 weeks followed by 1-week rest. The same dosage and schedule was repeated for two additional courses. Sixteen previously treated patients were registered, and 15 were evaluated. Common toxicities were fever and leukopenia; the abnormality of laboratory tests included elevations in aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, C-reactive protein, and beta2-microglobin. Dose-limiting toxicity was the grade 3 elevation of aminotransferases, and was observed in two of six patients at the 300-ng/kg dose level after the first course in one patient and after the third course in the other. Leukopenia was observed at all of the dose levels; two of six patients at 300 ng/kg experienced grade 3 leukopenia. Thus, 300 ng/kg was determined to be the maximum acceptable dose. Peak plasma levels of rhIL-12 decreased in the second courses, but the areas under the curve were almost the same in the first and second courses. Biological effects included increases of plasma levels of IFN-gamma, tumor necrosis factor-alpha, IL-6, IL-10, and neopterin. In two patients with renal cell carcinoma, complete response and partial response of metastatic tumors were observed with 50 and 300 ng/kg; the responses lasted for 5 and 3.5 months, respectively. Although immunological response to rhIL-12 varies depending on administration route and schedule and on patients' physiological conditions, the recommended dose for Phase II studies is 300 ng/kg s.c. three times a week for 2 weeks followed by 1-week rest.


Subject(s)
Interleukin-10/adverse effects , Interleukin-10/pharmacokinetics , Neoplasms/drug therapy , Adult , Aged , Carcinoma, Renal Cell/drug therapy , Cohort Studies , Dose-Response Relationship, Drug , Female , Humans , Infusions, Intravenous , Interferon-gamma/blood , Interleukin-10/administration & dosage , Interleukin-10/blood , Interleukin-6/blood , Japan , Kidney Neoplasms/drug therapy , Male , Middle Aged , Neoplasms/blood , Neoplasms/immunology , Neopterin/blood , Recombinant Proteins/administration & dosage , Recombinant Proteins/adverse effects , Recombinant Proteins/pharmacokinetics , Tumor Necrosis Factor-alpha/analysis
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