Development of a Pharmacokinetic Model Describing Neonatal Fc Receptor-Mediated Recycling of HL2351, a Novel Hybrid Fc-Fused Interleukin-1 Receptor Antagonist, to Optimize Dosage Regimen.

HL2351 (hIL-1Ra-hyFc) is a novel recombinant protein formed by the fusion of two human interleukin-1 receptor antagonist components into one antibody-derived fragment crystallizable portion. Although HL2351 has a pharmacological mechanism of action similar to that of anakinra as a commercialized biopharmaceutical drug, HL2351 has been desired to reduce the dose frequency and improve therapeutic efficacy due to its long circulation half-life. In this study, we aimed to develop a population pharmacokinetic (PK) model for HL2351 using a neonatal Fc receptor (FcRn)-mediated recycling model based on a quasi-steady-state approximation of target-mediated drug disposition (TMDD) for the description of interactions between the drug and FcRn. FcRn recycling was expected in the case of HL2351 because of PK related to the antibody portion. A TMDD model was also applied to describe interactions of IL1R with HL2351 or anakinra. PK data were collected from a phase I study conducted in six groups (1, 2, 4, 8, 12 mg/kg HL2351 and 100 mg anakinra single subcutaneous administration; n = 8 per group). In consequence, the PK of anakinra and HL2351 following administration of multiple doses at different dosages were simulated. Optimized doses were considered based on average concentrations of IL1R bound to anakinra and HL2351. HL2351 at doses of 326 mg or 4.267, 4.982, 5.288, 5.458, or 5.748 mg/kg once weekly or HL2351 at 1726 mg or 21.92, 26.86, 29.10, 30.36, or 32.53 mg/kg once biweekly would have similar therapeutic effects with anakinra at a dose of 100 mg or 1, 2, 3, 4, or 8 mg/kg administered once daily, respectively.

Injectable biomaterials for delivery of interleukin-1 receptor antagonist: Toward improving its therapeutic effect.

Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring anti-inflammatory cytokine that inhibits IL-1 activity and has been proposed to treat a wide variety of systemic and local inflammatory pathologies for multiple decades. However, the short half-life and high concentration required to inhibit IL-1 activity has limited its use in clinical applications. Many strategies have been developed with the goal of improving the therapeutic efficacy of IL-1Ra for a variety of pathologies, including fusing IL-1Ra to protein/peptide/polymer partners, releasing IL-1Ra from injectable polymer or mineral particles, and release of IL-1Ra from injectable coacervates and gels. This literature review examines injectable biomaterials engineered to improve IL-1Ra delivery, both locally and systemically, to increase its efficacy and ease of use in clinic. STATEMENT OF SIGNIFICANCE: Interleukin-1 receptor antagonist (IL-1Ra) is a therapeutic protein with the potential to treat numerous inflammatory conditions and diseases. However, its short biological half-life and high therapeutic concentration may limit its utility in all but a few clinical scenarios. In this review, we present the biomaterial based delivery strategies which have been explored to deliver IL-1Ra to improve its efficacy and applicability to treat inflammation.

Pharmacokinetics of Anakinra in Subjects of Heavier vs. Lighter Body Weights.

This trial (20010168) studied how body weight (BW) and body mass index (BMI) influenced the pharmacokinetics (PK) of anakinra. Subjects (n = 32) were assigned to four groups (n = 8) according to BW and BMI. Randomization was according to a four-treatment, four-period, four-sequence crossover design. The four anakinra injections were 100, 150, and 300 mg s.c. and 100 mg i.v. Plasma samples were measured by enzyme-linked immunosorbent assay and noncompartmental PK parameters estimated. BW demonstrated the following effects: after i.v. administration, significant effects (P < 0.05) were observed for exposure (area under the concentration-time curve from zero to infinity (AUC0-∞ )), peak plasma concentration (Cmax ), volume of distribution at steady state, and clearance; whereas after s.c. administration, significant effects (P < 0.05) were observed for Cmax , AUC0-∞ , terminal half-life, and estimated apparent clearance. Mean AUC was reduced 24% and 33% for heavier (BW ≥ 100 kg) vs. lighter subjects (BW ≤ 90 kg) after i.v. and s.c. administration, respectively. BMI increased clearance for heavier subjects. For example, mean (SD) plasma clearance of i.v. anakinra increased from 1.17 ± 0.29 to 1.62 ± 0.24 mL/minute/kg (P < 0.05) for larger (> 100 kg) obese (BMI > 36) vs. larger (> 100 kg) less obese (BMI < 35) subjects, respectively. Similarly, results following s.c. supported those after i.v. administration. Derived half-lives increased with higher BW and higher BMI ranging from 3.63 hour for less obese, lighter-weight subjects to 7.62 hour for obese, heavier-weight subjects. Absolute bioavailability ranged from 80-92% and was unrelated to BW or BMI. Anakinra exposure is statistically significantly related to BW and to a lesser extent BMI.

A peptide derived from melanotransferrin delivers a protein-based interleukin 1 receptor antagonist across the BBB and ameliorates neuropathic pain in a preclinical model.

Delivery of biologic drugs across the blood-brain barrier is becoming a reality. However, the solutions often involve the assembly of complex multi-specific antibody molecules. Here we utilize a simple 12 amino-acid peptide originating from the melanotransferrin (MTf) protein that has shown improved brain delivery properties. 3D confocal fluorescence microscopic analysis demonstrated brain parenchymal localisation of a fluorescently labelled antibody (NIP228) when chemically conjugated to either the MTf peptide or full-length MTf protein. Measurement of plasma kinetics demonstrated the MTf peptide fusions had very similar kinetics to an unmodified NIP228 control antibody, whereas the fusion to MTf protein had significantly reduced plasma exposure most likely due to a higher tissue distribution in the periphery. Brain exposure for the MTf peptide fusions was significantly increased for the duration of the study, exceeding that of the fusions to full length MTf protein. Using a neuropathic pain model, we have demonstrated that fusions to interleukin-1 receptor antagonist (IL-1RA) are able to induce significant and durable analgesia following peripheral administration. These data demonstrate that recombinant and chemically conjugated MTf-based brain delivery vectors can deliver therapeutic levels of drug to the central nervous system.

Pharmacokinetics and Safety of Recombinant Human Interleukin-1 Receptor Antagonist GR007 in Healthy Chinese Subjects.

BACKGROUND AND OBJECTIVES: The recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) GR007 is a candidate drug with the potential to prevent the toxicity induced by chemotherapy agents by blocking the IL-1 signaling pathway. The aim of this study was to assess the pharmacokinetics and safety of GR007 in healthy Chinese subjects. METHODS: Thirty subjects received a single intramuscular injection of 30 mg (n = 10), 90 mg (n = 10), or 150 mg (n = 10) GR007. After administration, the pharmacokinetic characteristics and safety were evaluated. RESULTS: No serious adverse events were reported in this study, and the adverse events reported showed no dose dependency. Pharmacokinetic analysis showed that the median time to maximum concentration (Tmax) of GR007 in the three groups was between 2.75 and 4.00 h and the geometric mean elimination half-life (T1/2) for each group was 2.38, 2.22, and 3.29 h, respectively. The area under the concentration vs time curve (AUC), but not the maximum concentration (Cmax), increased in a dose-proportional manner. CONCLUSIONS: The results showed that a single intramuscular injection of 30-150 mg GR007 had good safety and tolerability in healthy Chinese subjects. The results of the evaluation of the safety and pharmacokinetics of GR007 performed in this study support its use as a repeated daily injection in ongoing clinical trials focusing on patients with cancer.

Reduction of inflammation after administration of interleukin-1 receptor antagonist following aneurysmal subarachnoid hemorrhage: results of the Subcutaneous Interleukin-1Ra in SAH (SCIL-SAH) study.

OBJECTIVE Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating cerebrovascular event with long-term morbidity and mortality. Patients who survive the initial bleeding are likely to suffer further early brain injury arising from a plethora of pathological processes. These may result in a worsening of outcome or death in approximately 25% of patients and may contribute to longer-term cognitive dysfunction in survivors. Inflammation, mediated by the cytokine interleukin-1 (IL-1), is an important contributor to cerebral ischemia after diverse forms of brain injury, including aSAH. Its effects are attenuated by its naturally occurring antagonist, IL-1 receptor antagonist (IL-1Ra [anakinra]). The authors hypothesized that administration of additional subcutaneous IL-1Ra would reduce inflammation and associated plasma markers associated with poor outcome following aSAH. METHODS This was a randomized, open-label, single-blinded study of 100 mg subcutaneous IL-1Ra, administered twice daily in patients with aSAH, starting within 3 days of ictus and continuing until 21 days postictus or discharge from the neurosurgical center, whichever was earlier. Blood samples were taken at admission (baseline) and at Days 3-8, 14, and 21 postictus for measurement of inflammatory markers. The primary outcome was difference in plasma IL-6 measured as area under the curve between Days 3 and 8, corrected for baseline value. Secondary outcome measures included similar area under the curve analyses for other inflammatory markers, plasma pharmacokinetics for IL-1Ra, and clinical outcome at 6 months. RESULTS Interleukin-1Ra significantly reduced levels of IL-6 and C-reactive protein (p < 0.001). Fibrinogen levels were also reduced in the active arm of the study (p < 0.002). Subcutaneous IL-1Ra was safe, well tolerated, and had a predictable plasma pharmacokinetic profile. Although the study was not powered to investigate clinical effect, scores of the Glasgow Outcome Scale-extended at 6 months were better in the active group; however, this outcome did not reach statistical significance. CONCLUSIONS Subcutaneous IL-1Ra is safe and well tolerated in aSAH. It is effective in reducing peripheral inflammation. These data support a Phase III study investigating the effect of IL-1Ra on outcome following aSAH. Clinical trial registration no.: EudraCT: 2011-001855-35 ( www.clinicaltrialsregister.eu ).

RSM optimization of HSA/IL1Ra in Pichia pastoris overexpression strain and study of its in vivo activity in reducing hyperglycemia of GK rats.

Human serum albumin (HSA) and interleukin-1 receptor antagonist (IL1Ra) fusion protein is a potential long-acting drug in the treatment of type 2 diabetes. Previously, the expression level of HSA/IL1Ra in Pichia pastoris was successfully improved by increasing the gene copy number and coexpression with chaperone (protein disulfide isomerase) in our laboratory. However, the overexpression strain resulted in low production of high- cell-density fermentation. In this study, the culture medium was optimized in both flask and fermenter, and the optimum culture medium notably increased the productivity and stability of HSA/IL1Ra. To further improve the expression, response surface methodology was used to further optimize the culture condition through modeling three selected parameters (induction pH, induction temperature [T], and maximum methanol feed rate [Vm ]). The maximum yield of HSA/IL1Ra reached 1.1 g/L (10-fold higher than original fermentation condition) under the optimized culture condition (pH 7.0, T = 29 â„ƒ and Vm = 4.82 mL/L/H) in a 5-L fermenter. In addition, the degradation position of HSA/IL1Ra during fermentation was determined to be K571, serving as a potential target for genetic modification strategies to reduce the degradation. Finally, the in vivo activity study showed that HSA/IL1Ra maintained the therapeutic effect of IL1Ra in type 2 diabetes model rats meanwhile reducing the frequency of administration.

Staurosporine as an agonist for induction of GLUT4 translocation, identified by a pH-sensitive fluorescent IRAP-mOrange2 probe.

Insulin-stimulated GLUT4 translocation from GLUT4 storage vesicles (GSVs) to the plasma membrane (PM) constitutes a key process for blood glucose control. Therefore, compounds that could promote GLUT4 translocation into the PM represent potential drugs for the treatment of diabetes. In this research, we screened for agonists that induce GLUT4 translocation by using a novel pH-sensitive fluorescent probe, insulin-regulated aminopeptidase (IRAP)-mOrange2. We identified as well as validated one agonist, staurosporine, from a 64,000 compound library. Staurosporine promotes GSVs translocation into the PM and increases glucose uptake through the AMP-activated protein kinase (AMPK) pathway, serving as an effective insulin additive analogue in L6 cells. Our work highlights the convenience and efficiency of this novel pH-sensitive fluorescent probe and reveals the new biological activity of staurosporine as an agonist for GLUT4 translocation and as an effective insulin additive analogue.

Study design and population pharmacokinetic analysis of a phase II dose-ranging study of interleukin-1 receptor antagonist.

Interleukin-1 receptor antagonist, a naturally-occurring antagonist to the pro-inflammatory cytokine Interleukin-1, is already in clinical use. In experimental models of stroke, Interleukin-1 receptor antagonist in cerebrospinal fluid has been associated with cerebral neuroprotection and in a phase I clinical trial in patients with subarachnoid haemorrhage it crosses the blood-cerebrospinal fluid barrier. The aims of the current work were to design a dose-ranging clinical study in patients and to analyse the plasma and cerebrospinal fluid data obtained using a population pharmacokinetic modelling approach. The study was designed using prior information: a published population pharmacokinetic model and associated parameter estimates. Simulations were carried out to identify combinations of intravenous bolus and 4 h infusion doses that could achieve a concentration of 100 ng/ml in cerebrospinal fluid within approximately 30 min. The most informative time points for plasma and cerebrospinal fluid were obtained prospectively; optimisation identified five sampling time points that were included in the 15 time points in the present study design. All plasma and cerebrospinal fluid concentration data from previous and current studies were combined for updated analysis. The result of the simulations showed that a dosage regimen of 500 mg intravenous bolus and 10 mg/kg/h could achieve the target concentration, however four other regimens that represent a stepwise increase in maximum concentration were also selected. Analysis of the updated data showed improvement in parameter accuracy and predictive performance of the model; the percentage relative standard errors for fixed and random-effects parameters were <15 and 35% respectively. A dose-ranging study was successfully designed using modelling and simulation.

Central delivery of iodine-125-labeled cetuximab, etanercept and anakinra after perispinal injection in rats: possible implications for treating Alzheimer's disease.

INTRODUCTION: Alzheimer's disease is a debilitating condition, and the search for an effective treatment is ongoing. Inflammation, in reaction to amyloid deposition, is thought to accelerate cognitive decline. With tumor necrosis factor α being an important proinflammatory cytokine, a recent trial investigated the effect of the tumor necrosis factor α inhibitor etanercept after peripheral administration in patients with Alzheimer's disease. Although there was no significant effect, others have claimed spectacular effects of etanercept after perispinal injection. In the present study, the central delivery of drugs with a large molecular weight was evaluated after injection in the cervical perispinal region in rats. If successful, this strategy might increase therapeutic options for patients with Alzheimer's disease. METHODS: Nine male Sprague-Dawley rats were given injections of iodine-125-labeled cetuximab (146 kDa), etanercept (51 kDa), and anakinra (17 kDa). Each radioiodinated drug was injected in the perispinal region in two rats and into the dorsal tail vein in one rat. Directly after injection, the rats were placed in a head-down position for 3 minutes to direct blood flow into the valveless vertebral venous system. A single-positron emission computed tomography scan was acquired starting 5 minutes after injection, subsequently the rats were euthanized and bio-distribution was determined.   RESULTS: Intracranial delivery of the radiolabeled drugs could not be visualized in all but one of the rats. Injected drugs accumulated locally in the perispinal region. CONCLUSIONS: In this study, no evidence could be found for the delivery of drugs to the central nervous system after perispinal injection. Additional research is needed before this treatment can be used in patients with Alzheimer's disease.

Modeling and design of challenge tests: Inflammatory and metabolic biomarker study examples.

Given the complexity of pharmacological challenge experiments, it is perhaps not surprising that design and analysis, and in turn interpretation and communication of results from a quantitative point of view, is often suboptimal. Here we report an inventory of common designs sampled from anti-inflammatory, respiratory and metabolic disease drug discovery studies, all of which are based on animal models of disease involving pharmacological and/or patho/physiological interaction challenges. The corresponding data are modeled and analyzed quantitatively, the merits of the respective approach discussed and inferences made with respect to future design improvements. Although our analysis is limited to these disease model examples, the challenge approach is generally applicable to the vast majority of pharmacological intervention studies. In the present five Case Studies results from pharmacodynamic effect models from different therapeutic areas were explored and analyzed according to five typical designs. Plasma exposures of test compounds were assayed by either liquid chromatography/mass spectrometry or ligand binding assays. To describe how drug intervention can regulate diverse processes, turnover models of test compound-challenger interaction, transduction processes, and biophase time courses were applied for biomarker response in eosinophil count, IL6 response, paw-swelling, TNFα response and glucose turnover in vivo. Case Study 1 shows results from intratracheal administration of Sephadex, which is a glucocorticoid-sensitive model of airway inflammation in rats. Eosinophils in bronchoalveolar fluid were obtained at different time points via destructive sampling and then regressed by the mixed-effects modeling. A biophase function of the Sephadex time course was inferred from the modeled eosinophil time courses. In Case Study 2, a mouse model showed that the time course of cytokine-induced IL1ß challenge was altered with or without drug intervention. Anakinra reversed the IL1ß induced cytokine IL6 response in a dose-dependent manner. This Case Study contained time courses of test compound (drug), challenger (IL1ß) and cytokine response (IL6), which resulted in high parameter precision. Case Study 3 illustrates collagen-induced arthritis progression in the rat. Swelling scores (based on severity of hind paw swelling) were used to describe arthritis progression after the challenge and the inhibitory effect of two doses of an orally administered test compound. In Case Study 4, a cynomolgus monkey model for lipopolysaccharide LPS-induced TNFα synthesis and/or release was investigated. This model provides integrated information on pharmacokinetics and in vivo potency of the test compounds. Case Study 5 contains data from an oral glucose tolerance test in rats, where the challenger is the same as the pharmacodynamic response biomarker (glucose). It is therefore convenient to model the extra input of glucose simultaneously with baseline data and during intervention of a glucose-lowering compound at different dose levels. Typically time-series analyses of challenger- and biomarker-time data are necessary if an accurate and precise estimate of the pharmacodynamic properties of a test compound is sought. Erosion of data, resulting in the single-point assessment of drug action after a challenge test, should generally be avoided. This is particularly relevant for situations where one expects time-curve shifts, tolerance/rebound, impact of disease, or hormetic concentration-response relationships to occur.

Inhibition of interleukin-1ß-stimulated matrix metalloproteinases via the controlled release of interleukin-1Ra from chitosan microspheres in chondrocytes.

The aim of the study was to determine whether the controlled release of interleukin (IL)-1Ra from chitosan (CS) microspheres inhibits the IL-1ß-stimulated production of matrix metalloproteinases (MMPs) in chondrocytes. The CS-IL-1Ra microspheres were fabricated by an emulsification method using sodium tripolyphosphate as a crosslinker, and the controlled release of IL-1Ra was determined using an enzyme-linked immunosorbent assay. IL-1ß was added to normal rat chondrocytes to stimulate MMP production. The chondrocytes were incubated with CS-IL-1Ra microspheres to assess its effects on IL-1ß-induced MMP expression. Chondrocyte proliferation and glycosaminoglycan (GAG) content were also determined. The mRNA expression and protein levels of IL-1ß, MMP-1, MMP-3 and MMP-13 were detected using reverse transcription-polymerase chain reaction and western blotting analyses, respectively. The IL-1Ra release kinetics were characterized by an initial burst release, which was reduced to a linear release over seven days. The mRNA expression levels and protein levels of IL-1ß, MMP-1, MMP-3 and MMP-13 were reduced compared with the control group. The present study demonstrated the chondroprotective properties of CS microspheres as a controlled release system carrying IL-1Ra, due to the ability of the system to downregulate the expression of osteoarthritis-associated matrix-degrading proteinases in chondrocytes.

Protein HESylation for half-life extension: synthesis, characterization and pharmacokinetics of HESylated anakinra.

Half-life extension (HLE) is becoming an essential component of the industrial development of small-sized therapeutic peptides and proteins. HESylation(®) is a HLE technology based on coupling drug molecules to the biodegradable hydroxyethyl starch (HES). In this study, we report on the synthesis, characterization and pharmacokinetics of HESylated anakinra, where anakinra was conjugated to propionaldehyde-HES using reductive amination, leading to a monoHESylated protein. Characterization using size exclusion chromatography and dynamic light scattering confirmed conjugation and the increase in molecular size, while Fourier transform infrared spectroscopy showed that the secondary structure of the conjugate was not affected by coupling. Meanwhile, microcalorimetry and aggregation studies showed a significant increase in protein stability. Surface plasmon resonance and microscale thermophoresis showed that the conjugate retained its nanomolar affinity, and finally, the pharmacokinetics of the HESylated protein exhibited a 6.5-fold increase in the half-life, and a 45-fold increase in the AUC. These results indicate that HESylation(®) is a promising HLE technology.

Anakinra pharmacokinetics in children and adolescents with systemic-onset juvenile idiopathic arthritis and autoinflammatory syndromes.

BACKGROUND: Anakinra pharmacokinetics and pharmacodynamics were investigated in children and adolescents treated for systemic-onset juvenile idiopathic arthritis (SJIA) and autoinflammatory syndromes. METHODS: Anakinra was given subcutaneously at doses between 2 and 10 mg/kg (maximum 100 mg) per day. Anakinra concentrations were recorded in patients, as well as C-reactive protein (CRP) levels, on different occasions. The data were fitted to a pharmacokinetic-pharmacodynamic model via a population approach using Monolix. RESULTS: A total of 87 children and adolescents, 8 months to 21 years old, were available for pharmacokinetic evaluation. A one compartment model with linear absorption and elimination described the pharmacokinetics. Taking into account bodyweight to explain variations in apparent clearance (CL/F) and distribution volume (V/F) significantly reduced the associated between-subject and between-occasion variabilities. The final estimates were 6.24 L/h/70 kg and 65.2 L/70 kg for CL/F and V/F respectively. A mixture pharmacodynamic model described the CRP level change during anakinra treatment for the SJIA patients with 2 subpopulations, patients with high baseline and large CRP decrease and patients with low baseline and small CRP decrease followed by a re-increase in CRP levels. There was no significant effect of the combined anti-inflammatory treatment. The proportion of patients for which the development of a resistance to treatment was significant was 62% and the corresponding time was approximately 60 days. CONCLUSIONS: Based on effects in SJIA, a prospective dosage adjustment was proposed based on a 0.4 mg/L Css target in order to obtain a CRP decrease to 10 mg/L or below.

[Construction of IL-1Ra-HSA fusion protein and analysis of its bioactivity and pharmacokinetics].

In order to increase the plasma half-life and tissue specificity of IL-1 receptor antagonist, a recombinant fusion protein IL-1Ra-HSA, linked by a rigid peptide linker PAPAP, was engineered and expressed by the Pichia pastoris host cells. The fusion protein was secreted to the host cells culture, identified by Western blot, and purified by affinity chromatography. This was followed by a further examination of its bioactivity and pharmacokinetics. Our results demonstrated that the fusion protein retained the antagonist activity of IL-1Ra, capable of binding specifically to the IL-1 receptor on human melanoma A375.S2 cells, and inhibits the cytolytic effect of IL-1beta to A375.S2 cells. Albumin fusion dramatically extended the half-life of IL-1Ra and resulted in a specific accumulation of IL-1Ra in the arthritic paws and a lower distribution of IL-1Ra in other organs such as liver, kidney, spleen and lung in mice with collagen-induced arthritis. The findings reported herein indicate that the fusion protein is likely to have greater clinical applications in areas such as the treatment of rheumatoid arthritis.

IL-1ra delivered from poly(lactic-co-glycolic acid) microspheres attenuates IL-1ß-mediated degradation of nucleus pulposus in vitro.

INTRODUCTION: Inflammation plays a key role in the progression of intervertebral disc degeneration, a condition strongly implicated as a cause of lower back pain. The objective of this study was to investigate the therapeutic potential of poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with interleukin-1 receptor antagonist (IL-1ra) for sustained attenuation of interleukin-1 beta (IL-1ß) mediated degradative changes in the nucleus pulposus (NP), using an in vitro model. METHODS: IL-1ra was encapsulated in PLGA microspheres and release kinetics were determined over 35 days. NP agarose constructs were cultured to functional maturity and treated with combinations of IL-1ß and media conditioned with IL-1ra released from microspheres at intervals for up to 20 days. Construct mechanical properties, glycosaminoglycan content, nitrite production and mRNA expression of catabolic mediators were compared to properties for untreated constructs using unpaired Student's t-tests. RESULTS: IL-1ra release kinetics were characterized by an initial burst release reducing to a linear release over the first 10 days. IL-1ra released from microspheres attenuated the degradative effects of IL-1ß as defined by mechanical properties, glycosaminoglycans (GAG) content, nitric oxide production and mRNA expression of inflammatory mediators for 7 days, and continued to limit functional degradation for up to 20 days. CONCLUSIONS: In this study, we successfully demonstrated that IL-1ra microspheres can attenuate the degradative effects of IL-1ß on the NP for extended periods. This therapeutic strategy may be appropriate for treating early-stage, cytokine-mediated disc degeneration. Ongoing studies are focusing on testing IL-1ra microspheres in an in vivo model of disc degeneration, as a prelude to clinical translation.

Sustained delivery of IL-1Ra from pluronic F127-based thermosensitive gel prolongs its therapeutic potentials.

PURPOSE: Pluronic F-127 (PF127) has previously shown to prolong the sustained release of various proteinous drugs and their serum half-lives. Subsequently, we have extended this approach to look at in vitro release, in vivo efficacy and pharmacokinetics of interleukin-1 receptor antagonist (IL-1Ra). METHODS: Various concentrations of PF127 gels were prepared using cold method. In vitro drug release kinetic studies were performed using membraneless dissolution method. Stability of IL-1Ra was assessed by SDS-PAGE. In vivo studies and in vivo bioactivity of IL-1Ra were also performed on wistar rats. RESULTS: IL-1Ra loaded PF127 gels showed in vitro sustained release of IL-1Ra, depending on the concentration of gel used. SDS-PAGE confirmed the stability of protein during its in vitro release. PF127 gel also exhibited prolonged release of IL-1Ra in rats as compared to that of IL-1Ra aq. solution. In vivo bioactivity of IL-1Ra loaded in gel was confirmed by its ability to inhibit IL-1ß-stimulated induction of IL-6. CONCLUSIONS: When compared directly, IL-1Ra loaded PF127 gel exhibited prolonged in vitro and in vivo release, greater efficacy to induce hypoglycemia and inhibited IL-1ß-stimulated production of IL-6 as compared to IL-1Ra aq. solution. We believe that this methodology for sustained delivery of IL-1Ra probably be suitable for the convenience of patients to achieve desired therapeutic potentials without exceeding dose limits and frequent administration.

Characterization of 99mTc-labeled cytokine ligands for inflammation imaging via TNF and IL-1 pathways.

INTRODUCTION: TNFR2-Fc and IL-1ra-Fc are recombinant cytokine ligands that target TNF and IL-1. TNFR2-Fc-IL-1ra, a dual-domain agent that incorporates both ligands, allows bifunctional binding of IL-1 receptors and TNF. This study was designed to characterize (99m)Tc-labeled forms of these ligands, (99m)Tc-IL-1ra-Fc (IF), (99m)Tc-TNFR2-Fc (TF), and (99m)Tc-TNFR2-Fc-IL-1ra (TFI), for inflammation imaging. METHODS: The cytokine ligands were labeled with (99m)Tc by a direct approach via 2-iminothiolane (2-IT) reduction at various 2-IT/protein molar ratios. In vivo inflammation targeting studies were carried out in a mouse ear edema model created by topical application of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the right ear of ICR mice. RESULTS: Radiolabeling yields increased with increasing amounts of 2-IT. When the 2-IT/protein ratio reached 1000, the radiolabeling yield was greater than 90% without significant colloid production. TPA-treated ears showed high radioligand uptake, which was clearly detected by SPECT and autoradiographic imaging. The activities (%ID/g) in the inflamed and control ears at 3h after injection were 2.76 ± 0.20 vs. 0.69 ± 0.12 for IF, 5.86 ± 0.40 vs. 2.86 ± 0.61 for TF, and 7.61 ± 0.86 vs. 1.99 ± 0.31 for TFI (P<0.05 vs. controls). TFI showed significantly higher uptake in the inflamed ears compared to TF and IF (P<0.05). Blocking study results indicated specificity of radioligand binding with decreased radioactive uptake in the inflamed ears. Western blotting and ELISA analysis further confirmed a high expression of IL-1ß and TNF-α in the inflamed ears. CONCLUSIONS: (99m)Tc-labeled cytokine ligands are a promising approach for detecting and understanding the inflammatory process. TFI may be more useful than the single-domain ligands for noninvasive detection of inflammatory sites.

Delayed administration of interleukin-1 receptor antagonist reduces ischemic brain damage and inflammation in comorbid rats.

Many neuroprotective agents have been effective in experimental stroke, yet few have translated into clinical application. One reason for this may be failure to consider clinical comorbidities/risk factors in experimental models. We have shown that a naturally occurring interleukin-1 receptor antagonist (IL-1Ra) is protective against ischemic brain damage in healthy animals. However, protective effects of IL-1Ra have not been determined in comorbid animals. Thus, we tested whether IL-1Ra protects against brain injury induced by experimental ischemia in aged JCR-LA (corpulent) rats, which have clinically relevant risk factors. Male, aged, lean, and corpulent rats exposed to transient (90 minutes) occlusion of the middle cerebral artery (tMCAO) were administered two doses of IL-1Ra (25 mg/kg, subcutaneously) during reperfusion. Brain injury and neuroinflammatory changes were assessed 24 hours after tMCAO. Our results show that IL-1Ra administered at reperfusion significantly reduced infarct volume measured by magnetic resonance imaging (50%, primary outcome) and blood-brain barrier disruption in these comorbid animals. Interleukin-1Ra also reduced microglial activation, neutrophil infiltration, and cytokines levels in the brain. These data are the first to indicate that IL-1Ra protects against ischemic brain injury when administered via a clinically relevant route and time window in animals with multiple risk factors for stroke.

Interleukin-1 receptor antagonist: a new therapy for type 2 diabetes mellitus.

Various complex mechanisms and their multifactorial pathways decisively provoke low-grade local and systemic inflammation in ß-cells of pancreatic islets and peripheral tissues to induce ß-cells' dysfunction and apoptosis, insulin resistance, and ultimately, overt type 2 diabetes mellitus (T2DM). Conventional antidiabetic agents are being less popular, as they have some potential adverse effects. Currently, many anti-inflammatory therapeutic modalities are being investigated to abate the infuriating effects of inducers of T2DM and among them, interleukin-1 receptor antagonist (IL-1Ra) is the only one that has been approved by US Food and Drug Administration. We have compared IL-1Ra with other anti-inflammatory agents and conventional antidiabetic agents. Although, IL-1Ra has broad-spectrum anti-inflammatory activities, it also has some limitations due to its short half-life. To overcome the problem of short half-life of IL-1Ra, recently, we fused IL-1Ra in recombinant human serum albumin and expressed it in Pichia pastoris. Its bioactivity was also checked by IL-1-induced A375.S2 apoptotic cells. Furthermore, we have also formulated IL-1Ra with Pluronic F-127-based thermosensitive gel and investigated its in vitro characteristics to prolong its therapeutic effects. Further studies are required to investigate its therapeutic effects against diabetes and diabetes-associated complications.