Development and characterization of a fusion protein between thermally responsive elastin-like polypeptide and interleukin-1 receptor antagonist: sustained release of a local antiinflammatory therapeutic.
OBJECTIVE
Interleukin-1 receptor antagonist (IL-1Ra) has been evaluated for the intraarticular treatment of osteoarthritis. Such administration of proteins may have limited utility because of their rapid clearance and short half-life in the joint. The fusion of a drug to elastin-like polypeptides (ELPs) promotes the formation of aggregating particles that form a "drug depot" at physiologic temperatures, a phenomenon intended to prolong the presence of the drug. The purpose of this study was to develop an injectable drug depot composed of IL-1Ra and ELP domains and to evaluate the properties and bioactivity of the recombinant ELP-IL-1Ra fusion protein.


METHODS
Fusion proteins between IL-1Ra and 2 distinct sequences and molecular weights of ELP were overexpressed in Escherichia coli. Environmental sensitivity was demonstrated by turbidity and dynamic light scattering as a function of temperature. IL-1Ra domain activity was evaluated by surface plasmon resonance, and in vitro antagonism of IL-1-mediated lymphocyte and thymocyte proliferation, as well as IL-1-induced tumor necrosis factor alpha (TNFalpha) expression and matrix metalloproteinase 3 (MMP-3) and ADAMTS-4 messenger RNA expression in human intervertebral disc fibrochondrocytes. IL-1Ra immunoreactivity was assessed before and after proteolytic degradation of the ELP partner.


RESULTS
Both fusion proteins underwent supramolecular aggregation at subphysiologic temperatures and slowly resolubilized at 37 degrees C. Interaction with IL-1 receptor was slower in association but equivalent in dissociation as compared with the commercial antagonist. Anti-IL-1 activity was demonstrated by inhibition of lymphocyte and thymocyte proliferation and by decreased TNFalpha expression and ADAMTS-4 and MMP-3 transcription by fibrochondrocytes. ELP domain proteolysis liberated a peptide of comparable size and immunoreactivity as the commercial IL-1Ra. This peptide was more bioactive against lymphocyte proliferation, nearly equivalent to the commercial antagonist.


CONCLUSIONS
The ELP-IL-1Ra fusion protein proved to retain the characteristic ELP inverse phase-transitioning behavior as well as the bioactivity of the IL-1Ra domain. This technology represents a novel drug carrier designed to prolong the presence of bioactive peptides following intraarticular delivery.
Shamji
Mohammed F
MF
Duke University, Durham, North Carolina 27708, USA.
Betre
Helawe
H
Kraus
Virginia B
VB
Chen
Jun
J
Chilkoti
Ashutosh
A
Pichika
Rajeswari
R
Masuda
Koichi
K
Setton
Lori A
LA
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000493:pharmacokinetics*; D002455:Cell Division / Q000187:drug effects / Q000276:immunology; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D016503:Drug Delivery Systems / Q000379:methods; D015195:Drug Design; D004549:Elastin / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D053590:Interleukin 1 Receptor Antagonist Protein / Q000235:genetics*; D007403:Intervertebral Disc / Q000166:cytology; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D010455:Peptides / Q000235:genetics; D017472:Receptors, Interleukin-1 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000493:pharmacokinetics*; D013696:Temperature; D013950:Thymus Gland / Q000166:cytology; D020298:U937 Cells
D000893:Anti-Inflammatory Agents; D053590:Interleukin 1 Receptor Antagonist Protein; D010455:Peptides; D017472:Receptors, Interleukin-1; D011993:Recombinant Fusion Proteins; D004549:Elastin