Functions of interleukin 1 receptor antagonist in gene knockout and overproducing mice.

Interleukin 1 receptor antagonist (IL-1ra) is a cytokine whose only known action is competitive inhibition of the binding of interleukin 1 (IL-1) to its receptor. To investigate the physiological roles of endogenously produced IL-1ra, we generated mice that either lack IL-1ra or overproduce it under control of the endogenous promoter. Mice lacking IL-1ra have decreased body mass compared with wild-type controls. They are more susceptible than controls to lethal endotoxemia but are less susceptible to infection with Listeria monocytogenes. Conversely, IL-1ra overproducers are protected from the lethal effects of endotoxin but are more susceptible to listeriosis. Serum levels of IL-1 following an endotoxin challenge are decreased in IL-1ra nulls and increased in IL-1ra overproducers in comparison to controls. These data demonstrate critical roles for endogenously produced IL-1ra in growth, responses to infection and inflammation, and regulation of cytokine expression.

Suppression of experimental arthritis by gene transfer of interleukin 1 receptor antagonist cDNA.

Restoration of the impaired balance between pro- and antiinflammatory cytokines should provide effective treatment of rheumatoid arthritis. Gene therapy has been proposed as an approach for delivery of therapeutic proteins to arthritic joints. Here, we examined the efficacy of antiinflammatory gene therapy in bacterial cell wall-induced arthritis in rats. Human secreted interleukin 1 receptor antagonist (sIL-1ra) was expressed in joints of rats with recurrent bacterial cell wall-induced arthritis by using ex vivo gene transfer. To achieve this, primary synoviocytes were transduced in culture with a retroviral vector carrying the sIL-1ra cDNA. Transduced cells were engrafted in ankle joints of animals prior to reactivation of arthritis. Animals in control groups were engrafted with synoviocytes transduced with lacZ and neo marker genes. Cells continued to express transferred genes for at least 9 days after engraftment. We found that gene transfer of sIL-1ra significantly suppressed the severity of recurrence of arthritis, as assessed by measuring joint swelling and by the gross-observation score, and attenuated but did not abolish erosion of cartilage and bone. The effect of intraarticularly expressed sIL-1ra was essentially local, as there was no significant difference in severity of recurrence between unengrafted contralateral joints in control and experimental groups. We estimate that locally expressed sIL-1ra was about four orders of magnitude more therapeutically efficient than systemically administered recombinant sIL-1ra protein. These findings provide experimental evidence for the feasibility of antiinflammatory gene therapy for arthritis.

Identification of receptor-binding domains on human interleukin 5 and design of an interleukin 5-derived receptor antagonist.

A detailed structure-function analysis of human interleukin 5 (hIL5) has been performed. The hIL5 receptor is composed of two different polypeptide chains, the alpha and beta subunits. The alpha subunit alone is sufficient for ligand binding, but association with the beta subunit leads to a 2- to 3-fold increase in binding affinity. The beta chain is shared with the receptors for IL3 and granulocyte/macrophage-colony-stimulating factor--hence the descriptor beta C (C for common). All hIL5 mutants were analyzed in a solid-phase binding assay for hIL5R alpha interaction and in a proliferation assay using IL5-dependent cell lines for receptor-complex activation. Most residues affecting binding to the receptor alpha subunit were clustered in a loop connecting beta-strand 1 and helix B (mutants H38A, K39A, and H41A), in beta-strand 2 (E89A and R91A; weaker effect for E90A) and close to the C terminus (T109A, E110A, W111S, and I112A). Mutations at one position, E13 (Glu13), caused a reduced activation of the hIL5 receptor complex. In the case of E13Q, only 0.05% bioactivity was detected on a hIL5-responsive subclone of the mouse promyelocytic cell line FDC-P1. Moreover, on hIL5-responsive TF1 cells, the same mutant was completely inactive and proved to have antagonistic properties. Interactions of this mutant with both receptor subunits were nevertheless indistinguishable from those of nonmutated hIL5 by crosslinking and Scatchard plot analysis of transfected COS-1 cells.

Increased potency of a novel complement factor 5a receptor antagonist in a rat model of inflammatory bowel disease

We have previously shown that complement factor 5a(C5a) plays a role in the pathogenesis of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats by using the selective, orally active C5a antagonist AcF-[OP(D-Cha) WR]. This study tested the efficacy and potency of a new C5a antagonist, hydrocinnamate (HC)-[OP(D-Cha) WR], which has limited intestinal lumenal metabolism, in this model of colitis. Analogs of AcF-[OP(D-Cha) WR] were examined for their susceptibility to alimentary metabolism in the rat using intestinal mucosal washings. One metabolically stable analog, HC-[OP(D-Cha)WR], was then evaluated pharmacokinetically and investigated at a range of doses (0.03 - 10 mg/kg/ day p.o.) in the 8-day rat TNBS- colitis model, against the comparator drug AcF-[OP(D-Cha) WR]. Using various amino acid substitutions, it was determined that the AcF moiety of AcF-[OP(D-Cha) WR] was responsible for the metabolic instability of the compound in intestinal mucosal washings. The analog HC-[OP( D-Cha) WR], equiactive in vitro to AcF-[OP(D-Cha) WR], was resistant to intestinal metabolism, but it displayed similar oral bioavailability to AcF-[OP(D-Cha) WR]. However, in the rat TNBS- colitis model, HC-[OP(D-Cha) WR] was effective at reducing mortality, colon edema, colon macroscopic scores, and increasing food consumption and body weights, at 10- to 30- fold lower oral doses than AcF-[OP( D-Cha) WR]. These studies suggest that resistance to intestinal metabolism by HC-[OP(D-Cha) WR] may result in increased local concentrations of the drug in the colon, thus affording efficacy with markedly lower oral doses than AcF-[OP(D-Cha) WR] against TNBS-colitis. This large increase in potency and high efficacy of this compound makes it a potential candidate for clinical development against intestinal diseases such as inflammatory bowel disease.

Will the new CB1 cannabinoid receptor antagonist SR-147778 have advantages over rimonabant?

Obesity and alcoholism are two common modern-day diseases. The cannabinoid CB, receptor antagonist rimonabant is in Phase III clinical trial for the treatment of obesity with preliminary results showing that it decreases appetite and body weight. Animal studies have shown that rimonabant is effective in the treatment of alcoholism. SR-147778 is a new potent and selective CB1 receptor antagonist. In animals, SR-147778 has been shown to inhibit CB1 receptor-mediated hypothermia, analgesia and slowing of gastrointestinal transit. In rats trained to drink sucrose, the oral administration of SR-147778 3 mg/kg, before the presentation of sucrose, decreased the consumption of sucrose. SR-147778 3 mg/kg also reduced spontaneous feeding in rats deprived of food and also in non-deprived rats. In Sardinian alcohol-preferring (sP) rats, in the alcohol-naive state, SR-147778 slowed the development of a preference for alcohol. in alcohol-experienced sP rats SR-147778 (2.5, 5 and 10 mg/kg p.o.) reduced the alcohol intake. When alcohol-experienced sP rats are deprived of alcohol for 15 days, there is a large intake of alcohol on reintroduction of alcohol, and this response was almost abolished by treatment with SR-147778. From the preclinical studies published to date, there is no obvious major point of difference between rimonabant and SR-147778, and both are promising agents for the treatment of obesity and alcoholism.