Hepatocyte clearance and pharmacokinetics of recombinant factor IX glycosylation variants.

Addition of N-linked glycosylation sites has been shown to increase serum half-life and decrease clearance for proteins such as recombinant erythropoietin (EPO). However, factor IX (FIX) variants with additional N-linked glycans ("HG" variants) that were expressed in HKB11 cells showed increased clearance in rat in vivo pharmacokinetic studies relative to FIX variants with no additional glycans. Variants with multiple additional glycans were the most rapidly cleared. A rat hepatocyte clearance assay was developed to measure intrinsic clearance of these FIX variants in vitro. The rank order of clearance of the variants was the same both in vivo and in the in vitro hepatocyte assay. In the in vitro assay, heparin, galactose, and asialo-orosomucoid inhibited clearance of a FIX HG variant by hepatocytes, and asialo-FIX was rapidly cleared, suggesting roles for the asialoglycoprotein receptor (ASGPR) and cell surface proteoglycans in FIX clearance. Thus the in vitro hepatocyte intrinsic clearance assay is both useful and predictive for identifying rapidly cleared recombinant proteins and for helping to identify receptors involved in clearance of proteins by the liver.

Beneficial role of the GPR30 agonist G-1 in an animal model of multiple sclerosis.

The beneficial effects of estrogens in multiple sclerosis are thought to be mediated exclusively by the classical nuclear estrogen receptors ERalpha and ERbeta. However, recently many reports revealed that estrogens are able to mediate rapid signals through a G protein-coupled receptor (GPCR), known as GPR30. In the present study, we set out to explore whether effects mediated through this receptor were anti-inflammatory and could account for some of the beneficial effects of estrogen. We demonstrate that GPR30 is expressed in both human and mouse immune cells. Furthermore a GPR30-selective agonist, G-1, previously described by us, inhibits the production of lipopolysaccharide (LPS)-induced cytokines such as TNF-alpha and IL-6 in a dose-dependent manner in human primary macrophages and in a murine macrophage cell line. These effects are likely mediated solely through the estrogen-specific receptor GPR30 since the agonist G-1 displayed an IC(50) far greater than 10 microM on the classical nuclear estrogen receptors as well as a panel of 25 other GPCRs. Finally, we show that the agonist G-1 is able to reduce the severity of disease in both active and passive EAE models of multiple sclerosis in SJL mice and that this effect is concomitant with a G-1-mediated decrease in proinflammatory cytokines, including IFN-gamma and IL-17, in immune cells harvested from these mice. The effect of G-1 appears indirect, as the GPR30 agonist did not directly influence IFN-gamma or IL-17 production by purified T cells. These data indicate that G-1 may represent a novel therapeutic agent for the treatment of chronic autoimmune, inflammatory diseases.

5th annual conference on cytokines and inflammation 29-30 January 2007, Breckenridge, Colorado, USA.

The concept of anticytokine therapies for the treatment of inflammatory diseases has been proven with the successful launch of therapeutics targeting TNF and IL-1, and with numerous additional anticytokine strategies in development. The 5th annual conference on Cytokines and Inflammation provided a timely update on this topic with sections devoted to cytokine biology, chemokines, new technologies for cytokine-based therapy and small-molecule agonists and antagonists. This brief review summarizes key findings from the conference.

Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

Mechanistic studies with potent and selective inducible nitric-oxide synthase dimerization inhibitors.

A series of potent and selective inducible nitric-oxide synthase (iNOS) inhibitors was shown to prevent iNOS dimerization in cells and inhibit iNOS in vivo. These inhibitors are now shown to block dimerization of purified human iNOS monomers. A 3H-labeled inhibitor bound to full-length human iNOS monomer with apparent Kd approximately 1.8 nm and had a slow off rate, 1.2 x 10(-4) x s(-1). Inhibitors also bound with high affinity to both murine full-length and murine oxygenase domain iNOS monomers. Spectroscopy and competition binding with imidazole confirmed an inhibitor-heme interaction. Inhibitor affinity in the binding assay (apparent Kd values from 330 pm to 27 nm) correlated with potency in a cell-based iNOS assay (IC50 values from 290 pm to 270 nm). Inhibitor potency in cells was not prevented by medium supplementation with l-arginine or sepiapterin, but inhibition decreased with time of addition after cytokine stimulation. The results are consistent with a mechanism whereby inhibitors bind to a heme-containing iNOS monomer species to form an inactive iNOS monomer-heme-inhibitor complex in a pterin- and l-arginine-independent manner. The selectivity for inhibiting dimerization of iNOS versus endothelial and neuronal NOS suggests that the energetics and kinetics of monomer-dimer equilibria are substantially different for the mammalian NOS isoforms. These inhibitors provide new research tools to explore these processes.