Prophylactic factor VIII (FVIII) replacement therapy in hemophilia A requires intravenous administration up to every other day due to the short half-life of FVIII in plasma. Plasma half-life extension of FVIII by polyethylene glycol (PEG) conjugation is thought to be mediated by decreasing hepatic clearance of FVIII. BAY 94-9027 is a rationally designed B-domain–deleted (BDD) FVIII molecule, in which a single 60-kDa PEG molecule was attached to a specific amino acid (1804) to increase its circulating half-life and reduce the exposure to epitopes reported to cause immunogenicity in the A3 domain while preserving full biological function. BAY 94-9027 is currently in clinical trials and has prolonged half-life and improved efficacy in animal models and humans.

Abstract Background Reduced frequency of administration as well as subcutaneous (s.c.) injection would improve the treatment of Hemophilia B. Conjugation to polyethylene glycol (PEG) has been shown to increase the half-life of i.v. dosed Factor IX (FIX), but s.c. dosing of PEGylated FIX was not previously evaluated. Because s.c. dosing is limited by volume and bioavailability, we evaluated the combination of PEGylation with the increased specific activity variant R338A to reduce the amount of protein needed to provide therapeutic levels of FIX. Methods FIX-R338A was PEGylated on N-linked glycans in the activation peptide by periodate oxidation of sialic acid residues followed by conjugation to amino-oxy functionalized PEG. Pharmacokinetic (PK) profiles were determined in hemophilia B mice and cynomologous monkeys. Allometric scaling was used to predict dose regimens in humans. Prophylactic efficacy was determined in a Hemophilia B mouse tail bleeding model. Results 60kDaPEG-R338A had prolonged terminal half-life in mice (3-fold) and monkeys (5-fold) and the s.c. bioavailability was 44% and 35%, respectively. The volume of distribution was reduced 5-fold. To achieve a trough level of 3% FIX activity, s.c. dosing at weekly, bi-monthly and monthly intervals was predicted to require doses of 7, 25 and 220 IU/kg in patients. However, in a tail vein transection injury model, approximately 10-fold higher plasma FIX activity levels of PEGylated proteins were found to be needed to protect hemophilia B mice against bleeding than was required for i.v. dosed un-PEGylated recombinant FIX. This difference was observed for PEGylated wild-type and R338A proteins, dosed i.v or s.c. We hypothesize that this is related to the reduced distribution of PEGylated FIX to the extravascular compartment. Trough levels of 30% FIX activity were predicted to be achievable in humans after weekly and bi-monthly s.c. dosing at 70 and 260 IU/kg. Conclusions The PEGylation of FIX led to a significant improvement in both i.v. and s.c. PK. Unexpectedly, a 10-fold higher plasma activity was needed for PEGylated FIX to provide protection against bleeding in Hemophilia B mice, suggesting that trough levels of 10 to 30% of PEGylated FIX activity may be needed in patients to provide efficacy equivalent to current therapy of recombinant or plasma derived FIX. Nevertheless, 60kDaPEG-R338A has the potential to treat hemophilia B patients with once weekly or twice monthly subcutaneous injection. Disclosures: Sim: Bayer HealthCare: Employment. Brooks:Bayer HealthCare: Employment. Mallari:Bayer HealthCare: Employment. Xu:Bayer HealthCare: Employment. Feldman:Bayer HealthCare: Employment. Schneider:Bayer HealthCare: Employment. Patel:Bayer HealthCare: Employment. Blasko:Bayer HealthCare: Employment. Ho:Bayer HealthCare: Employment. Su:Bayer HealthCare: Employment. Liu:Bayer HealthCare: Employment. Laux:Bayer HealthCare: Employment. Murphy:Bayer HealthCare: Employment.

SummaryBackgroundThe rapid clearance of factor IX (FIX) necessitates frequent intravenous administration to achieve effective prophylaxis for patients with hemophilia B. Subcutaneous administration would be a preferred route of administration but is limited by bioavailability.ObjectivesTo improve the pharmacokinetics (PK) and bioavailability of FIX, a screen was performed to identify positions for the introduction of novel glycosylation sites with maximal effect on PK and maintenance of coagulation activity.MethodsTwo hundred fifty-one variants, each containing one additional N-linked glycosylation site, were screened in vitro, and the PK profiles of selected variants mapping to spatially distinct regions of FIX were evaluated in mice. Optimal variants were combined, and their PK and efficacy were determined in mice with hemophilia B.ResultsVariants that mapped to spatially distinct regions of the FIX structure exhibited different degrees of improved PK and enabled selection of optimized sites while minimizing the loss of FIX activity. Combining the most effective N-glycan sites in the same FIX molecule resulted in further improvements in PK. An optimized variant containing three novel N-glycan sites (at amino acids 103, 151, and 228), and the activity enhancing 338A variant had double the specific activity of wild-type FIX, exhibited 4.5-fold reduced clearance and 2.4-fold increased subcutaneous bioavailability, and was efficacious at a fivefold lower mass dose than wild-type FIX after subcutaneous injection in a bleeding model in mice with hemophilia B.ConclusionsGlycoengineering was used to significantly improve the subcutaneous PK and efficacy of FIX and may have advantages for subcutaneous dosing.

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.

L'invention porte de maniere generale sur des procedes de detection de facteurs de coagulation sanguine pegyles dans un echantillon. Ces procedes utilisent des agents de capture anti-polyethylenes glycols (PEG) et des agents de sonde anti-facteurs de coagulation sanguine pour la detection. L'invention porte egalement sur des procedes de determination de l'activite de facteurs de coagulation sanguine pegyles dans un echantillon.

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.

Gene delivery of angiogenic growth factors is a promising approach for the treatment of ischemic cardiovascular diseases. However, success of this new therapeutic principle is hindered by the lack of critical understanding as to how disease pathology affects the efficiency of gene delivery and/or the downstream signaling pathways of angiogenesis. Critical limb ischemia occurs in patients with advanced atherosclerosis often exhibiting deficiency in endothelial nitric oxide production. Similar to these patients, segmental femoral artery resection progresses into severe ischemic necrosis in mice deficient in endothelial nitric oxide synthase (ecNOS-KO) as well as in balb/c mice. We used these models to evaluate the influence of severe ischemia on transfection efficiency and duration of transgene expression in the skeletal muscle following plasmid injection in combination with electroporation. Subsequently, we also explored the potential therapeutic effect of the phosphomimetic mutant of ecNOS gene (NOS1177D) using optimized delivery parameters, and found significant benefit both in ecNOS-KO and balb/c mice. Our results indicate that NOS1177D gene delivery to the ischemic skeletal muscle can be efficient to reverse critical limb ischemia in pathological settings, which are refractory to treatments with a single growth factor, such as vascular endothelial growth factor.

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-1α, is hydroxylated on specific proline residues to target HIF-1α 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-1α specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

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 K d ∼1.8 nm and had a slow off rate, 1.2 × 10−4s−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 K d 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- andl-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.

Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC ) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC(50) values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (K(d) approximately 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor-heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein-protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED(50) values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies.

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Thrombomodulin (TM) is a cofactor for protein C activation by thrombin and each residue of a consensus Ca2+ site in the sixth epidermal growth factor domain (EGF6) is essential for this cofactor activity [Nagashima, M., Lundh, E., Leonard, J.C., Morser, J. & Parkinson, J.F. (1993) J. Biol. Chem. 268, 2888-2892]. Three soluble analogs of the extracellular domain of TM, solulin (Glu4-Pro490), TME1-6 (Cys227-Cys462) and TMEi4-6 (Val345-Cys462) were prepared for equilibrium dialysis experiments by exhaustive dialysis against Ca2+-depleted buffer. However, all three analogs still contained one tightly bound Ca2+ (Kd approximately 2 microm), which could only be removed by EDTA. Epitope mapping with Ca2+-dependent monoclonal antibodies to EGF6 provided further localization of this tight Ca2+ site. Equilibrium dialysis of the soluble TM analogs in [45Ca2+] between 10 and 200 microm revealed a second Ca2+ site (Kd = 30 +/- 10 microm) in both solulin and TME1-6, but not in TMEi4-6. Ca2+ binding to this second site was unaffected by bound thrombin and we attribute it to the consensus Ca2+ site in EGF3. A 75-fold decrease in the binding affinity of thrombin to TM was observed with immobilized solulin treated with EDTA to remove the high affinity Ca2+ by measuring kassoc and kdiss rates in a BIAcoretrade mark instrument. Ca2+-dependent conformational transitions detected by CD spectroscopy in the far UV indicate a more ordered structure upon Ca2+ binding. Bound Ca2+ stabilized soluble TM against protease digestion at a trypsin-like protease-sensitive site between Arg456 and His457 in EGF6 compared with protease treatment in EDTA. Finally, TM containing EGF domains 4-6, but lacking the interdomain loop between EGF3 and 4 (TME4-6), has an identical Ca2+ dependence for the activation of protein C as found for TMEi4-6, indicating this interdomain loop is not involved in Ca2+ binding.

The crystal structures of the heme domain of human inducible nitric-oxide synthase (NOS-2) in zinc-free and -bound states have been solved. In the zinc-free structure, two symmetry-related cysteine residues form a disulfide bond. In the zinc-bound state, these same two cysteine residues form part of a zinc-tetrathiolate (ZnS4) center indistinguishable from that observed in the endothelial isoform (NOS-3). As in NOS-3, ZnS4 plays a key role in stabilizing intersubunit contacts and in maintaining the integrity of the cofactor (tetrahydrobiopterin) binding site of NOS-2. A comparison of NOS-2 and NOS-3 structures illustrates the conservation of quaternary structure, tertiary topology, and substrate and cofactor binding sites, in addition to providing insights on isoform-specific inhibitor design. The structural comparison also reveals that pterin binding does not preferentially stabilize the dimer interface of NOS-2 over NOS-3.

The crystal structures of the heme domain of human inducible nitric-oxide synthase (NOS-2) in zinc-free and -bound states have been solved. In the zinc-free structure, two symmetry-related cysteine residues form a disulfide bond. In the zinc-bound state, these same two cysteine residues form part of a zinc-tetrathiolate (ZnS4) center indistinguishable from that observed in the endothelial isoform (NOS-3). As in NOS-3, ZnS4 plays a key role in stabilizing intersubunit contacts and in maintaining the integrity of the cofactor (tetrahydrobiopterin) binding site of NOS-2, A comparison of NOS-2 and NOS-3 structures illustrates the conservation of quaternary structure, tertiary topology, and substrate and cofactor binding sites, in addition to providing insights on isoform-specific inhibitor design. The structural comparison also reveals that pterin binding does not preferentially stabilize the dimer interface of NOS-2 over NOS-3.