Glycoengineered factor IX variants with improved pharmacokinetics and subcutaneous efficacy.

BACKGROUND: The 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. OBJECTIVES: To 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. METHODS: Two 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. RESULTS: Variants 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. CONCLUSIONS: Glycoengineering was used to significantly improve the subcutaneous PK and efficacy of FIX and may have advantages for subcutaneous dosing.

Effective treatment of vascular endothelial growth factor refractory hindlimb ischemia by a mutant endothelial nitric oxide synthase gene.

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.

Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry.

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.

Crystal structures of zinc-free and -bound heme domain of human inducible nitric-oxide synthase. Implications for dimer stability and comparison with endothelial nitric-oxide synthase.

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 (ZnS(4)) center indistinguishable from that observed in the endothelial isoform (NOS-3). As in NOS-3, ZnS(4) 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 interaction of thrombomodulin with Ca2+.

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.

Management of anomalous rotation in adults.

Rotational anomalies of the gut are infrequently encountered in the adult population. Management of this population is debatable because of a few anecdotal reports and small patient series. We present the successful surgical correction of a patient with symptomatic nonrotation and review our experience with six asymptomatic patients with anomalous rotation discovered incidentally at laparotomy for another disease process. The incidence of anomalous rotation is reported as high as 0.5% in autopsy series. Therefore, a large majority of subjects are clinically silent throughout life. Although a surgical emergency in the pediatric population, serendipituous discovery in the asymptomatic adult does not require surgical intervention. It is important, however, that the patient have a thorough understanding of his abnormality so that should symptoms arise, urgent surgical intervention may be warranted. We recommend appendectomy in those patients undergoing laparotomy for other conditions. In patients with chronic abdominal symptoms, surgical intervention is warranted.

The short loop between epidermal growth factor-like domains 4 and 5 is critical for human thrombomodulin function.

Thrombomodulin (TM) is a cofactor for activation of protein C by thrombin. We showed that 80-90% of this cofactor activity is lost by oxidation of Met388, located within the short interdomain loop between epidermal growth factor-like domains 4 and 5 (Glaser, C. B., Morser, J., Clarke, J. H., Blasko, E., McLean, K., Kuhn, I., Chang, R.-J., Lin, J.-H., Vilander, L., Andrews, W. H., and Light, D. R. (1992) J. Clin. Invest. 90, 2565-2573). For each of the 3 amino acids of the loop, site-specific mutants are described in which, 1) all possible single amino acid substitutions are made, 2) deletions are made, or 3) alanine is inserted adjacent to each residue of the loop. Most substitutions within the loop (38/57) result in a > 50% decrease in cofactor activity, while changes in the length of this region result in > 90% loss of activity. Only the Met388-->Leu mutant has higher cofactor activity (2-fold) than wild-type TM. A number of soluble and full-length TM analogs with the Met388-->Leu substitution are improved thrombin cofactors, whether produced in bacteria, insect, or mammalian cells. Detailed kinetic analysis of a soluble TM analog consisting of the six EGF-like domains secreted from insect cells shows that the enhanced activity of the Met388-->Leu mutant results from an increased catalytic efficiency (kcat/Km). This enhancement is maximal at physiological concentrations of calcium. The loss of activity following Met388 oxidation in the wild-type protein is the result of both decreased binding to thrombin (Kd effect) and a decreased interaction of the TM.thrombin complex with protein C (Km effect). We demonstrate the critical role of this interdomain loop in the biological anticoagulant properties of TM.

Oxidation of a specific methionine in thrombomodulin by activated neutrophil products blocks cofactor activity. A potential rapid mechanism for modulation of coagulation.

Endothelial thrombomodulin (TM) plays a critical role in hemostasis as a cofactor for thrombin-dependent formation of activated protein C, a potent anticoagulant. Chloramine T, H2O2, or hypochlorous acid generated from H2O2 by myeloperoxidase rapidly destroy 75-90% of TM cofactor activity. Activated PMN, the primary in vivo source of biological oxidants, also rapidly inactivate TM. Oxidation of TM by PMN is inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase. Both Met291 and Met388 in the six epidermal growth factor-like repeat domain are oxidized; however, only substitutions of Met388 lead to TM analogues that resist oxidative inactivation. We suggest that in inflamed tissues activated PMN may inactivate TM and demonstrate further evidence of the interaction between the inflammatory process and induction of thrombotic potential.

Acute, complete, extrinsic obstruction of the Björk-Shiley valve in the immediate postoperative period.

The following report describes a case in which acute, complete, extrinsic obstruction of a Björk-Shiley prosthetic aortic valve by suture material occurred in the immediate postoperative period.