Pharmacokinetics and Pharmacodynamics of Canakinumab in Patients With Systemic Juvenile Idiopathic Arthritis.

The characterization of the pharmacokinetic (PK) and pharmacodynamic (PD) properties in pediatric patients is essential in supporting the recommended dosage of canakinumab in the relevant population. Here the PK and PD properties of canakinumab-a monoclonal antibody-in pediatric patients with systemic juvenile idiopathic arthritis (SJIA) are presented. Blood samples were obtained from 4 phase 2/3 clinical studies in patients with SJIA. Canakinumab PK properties and total interleukin (IL)-1ß kinetic properties were characterized by a population-based PK-binding model. On administration, canakinumab increased total IL-1ß complex in SJIA patients. Canakinumab clearance and volume of distribution were not impacted by age in pediatric patients after correction for the patient's body weight. The estimated serum clearance of canakinumab was 0.106 ± 0.00689 L/day, with a corresponding volume of distribution at steady state of 3.2 L and an estimated half-life of 22 days, based on a model typical body weight of 33 kg. Body-weight-based dosing provided comparable canakinumab exposure across the age groups in patients 2 to <20 years with SJIA. In younger children, a modest increase in the turnover rate of IL-1ß was observed. Compared to other indications, IL-1ß production rate was higher and clearance was slower in patients with SJIA. Low immunogenicity incidence of 3.1% was observed, and none of the patients had neutralizing antibodies. In conclusion, the PK/PD findings further support dose selection of canakinumab in patients with SJIA.

Pharmacokinetic and pharmacodynamic properties of canakinumab in patients with gouty arthritis.

Pharmacokinetics and pharmacodynamics of the anti-interleukin (IL)-1ß monoclonal antibody, canakinumab, in gouty arthritis patients from three studies are reported. Canakinumab has low serum clearance (0.214 L/day), low steady-state volume of distribution (7.44 L), a 25.8-day half-life, and approximately 60% subcutaneous absolute bioavailability in a typical 93-kg patient. Creatinine clearance had a small positive impact on serum canakinumab clearance that is not likely to be clinically relevant. Binding to circulating IL-1ß was demonstrated by increases in total serum IL-1ß following canakinumab dosing. Total IL-1ß kinetics and canakinumab pharmacokinetics were characterized by a population-based pharmacokinetic-binding model, where the estimated apparent in vivo dissociation constant (signifying binding affinity of canakinumab to circulating IL-1ß) was 0.99 nmol/L in gouty arthritis patients. Canakinumab treatment provided rapid, sustained decreases in C-reactive protein and serum amyloid A, provided superior pain relief to triamcinolone acetonide, and increased time to first recurrent attack (P ≤ 0.01 favoring all canakinumab doses vs. triamcinolone acetonide).

SUMO-dependent regulation of centrin-2.

Centrins are multifunctional Ca(2+)-binding proteins that are highly conserved from yeast to humans. Centrin-2 is a core component of the centrosome of higher eukaryotes. In addition, it is present within the nucleus, in which it is part of the xeroderma pigmentosum group C (XPC) complex, which controls nucleotide excision repair (NER). Regulation of the subcellular distribution of centrin-2 has so far remained elusive. Here we show that centrin-2 is a substrate of SUMOylation in vitro and in vivo, and that it is preferentially modified by SUMO2/3. Moreover, we identify the SUMO E3-like ligase human polycomb protein 2 (PC2; also known as hPC2) as essential for centrin-2 modification. Interference with the SUMOylation pathway leads to a striking defect in nuclear localization of centrin-2 and accumulation in the cytoplasm, whereas centrosomal recruitment of centrin-2 is unaffected. Depletion of the XPC protein mimics this situation and we provide evidence that SUMO conjugation of centrin-2 enhances its binding to the XPC protein. These data show that the nucleocytoplasmic shuttling of centrin-2 depends on the SUMO system and indicates that localization of centrin-2 within the nucleus depends on its ability to bind to the XPC protein.

RanBP2 and SENP3 function in a mitotic SUMO2/3 conjugation-deconjugation cycle on Borealin.

The ubiquitin-like SUMO system controls cellular key functions, and several lines of evidence point to a critical role of SUMO for mitotic progression. However, in mammalian cells mitotic substrates of sumoylation and the regulatory components involved are not well defined. Here, we identify Borealin, a component of the chromosomal passenger complex (CPC), as a mitotic target of SUMO. The CPC, which additionally comprises INCENP, Survivin, and Aurora B, regulates key mitotic events, including chromosome congression, the spindle assembly checkpoint, and cytokinesis. We show that Borealin is preferentially modified by SUMO2/3 and demonstrate that the modification is dynamically regulated during mitotic progression, peaking in early mitosis. Intriguingly, the SUMO ligase RanBP2 interacts with the CPC, stimulates SUMO modification of Borealin in vitro, and is required for its modification in vivo. Moreover, the SUMO isopeptidase SENP3 is a specific interaction partner of Borealin and catalyzes the removal of SUMO2/3 from Borealin. These data thus delineate a mitotic SUMO2/3 conjugation-deconjugation cycle of Borealin and further assign a regulatory function of RanBP2 and SENP3 in the mitotic SUMO pathway.

Structure of a Survivin-Borealin-INCENP core complex reveals how chromosomal passengers travel together.

The chromosomal passenger complex (CPC) is a key regulator of chromosome segregation and cytokinesis. CPC functions are connected to its localization. The complex first localizes to centromeres and later associates with the central spindle and midbody. Survivin, Borealin, and INCENP are the three components of the CPC that regulate the activity and localization of its enzymatic component, the kinase Aurora B. We determined the 1.4 A resolution crystal structure of the regulatory core of the CPC, revealing that Borealin and INCENP associate with the helical domain of Survivin to form a tight three-helical bundle. We used siRNA rescue experiments with structure-based mutants to explore the requirements for CPC localization. We show that the intertwined structural interactions of the core components lead to functional interdependence. Association of the core "passenger" proteins creates a single structural unit, whose composite molecular surface presents conserved residues essential for central spindle and midbody localization.

Raf kinase inhibitory protein regulates aurora B kinase and the spindle checkpoint.

Raf kinase inhibitory protein (RKIP or PEBP) is an inhibitor of the Raf/MEK/MAP kinase signaling cascade and a suppressor of cancer metastasis. We now show that RKIP associates with centrosomes and kinetochores and regulates the spindle checkpoint in mammalian cells. RKIP depletion causes decreases in the mitotic index, the number of metaphase cells, and traversal times from nuclear envelope breakdown to anaphase, and an override of mitotic checkpoints induced by spindle poisons. Raf-1 depletion or MEK inhibition reverses the reduction in the mitotic index, whereas hyperactivation of Raf mimics the RKIP-depletion phenotype. Finally, RKIP depletion or Raf hyperactivation reduces kinetochore localization and kinase activity of Aurora B, a regulator of the spindle checkpoint. These results indicate that RKIP regulates Aurora B kinase and the spindle checkpoint via the Raf-1/MEK/ERK cascade and demonstrate that small changes in the MAP kinase (MAPK) pathway can profoundly impact the fidelity of the cell cycle.

Centromere targeting of the chromosomal passenger complex requires a ternary subcomplex of Borealin, Survivin, and the N-terminal domain of INCENP.

The chromosomal passenger complex (CPC), consisting of the serine/threonine kinase Aurora B, the inner centromere protein INCENP, Survivin, and Borealin/DasraB, has essential functions at the centromere in ensuring correct chromosome alignment and segregation. Despite observations that small interfering RNA-mediated knockdown of any one member of the CPC abolishes localization of the other subunits, it remains unclear how the complex is targeted to the centromere. We have now identified a ternary subcomplex of the CPC comprising Survivin, Borealin, and the N-terminal 58 amino acids of INCENP in vitro and in vivo. This subcomplex was found to be essential and sufficient for targeting to the centromere. Notably, Aurora B kinase, the enzymatic core of the CPC, was not required for centromere localization of the subcomplex. We demonstrate that CPC targeting to the centromere does not depend on CENP-A and hMis12, two core components for kinetochore/centromere assembly, and provide evidence that the CPC may be directed to centromeric DNA directly via the Borealin subunit. Our findings thus establish a functional module within the CPC that assembles on the N terminus of INCENP and controls centromere recruitment.

Cooperation between mitotic kinesins controls the late stages of cytokinesis.

Cell division is regulated by protein kinases of the Cdk, Polo, and Aurora families. Although it has long been established that temporal control is central to the coordinated action of these kinases, the importance of spatial regulation has only recently been appreciated and is still poorly understood. The kinesin-6 family motor protein MKlp1 is a key regulator of cytokinesis and an ideal substrate for studying spatially regulated protein-phosphorylation events. MKlp1 is negatively regulated by Cdk1 phosphorylation during metaphase and becomes activated in anaphase when cleavage-furrow assembly commences. Aurora B phosphorylates MKlp1 during anaphase and is required for its function in cytokinesis. Another kinesin-6 family motor, MKlp2, mediates the relocation of Aurora B from the centromeres to the central spindle at the onset of anaphase. We now demonstrate that this process is required for the phosphorylation of MKlp1 at S911, an Aurora B consensus site overlapping a bipartite nuclear localization sequence (NLS). MKlp1(S911A) targets to the central spindle but is prematurely imported into the nucleus and fails to support cytokinesis. Spatial restriction of Aurora B to the central spindle by MKlp2 therefore regulates MKlp1 during cytokinesis in human cells.