Pharmacological Characterization of ZYAN1, a Novel Prolyl Hydroxylase Inhibitor for the Treatment of Anemia.

Prolyl hydroxylase (PHD) inhibitors stabilize hypoxia inducible factor (HIF), and exert antianemic effect by potentiating erythropoietin (EPO) expression and down-regulation of hepcidin. ZYAN1 is a novel PHD inhibitor under clinical development for the treatment of anemia. The pharmacodynamic effects of acute and chronic dosing of ZYAN1 were assessed in normal and 5/6 nephrectomized Wistar rats. The effect of ZYAN1 was also investigated in cisplatin-induced anemia using C57 mice. Acute treatment with ZYAN1 increased circulating EPO levels (10.3 ± 3.7 and 40.0 ± 8.5 fold rise at 15 and 30 mg/kg, respectively), reticulocyte count (4.2 ± 0.5 and 6.0 ± 0.2 fold rise at 15 and 30 mg/kg, respectively) and stabilized HIF (28% increase at 45 mg/kg) in normal rats. Nephrectomized rats showed similar dose-related pharmacodynamic effects. In a 28-day study in nephrectomized rats, ZYAN1 administered every alternate day, caused increase in hemoglobin (1.9 ± 0.3 and 2.5 ± 0.4 g/dL) and RBC count (10.7 ± 4.0 and 14.0 ± 4.1%) at 15 and 30 mg/kg respectively. In cisplatin-treated mice also an increase in hemoglobin (3.4 ± 0.2 and 5.9 ± 0.2 g/dL) and RBC count (22.5 ± 2.2 and 37.3 ± 1.7%) at 15 and 30 mg/kg respectively was observed. ZYAN1's effects on hemoglobin and RBC count were distinct from darbepoietin. ZYAN1 demonstrated hematinic potential by combined effects on EPO release and efficient iron utilization. The efficacy of ZYAN1 in disease models of different etiologies suggests that it will be useful in treating wide spectrum of anemia patients.

The C2B domain of synaptotagmin is a Ca(2+)-sensing module essential for exocytosis.

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca(2+) sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca(2+) ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca(2+)-sensing module. Here, we report that Ca(2+) drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca(2)+ are mediated by a set of conserved acidic Ca(2)+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca(2)+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.

Delineation of the oligomerization, AP-2 binding, and synprint binding region of the C2B domain of synaptotagmin.

Biochemical and genetic studies indicate that synaptotagmin I functions as a Ca2+ sensor during synaptic vesicle exocytosis and as a membrane receptor for the clathrin adaptor complex, AP-2, during endocytosis. These functions involve the interaction of two conserved domains, C2A and C2B, with effector proteins. The C2B domain mediates Ca2+-triggered synaptotagmin oligomerization, binds AP-2 and is important for the interaction of synaptotagmin with Ca2+ channels. Here, we report that these are conserved biochemical properties: Ca2+ promoted the hetero-oligomerization of synaptotagmin I with synaptotagmins III and IV, and all three synaptotagmin isoforms bound the synprint region of the alpha1B subunit of N-type Ca2+ channels. Using chimeric and truncated C2 domains, we defined a common region of C2B that mediates oligomerization and AP-2 binding. Within this region, two adjacent lysine residues were identified that were critical for synaptotagmin oligomerization, AP-2, and synprint binding. Competition experiments demonstrated that the synprint fragment was an effective inhibitor of synaptotagmin oligomerization and also blocked binding of synaptotagmin to AP-2. In a model for the structure of C2B, the common effector binding site localized to a putative Ca2+-binding loop and a concave region formed by two beta-strands. These studies provide the first structural information regarding C2B target protein recognition and provide the means to selectively disrupt synaptotagmin-effector interactions for functional studies.

Phosphonates and phosphinates: novel leaving groups for benzisothiazolone inhibitors of human leukocyte elastase.

A novel class of alkyl and aryl phosphonate and phosphinate acid-based leaving groups has been developed for utilization in the synthesis of benzoisothiazolone (BIT) inhibitors of human leukocyte elastase (HLE). A number of BITs were synthesized with phosphonate and phosphinate acid-based leaving groups and were found to be potent inhibitors of HLE. Compound 3c with a diethyl phosphonate leaving group is the most potent inhibitor synthesized in this series with Ki* = 0.035 nM and ED50 = 2.0 mg/kg.

Orally bioavailable benzisothiazolone inhibitors of human leukocyte elastase.

Human leukocyte elastase (HLE) has been proposed as a primary mediator of pulmonary emphysema and other inflammatory airway diseases. HLE is capable of cleaving many proteins, including elastin, other components of connective tissue, certain complement proteins, and receptors. Under normal conditions an appropriate balance exists in the lung between HLE and endogenous inhibitors, which scavenge the released enzyme before it exerts deleterious effects in the lung. Emphysema is thought to result from an imbalance in the lung between HLE and endogenous inhibitor (elevated elastase or insufficient inhibitor) that leads to the destruction of alveoli. We have identified WIN 64733 (2) and WIN 63759 (3) as potent (Ki* = 14 and 13 pM, respectively), selective, mechanism-based inhibitors of HLE which are orally bioavailable in the dog (absolute bioavailability 46% and 21%, respectively). In this series the in vitro stabilities of the inhibitors in blood, jejunal homogenates, and liver S9 homogenates are useful predictors of oral bioavailability. After being administered orally (30 mg/kg) to dogs, compounds 2 and 3 are found in the lung, being detected in the epithelial lining fluid obtained by bronchoalveolar lavage (Cmax of 2.5 and 0.47 microgram/mL, respectively).

Diabetes insipidus following cardiorespiratory arrest.

Diabetes insipidus (DI) and other features of hypothalamic dysfunction were observed in two patients resuscitated following cardio-respiratory arrest (CRA). Both patients also developed marked hyperglycemia probably related to hypertonic dehydration. The development of DI in survivors of CRA suggests severe hypothalamic hypoxic injury and poor prognosis.