Postnatal development and adult function of the central nervous system are dependent on the capacity of neurons to effect long-term changes of specific properties in response to neural activity. This neuronal response has been demonstrated to be tightly correlated with the expression of a set of regulatory genes which include transcription factors as well as molecules that can directly modify cellular signaling. It is hypothesized that these proteins play a role in activitydependent responses. Previously, we described the expression and regulation in brain of an inducible form of prostaglandin synthase/cyclooxygenase, termed COX-2. COX-2 is a ratelimiting enzyme in prostanoid synthesis and its expression is rapidly regulated in developing and adult forebrain by physiological synaptic activity. Here we demonstrate that COX-2 immunoreactivity is selectively expressed in a subpopulation of excitatory neurons in neo-and allocortices, hippocampus, and amygdala and is compartmentalized to dendritic arborizations. Moreover, COX-2 immunoreactivity is present in dendritic spines, which are specialized structures involved in synaptic signaling. The developmental profile of COX-2 expression in dendrites follows well known histogenetic gradients and coincides with the critical period for activitydependent synaptic remodeling. These results suggest that COX-2, and its diffusible prostanoid products, may play a role in postsynaptic signaling of excitatory neurons in cortex and associated structures.Neural activity results in specific structural and functional modifications of the cerebral cortex. This activity-dependent process is essential for achieving the appropriate synaptic relationships during development and for normal function of the mature cortex (1). Recent studies are beginning to identify molecular mechanisms underlying activity-dependent changes (2). There is abundant correlative evidence linking neural activity and transcription factor (TF) expression (3). Members of the Fos, Jun, and zinc finger TF families are naturally expressed at high levels in specific populations of cortical neurons and this expression is tightly regulated by synaptic activity (4, 5). Furthermore, these TFs are also rapidly and transiently induced in different paradigms of synaptic plasticity consistent with the notion that they regulate the expression of specific effector genes that underlie long-term plasticity (3).In addition to TFs, the initial genomic response to neural activity includes proteins that can directly modify cellular function. Among them is an inducible form of the enzyme prostaglandin synthase/cyclooxygenase, termed COX-2 (6-9). Cyclooxygenase is the first enzyme in the prostaglandin/ prostacyclin/thromboxane pathway and converts arachidonic acid to prostaglandin G2/prostaglandin H2. There are presently two known forms of cyclooxygenase: a constitutively expressed form termed COX-1 (10) and the inducible formThe publication costs of this article were defrayed in part by page charge payment. This article must the...

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The preparation of antibody drug conjugates (ADCs), particularly those containing hydrophobic payloads, may promote aggregate formation. The aggregate (high molecular mass species, HMMS) is an impurity that must be controlled in the final drug substance and drug product formulation to meet product safety and efficacy. Although there are numerous methods designed to mitigate protein aggregation, some of the reasons for the formation of aggregates in ADCs are unique and specific to the synthetic preparation and structure of each conjugate. Proper structural characterization and identification of causes of aggregation are key to improving the process for preparation of ADCs for minimizing aggregate formation. In this article, we have characterized aggregates generated during the preparation of a lysine conjugate, gemtuzumab ozogamicin, prepared from an IgG4 antibody (hP67.6) and a calicheamicin-based linker-payload. Using analytical and biophysical techniques, structural details of the aggregates formed in the conjugation process are elucidated. The aggregates are predominantly composed of a mixture of dimers and multimers with high drug loaded species. The level of conjugation to the N-termini of the mAb was higher in aggregates than in the monomeric ADC. Studies performed on the biological activity revealed differences in binding affinity and cytotoxicity between aggregates and monomeric ADC species.

These fully human antibodies therefore have the potential to provide an effective anti-tumor biological therapy in the human clinical setting.

Aldose reductase (AKR1B1) mediates the first and rate limiting step in the conversion of glucose to polyols that contribute to redox imbalance, formation of reactive oxygen species, and activation of signal transduction pathways that can promote tumor formation and tumor growth. Aldose reductases are overexpressed in a variety of human tumors, and genetic or pharmacological blockade of AKR1B1 has been reported to attenuate these processes. CP-744809 is a highly potent and selective aldose reductase enzyme inhibitor which has been previously shown to provide efficacy in an experimental model of diabetic retinopathy. The compound has properties of high oral bioavailability, very high protein binding, and a very long half life in vivo. In this study, we have characterized the activity of this agent in animal models of angiogenesis and tumor growth. CP-744809 treatment significantly reduced VEGF-induced angiogenesis in a dose-dependent manner in the rat corneal micropocket assay, suggesting a heretofore unrecognized role for aldose reductase in this process. Interestingly, when the related aldose reductase inhibitor Zopolrestat was administered to normal rats, a significantly decreased level of the angiogenic lipid mediator sphingosine-1-phosphate was observed along with other lipid changes. In xenograft models, the CP-744809 treatment was associated with significant and reproducible reduction in tumor size that was dose dependent and superior to treatment with Zopolrestat. Final tumor mass in individual animals was highly correlated with the corresponding intratumoral drug concentration. In contrast, drug concentrations in serum were poorly correlated with drug concentrations in tumor or with tumor mass. Serum drug concentrations at every dose were consistently 7-10 fold higher than those observed in tumor, suggesting a barrier to optimal penetration of tumor tissue by the compound. Pooling data from multiple in vivo studies, the total intra-tumoral drug concentration (protein bound and unbound) required to achieve half maximal efficacy (EC50) was estimated to be 77 micrograms/ml. These results confirm the therapeutic potential of aldose reductase as an interesting cancer target, and have uncovered a novel antiangiogenic activity. They also highlight the obstacle that adequate tumor tissue penetration may pose for clinical development of agents such as CP-744809. Combination treatments with other agents are being evaluated to assess the potential for synergistic activity with the vascular and inflammation modulating properties of aldose reductase inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4540.