Nanosphere pharmacodynamics improves safety of immunostimulatory cytokine therapy.

Systemic administration of interleukin (IL)-12 induces potent anti-tumor immune responses in preclinical cancer models through the systemic activation of effector immune cells and release of proinflammatory cytokines. IL-12-loaded PLGA nanospheres (IL12ns) are hypothesized to improve therapeutic efficacy and thwart unwanted side effects observed in previous human clinical trials. Through the investigation of peripheral blood and local tissue immune responses in healthy BALB/c mice, the immune-protective pharmacodynamics of IL12ns were suggested. Nanospheres increased pro-inflammatory plasma cytokines/chemokines (IFN-γ, IL-6, TNF-α, and CXCL10) without inducing maladaptive transcriptomic signatures in circulating peripheral immune cells. Gene expression profiling revealed activation of pro-inflammatory signaling pathways in systemic tissues, the likely source of these effector cytokines. These data support that nanosphere pharmacodynamics, including shielding IL-12 from circulating immune cells, depositing peripherally in systemic immune tissues, and then slowly eluting bioactive cytokine, thereafter, are essential to safe immunostimulatory therapy.

Revisiting the Therapeutic Potential of Bothrops jararaca Venom: Screening for Novel Activities Using Connectivity Mapping.

Snake venoms are sources of molecules with proven and potential therapeutic applications. However, most activities assayed in venoms (or their components) are of hemorrhagic, hypotensive, edematogenic, neurotoxic or myotoxic natures. Thus, other relevant activities might remain unknown. Using functional genomics coupled to the connectivity map (C-map) approach, we undertook a wide range indirect search for biological activities within the venom of the South American pit viper Bothrops jararaca. For that effect, venom was incubated with human breast adenocarcinoma cell line (MCF7) followed by RNA extraction and gene expression analysis. A list of 90 differentially expressed genes was submitted to biosimilar drug discovery based on pattern recognition. Among the 100 highest-ranked positively correlated drugs, only the antihypertensive, antimicrobial (both antibiotic and antiparasitic), and antitumor classes had been previously reported for B. jararaca venom. The majority of drug classes identified were related to (1) antimicrobial activity; (2) treatment of neuropsychiatric illnesses (Parkinson's disease, schizophrenia, depression, and epilepsy); (3) treatment of cardiovascular diseases, and (4) anti-inflammatory action. The C-map results also indicated that B. jararaca venom may have components that target G-protein-coupled receptors (muscarinic, serotonergic, histaminergic, dopaminergic, GABA, and adrenergic) and ion channels. Although validation experiments are still necessary, the C-map correlation to drugs with activities previously linked to snake venoms supports the efficacy of this strategy as a broad-spectrum approach for biological activity screening, and rekindles the snake venom-based search for new therapeutic agents.

Connectivity maps for biosimilar drug discovery in venoms: the case of Gila monster venom and the anti-diabetes drug Byetta®.

Like most natural product libraries animal venoms have long been recognized as potentially rich source of biologically active molecules with the potential to be mined for the discovery of drugs, drug leads and/or biosimilars. In this work we demonstrate as a proof of concept a novel approach to explore venoms for potential biosimilarity to other drugs based on their ability to alter the transcriptomes of test cell lines followed by informatic searches and Connectivity Mapping to match the action of the venom on the cell gene expression to that of other drugs in the Connectivity Map (C-Map) database. As our test animal venom we chose Heloderma suspectum venom (Gila monster) since exendin-4, a glucagon-like peptide 1 receptor agonist, isolated from the venom is currently on the market to treat type 2 diabetes. The action of Byetta(®) (exentide, synthetic exendin-4), was also used in transcriptome studies. Analysis of transcriptomes from cells treated with the venom or the drug showed similarities as well as differences. The former case was primarily attributed to the fact that Gila monster venom likely contains a variety of biologically active molecules that could alter the MCF7 cell transcriptome compared to that of the single perturbant Byetta(®). Using Ingenuity Pathway Analysis software, insulin-like growth factor 1 signaling was identified in the category of "Top Canonical Pathways" for both the venom and Byetta(®). In the category of "Top Molecules" up-regulated, both venom and Byetta(®) shared IL-8, cyclic AMP-dependent transcription factor 3 (ATF-3), neuron-derived orphan receptor 1 (NR4A3), dexamethasone-induced Ras-related protein 1 (RASD1) and early growth response protein 1, (EGR-1) all with potential relevance in diabetes. Using Connectivity Mapping, Gila monster venom showed positive correlation with 1732 instances and negative correlation with 793 instances in the Connectivity database whereas Byetta(®) showed positive correlation with 1692 instances and negative correlation with 868 instances. Interestingly, the Gila monster venom and Byetta(®) both showed positive correlation with the anti-diabetic drugs troglitazone, of the thiazolidinedione class, and metformin, of the biguanide class, although Byetta(®) as a glucagon-like peptide-1 (GLP-1) agonist functions in a different manner than either of these two classes of anti-diabetic drugs. In summary, despite the fact that Gila monster venom contains a mixture of biologically active molecules, similarities in terms of perturbation of gene expression profiles on MCF7 cells were observed between the venom and the drug Byetta(®). Furthermore, using Connectivity Mapping the Gila monster venom was demonstrated to have nodes of positive correlation to several anti-diabetic drugs two of which were the same as observed with Byetta(®). Therefore, this study suggests that by using this approach novel drug activities heretofore unconsidered may be discovered in venoms using informatic tools and Connectivity Mapping.

Gene expression profiling reveals cross-talk between melanoma and fibroblasts: implications for host-tumor interactions in metastasis.

Host-tumor interaction is considered critical in carcinogenesis, tumor invasion, and metastasis. To explore the reciprocal effects of host-tumor interaction, we developed a system to assess the gene expression patterns of A2058 human melanoma cells cocultured in fibrillar collagen with HS-68 primary human fibroblasts. The gene expression pattern of the cocultured A2058 cells was only modestly affected, whereas the HS-68 fibroblast gene expression pattern was significantly altered. Interleukin-11 and inhibitor of DNA-binding domain-1 gene expression in the cocultured A2058 cells was down-regulated, indicative of a proinflammatory response and resistance to apoptosis, respectively. The overall pattern of up-regulated genes indicated triggering of the proinflammatory process. In addition, the melanoma growth and migration stimulatory chemokines CXCL1 and CXCL2 were significantly up-regulated in the cocultured fibroblasts. These results were corroborated by additional coculture experiments with the melanoma cell lines WM-164, BLM, and SK-Mel-28 and immunohistochemistry on invasive human melanoma sections. Taken together, these results indicate that tumor cells cause a proinflammatory and melanoma growth-promoting response in stromal fibroblasts. The role of inflammation in carcinogenesis, tumor promotion, invasion, and metastasis is viewed as being increasingly important and the results of these studies underscore this as well as identify certain key proteins that are expressed as a result of the complex interactive processes in the host-tumor microenvironment.

Vascular protection by estrogen in ischemia-reperfusion injury requires endothelial nitric oxide synthase.

Estrogen increases nitric oxide (NO) production by inducing the activity of endothelial NO synthase (eNOS) (Simoncini et al. Nature 407: 538, 2000). Ischemia (30 min) and reperfusion (I/R) increased the number of adherent leukocytes and decreased their rolling velocities in mouse cremaster muscle venules with a strong dependence on wall shear rate. Minimum rolling velocity at approximately 5 min after the onset of reperfusion was accompanied by increased P-selectin expression. This preceded the peak in leukocyte adhesion (at 10-15 min). In untreated wild-type mice, I/R caused a decrease of leukocyte rolling velocity from 37 to 26 microm/s and a 2.0-fold increase in leukocyte adhesion. Both were completely abolished by 0.25 mg ip estrogen 1 h before surgery. In eNOS(-/-) mice, the decrease of leukocyte rolling velocity and increase in adhesion were similar but were only marginally improved by estrogen. We conclude that the protective effect of estrogen, as measured by leukocyte rolling and adhesion, is significantly reduced in eNOS(-/-) mice, suggesting that induction of eNOS activity is the major mechanism of vasoprotection by estrogen in this model.

Acute cardiovascular protective effects of corticosteroids are mediated by non-transcriptional activation of endothelial nitric oxide synthase.

Corticosteroids have been shown to exert beneficial effects in the treatment of acute myocardial infarction, but the precise mechanisms underlying their protective effects are unknown. Here we show that high-dose corticosteroids exert cardiovascular protection through a novel mechanism involving the rapid, non-transcriptional activation of endothelial nitric oxide synthase (eNOS). Binding of corticosteroids to the glucocorticoid receptor (GR) stimulated phosphatidylinositol 3-kinase and protein kinase Akt, leading to eNOS activation and nitric oxide dependent vasorelaxation. Acute administration of pharmacological concentrations of corticosteroids in mice led to decreased vascular inflammation and reduced myocardial infarct size following ischemia and reperfusion injury. These beneficial effects of corticosteroids were abolished by GR antagonists or eNOS inhibitors in wild-type mice and were completely absent in eNOS-deficient (Nos3(-/-)) mice. The rapid activation of eNOS by the non-nuclear actions of GR, therefore, represents an important cardiovascular protective effect of acute high-dose corticosteroid therapy.