Friday, May 29, 2009

I have several write-ups about some of these abstracts that I'm working on. I'm shutting it down for the rest of today and heading up to Hollywood for the weekend for some R & R.

Check back, when I'm laying by the pool at the London West Hollywood, there may be a post or two. Cheers.

BTW, NVLT is trading now at 74 cents, up a nickel. I see a trend here.....

Wednesday, May 27, 2009

In vivo pharmacokinetic and pharmacodynamic behavior of NOV-002, a redox-modulating glutathione disulfide mimetic

Abstract #4. Session Title: Biochemical Modulators

NOV-002 is a novel formulation of oxidized glutathione (GSSG) currently in a pivotal Phase 3 clinical trial in advanced non-small cell lung cancer. In clinical trials conducted to date, NOV-002 administered in combination with standard chemotherapeutic regimens has resulted in increased efficacy (survival, tumor response) and improved tolerance of standard chemotherapy (e.g. enhanced hematological recovery, immune stimulation). Recently, we showed that the proliferative effects of NOV-002 in the pre-myeloid HL-60 cell line is commensurate with stress-induced S-glutathionylation and activation of kinase pathways (AKT, JAK2 and STAT5) that are known to regulate hematopoiesis. The present investigation was undertaken to characterize the plasma pharmacokinetics of NOV-002 in C57 black mice and to determine if the drug caused S-glutathionylation of plasma proteins that might be used as surrogate biomarkers for drug efficacy. Using HPLC-MS, NOV-002 was measured in plasma at various times following a single intraperitoneal dose. Peak plasma concentrations were observed 10 min following administration of NOV-002 and were diminished by 60 min. Fluorescence based methods were used for quantification of protein thiol content and three S-glutathionylated plasma proteins were identified by mass spectrometry, serine proteinase inhibitor, contrapsin, and a-1-antitrypsin1-6 precursor. These preclinical pharmacokinetic and pharmacodynamic results provide evidence that, as in cell systems, NOV-002 administration in vivo results in generation of a transient oxidative signal that may trigger a variety of redox-regulated biochemical cascades. Such correlates could help to understand and predict clinical responses to treatment with NOV-002.

NOV-002, a redox-modulating glutathione disulfide mimetic, leads to calcium influx and nitric oxide generation through eNOS activation in myeloid c

Abstract 3: Session: NF-kB, Oxidative Stress, and Other Pathways in Cellular Drug Responses


NOV-002, a glutathione disulfide mimetic, is currently in a pivotal Phase 3 clinical trial in advanced non-small cell lung cancer. In clinical trials conducted to date, NOV-002 administered in combination with standard chemotherapeutic regimens has resulted in increased efficacy (survival, tumor response) and improved tolerance of standard chemotherapy (e.g. enhanced hematological recovery, immune stimulation). Recently, we showed that the myeloproliferative effect of NOV-002 in the pre-myeloid HL-60 cell line occurs in parallel with stress-induced S-glutathionylation and activation of kinase pathways (AKT, JAK2 and STAT5) that are known to regulate hematopoiesis. NOV-002 treatment produces oxidant signals intracellularly and at the cell surface. HL60 cells express the cell surface enzyme gamma-glutamyl transpeptidase (GGT). NOV-002 is a substrate for GGT and its cleavage alters the intracellular redox potential. Here we show that NOV-002 treatment in HL60 cells mediates changes of the plasma membrane electrical potential as detected by the fluorescent probe, bisOxonal. These changes were concurrent with time- and dose-dependent increases in the accumulation of intracellular Ca2+ as detected by Fura-2-AM and Fluo-3-AM fluorescent dyes. These effects were abolished by the addition of an extracellular Ca2+ chelator (EGTA) and decreased by the intracellular chelator, BAPTA-AM. These data suggest that NOV-002 induced calcium signaling involves entry of extracellular Ca2+ across the plasma membrane. Microarray analyses highlighted that NOV-002 treatment impacts expression of proteins involved in calcium signaling and nitric oxide (NO) metabolism pathways. NO generation following NOV-002 treatment was decreased by ~80% in HL60 cells transfected with siRNA to deplete eNOS levels. Overall, NOV-002 mediated alteration of cell surface redox status and transmembrane potential resulted in an induction of calcium influx, followed by NO generation through eNOS activation. These events may be pertinent to myeloproliferation that has been observed in cancer patients treated with NOV-002 plus standard chemotherapy.

The role of polymorphisms of glutathione S-transferase pi in redox regulation through protein S-glutathionylation

Abstract #2: Session: Resistance to Antitubulin Drugs, Resistance to Platinums, and Glutathione Metabolism

Glutathione S-transferase P1-1 (GSTP) is a prominent and ubiquitously expressed enzyme protein in many human cancers. Its expression is increased in response to a variety of apparently unrelated stress conditions and high levels (sometimes ~1-2% of cytosolic protein) are frequently linked to drug resistance phenotypes, even when the selecting drug is not a substrate for the enzyme. While recent results have shown that GSTP interacts non-covalently with kinases, it has always seemed probable that a more prevalent cellular function may be ascribed to such a common protein. Recently, we have demonstrated by in vitro and in vivo approaches that the catalytic properties of GSTP are critical to the post-translational S-glutathionylation of cysteine residues in a number of different target proteins (Townsend et al, JBC 2009). Genetic polymorphisms of GSTP have differing catalytic constants for some small molecule substrates. In the present studies, we evaluated the role of GSTP polymorphisms in regulating S-glutathionylation reactions. We generated stably transfected HEK293 cells expressing the four human GSTP*A-D alleles (Ile105/Ala114; Val105/Ala114; Val105/Val114; and Ile105/Val114) and a catalytically inactive mutant. In response to two drugs, NOV-002, a glutathione disulfide mimetic and PABA/NO, a nitric oxide releasing prodrug, cells mount a rapid S-glutathionylation of specific target proteins. The rate of protein S-glutathionylation is significantly enhanced by the presence of a catalytically active (intact tyr7 residue) GSTP and is also is greater for the GSTP*A and *D alleles. A role for GSTP in the catalytic formation of a disulfide between the thiolate of GSH and a low pK cysteine thiol in a target protein is a novel property for GSTP. The differential susceptibility to cancer initiation and drug response associated with some GSTP polymorphisms may be attributable to the their capacity to respond to oxidative and nitrosative stress through protein S-glutathionylation.


Inhibition of tumor cell invasion and ErbB2/PI3K signaling pathways by the glutathione disulfide-mimetic NOV-002

Abstract #1:

NOV-002, a glutathione disulfide-mimetic, is in advanced clinical development for oncology indications. It is believed to act by modulating intracellular and cell surface redox status resulting in a pleiotropic pharmacologic profile. Here we report the ability of NOV-002 to inhibit the invasiveness of human tumor cells in vitro by suppressing the activation of a well-characterized, redox sensitive signaling pathway that is critical for tumor cell growth and invasion. In the Matrigel invasion assay, NOV-002 inhibited the invasion of a variety of human tumor cell lines including A549 (non-small cell lung), MDA-MB436 (breast), SKOV3 (ovarian) and HCT-15, HCT-116 and Colo205 (colorectal) in a dose-dependent manner with IC50s between 17.7mM and 91mM. In contrast, cell migration was only inhibited in the colorectal tumor cell lines suggesting that NOV-002 may affect pathways which are specific for invasion. 1 mM NOV-002 (the highest concentration tested in the invasion and migration assays) was not toxic to any of the tumor cell lines studied even after 72 hours of culture using MTT viability assay. These effects were shown to involve the suppression of ErbB2 and phosphoinositide-3 kinase (PI3K) pathways by NOV-002. Immunoblot analysis demonstrated that NOV-002 reduced the expression of the phosphorylated (active) forms of signaling proteins ErbB2 and PI3k, known to regulate tumor cell invasion in A549 and Colo205 cells, without affecting the total amount of these proteins. Activation of Akt and RhoA, downstream molecules of the ErbB2/PI3K pathway, was also inhibited by NOV-002. Thus in both A549 and Colo205 cell lines, the active form of both upstream (ErbB2 and PI3K) and downstream (Akt and RhoA) signaling proteins were suppressed by NOV-002 at concentrations between 30 mM and 1 mM in a dose-dependent manner. Inhibition was more pronounced at 24 hours post-exposure compared to 8 or 16 hours. In prostate cancer PC3 cells which are not affected by NOV-002 in the invasion assay, suppression of ErbB2/PI3K signaling pathway was not observed. Thus, inhibition of tumor cell invasion by NOV-002 may be due to the inhibition of the ErbB2/PI3K signaling pathway activation that controls this process. We have previous shown that ERp5, a member of the redox-regulated protein disulfide isomerase family, promotes tumor cell invasion by activating the ErbB2/PI3K pathway. Thus ERp5 may represent a direct target for NOV-002. These results also indicate that NOV-002 may have particular therapeutic benefit in the treatment of cancers that utilize ErbB2 and PI3K pathway activation.

I'll get them all posted and then try to explain some science.

NVLT.OB at AACR 2009

I have the abstracts they presented, sans the figures. I will post them shortly as they download.

NVLT is trading at 64 cents today.

Monday, May 25, 2009

I have the data from AACR meeting, NVLT, will post tomorrow.