nal-IRI+5-FU/LV versus 5-FU/LV in post-gemcitabine metastatic pancreatic cancer: Randomized phase 2 trial in Japanese patients.

BACKGROUND: In the NAPOLI-1 phase 3 trial, liposomal irinotecan (nal-IRI) +5-fluorouracil/leucovorin (5-FU/LV) significantly increased mPFS versus 5-FU/LV (3.1 vs. 1.5 months [unstratified HR = 0.56, p = 0.0001]) in patients with mPAC that progressed on prior gemcitabine-based therapy. This randomized phase 2 trial evaluated nal-IRI+5-FU/LV tolerability (Part 1), safety, and efficacy (Part 2; outcomes reported here) in Japanese patients with mPAC that progressed on gemcitabine-based therapy. METHODS: Patients were randomized 1:1 and stratified by KPS (70 and 80 vs. ≥90) and baseline albumin (≥4.0 g/dl vs. <4.0 g/dl). Primary endpoint was PFS; secondary endpoints were ORR, DCR, OS, TTF, CA19-9 response, and QoL. The ITT population comprised all randomized patients. RESULTS: Patient characteristics differed between nal-IRI+5-FU/LV (n = 40) and 5-FU/LV (n = 39) arms, including baseline hepatic lesions (63% vs. 51%), stage IV disease at diagnosis (78% vs. 51%), and post-study anticancer therapy (55% vs. 72%). Investigator-assessed mPFS increase with nal-IRI+5-FU/LV was clinically meaningful and statistically significant versus 5-FU/LV (2.7 vs. 1.5 months, HR = 0.60). Independently assessed mPFS showed similar trends (1.7 vs. 1.6 months, HR = 0.79). mOS was 6.3 months with nal-IRI+5-FU/LV and not reached with 5-FU/LV. ORR increased significantly with nal-IRI+5-FU/LV versus 5-FU/LV (18% vs. 0, rate difference 17.5). Commonly reported grade ≥3 treatment-emergent AEs were decreased neutrophil count (37% vs. 3%), decreased white blood cell count (20% vs. 0), and diarrhea (17% vs. 3%). CONCLUSIONS: In conclusion, clinically meaningful and statistically significant gains in investigator-assessed PFS and ORR were observed with nal-IRI+5-FU/LV versus 5-FU/LV in Japanese patients, with no new or unexpected safety signals. (Clinicaltrials.gov ID: NCT02697058).

Constitutive NF-kappaB activation, normal Fas-induced apoptosis, and increased incidence of lymphoma in human herpes virus 8 K13 transgenic mice.

Human herpesvirus 8 (HHV-8, also called Kaposi's sarcoma-associated herpes virus) has been linked to Kaposi's sarcoma and primary effusion lymphoma. HHV-8-encoded viral Fas-associated death domain-like IL-1-converting enzyme inhibitory protein (vFLIP) is one of the few viral proteins to be expressed in latently infected cells and plays a key role in the survival and proliferation of primary effusion lymphoma cells. Two main functions have been ascribed to HHV-8 vFLIP, inhibition of caspase 8/Fas-associated death domain-like IL-1-converting enzyme and activation of NF-kappaB. In this article, we demonstrate that vFLIP-expressing transgenic mice lack any of the features seen in mice deficient in caspase 8 or Fas-associated death domain protein and are not resistant to Fas-induced apoptosis. On the other hand, these mice display constitutive activation of classical and alternative NF-kappaB pathways, enhanced response to mitogenic stimuli, and increased incidence of lymphoma. Collectively, our results demonstrate that HHV-8 vFLIP is an oncogenic protein that mimics the signaling activities of caspase 8 during antigen receptor signaling and could contribute to the development of lymphoproliferative disorders via constitutive NF-kappaB activation independent of inhibition of Fas-induced apoptosis.

Inhibition of neuronal apoptosis by the cyclin-dependent kinase inhibitor GW8510: identification of 3' substituted indolones as a scaffold for the development of neuroprotective drugs.

Increasing evidence suggests that neuronal apoptosis is triggered by the inappropriate activation of cyclin-dependent kinases leading to an abortive re-entry of neurons into the cell cycle. Pharmacological inhibitors of cell-cycle progression may therefore have value in the treatment of neurodegenerative diseases in humans. GW8510 is a 3' substituted indolone that was developed recently as an inhibitor of cyclin-dependent kinase 2 (CDK2). We found that GW8510 inhibits the death of cerebellar granule neurons caused by switching them from high potassium (HK) medium to low potassium (LK) medium. Although GW8510 inhibits CDK2 and other CDKs when tested in in vitro biochemical assays, when used on cultured neurons it only inhibits CDK5, a cytoplasmic CDK that is not associated with cell-cycle progression. Treatment of cultured HEK293T cells with GW8510 does not inhibit cell-cycle progression, consistent with its inability to inhibit mitotic CDKs in intact cells. Neuroprotection by GW8510 is independent of Akt and MEK-ERK signaling. Furthermore, GW8510 does not block the LK-induced activation of Gsk3beta and, while inhibiting c-jun phosphorylation, does not inhibit the increase in c-jun expression observed in apoptotic neurons. We also examined the effectiveness of other 3' substituted indolone compounds to protect against neuronal apoptosis. We found that like GW8510, the VEGF Receptor 2 Kinase Inhibitors [3-(1H-pyrrol-2-ylmethylene)-1,3-dihydroindol-2-one], {(Z)-3-[2,4-Dimethyl-3-(ethoxycarbonyl)pyrrol-5-yl)methylidenyl]indol-2-one} and [(Z)-5-Bromo-3-(4,5,6,6-tetrahydro-1H-indol-2-ylmethylene)-1,3-dihydroindol-2-one], the Src family kinase inhibitor SU6656 and a commercially available inactive structural analog of an RNA-dependent protein kinase inhibitor 5-Chloro-3-(3,5-dichloro-4-hydroxybenzylidene)-1,3-dihydro-indol-2-one, are all neuroprotective when tested on LK-treated neurons. Along with our recent identification of the c-Raf inhibitor GW5074 (also a 3' substituted indolone) as a neuroprotective compound, our findings identify the 3' substituted indolone as a core structure for the designing of neuroprotective drugs that may be used to treat neurodegenerative diseases in humans.

Induction of broad-based immune response against HIV-1 subtype C gag DNA vaccine in mice.

Prevention of HIV infection through an effective vaccine is need of the hour as per the AIDS pandemic scene, particularly in the developing world. Here we report the work done with gag gene construct pJWgagprotease49587 from HIV-1subtype C Indian strain. The construct pJWgagprotease49587 was tested positive for expression in COS-7 cells by p24 antigen capture ELISA, immunoblotting and by transmission electron microscopy that revealed virus like particle formation. Immunogenicity studies showed induction of good lymphoproliferative and cytotoxic (CTL) responses in Balb/c mice. The cytokine repertoire elicited showed a TH1 type of immune response. In an epitope mapping study, IFNgamma secretion by spleen cells from immunized mice was observed to seven peptides from different regions of gag. Recognition of multiple epitopes demonstrates elicitation of a broad based immune response against gag following immunization with the construct. In view of the high propensity of escape mutant induction during the course of HIV infection, it is encouraging to use immunogens eliciting viable immune responses to a broad spectrum of epitopes. Hence the construct pJWgagprotease49587 is a good candidate for immunogenecity testing in nonhuman primates as a probable vaccine candidate.

Use of lentiviral vectors for delivery of small interfering RNA.

Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis of mammalian cells. Although in the original studies expression of siRNA in mammalian cells was achieved via the transfection of double stranded oligonucleotides, subsequent studies described the use of plasmids to achieve long-term and stable expression of siRNA. Recently, several groups have described the use of retroviral vectors for siRNA delivery. However, retroviral vectors require active cell division for gene transfer and also suffer from the problem of gene-silencing. In this report we have modified a commercially available self-inactivating lentiviral vector for the delivery of siRNA into mammalian cells. We demonstrate the ability of this modified vector to efficiently transfer siRNA into HeLa S3 cells and downregulate p53 expression. Our results suggest that lentiviruses are efficient vectors for delivery of siRNA into mammalian cells. Based on the known ability of these vectors to infect both dividing and non-dividing cells and achieve long-term multilineage gene expression, their use as a therapeutic tool for the delivery of siRNA deserves further study.