Efficacy of N-methanocarbathymidine in treating mice infected intranasally with the IHD and WR strains of vaccinia virus.

N-Methanocarbathymidine [(N)-MCT] is a newly identified inhibitor of orthopoxvirus replication in cell culture and in mice. Limited published animal studies indicated the compound is effective by intraperitoneal (i.p.) route at 10-100 mg/(kg day). More extensive studies using different treatment regimens in intranasally infected mice were conducted in order to further explore the potential of this compound compared to cidofovir in treating vaccinia virus infections. (N)-MCT was given twice a day for 7 days, whereas cidofovir was administered once a day for 2 days, each starting 24h after virus exposure for most experiments. (N)-MCT was not toxic up to 1000 mg/(kg day) by the i.p. treatment route. Oral and i.p. treatment regimens with (N)-MCT were directly compared during a vaccinia virus (IHD strain) infection, indicating that the nucleoside has good oral bioavailability in mice. Treatments by i.p. route with (N)-MCT (100 mg/(kg day)) reduced lung, nasal, and brain virus titers during an IHD virus infection, but not nearly to the same extent as i.p. cidofovir (100 mg/(kg day)). Treatment with both compounds decreased liver, spleen, and kidney virus titers, as well as reduced lung consolidation scores and lung weights. Onset of treatment could be delayed by 2 days with (N)-MCT and by 3 days with cidofovir, providing significant survival benefit during the IHD virus infection. Against a vaccinia virus (WR strain) infection in mice, i.p. (N)-MCT treatment prevented death at 500 mg/(kg day), which was comparable in activity to i.p. cidofovir (100 mg/(kg day)). Significant reductions in tissue virus titers occurred with both treatment regimens. (N)-MCT could be further pursued for its potential to treat orthopoxvirus infections in humans.

Interaction and stabilization of X-linked inhibitor of apoptosis by Raf-1 protein kinase.

Raf-1 serine/threonine protein kinase plays an important role in cell growth, differentiation and cell survival. Recent reports using c-raf-1 gene-knockouts have observed MEK/ERK independent functions of Raf-1 in cell survival and protection from apoptosis. Raf-1 has also been shown to be involved in counteracting specific apoptotic pathways by restraining caspase activation, although the precise mechanism is unknown. XIAP is a potent inhibitor of apoptosis that blocks both the mitochondria and death receptor mediated pathways of apoptosis by directly binding to and inhibiting the initiator and effector caspases. In our efforts to understand the mechanism by which Raf-1 inhibits caspase activation, we discovered a novel interaction between Raf-1 and XIAP. In this study, we describe the physical interaction between Raf-1 and XIAP in vitro and in vivo in mammalian cells. We also demonstrate that Raf-1 phosphorylates XIAP in vitro and in vivo. Additionally, Raf-1 prevents XIAP degradation in response to different apoptotic triggers. Our studies identify XIAP as a new substrate of Raf-1 and provide potentially important insight into mechanisms underlying Raf-1 effects on cell survival.

Gene expression profile by inhibiting Raf-1 protein kinase in breast cancer cells.

Raf-1 protein serine-threonine kinase plays an important role in cell growth, proliferation, and cell survival. Previously, we and others have demonstrated that antisense raf oligonucleotide-mediated inhibition of Raf-1 expression leads to tumor growth arrest, radiosensitization and chemosensitization in vivo. Raf-1 inhibition is also associated with apoptotic cell death. In this study, we inhibited Raf-1 using an antisense raf oligonucleotide (AS-raf-ODN) to identify downstream targets of Raf-1 using microarray gene expression analysis. Treatment of MDA-MB-231 breast cancer cells with 250 nM AS-raf-ODN led to significant inhibition of Raf-1 protein (75.2 +/- 9.6%) and c-raf-1 mRNA levels (86.2 +/- 3.3%) as compared to untreated control cells. The lipofectin control or mismatch oligonucleotide had no effect on Raf-1 expression. To determine the changes in gene expression profiles that were due to inhibition of Raf-1, we simultaneously compared the gene expression patterns in AS-raf-ODN treated cells with untreated control cells and cells treated with lipofectin alone or MM-ODN. A total of 17 genes (4 upregulated and 13 down-regulated) including c-raf-1 were identified that were altered after AS-raf-ODN treatment. Functional clustering analysis revealed genes involved in apoptosis (Bcl-XL), cell adhesion (paxillin, plectin, Rho GDIalpha, CCL5), metabolism (GM2A, SLC16A3, PYGB), signal transduction (protein kinase C nu), and transcriptional regulation (HMGA1), and membrane-associated genes (GNAS, SLC16A3). Real-time PCR, Northern analysis and Western analysis confirmed the microarray findings. Our study provides insight into Raf-1 related signaling pathways and a model system to identify potential target genes.

Pharmacokinetics, toxicity, and efficacy of ends-modified raf antisense oligodeoxyribonucleotide encapsulated in a novel cationic liposome.

Raf-1 protein serine threonine kinase plays an important role in cell survival and proliferation. Antisense inhibition of Raf-1 expression has been shown to enhance the cytotoxic effects of radiation and anticancer drugs. Here we have evaluated the toxicity, pharmacokinetics, and antitumor efficacy of a novel formulation of liposome-entrapped raf antisense oligodeoxyribonucleotide (LErafAON). The LErafAON preparation showed high liposome entrapment efficiency of rafAON (>85%) and stability at room temperature. In CD2F1 mice, administration of LErafAON produced no morbidity/mortality (5-35 mg/kg/dose, i.v., x12). Dose-related elevations in liver enzymes (alanine aminotransferase and aspartate aminotransferase) and histopathological changes in liver were noted in LErafAON and blank liposome groups. No morbidity/mortality and changes in clinical chemistry or histopathology were observed in New Zealand white rabbits (3.75 mg/kg/dose, i.v., x8; 6.5 mg/kg/dose, i.v., x6) or in cynomolgous monkeys (3.75 or 6.25 mg/kg/dose, i.v., x9). Transient decrease in total hemolytic complement activity (approximately 62-74%) and increases in C3a (approximately 3-fold) and Bb levels (approximately 5-12-fold) were observed in LErafAON and blank liposome groups of monkeys. A 30 mg/kg i.v. dose of LErafAON in human prostate tumor (PC-3)-bearing BALB/c athymic mice gave a terminal plasma half-life of 27 h, and intact rafAON could be detected in plasma and in normal and tumor tissues for up to at least 48 h. In monkeys, the terminal plasma half-life of 30.36 +/- 23.87 h was observed at an i.v. dose of 6.25 mg/kg. LErafAON (25 mg/kg/dose, i.v., x10) or ionizing radiation (3.8 Gy/day, x5) treatment of PC-3 tumor-bearing athymic mice led to tumor growth arrest, whereas a combination of LErafAON and ionizing radiation treatments resulted in tumor regression. LErafAON treatment caused inhibition of Raf-1 protein expression in normal and tumor tissues in these mice (>50%, versus controls). These data have formed a basis of the clinical Phase I studies of LErafAON for cancer treatment.

Efficacy of N-methanocarbathymidine against genital herpes simplex virus type 2 shedding and infection in guinea pigs.

BACKGROUND: Current approved nucleoside therapies for genital herpes simplex virus (HSV) infections are effective but improved therapies are needed for treatment of both acute and recurrent diseases. METHODS: The effects of N-methanocarbathymidine were evaluated and compared to acyclovir using guinea pig models of acute and recurrent infection. For acute disease following intravaginal inoculation of 10(6 )pfu HSV-2 (MS strain), animals were treated intraperitoneally beginning 24 h post-infection, and the effects on disease severity, vaginal virus replication, subsequent recurrences, and latent virus loads were evaluated. For evaluation of recurrent infection, animals were treated for 21 days beginning 14 days after infection and disease recurrence and recurrent shedding were evaluated. RESULTS: Treatment of the acute disease with N-methanocarbathymidine significantly reduced the severity of acute disease and decreased acute vaginal virus shedding more effectively than acyclovir. Significantly, none of the animals developed visible disease in the high-dose N-methanocarbathymidine group and this was the only group in which the number of days with recurrent virus shedding was reduced. Treatment of recurrent disease was equivalent to acyclovir when acyclovir was continuously supplied in the drinking water. CONCLUSION: N-methanocarbathymidine was effective as therapy for acute and recurrent genital HSV-2 disease in the guinea pig models.

Enhanced therapeutic effects of doxorubicin and paclitaxel in combination with liposome-entrapped ends-modified raf antisense oligonucleotide against human prostate, lung and breast tumor models.

Raf-1 protein kinase plays an important role in cell growth, proliferation and cell survival. We have previously described the use of liposome-entrapped antisense raf oligonucleotide (LErafAON) to inhibit Raf-1 expression resulting in tumor growth inhibition and radiosensitization. The present study was undertaken to evaluate the chemosensitization effects of LErafAON in combination with doxorubicin or paclitaxel on a panel of human tumor xenografts. LErafAON (25.0 mg/kg i.v. x 10) displayed significant antitumor activity (P<0.05) when administered as a single agent in prostate (PC-3), lung (A549) and breast (MDA-MB 231) carcinoma models. Doxorubicin (1.0-4.0 mg/kg i.v. per week x 3) and paclitaxel (1.0-4.0 mg/kg i.v. on alternate days x 3) were administered as single agents at non-toxic doses that led to only minimal to moderate antitumor activity. However, a combination of LErafAON with doxorubicin or paclitaxel led to significantly enhanced antitumor activity in all the tumor types tested (PC-3, P<0.03; A549, P<0.035; MDA-MB 231, P<0.045) as compared with LErafAON alone or chemotherapeutic agents alone treated groups. This effect of chemosensitization appeared to be sequence-specific because a mismatch control oligonucleotide continued to show significant tumor growth. Additionally, no inhibition in Raf-1 expression in MDA-MB 231 tumor tissue was observed with mismatch oligonucleotide treatment. On the other hand, LErafAON treatment led to >75% inhibition of Raf-1 expression in tumor tissue. These preclinical observations support the use of LErafAON in combination with chemotherapeutic agents to improve the treatment of human cancers.

A phase I study of liposomal-encapsulated docetaxel (LE-DT) in patients with advanced solid tumor malignancies.

BACKGROUND: Docetaxel is a taxane anticancer drug used in a wide variety of solid tumors. Liposomes are versatile drug carriers that may increase drug solubility, serve as sustained release systems, provide protection from drug degradation and toxicities, and help overcome multidrug resistance. This phase I study was conducted to determine the maximum tolerated dose, dose-limiting toxicities (DLTs), pharmacokinetics (PK), and clinical response of liposomal-encapsulated docetaxel (LE-DT) in patients with advanced solid tumor malignancies. METHODS: LE-DT was administered using a standard 3 + 3 dose escalation schema with dose levels of 50, 65, 85, 110, and 132 mg/m(2) IV on a 3-week cycle. Toxicities were assessed using the NCI-CTCAE version 3.0, and response was assessed using RECIST criteria (version 1.0). PK samples were drawn during cycle 1 and analyzed using a non-compartmental analysis. RESULTS: Twenty-four patients were treated for 1-30 cycles (median = 4). No DLTs were experienced through dose levels of 50, 65, 85, and 110 mg/m(2). Two out of two patients experienced grade 4 neutropenia at the 132 mg/m(2) dose level. When an additional three patients were treated at the expanded 110 mg/m(2) dose level, two experienced grade 4 neutropenia. The 85 mg/m(2) dose level was reassessed with an expanded group of three additional patients, and only one of three patients experienced grade 4 neutropenia. The protocol was amended to allow G-CSF during cycle 1, and an additional three patients were treated at 110 mg/m(2) with no DLTs experienced. No patient experienced significant neuropathy, even patients treated for 19, 20, and 30 cycles. PK followed a two-compartment elimination pattern; there was no correlation between PK and toxicity. Two patients with thyroid and neuroendocrine cancer had partial responses (PR, 8%), and one patient with non-small-cell lung cancer had an unconfirmed PR. Eight patients (33%) had stable disease lasting more than 3 months, for a clinical benefit rate of 41%. CONCLUSION: LE-DT was well tolerated with expected toxicities of neutropenia, anemia, and fatigue, but without neuropathy or edema. Clinical benefit (SD + PR) was observed in 41% of the patients. The recommended phase II dose of LE-DT is 85 mg/m(2) without G-CSF or 110 mg/m(2) with G-CSF.

Efficacy of orally administered low dose N-methanocarbathymidine against lethal herpes simplex virus type-2 infections of mice.

BACKGROUND: N-methanocarbathymidine (N-MCT) has previously been shown to be effective against lethal orthopoxvirus and herpes simplex virus type-1 infections in mice. In this investigation, the antiviral activity of N-MCT was assessed against herpes simplex virus type-2 (HSV-2) in BALB/c mice. METHODS: BALB/c mice were infected intranasally with a lethal challenge dose of HSV-2. N-MCT was administered orally twice daily to mice using doses of 0.01 to 100 mg/kg to determine effects on survival and on viral replication in organ and central nervous system (CNS) samples. RESULTS: N-MCT provided significant protection from mortality even when treatments were delayed until 3 days after viral infection. Viral replication in organ and CNS samples from N-MCT-treated mice was reduced below the limit of detection after 4 days of treatment. CONCLUSIONS: These results indicated that low dose N-MCT treatment was more effective than acyclovir therapy. N-MCT may be effective against HSV disease in humans and is currently undergoing preclinical evaluation. In particular, its potential use as a combination therapy for HSV, with its differing metabolism from acyclovir, make it a promising compound to develop for human use.

N-Methanocarbathymidine is more effective than acyclovir for treating neonatal herpes simplex virus infection in guinea pigs.

The outcome of neonatal herpes simplex (HSV) infection, even after therapy with high dose acyclovir (ACV), is not optimum. We therefore evaluated N-Methanocarbathymidine ((N)-MCT) using the guinea pig model of neonatal herpes. Treatment with ACV (60 mg/kg/day) was compared to doses of 1, 5, and 25 mg/kg/day of (N)-MCT initiated 1, 2, or 3 days postinoculation (dpi). Both ACV and (N)-MCT significantly improved survival, but only (N)-MCT significantly reduced the number of animals with symptoms when begun at 1 dpi. When therapy was begun at 2 dpi, only (N)-MCT (1, 5, or 25 mg/kg/day) significantly increased survival. In fact, (N)-MCT improved survival up to 3 dpi, the last time point evaluated. (N)-MCT was highly effective and superior to high dose ACV therapy for the treatment of neonatal herpes in the guinea pig model.

Therapeutic efficacy of Cintredekin Besudotox (IL13-PE38QQR) in murine lung fibrosis is unaffected by immunity to Pseudomonas aeruginosa exotoxin A.

BACKGROUND: We have previously explored a therapeutic strategy for specifically targeting the profibrotic activity of IL-13 during experimental pulmonary fibrosis using a fusion protein comprised of human IL-13 and a mutated form of Pseudomonas aeruginosa exotoxin A (IL13-PE) and observed that the intranasal delivery of IL13-PE reduced bleomycin-induced pulmonary fibrosis through its elimination of IL-13-responsive cells in the lung. The aim of the present study was to determine whether the presence of an immune response to P. aeruginosa and/or its exotoxin A (PE) would diminish the anti-fibrotic properties of IL13-PE. METHODOLOGY/PRINCIPAL FINDINGS: Fourteen days after P. aeruginosa infection, C57BL/6 mice were injected with bleomycin via the intratracheal route. Other groups of mice received 4 doses of saline or IL13-PE by either intranasal or intraperitoneal application, and were challenged i.t. with bleomycin 28 days later. At day 21 after bleomycin, all mice received either saline vehicle or IL13-PE by the intranasal route and histopatological analyses of whole lung samples were performed at day 28 after bleomycin. Intrapulmonary P. aeruginosa infection promoted a neutralizing IgG2A and IgA antibody response in BALF and serum. Surprisingly, histological analysis showed that a prior P. aeruginosa infection attenuated the development of bleomycin-induced pulmonary fibrosis, which was modestly further attenuated by the intranasal administration of IL13-PE. Although prior intranasal administration of IL13-PE failed to elicit an antibody response, the systemic administration of IL13-PE induced a strong neutralizing antibody response. However, the prior systemic sensitization of mice with IL13-PE did not inhibit the anti-fibrotic effect of IL13-PE in fibrotic mice. CONCLUSIONS: Thus, IL13-PE therapy in pulmonary fibrosis works regardless of the presence of a humoral immune response to Pseudomonas exotoxin A. Interestingly, a prior infection with P. aeruginosa markedly attenuated the pulmonary fibrotic response suggesting that the immune elicitation by this pathogen exerts anti-fibrotic effects.

BRCC2, a novel BH3-like domain-containing protein, induces apoptosis in a caspase-dependent manner.

We report here the structure-functional characterization of a novel intronless gene, BRCC2, located on human chromosome 11q24.1. BRCC2 open reading frame (327 bp) codes for an approximately 12-kDa protein (108 amino acids (aa)) localized predominantly in the cytosol and to a lesser extent in the mitochondria. Ectopic expression of BRCC2 cDNA also was found in both the cytosol and mitochondria. Exogenous expression of BRCC2 caused apoptotic cell death in three different cell lines as evidenced by enhanced chromatin condensation, DNA fragmentation, or an enhanced number of cells in the sub-G(1) phase. In human prostate cancer cells (PC-3), BRCC2-induced DNA fragmentation was blocked efficiently by coexpression of the anti-apoptotic molecule, Bcl-X(L). Transient transfection of BRCC2 cDNA into PC-3 cells in the presence of a broad-range caspase inhibitor, Z-VAD-fmk (100 microM, 24 h), abrogated DNA fragmentation. Consistently, BRCC2 expression correlated with the activation of caspase-3 and caspase-9. An N-terminal deletion mutant of BRCC2 (10.2 kDa, Delta1-16 aa) lacking a BH3-like domain (5-12 aa, LPIEGQEI) or BRCC2 containing a mutant BH3-like domain (leucine 5-->glutamate) failed to induce apoptosis, whereas a C-terminal deletion mutant (6.8 kDa, Delta62-108 aa) retained the apoptotic activity comparable to the full-length BRCC2. Finally, the treatment of HeLa cells with doxorubicin or hydrogen peroxide (H(2)O(2)) led to an increase in the mitochondrial (heavy membrane) level of endogenous BRCC2 (doxorubicin (100 ng/ml), 5 h, approximately 2-fold; H(2)O(2) (200 microM), 2 h, approximately 2-fold). These findings demonstrate that BRCC2 functions as a proapoptotic molecule and suggest that BRCC2 induces a caspase-dependent mitochondrial pathway of cell death.

Combination with liposome-entrapped, ends-modified raf antisense oligonucleotide (LErafAON) improves the anti-tumor efficacies of cisplatin, epirubicin, mitoxantrone, docetaxel and gemcitabine.

Raf-1 protein serine/threonine kinase plays an important role in cell proliferation and cell survival. We have previously described a novel cationic liposome-entrapped formulation of raf antisense oligodeoxyribonucleotide (LErafAON) and its use as a radiosensitizer. The aim of this study was to examine the effect of combination of LErafAON and a chemotherapeutic agent on growth of human prostate (PC-3) and pancreatic tumor xenografts in athymic mice (Aspc-1 and Colo 357). In PC-3 tumor-bearing mice, administration of a combination of LErafAON (i.v., 25 mg/kg/dose, x10/16) and cisplatin (i.v., 11.0 mg/kg/dose, x3), epirubicin (EPI) (i.v., 9.0 mg/kg/dose, x3) or mitoxantrone (MTO) (i.v., 2.5 mg/kg/dose, x3) led to enhanced tumor growth inhibition as compared with single agents (LErafAON+cisplatin versus cisplatin, p<0.0002, n=8; LErafAON+EPI versus EPI, p<0.0001, n=6; LErafAON+MTO versus MTO, p<0.05, n=5). In prostate or pancreatic tumor-bearing mice, combination of LErafAON (i.v., 25 mg/kg/dose, x10/13) with docetaxel (Taxotere) (i.v., 5, 7.5 or 10 mg/kg/dose, x2/4) led to tumor regression or enhanced growth inhibition as compared with single agents (PC-3: LErafAON+Taxotere versus Taxotere, p<0.02, n=7; Aspc-1: LErafAON+Taxotere versus Taxotere, p<0.03, n=5; Colo 357: LErafAON+Taxotere versus Taxotere, p<0.04, n=7). Combination of LErafAON (i.v., 25 mg/kg/dose, x10/13) with gemcitabine (i.v., 75 mg/kg/dose, x4/6) also caused a significant tumor growth inhibition in the two pancreatic carcinoma models studied (Aspc-1: LErafAON+gemcitabine versus gemcitabine, p<0.0001, n=7; Colo 357: LErafAON+gemcitabine versus gemcitabine, p<0.002, n =5). LErafAON treatment (i.v., 25 mg/kg/dose, x10) caused inhibition of Raf-1 protein expression in these tumor tissues (around 25-60%, n=4-7). Interestingly, Taxotere treatment per se also led to decreased steady state level of Raf-1 protein in PC-3 and Aspc-1 tumor tissues (i.v., 10 mg/kg/dose, x1 or 7.5 mg/kg/dose, x2; around 25-80%, n=2/6). Present studies demonstrate enhanced tumor growth inhibition or regression in response to a combination of a chemotherapeutic drug and LErafAON. These data provide a proof-of-principle for the clinical use of LErafAON in combination with chemotherapy for cancer treatment.