Interfering with Fos expression in rat perirhinal cortex impairs recognition memory.

Previous work has shown that immunohistochemical imaging of Fos protein is a reliable marker for changes in activity related to recognition memory in the perirhinal (PRH) cortex of the medial temporal lobe; however, whether PRH Fos expression is necessary for recognition memory had not been established. To investigate this potential requirement, antisense Fos oligodeoxynucleotide (ODN) was infused locally into PRH cortex to interfere with Fos production. As in previous studies, differential Fos expression produced by viewing novel or familiar visual stimuli was measured by immunohistochemistry: antisense Fos ODN infusion into PRH cortex disrupted the normal pattern of differential Fos expression in PRH cortex. The effect of antisense Fos ODN infusion into PRH cortex was therefore sought on recognition memory. Infusion before or immediately after acquisition impaired recognition memory for objects when the memory delay was 3 or 24 h, but not when the delay was 20 min, or when the ODN was infused before retrieval after a 24-h delay. The findings indicate a role for Fos in consolidation processes underlying long-term recognition memory for objects and establish that interfering with its expression impairs recognition memory. Antisense Fos ODN infusion also impaired object-in-place recognition memory. The results demonstrate that Fos is necessary for neuronal mechanisms in PRH cortex essential to recognition memory.

c-Met antisense oligodeoxynucleotides as a novel therapeutic agent for glioma: in vitro and in vivo studies of uptake, effects, and toxicity.

BACKGROUND: c-Met, a receptor tyrosine kinase, and its ligand, hepatocyte growth factor, are critical in cellular proliferation, motility, and invasion and are known to be overexpressed in gliomas. The aim of our study was to investigate the uptake and effects of c-Met antisense oligodeoxynucleotides (ASODNs) on rat and human glioma cells in vitro and the uptake and toxicity of these nucleotides in rat carcinomatosis and brain tumor models. MATERIALS AND METHODS: The three human cell lines (U87, BT325, SHG44) and the C6 rat glioma cell line were cultured. To study the uptake of oligodeoxynucleotides (ODNs) by glioma cells in vitro, cultured glioma cells readily incorporated caroboxyfluorescein-5-succimidyl ester (FAM) labeled phosphorothioate oligodeoxynucleotides, as demonstrated by immunofluorescence microscopy and flow cytometry. To study the effect of ASODNs treatment on c-Met expression in vitro, Expression of c-Met was assessed by immunofluorescence microscopy and reverse transcriptase polymerase chain reaction (RT-PCR) analysis. For animal studies of ODNs toxicity and uptake, eight rats underwent placement of cisternal catheters, under general anesthesia. Four rats were given 24 mug FAM-labeled ASODNs while the others were given a saline control injection. After a 24 h observation period, rats were sacrificed by barbiturate overdose, and their brains were studied. RESULTS: For all cell lines, fluorescence was seen to increase with increasing ASODNs concentration. Cells treated in similar fashion were also analyzed by flow cytometry to graphically illustrate the differing fluorescence. Multiple glioma cell lines were tested, with similar results. c-Met ASODNs was found to be successfully incorporated from the media into cultured human glioma cells, even at concentrations as low as 2 muM. In addition, maintenance of the pH-dependent green fluorescence color, as seen by immunofluorescence microscopy and by using flow cytometry, indicated that the FAM was not contained within lysosomes. Immunofluorescence microscopy and RT-PCR analysis showed decreases in c-Met expression with oligodeoxynucleotides treatment. Uptake into tumor cells was also demonstrated in vivo, with no detectable toxicity at concentrations exceeding expected therapeutic levels. CONCLUSION: These data are encouraging for further study of c-Met antisense oligodeoxynucleotides as a therapeutic modality for glioma.

The small heart mutation reveals novel roles of Na+/K+-ATPase in maintaining ventricular cardiomyocyte morphology and viability in zebrafish.

Forward genetic screens in zebrafish have been used to identify mutations in genes with important roles in organogenesis. One of these mutants, small heart, develops a diminutive and severely malformed heart and multiple developmental defects of the brain, ears, eyes, and kidneys. Using a positional cloning approach, we identify that the mutant gene encodes the zebrafish Na+/K+-ATPase alpha1B1 protein. Disruption of Na+/K+-ATPase alpha1B1 function via morpholino "knockdown" or pharmacological inhibition with ouabain phenocopies the mutant phenotype, in a dose-dependent manner. Heterozygosity for the mutation sensitizes embryos to ouabain treatment. Our findings present novel genetic and morphological details on the function of the Na+/K+-ATPase alpha1B1 in early cardiac morphogenesis and the pathogenesis of the small heart malformation. We demonstrate that the reduced size of the mutant heart is caused by dysmorphic ventricular cardiomyocytes and an increase in ventricular cardiomyocyte apoptosis. This study provides a new insight that Na+/K+-ATPase alpha1B1 is required for maintaining ventricular cardiomyocyte morphology and viability.

Oligodeoxynucleotide studies in primates: antisense and immune stimulatory indications.

Antisense oligodeoxynucleotide compounds (AS ODN) are being developed as therapeutics for various disease indications. Their safety and pharmacokinetics are most commonly evaluated in rodents and nonhuman primates. Traditional AS ODN are short, single strands of DNA, and they target specific mRNA sequences. Plasma clearance of AS ODN is rapid, broad tissue distribution occurs, and elimination is by nuclease metabolism. Structural modifications to AS ODN have been made to enhance their efficacy and improve their safety. A number of class effects are observed with AS ODN that are unrelated to the specific targeted mRNA sequence. Acute effects include activation of the alternative complement pathway and inhibition of the intrinsic coagulation pathway. In monkeys, rodents, and dogs given repeated doses of AS ODN, accumulation of AS ODN and/or metabolites occurs in the form of basophilic granules in various tissues, including the kidney, lymph nodes and liver. A new potential therapeutic application of ODN is that of immune stimulation. Immunostimulatory ODN (IS ODN) are being investigated for use in treating cancer, infectious disease, and allergy. For the development of both AS and IS ODN, primates will continue to be important for safety assessment.

Potentiation of dexamethasone-, paclitaxel-, and Ad-p53-induced apoptosis by Bcl-2 antisense oligodeoxynucleotides in drug-resistant multiple myeloma cells.

Overexpression of Bcl-2 in myeloma cells results in resistance to drugs such as dexamethasone (DEX), adenovirus-mediated delivery of p53 (Ad-p53), and paclitaxel (TAX), which work through the intrinsic apoptotic pathway. Bcl-2 antisense oligodeoxynucleotides (Bcl-2-ASO) have been shown to induce apoptosis in cancer cells, as a single agent or, better, in combination with chemotherapy. We hypothesized that down-regulation of Bcl-2 by Bcl-2-ASO will sensitize drug-resistant myeloma cells to undergo apoptosis. In this paper we report a detailed time/dose study of the effect of Bcl-2-ASO on myeloma cells with varying levels of Bcl-2. Treatment of myeloma cells expressing relatively low levels of Bcl-2 with Bcl-2-ASO resulted in a substantial apoptosis concomitant with a substantial depletion of Bcl-2 protein. Maximal apoptosis was observed at 5 to 10 microg/mL Bcl-2-ASO, following 4 days of treatment. Down-regulation of Bcl-2 and apoptosis were time and dose dependent and were sequence specific. In these cell lines, apoptosis was accompanied by activation of caspase-9 and caspase-3 and by release of cytochrome c to the cytosol. In contrast, high Bcl-2-expressing myeloma cells were practically resistant to Bcl-2-ASO. Most important, however, pretreatment of myeloma cells expressing high levels of Bcl-2 with Bcl-2-ASO increased the extent of DEX-, TAX-, and Ad-p53-induced apoptosis from 10%-20% to 70%-90%. Increased apoptosis was accompanied by additional decrease in Bcl-2 protein. Similar results for down-regulation of Bcl-2 and apoptosis were obtained with freshly isolated myeloma cells. These data support development of clinical trials with combinations of Bcl-2-ASO and DEX, TAX, or Ad-p53 in the treatment of refractory myeloma patients.

Antisense oligodeoxynucleotide directed against c-myb has anticancer activity and potentiates the antiproliferative effect of conventional anticancer drugs acting by different mechanisms in human colorectal cancer cells.

The C-MYB proto-oncogene encodes a DNA-binding protein with transactivation properties that plays an important regulatory role in cell proliferation and differentiation. Overexpression of C-MYB in colonic tumors compared to normal mucosa suggests that c-myb may play a role in the malignant transformation of colonic mucosa and that inhibition of c-myb expression may suppress, to some extent, the proliferation of neoplastic cells. Complete suppression of tumor cell proliferation may require inhibition of multiple growth-promoting genes. Alternatively, the combination of oncogene-targeted oligodeoxynucleotides (ODNs) with standard cytotoxic agents might represent a useful therapeutic approach to improving cancer treatment. In the present study, we have investigated whether the inhibition of a growth-promoting gene, namely c-myb, affects the sensitivity of human colorectal cancer cells, in vitro, to conventional chemotherapeutic drugs: taxol, 5-fluorouracil, vinblastine and doxorubicin. We show that c-myb antisense phosphorothioate (S) ODN treatment induces growth arrest in the G(1)/G(2) phases of the cell cycle and inhibits cell proliferation in a dose- and time-dependent manner. Also, treatment with c-myb antisense (S)ODN decreases c-myb mRNA and protein expression. A greater inhibition of cell proliferation in vitro was obtained with the combination of c-myb (S)ODN and cytotoxic drugs. The combinations exerted additive and synergistic effects on human colorectal cancer cells. This study suggests that c-myb antisense (S)ODN might be useful in the therapy of colorectal cancer in combination with chemotherapeutic drugs.

Enhanced in vivo tumour response from combination of carboplatin and low-dose c-myc antisense oligonucleotides.

BACKGROUND: Phosphorothioate oligonucleotides ([S]ODNs) contain a modified phosphate backbone. Antisense [S]ODNs targeted to specific oncogenes have been used to varying success in vivo. Carboplatin is a commonly used chemotherapeutic and is associated with chemoresistance in some human tumours. The potential for combined antisense [S]ODNs and carboplatin chemotherapy has only recently been explored in vivo. MATERIALS AND METHODS: This study examines the effect of c-myc antisense oligomers delivered in isolation as naked DNA and in combination with carboplatin upon the growth kinetics of an in vivo transplantable adenocarcinoma using rodents. RESULTS: Tumours treated with a combination of 600 microg of 15-mer c-myc phosphorothioate antisense oligodeoxyribonucleotide and an intravenous administration of carboplatin (3 mg/kg), demonstrated a significant (p<0.05) retardation in tumour growth kinetics relative to a control. Two mismatch antisense controls did not significantly inhibit tumour growth. C-myc protein studies in tumour sections failed to show significant differences in c-myc expression in any of the treated tumours. CONCLUSION: This study demonstrates that carboplatin affects the relative abundance of c-myc and that combination treatment of carboplatin and c-myc phosphorothioate antisense oligonucleotides in vivo results in synergistic tumour retardation.

A randomized phase II and pharmacokinetic study of the antisense oligonucleotides ISIS 3521 and ISIS 5132 in patients with hormone-refractory prostate cancer.

PURPOSE: Protein kinase C (PKC)-alpha and Raf-1 are important elements of proliferative signal transduction pathways in both normal and malignant cells. Abrogation of either Raf-1 or PKC-alpha function can both inhibit cellular proliferation and induce apoptosis in several experimental cancer models including prostate cancer cell lines. ISIS 3521 and ISIS 5132 are antisense phosphorothioate oligonucleotides that inhibit PKC-alpha and Raf-1 expression, respectively, and induce a broad spectrum of antiproliferative and antitumor effects in several human tumor cell lines. In Phase I evaluation both ISIS 3521 and ISIS 5132 could be safely administered on 21-day i.v. infusion schedules and demonstrated preliminary evidence of antitumor activity. On the basis of these findings, a randomized Phase II study of ISIS 3521 and ISIS 5132 was performed in two comparable cohorts of patients who had chemotherapy-naïve, hormone-refractory prostate cancer (HRPC). PATIENTS AND METHODS: Patients with documented evidence of metastatic HRPC and a prostate-specific antigen (PSA) value > or =20 ng/ml were randomized to receive treatment with either ISIS 3521 or ISIS 5132 as a continuous i.v. infusion for 21 days repeated every 4 weeks. Patients were stratified according to the presence or absence of bidimensionally measurable disease at the time of randomization. The principal endpoints included PSA response, objective response in patients with bidimensionally measurable disease, and treatment failure defined as new or worsening symptoms; a fall in performance status of 2 levels; new or objective progression of disease; or a rise in PSA for 12 weeks without symptom improvement. Plasma samples were collected to assess individual steady-state concentrations and to relate this pharmacokinetic parameter to observed toxicities and responses. RESULTS: Thirty-one patients were randomized in this study; 15 patients received 43 courses of ISIS 3521 and 16 patients received 48 courses of ISIS 5132. The most common toxicities observed were mild to moderate (grade 1 or 2) fatigue and lethargy in 21% and 56% of patients treated with ISIS 3521 and ISIS 5132, respectively. Although no objective or PSA responses were observed in any patient treated with ISIS 3521 or ISIS 5132, persistent stable disease was observed in 3 patients for 5 or more months, and in 5 patients the PSA values did not rise >25% for 120 days or longer. CONCLUSIONS: The antisense oligonucleotides ISIS 3521 and ISIS 5132, at these doses and on this schedule, do not possess clinically significant single-agent antitumor activity in HRPC. Protracted stable disease in some patients may indicate a cytostatic effect. Additional work is required to define the optimal role of PKC-alpha or Raf-1 inhibition in the treatment of HRPC.

Influence of polymer architecture on the structure of complexes formed by PEG-tertiary amine methacrylate copolymers and phosphorothioate oligonucleotide.

The influence of polymer structure on the characteristics of complexes of a phosphorothioate antisense oligonucleotide (ISIS 5132) was studied, using well-defined cationic copolymers based on 2-(dimethylamino) ethyl methacrylate (DMAEMA) and poly(ethylene glycol) (PEG). The three related copolymer structures were: DMAEMA-PEG (a diblock copolymer) DMAEMA-OEGMA 7 (a brush-type copolymer), DMAEMA-stat-PEGMA (a comb-type copolymer); each of these were examined together with DMAEMA homopolymer, which served as a control. The results revealed that all the polymers exhibited good binding ability with the oligonucleotide (ON). Interestingly, the comb-type polymer DMAEMA-stat-PEGMA demonstrated the highest binding ability and DMAEMA homopolymer the lowest, as judged by a dye displacement assay. DMAEMA homopolymer produced large agglomerates of smaller individual complexes as observed by optical density, photon correlation spectroscopy and transmission electron microscopy studies. In contrast, two PEG-block copolymers, DMAEMA-PEG and DMAEMA-OEGMA 7, formed compact complexes of 80-150 nm which had good long-term colloidal stability. This is attributed to the steric stabilisation effect of the PEG chains on the ON-copolymer complexes. These two copolymers are believed to form complexes with ON that have a micellar structure. Comb-type DMAEMA-stat-PEGMA copolymer formed highly soluble complexes with the ON that did not phase separate from the buffer solution. This study clearly demonstrates that varying the copolymer architecture allows access to a range of ON complexes. In vitro cytotoxicity experiments on HepG2 cells showed that all of the tertiary amine methacrylate copolymers displayed lower cytotoxicity than the control poly(L-lysine).

Assessment of the genotoxic potential of ISIS 2302: a phosphorothioate oligodeoxynucleotide.

ISIS 2302, a phosphorothioate oligodeoxynucleotide with antisense activity against human ICAM-1 mRNA, was evaluated in a battery of tests to assess genotoxic potential. There was no evidence of genotoxicity in three in vitro studies performed: (i) a bacterial reverse mutation test; (ii) a chromosomal aberration test in Chinese hamster ovary cells; (iii) a mammalian cell gene mutation assay in L5187Y cells. Additionally, there was no in vivo evidence of genetic toxicity in a bone marrow micronucleus study in male and female mice. For all tests, top concentrations or doses assessed met harmonized regulatory guidelines. The cellular uptake of ISIS 2302 into target cells was confirmed using capillary gel electrophoresis and immunohistochemistry. Intracellular uptake into CHO cells, L5187Y cells, Salmonella typhimurium TA98 and bone marrow was concentration- and time-dependent. Consistent with what is known about the physical and chemical properties of phosphorothioate oligodeoxynucleotides, there was no evidence of genotoxicity in any of the assessed end-points. Furthermore, the absence of genotoxicity could not be ascribed to test system insensitivity or to an absence of exposure of the test system to ISIS 2302.

Inhibition of Bcl-xL expression sensitizes T-cell acute lymphoblastic leukemia cells to chemotherapeutic drugs.

We have examined the effects of antisense oligonucleotides to bcl-x on the survival and chemosensitivity of CEM cells, a T-acute lymphoblastic leukemia (T-ALL) cell line. Also, we have measured the levels of Bcl-2, Bcl-x, and Bax in 20 cases of T-ALL. By 18 h after the bcl-x antisense treatment, CEM cells showed over a 75% reduction in the levels of Bcl-xL protein and over 30% decreased viable cell counts compared with cells treated with the control oligonucleotide. The combination of bcl-x antisense plus either dexamethasone or doxorubicin showed either strong synergistic or additive killing of CEM cells, respectively. These findings indicate that bcl-x antisense has cytotoxic activity and increases chemotherapy-induced cell death in CEM cells, a model for T-ALL.

Synergistic chemsensitization and inhibition of tumor growth and metastasis by the antisense oligodeoxynucleotide targeting clusterin gene in a human bladder cancer model.

Clusterin expression is highly up-regulated in several normal and malignant tissues undergoing apoptosis. Although recent studies have demonstrated a protective role of clusterin expression against various kinds of apoptotic stimuli, the functional role of clusterin in the acquisition of a therapy-resistant phenotype in bladder cancer remains unknown. The objectives of this study were to determine whether antisense (AS) oligodeoxynucleotide (ODN) targeting the clusterin gene enhances apoptosis induced by cisplatin and to evaluate the usefulness of combined treatment with AS clusterin ODN and cisplatin in the inhibition of KoTCC-1 tumor growth and metastasis in a human bladder cancer KoTCC-1 model. We initially revealed the dose-dependent and sequence-specific inhibition of clusterin expression by AS clusterin ODN treatment in KoTCC-1 cells at both mRNA and protein levels. Clusterin mRNA was increased in a dose-dependent manner by cisplatin treatment at concentrations < or =10 mg/ml, and clusterin mRNA up-regulation induced by 10 mg/ml cisplatin peaked by 48-h post-treatment and began decreasing by 72-h post-treatment. Although there was no significant effect on growth of KoTCC-1 cells, AS clusterin ODN treatment significantly enhanced cisplatin chemosensitivity of KoTCC-1 cells in a dose-dependent manner, reducing the IC(50) by >50%. Characteristic apoptotic DNA ladder formation and cleavage of poly(ADP-ribose) polymerase protein were detected after combined treatment with AS clusterin ODN and cisplatin but not either agent alone. In vivo systemic administration of AS clusterin and cisplatin significantly decreased the s.c. KoTCC-1 tumor volume compared with mismatch control ODN plus cisplatin. Furthermore, after the orthotopic implantation of KoTCC-1 cells, combined treatment with AS clusterin and cisplatin significantly inhibited the growth of primary KoTCC-1 tumors, as well as the incidence of lymph node metastasis. Collectively, these findings demonstrated that clusterin helps confer a chemoresistant phenotype through inhibition of apoptosis and that combined AS clusterin ODN may be useful in enhancing the effects of cytotoxic chemotherapy in patients with bladder cancer.

Bcl-2 antisense oligonucleotides enhance the cytotoxicity of chlorambucil in B-cell chronic lymphocytic leukaemia cells.

We have previously shown that the Bcl-2 antisense oligonucleotide ODN 2009 can induce apoptosis in B-cell chronic lymphocytic leukaemia (B-CLL) cells. In this study we evaluated whether ODN 2009 could increase the sensitivity of B-CLL cells to Chlorambucil-induced cell death in vitro in order to establish whether the notion of antisense-mediated chemosensitisation could be applied to B-CLL. Bcl-2 antisense in combination with Chlorambucil resulted in a more marked reduction in Bcl-2 protein expression (p = 0.003), enhanced Bax expression (p < 0.0001) and increased apoptosis when compared to cells incubated with Chlorambucil alone (p = 0.03). This increased in vitro cytotoxicity demonstrates a proof of the concept that a combination of Bcl-2 antisense oligonucleotides with conventional chemotherapeutic drugs may elicit an enhanced therapeutic effect in B-CLL and should therefore be considered for further investigation in the form of a clinical trial.

Toxicity and toxicokinetics of a phosphorothioate oligonucleotide against the c-myc oncogene in cynomolgus monkeys.

A 2-week toxicity and toxicokinetic study of a 15-mer phosphorothioate oligonucleotide, INX-3280, against the c-myc oncogene was performed in cynomolgus monkeys. As this oligonucleotide readily adopts an aggregate structure, a quadruplex, which may be associated with adverse physiologic effects, this study was performed using INX-3280 that had been converted to its monomeric form. Animals received intravenous (i.v.) infusions of monomeric INX-3280 three times per week for 2 weeks at doses of 3 or 15 mg/kg per administration. The monkeys were examined for clinical signs: changes in hematology, serum chemistry, coagulation, and urinalysis parameters; complement activation; macroscopic findings at necropsy; and histopathologic alterations. In addition, the toxicokinetics of INX-3280 were evaluated, using a validated HPLC assay, after the first and last (sixth) doses. No treatment-related clinical signs of any adverse effects were observed, and there were no test article-related changes in hematology, serum chemistry, or complement activation parameters. The only alteration in clinical pathology parameters was a minor (30%) prolongation of the activated partial thromboplastin time (aPTT), reflecting slight inhibition of the intrinsic coagulation pathway, which was less than that reported with other oligonucleotides given at similar doses. Treatment-related histopathologic alterations consisted of characteristic accumulation of basophilic material in the cytoplasm of tubular epithelial cells in the kidney, resident macrophages in the lymph nodes, and Kupffer cells in the liver. These changes were graded as minimal in all cases. The basophilic material is believed to reflect accumulation of the oligonucleotide or metabolites or both. The pharmacokinetic parameters of INX-3280 were identical on the first and sixth administrations and were similar to those reported for other phosphorothioate oligonucleotides. Maximum concentration (Cmax) values for INX-3280 (101-119 microg/ml) were in excess of the threshold plasma concentrations reported to trigger complement activation by phosphorothioate oligonucleotides. It is concluded that the safety profile of monomeric INX-3280 in cynomolgus monkeys is quite favorable relative to the known effects of other phosphorothioate oligonucleotides, particularly with respect to the blood level-related toxicities of this class of compounds, including complement activation and inhibition of coagulation. This study found no toxicities that were expected to be clinically significant.

Human ovarian cancer and cisplatin resistance: possible role of inhibitor of apoptosis proteins.

The inhibitor of apoptosis proteins (IAPs) constitutes a family of highly conserved apoptosis suppressor proteins that were originally identified in baculoviruses. Although IAP homologs have recently been demonstrated to suppress apoptosis in mammalian cells, their expression and role in human ovarian epithelial cancer and chemotherapy resistance are unknown. In the present study we used cisplatin-sensitive and -resistant human ovarian surface epithelial (hOSE) cancer cell lines and adenoviral antisense and sense complementary DNA expression to examine the role of IAP in the regulation of apoptosis in human ovarian cancer cells and chemoresistance. Antisense down-regulation of X-linked inhibitor of apoptosis protein (Xiap), but not human inhibitor of apoptosis protein-2 (Hiap-2), induced apoptosis in cisplatin-sensitive and, to a lesser extent, in -resistant cells. Cisplatin consistently decreased Xiap content and induced apoptosis in the cisplatin-sensitive, but not cisplatin-resistant, cells. Hiap-2 expression was either unaffected or inhibited to a lesser extent. The inhibition of IAP protein expression and induction of apoptosis by cisplatin was time and concentration dependent. Infection of cisplatin-sensitive cells with adenoviral sense Xiap complementary DNA resulted in overexpression of Xiap and markedly attenuated the ability of cisplatin to induce apoptosis. Immunohistochemical localization of the IAPs in hOSE tumors demonstrated the presence of Xiap and Hiap-2, with their levels being highest in proliferative, but not apoptotic, epithelial cells. These studies indicate that Xiap is an important element in the control of ovarian tumor growth and may be a point of regulation for cisplatin in the induction of apoptosis. These results suggest that the ability of cisplatin to down-regulate Xiap content may be an important determinant of chemosensitivity in hOSE cancer.

Alicaforsen. Isis Pharmaceuticals.

Alicaforsen (ISIS-2302) is an RNase H-dependent antisense inhibitor of the intercellular adhesion molecule ICAM-1 under development by Isis Pharmaceuticals, for the potential treatment of a variety of inflammatory disorders [175741]. As of April 1997 it was in phase III trials for Crohn's disease (CD); however, the trial failed and, in December 1999, the company suspended development for this indication [352801]. In October 2000, the company re-initiated development in CD [384820] and new phase III trials had begin by May 2001 [409704]. In August 2000, phase II studies of alicaforsen in an enema formulation for ulcerative colitis and a topical formulation for psoriasis were ongoing [378715]. Development of the compound for the potential treatment of rheumatoid arthritis (RA) was discontinued in 1999 [347579]. By the end of 1998, alicaforsen was in phase II trials for kidney transplant rejection. At this time, these trials were expected to finish in mid-1999 [343460]. However, they were ongoing in September 1999, although no further development has been reported for this indication since that time [338672]. In February 1995, Isis Pharmaceuticals and Boehringer Ingelheim (BI) signed a collaborative agreement on cell adhesion inhibitors, including alicaforsen [174111]. By early 1999, Isis and BI were to decide on the next developmental step for alicaforsen following further analyses of its performance against CD [292915], [315439]. Their joint development agreement was terminated in 1999; Isis regained rights to the product and by September 1999 was in talks to license alicaforsen to another partner for CD [338672]. In June 2000, Cytogenix entered into a sponsored research agreement with Baylor College of Medicine at the Texas Medical Center Houston for the use of its ssDNA expression system for the development of antisense strategies directed against intercellular adhesion molecules for the purpose of reducing lung inflammation and injury in disease states and conditions [369677]. US-05514788, and other patents, cover antisense cell adhesion molecule inhibitors [212289], [234792].

ISIS-3521. Isis Pharmaceuticals.

ISIS-3521 is a 20-mer antisense phosphorothioate oligonucleotide PKCa expression inhibitor, under development by Isis (formerly in collaboration with Novartis) for the potential treatment of solid tumors that are refractory to, or recurrent with, standard treatment regimens [175741]. In November 1999, Novartis announced that it would end its codevelopment of ISIS-3521 [348221], [348222]. In August 2001, Eli Lilly in-licensed ISIS-3521 [420062]. In October 2000, phase III trials of ISIS-3521, in combination with carboplatin and paclitaxel, were initiated for the treatment of non-small cell lung cancer (NSCLC) [386128]. The FDA granted ISIS-3521 Fast Track review status for NSCLC in November 2000 [388930]. In April 2001, Bear Sterns & Co predicted US approval of ISIS-3521 in 2002 [411081]. In August 2001, Eli Lilly and Isis entered into a four-year strategic alliance that includes ISIS-3521. For the license of ISIS-3521, Isis will receive $25 million in upfront fees and will be reimbursed for remaining phase III development and registration costs [420062].

In vitro and in vivo suppression of growth of rat liver epithelial tumor cells by antisense oligonucleotide against protein kinase C-alpha.

BACKGROUND/AIMS: It has been hypothesized that liver stem cells may be activated and proliferate upon liver injury and may participate in the development of liver cancer. GP7TB, a rat liver epithelial tumor cell line, possesses characteristics of liver stem-like cells and can develop into a tumor in syngeneic Fischer 344 rat. We found that protein kinase C-alpha (PKC-alpha) is overexpressed in GP7TB cells. The importance of PKC-alpha for this liver tumor cell was elucidated. METHODS: Antisense oligonucleotide (ODN) was applied to suppress the production of PKC-alpha in GP7TB cells in vitro and in vivo. Cell viability was measured by acid phosphatase assay. The cellular levels of PKC-alpha and Bcl-2 were monitored by Western-blot analysis. Activation of nuclear factor NF-kappaB was analyzed by electrophoretic mobility shift assay. Cell cycle phase distribution was monitored by FACScan. Cell apoptosis was detected by TUNEL assay and histochemical staining of tumor tissue sections. The in vivo experiment was conducted by implanting tumor mass of GP7TB in the liver of F-344 rat and continuous delivery of the ODN by a mini-osmotic pump. RESULTS: Antisense ODN effectively suppressed the level of PKC-alpha that resulted in the decrease of Bcl-2 and nuclear NF-kappaB. The cumulative viable cells also decreased dramatically for the antisense-treated group. FACScan showed that the cells were arrested at early S-phase. These cells in turn went into apoptosis without completing a cell cycle. It was found that growth of the tumor was suppressed efficiently by antisense ODN. Cell apoptosis was found in the orthotopic tumor. The normal liver cells were not affected. CONCLUSIONS: A lethal effect of depressing the level of PKC-alpha in GP7TB cells and success in suppressing orthotopic tumor growth in vivo suggests that PKC-alpha antisense ODN would be a promising therapeutic agent for some liver cancers.

Taxol and anti-stathmin therapy: a synergistic combination that targets the mitotic spindle.

Stathmin is an abundant cytosolic phosphoprotein that plays an important role in the regulation of cellular proliferation. Its major function is to promote depolymerization of the microtubules that make up the mitotic spindle. Taxol is an effective chemotherapeutic agent whose activity is mediated through stabilization of the microtubules of the mitotic spindle. We demonstrate that antisense inhibition of stathmin expression chemosensitizes K562 leukemic cells to the antitumor effects of Taxol and results in a synergistic inhibition of their growth and clonogenic potential. In the presence of stathmin inhibition, exposure to Taxol results in more severe mitotic abnormalities (hypodiploidy and multinucleation). This, in turn, results in increased apoptosis of the aneuploid cells during subsequent cell division cycles. This novel molecular-based therapeutic approach may provide an effective form of cancer therapy that would avoid the severe toxicities associated with the use of multiple chemotherapeutic agents with overlapping toxicity profiles.

Raf-1 kinase activity predicts for paclitaxel resistance in TP53mut, but not TP53wt human ovarian cancer cells.

We have recently reported that there is a significant Raf-1 kinase dependency of paclitaxel resistance in human cervical tumor cell lines. In light of the possibility that Raf-1 kinase inhibitors could be used to enhance paclitaxel responsiveness in ovarian cancer, we have characterized the Raf-1 kinase dependency of paclitaxel resistance in ovarian cancer cells. The relationship between Raf-1 kinase activity and the sensitivity to clinically relevant paclitaxel concentrations was determined in four ovarian cancer cell lines (CA-OV3, SK-OV3, 2780/WT and OAW42/WT). Furthermore, in recognition that such a drug combination would initially be used in patients whose tumors have recurred following cisplatin/paclitaxel treatment, we also determined the Raf-1 kinase dependency of paclitaxel cytotoxicity in cisplatin resistant variants of two of the ovarian cell lines (2780/CP and OAW42/CP). In the two cell lines (2780/WT and OAW42/WT) that possess a wild-type TP53 (TP53wt), the relationship between Raf-1 kinase activity and paclitaxel resistance was different from that observed in the cervical tumor cell lines. In these cell lines, paclitaxel-induced far more cell killing than would have been predicted from their Raf-1 kinase activity. However, in the ovarian cancer cell lines (CA-OV3, SK-OV3, 2780/CP and OAW42/CP) that have a mutant TP53 (TP53mut), the cytotoxicity induced by 60 nM paclitaxel exhibited the same relationship to Raf-1 kinase activity as previously observed in cervical tumor cell lines. These data suggest that the therapeutic efficacy of paclitaxel in ovarian cancer patient whose tumors have TP53mut might be increased if it is administered in combination with Raf-1 kinase inhibitors, e.g., ISIS 5132.