Targeting Listeria monocytogenes consensus sequence of internalin genes using an antisense molecule.

As an intracellular pathogen, Listeria monocytogenes can enter host cells where it can replicate and escape detection and eradication by the host immune response making the clearance of infection very challenging. Furthermore, with the advent of antimicrobial resistance, the need for alternative targets is inevitable. Internalin proteins are crucial to this bacterium as they contribute to bacterial entry to the systemic circulation. In this study, we targeted a highly conserved region of these proteins by an antisense sequence that was covalently conjugated to the cell penetrating peptides (CPP) to overcome the challenging delivery barriers. Then, we evaluated the efficiency of this construct in vitro. We also assessed the antigenicity, cytotoxicity, and probability of apoptosis induction by this construct. The studied CPP-PNA inhibited bacterial growth and suppressed the mRNA expression of internalins in a dose-dependent manner. In addition, at all studied concentrations, CPP-PNA significantly reduced the invasion rate of L. monocytogenes in the examined cell lines. Moreover, different concentrations of CPP-PNA did not have a significant antigenic, cytotoxic, and apoptotic properties compared to the control. These results suggest the effectiveness of CPP-antisense in targeting the mRNAs of internalins for various research, therapeutic and preventive purposes. However, additional research is required to evaluate the potency, safety, and pharmacokinetics of this compound for the prevention and treatment of listeriosis.

Serial incorporation of a monovalent GalNAc phosphoramidite unit into hepatocyte-targeting antisense oligonucleotides.

The targeting of abundant hepatic asialoglycoprotein receptors (ASGPR) with trivalent N-acetylgalactosamine (GalNAc) is a reliable strategy for efficiently delivering antisense oligonucleotides (ASOs) to the liver. We here experimentally demonstrate the high systemic potential of the synthetically-accessible, phosphodiester-linked monovalent GalNAc unit when tethered to the 5'-terminus of well-characterised 2',4'-bridged nucleic acid (also known as locked nucleic acid)-modified apolipoprotein B-targeting ASO via a bio-labile linker. Quantitative analysis of the hepatic disposition of the ASOs revealed that phosphodiester is preferable to phosphorothioate as an interunit linkage in terms of ASGPR binding of the GalNAc moiety, as well as the subcellular behavior of the ASO. The flexibility of this monomeric unit was demonstrated by attaching up to 5 GalNAc units in a serial manner and showing that knockdown activity improves as the number of GalNAc units increases. Our study suggests the structural requirements for efficient hepatocellular targeting using monovalent GalNAc and could contribute to a new molecular design for suitably modifying ASO.

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.

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.

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.

Potential role for heat shock protein 72 in antagonizing cerebral vasospasm after rat subarachnoid hemorrhage.

BACKGROUND: Cerebral vasospasm can be defined as delayed-onset narrowing of the cerebral arteries that can occur after a spontaneous aneurysmal subarachnoid hemorrhage (SAH). Despite a large number of experimental and clinical investigations, the exact pathophysiology of vasospasm remains unknown. Using a fluorescence differential-display system, we have identified the gene encoding heat shock protein 72 (HSP72) as being highly upregulated by cerebral vasospasm. We therefore elucidated the role of the HSP72 gene in cerebral vasospasm in a rat experimental SAH model. METHODS AND RESULTS: By angiography, cerebral vasospasm was detected from day 1, with maximal narrowing detected on day 2. Intracisternal injection of antisense HSP72 oligodeoxynucleotide led to specific inhibition of HSP72 gene expression and significantly aggravated cerebral vasospasm on days 2 and 3 of the angiographic studies. Oral administration of geranylgeranylacetone (GGA), an antiulcer drug, enhanced HSP72 induction and reduced cerebral vasospasm. CONCLUSIONS: These results suggest HSP72 plays a novel role in antagonizing delayed cerebral vasospasm after SAH and that GGA provides protective effects against delayed cerebral vasospasm, at least partly via induction of HSP72.

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.

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.

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.

Phase I and phase II safety and efficacy trial of intercellular adhesion molecule-1 antisense oligodeoxynucleotide (ISIS 2302) for the prevention of acute allograft rejection.

BACKGROUND: ISIS 2302, an antisense oligonucleotide that inhibits the expression of human intercellular adhesion molecule (ICAM)-1, was evaluated in combination with a cyclosporine (CsA)-prednisone (Pred) regimen first in a phase I safety and pharmacokinetic study and then in a phase II assessment of prophylaxis of acute rejection episodes in deceased donor renal allografts. METHODS: Both phase I and phase II trials were double-blinded and placebo-controlled, including 17 stable and 39 de novo patients, respectively, in time-lagged, ascending-dose regimens. Each study compared the outcomes of 8 alternate-day intravenous infusions of four ISIS 2302 dose levels (0.05, 0.5, 1.0, or 2.0 mg/kg) versus placebo (3:1 ratio). Patients were followed for 34 days (phase I) or 6 months (phase II). All transplant patients were followed for 3 years. RESULTS: ISIS 2302 produced no evident toxicity; a significant, dose-related increase in activated partial thromboplastin time was accompanied by a trend toward a decreased platelet count. ISIS 2302 did not alter the pharmacokinetic behavior of CsA. At 6 months, the rates of acute rejection episodes were 38.1% in the ISIS 2302 group versus 20.0% in the placebo group. Three-year graft survivals were similar. The mean creatinine values at 1, 2, and 3 years for all ISIS dose groups combined versus placebo over 3 years showed no significant differences. CONCLUSIONS: ISIS 2302 did not evoke side-effects and produced slightly improved renal function. However, in this pilot study, it did not further reduce the rate of acute rejection episodes or increase graft survival compared to a concentration-controlled CsA-Pred regimen.

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.

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].

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].

Antisense bcl-2 treatment increases programmed cell death in non-small cell lung cancer cell lines.

Programmed cell death (PCD) is a genetically regulated pathway that is altered in many cancers. This process is, in part, regulated by the ratio of PCD inducers (Bax) or inhibitors (Bcl-2). An abnormally high ratio of Bcl-2 to Bax prevents PCD, thus contributing to resistance to chemotherapeutic agents, many of which are capable of inducing PCD. Non-small cell lung cancer (NSCLC) cells demonstrate resistance to these PCD-inducing agents. If Bcl-2 prevents NSCLC cells from entering the PCD pathway, then reducing the amount of endogenous Bcl-2 product may allow these cells to spontaneously enter the PCD pathway. Our purpose was to determine the effects of bcl-2 antisense treatment on the levels of programmed cell death in NSCLC cells. First, we determined whether bcl-2 and bax mRNA were expressed in three morphologically distinct NSCLC cell lines: NCI-H226 (squamous), NCI-H358 (adenocarcinoma), and NCI-H596 (adenosquamous). Cells were then exposed to synthetic antisense bcl-2 oligonucleotide treatment, after which programmed cell death was determined, as evidenced by DNA fragmentation. Bcl-2 protein expression was detected immunohistochemically. All three NSCLC cell lines expressed both bcl-2 and bax mRNA and had functional PCD pathways. Synthetic antisense bcl-2 oligonucleotide treatment resulted in decreased Bcl-2 levels, reduced cell proliferation, decreased cell viability, and increased levels of spontaneous PCD. This represents the first evidence that decreasing Bcl-2 in three morphologically distinct NSCLC cell lines allows the cells to spontaneously enter a PCD pathway. It also indicates the potential therapeutic use of antisense bcl-2 in the treatment of NSCLC.

Technology evaluation: leukemia therapy, University of Pennsylvania.

The University of Pennsylvania is developing an antisense oligonucleotide (AS ON) as a potential treatmentfor myelogenous leukemia. The 24-mer phosphorothioate (PS) ON targets the c-myb gene (codons 2 to 9), a regulator of transcription. In a pilot study, patient bone marrow was purged with the PS ON before being returned to the patient. In January 1997, it was reported that out of six evaluable patients, four demonstrated marked hematological remission with normalized white blood cell counts. A second phase I trial was initiated, in which myelogenous leukemia patients were treated with systemic infusions of the PS ON at doses of 0.3 to 2.0 mg/kg/dayfor 7 days. By January 1997, 18 patients had been treated, 12 showed stable disease and one patient in blast crisis experienced a transient reversal to the chronic phase of the disease. No dose-related toxicity was noted and c-myb mRNA and protein levels were halved. Preclinical studies in leukemic mice showed that the myb AS PS ON increased survival times 2- to 4-fold and reduced leukemic proliferation in the brain [229790]. The ON was originally developed and patented at Temple University and was being jointly developed by Lynx Therapeutics, however, this collaboration was terminated in 1996 [264351]. New phase I studies are starting in 1999, with INX-3001 (University of Pennsylvania c-myb AS PS) supported by the NIH and Inex Pharmaceuticals Corporation.

Oligonucleotides as inhibitors of human immunodeficiency virus.

Inhibition of human immunodeficiency virus (HIV) replication by oligonucleotides is a complex process and may be implemented by an array of antiviral mechanisms. These include inhibition of virus adsorption to the host cell, inhibition of transcription via antisense or as the result of triple helix formation, and inhibition of viral encoded enzymes such as reverse transcriptase and integrase. Since the particular mechanism of HIV inhibition depends on the oligonucleotide (ON) sequence and the ON chemical modifications, the design and synthesis of potent HIV inhibitors has been an important and rewarding focus of ON research. In this era of great concern that HIV may rapidly display resistance to any antiviral compound with one mechanism of viral inhibition, oligonucleotides are potentially attractive alternatives for therapy. Several ONs have entered clinical evaluation in AIDS patients. At present Zintevir, which inhibits both HIV adsorption and HIV integrase, is in phase I/II clinical trials.

Toward antisense oligonucleotide therapy for cancer: ISIS compounds in clinical development.

Antisense oligonucleotides offer the promise of therapeutic effect with few toxic effects, by virtue of their high selectivity. Preclinical studies have provided evidence of antisense effects in vitro and in vivo, and phase I clinical trials have demonstrated safety, feasibility and activity of antisense oligonucleotides for the treatment of cancer. This review summarizes the status of development of three anticancer antisense oligonucleotides from ISIS Pharmaceuticals.

Preclinical evaluation of the effects of a novel antisense compound targeting C-raf kinase in mice and monkeys.

CGP 69846A (ISIS 5132) is an antisense phosphorothioate oligodeoxynucleotide which targets human C-raf kinase and is currently being developed as an antineoplastic agent. The toxicity of this compound was evaluated in mice and monkeys following repeated i.v. injections or infusions for 4 weeks at doses up to 100 mg/kg. Because CGP 69846A is inactive in the mouse, ISIS 11061, the murine-specific homologue targeting C-raf kinase mRNA was evaluated concurrently with CGP 69846A to assess the potential toxicity associated with reduced C-raf expression. There were no toxicities that differentiated ISIS 11061 from CGP 69846A in mice. Effects in mice included hepatomegaly and hepatocellular degeneration at the high dose of 100 mg/kg CGP 69846A that potentially resulted in lethality. Other effects which were observed at 20 and 100 mg/kg included mononuclear cell infiltrates in multiple organs, extramedullary hematopoiesis in the spleen and liver, an increase in bone marrow cellularity, an increase in white blood cells, a decrease in platelet counts, and Kupffer cell hyperplasia. These alterations were reversible following a recovery period. No adverse effects in mice were observed with doses < or = 10 mg/kg. In monkeys, administration of 10 mg/kg of CGP 69846A was associated with effects observed with other P = S ODNs, namely, prolongation of activated partial thromboplastin time (APTT) and activation of complement. These effects were transient and correlated with plasma concentrations of CGP 69846A. Below a concentration of 35 micrograms/ml of intact CGP 69846A the prolongation of APTT was less than 50% and levels of complement split products were not increased. All monkeys tolerated complement activation with no evidence of treatment-related clinical signs. Complement and coagulation were not affected by the lower doses of 1 and 3 mg/kg. No histopathology or alteration in hematology or serum chemistry was induced by doses up to 10 mg/kg in monkeys. The plasma and tissue deposition of CGP 69846A were characterized in mice and monkeys and toxicity was dependent on dose of CGP 69846A. In the present preclinical evaluation of toxicity in mice and monkeys, CGP 69846A is well tolerated at doses targeted for clinical trials. Toxicities induced by CGP 69846A in monkeys and mice occurred at doses of 10 mg/kg and greater. Effects induced by CGP 69846A were not unique and have been observed previously with other phosphorothioate oligodeoxynucleotides.

Hippocampal administration of kappa-opioid receptor antisense exacerbates isolation-induced hypertension.

Isolation of young male Sprague Dawley rats for 7 days provoked hypertension which was exacerbated by hippocampal administration of an antisense oligodeoxynucleotide targeted to the kappa-opioid receptor (1 microg/0.5 microL bilaterally, twice daily, during the isolation or grouping period). Systolic blood pressure rose from a mean of 134 to 162 mmHg in isolated rats treated with antisense and only 139 to 151 mmHg in grouped rats treated with antisense. In grouped rats treated with either vehicle or missense, isolation caused a mean increase in systolic blood pressure of only 16 and 14 mmHg respectively. Neither the missense oligodeoxynucleotide nor the vehicle had any significant effects upon systolic pressure in grouped rats, which had not been isolated. Pharmacological studies indicated that rats previously treated with the antisense had no significant depressor response to U62, 066E (a non-peptide kappa agonist known to reduce blood pressure acutely when administered into the hippocampus), however rats previously treated with vehicle or missense exhibited a significant hypotensive response to the drug. This implies that the antisense had reduced the density or activity of the kappa receptors within the hippocampal formation. These data are in accordance with our previous studies, i.e. in several rat models of hypertension, the increase in blood pressure may be modulated via hippocampal kappa opioid receptors.

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.