Antisense properties of 2'-O-dimethylaminooxyethyl (2'-O-DMAOE) oligonucleotides.

Antisense oligonucleotides with 2'-O-(2-[N,N-dimethyl)aminooxy]ethyl) or (2'-O-DMAOE) modification were synthesized and evaluated for nuclease resistance and pharmacology both in vitro and in vivo. This modification exhibits very high nuclease resistance and efficacy in various biological (ICAM-1, C-raf and PKC-alpha) targets.

A Phase I trial of H-ras antisense oligonucleotide ISIS 2503 administered as a continuous intravenous infusion in patients with advanced carcinoma.

BACKGROUND: Abnormal expression of Ras proteins frequently is found with oncogenic transformation making ras a promising therapeutic target. ISIS 2503 is a 20-base antisense phosphorothioate oligodeoxyribonucleotide that specifically downregulates H-ras expression and inhibits tumor cell growth in preclinical studies. Here, the authors report an initial clinical study of the safety and tolerability of an intravenous infusion of ISIS 2503 in patients with advanced cancer. METHODS: A continuous intravenous infusion of ISIS 2503 was administered for 14 days every 3 weeks to 23 patients with a variety of solid tumors refractory to standard therapy. The dose of ISIS 2503 was increased in sequential cohorts of patients, as toxicity allowed, until a final dose of 10.0 mg/kg/day of body weight was reached. Toxicity was scored by the National Cancer Institute's Common Toxicity Criteria, and tumor response was monitored after every two treatment cycles. Pharmacokinetic studies were performed in some of the patients up to, and including, the final dose of 10 mg/kg/day/day of body weight. Levels of H-ras mRNA expression also were determined in the circulating lymphocytes of some patients by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: A total of 23 patients received 63 cycles of ISIS 2503 at escalating doses to 10.0 mg/kg/day without dose-limiting toxicity and only minimal side effects. Four patients had stabilization of their disease for 6-10 cycles. No consistent decreases in H-ras mRNA levels were observed in peripheral blood lymphocytes. CONCLUSIONS: ISIS 2503, an antisense oligonucleotide against H-ras, was well tolerated as a single agent at doses up to 10.0 mg/kg/day by 14-day continuous intravenous infusion. Several patients had stabilization of disease, suggesting that ISIS 2503 had some tumor growth inhibitory effects and future trials of ISIS 2503 in combination with chemotherapy should be considered.

Detection of progesterone receptor forms A and B by immunohistochemical analysis.

AIM: The measurement of progesterone receptors (PR) is recommended as part of the clinical management of breast and endometrial cancers, and immunohistochemistry on formalin fixed tissue is now the method of choice. PR is expressed as two isoforms, PRA and PRB, and although both these proteins are expressed in hormone dependent cancers, there is evidence that a large proportion of tumours express a predominance of one isoform. Therefore, it is essential to document the individual detection of PRA and PRB by the presently available anti-PR antibodies. The aim of this study is to investigate the detection of PR isoforms A and B in formalin fixed, paraffin wax embedded cell lines and tissue sections by immunohistochemistry, using a panel of commercial and in house antibodies to human PR. METHODS: PR negative cell lines stably transfected to express only PRA (MCF-7Mll/PRA) or PRB (MDA-MB-231/PRB), and tissue sections of human breast carcinoma and normal endometrium were stained using an immunoperoxidase method. A panel of primary PR specific antibodies was evaluated for ability to detect both PRA and PRB proteins, and for intensity and distribution of positive staining under optimal conditions. RESULTS: Of the 11 antibodies assessed, only four recognised PRA and PRB similarly. Six recognised PRA proteins but were unable to detect PRB expression in the cell lines expressing only PRA or PRB. In tissues expressing high amounts of PRA and PRB, all antibodies tested demonstrated positive PR staining. However, in tissues expressing a predominance of PRB, differential staining patterns were observed, with variations in staining intensity and in the proportion of cells positive for PR. CONCLUSIONS: Most PR specific antibodies tested failed to detect PRB in formalin fixed tissue by immunohistochemical techniques, despite their ability to do so by immunoblot analysis. These observations suggest that there are conformational differences between PRA and PRB that mask epitopes on the PRB protein recognised by most anti-PR antibodies. The selection of antibodies that recognise both PRB and PRA in formalin fixed tissue is essential for the accurate evaluation of PR positivity in clinical specimens.

Phase I Trial of ISIS 5132, an antisense oligonucleotide inhibitor of c-raf-1, administered by 24-hour weekly infusion to patients with advanced cancer.

Raf-1 is a serine/threonine kinase that functions as a critical effector of Ras-mediated signal transduction via the mitogen-activated protein kinase pathway. Constitutive activation of this pathway directly contributes to malignant transformation in many human tumors. A 20-base phosphorothioate oligonucleotide complementary to c-raf-1 mRNA (ISIS 5132; CGP 69846A) has been shown to specifically suppress Raf-1 expression both in vitro and in vivo. This Phase I trial, involving 22 patients with advanced cancer, was designed to evaluate the safety, feasibility, and maximum tolerated dose of ISIS 5132 administration as a weekly 24-h i.v. infusion. Pharmacokinetic analysis was performed, and c-raf-1 mRNA levels in peripheral blood mononuclear cells were assessed using quantitative reverse transcription-PCR. This trial defined a maximum tolerated dose of 24 mg/kg/week on this schedule. Two of four patients treated at 30 mg/kg/week had serious adverse events after the first dose of ISIS 5132, including acute hemolytic anemia and acute renal failure and anasarca. There were no major responses documented. Dose-dependent complement activation was demonstrated on this schedule, but not on previously evaluated schedules, of ISIS 5132 administration. In contrast to other trials of ISIS 5132, there appeared to be no consistent suppression of peripheral blood mononuclear cell c-raf-1 mRNA level on this schedule at any of the dose levels analyzed. These data suggest that the efficacy and toxicity profiles of antisense oligonucleotides may be highly dependent on the schedule of administration and support the analysis of the putative molecular target in the evaluation of novel therapeutics.

Inhibition of C-raf expression by antisense oligonucleotides extends heart allograft survival in rats.

BACKGROUND: C-raf is a well-characterized serine/ threonine (Ser/Thr) protein kinase that is involved in the transduction of multiple signals of T cells. We demonstrate that the inhibition of C-raf mRNA expression prolongs heart allograft survival. METHODS: Three 20-mer C-raf antisense oligonucleotides, each with identical sequences, were synthesized with different chemical modifications: one as a uniform phosphorothioate oligodeoxynucleotide (PS oligo), a second with a PS backbone and 2'-methoxyethyl (ME) substitutions at the 2'-sugar positions in the first and last five nucleotides, and a third with a mixed PS and phosphodiester (PD) backbone and ME modifications on the first and last five nucleotides. RESULTS: Both ME-modified C-raf antisense oligos were at least 5-fold more effective than the PS C-raf antisense oligo in blocking C-raf mRNA expression in two cell lines. Similarly, each of the ME C-raf antisense oligos produced better heart allograft survival rates than did PS C-raf oligo. Furthermore, although the combination of PS C-raf antisense oligo with sirolimus (SRL) acted synergistically to extend heart allograft survival, the effect was potentiated by either of the ME-modified oligos. CONCLUSIONS: C-raf inhibition extends heart allograft survival, and ME-modification potentiates antisense activity.

Biophysical and antisense properties of oligodeoxynucleotides containing 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines.

The synthesis of 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines is described. The nucleosides were synthesized, functionalized into the phosphoramidites and incorporated into oligodeoxynucleotides. Spectroscopic melting experiments against complementary RNA showed increases of 3-4 degreesC per modification for single substitutions and smaller increases per incorporation for multiple substitutions relative to unmodified control sequences. The 7-propyne and 7-iodo nucleosides were incorporated into antisense sequences targeting the 3'-UTR of murine C- raf mRNA. Both nucleosides demonstrated substitution-dependent potency. The sequences with three and four substitutions of the 7-propyne-7-deaza-2-amino-2'-deoxyadenosine exhibited a 2-3-fold increase in potency over unmodifed controls.

Selective inhibition of A-Raf and C-Raf mRNA expression by antisense oligodeoxynucleotides in rat vascular smooth muscle cells: role of A-Raf and C-Raf in serum-induced proliferation.

Raf kinases, cytoplasmic serine/threonine protein kinases, have been proposed as important participants in mitogen-induced signal transduction. However, the precise role that Raf kinase isozymes play in cellular responses such as proliferation has not been resolved. The present study investigates the ability of antisense phosphorothioate oligodeoxynucleotides (ODNs), targeted against rat C-Raf and A-Raf kinases, to reduce gene expression and proliferation of cultured rat A10 smooth muscle cells (SMCs). Exposure of A10 cells to ISIS 11061, an active C-Raf antisense ODN, resulted in a potent, dose-dependent inhibition (IC50 = 55 nM) of C-Raf mRNA and protein expression. This inhibition was completely dependent on ODN sequence because the incorporation of increasing numbers of mismatches (up to six) into the sequence resulted in sequential loss of potency. Similarly, a dose-dependent reduction (IC50 = 125 nM) in A-Raf gene expression was observed after treatment of cells with the active A-Raf ODN, ISIS 9069, whereas two scrambled controls were without effect. These results demonstrate that ISIS 11061 and ISIS 9069 reduced gene expression in a sequence-specific and isozyme-specific manner. Moreover, administration of ISIS 11061 and ISIS 9069 to rat SMCs resulted in a significant and potent diminution of serum-induced proliferation with corresponding IC50 values of 216 and 273 nM, respectively. Taken together, these results indicate that A-Raf and C-Raf kinases play an important role in regulating vascular SMC proliferation and that antisense-mediated inhibition of Raf kinase activity may serve as a therapeutic modality in the treatment of vascular proliferative disorders.

Sequence-specific antitumor activity of a phosphorothioate oligodeoxyribonucleotide targeted to human C-raf kinase supports an antisense mechanism of action in vivo.

To determine the mechanism of action responsible for the in vivo antitumor activity of a phosphorothioate antisense inhibitor targeted against human C-raf kinase (ISIS 5132, also known as CGP69846A), a series of mismatched phosphorothioate analogs of ISIS 5132 or CGP69846A were synthesized and characterized with respect to hybridization affinity, inhibitory effects on C-raf gene expression in vitro, and antitumor activity in vivo. Incorporation of a single mismatch into the sequence of ISIS 5132 or CGP69846A resulted in reduced hybridization affinity toward C-raf RNA sequences and reduced inhibitory activity against C-raf expression in vitro and tumor growth in vivo. Moreover, incorporation of additional mismatches resulted in further loss of in vitro and in vivo activity in a manner that correlated well with a hybridization-based (i.e., antisense) mechanism of action. These results provide important experimental evidence supporting an antisense mechanism of action underlying the in vivo antitumor activity displayed by ISIS 5132 or CGP69846A.

Destabilization of Raf-1 by geldanamycin leads to disruption of the Raf-1-MEK-mitogen-activated protein kinase signalling pathway.

The serine/threonine kinase Raf-1 functions downstream of Rats in a signal transduction cascade which transmits mitogenic stimuli from the plasma membrane to the nucleus. Raf-1 integrates signals coming from extracellular factors and, in turn, activates its substrate, MEK kinase. MEK activates mitogen-activated protein kinase (MAPK), which phosphorylates other kinases as well as transcription factors. Raf-1 exists in a complex with HSP90 and other proteins. The benzoquinone ansamycin geldanamycin (GA) binds to HSP90 and disrupts the Raf-1-HSP90 multimolecular complex, leading to destabilization of Raf-1. In this study, we examined whether Raf-1 destabilization is sufficient to block the Raf-1-MEK-MAPK signalling pathway and whether GA specifically inactivates the Raf-1 component of this pathway. Using the model system of NIH 3T3 cells stimulated with phorbol 12-myristate 13-acetate (PMA), we show that GA does not affect the ability of protein kinase C alpha to be activated by phorbol esters, but it does block activation of MEK and MAPK. Further, GA does not decrease the activity of constitutively active MEK in transiently transfected cells. Finally, disruption of the Raf-1-MEK-MAPK signalling pathway by GA prevents both the PMA-induced proliferative response and PMA-induced activation of a MAPK-sensitive nuclear transcription factor. Thus, we demonstrate that interaction between HSP90 and Raf-1 is a sine qua non for Raf stability and function as a signal transducer and that the effects observed cannot be attributed to a general impairment of protein kinase function.

Nuclease resistance and antisense activity of modified oligonucleotides targeted to Ha-ras.

We have previously described structure-activity studies on a 17-mer uniform phosphorothioate antisense sequence targeted to human Ha-ras. In an effort to further improve the pharmacological properties of antisense oligonucleotides, structure-activity studies on this 17-mer sequence were expanded to examine both the effects of replacing phosphorothioate backbone linkages with phosphodiester linkages and the effects of incorporating various 2'-sugar modifications into phosphorothioate and phosphodiester oligonucleotides on oligonucleotide stability against nucleases in vitro and on antisense activity in cells. Replacement of three or more phosphorothioate linkages with phosphodiester linkages greatly compromised both nuclease resistance and antisense activity, and these effects correlated directly with the number of phosphodiester linkages incorporated into the oligonucleotide. However, substantial nuclease resistance, sufficient for obtaining potent antisense effects in cells, was conferred to phosphodiester oligonucleotides by incorporation of appropriate 2'-alkoxy sugar modifications. Nuclease stability and antisense activity imparted by these sugar modifications in phosphodiester backbones correlated with the size of the 2'-alkoxy substituent (pentoxy > propoxy > methoxy > deoxy). Furthermore, antisense activity mediated by oligonucleotides that exhibit partial resistance to nucleolytic degradation was dependent on both oligonucleotide concentration and the duration of oligonucleotide treatment.

Antitumor activity of a phosphorothioate antisense oligodeoxynucleotide targeted against C-raf kinase.

Substantial evidence exists supporting a direct role for raf kinases in the development and maintenance of certain human malignancies. Here we test the potential of phosphorothioate antisense oligodeoxynucleotides targeted against human C-raf-1 kinase to specifically inhibit C-raf-1 kinase gene expression and tumor progression in cell culture and in vivo, using human tumor xenograft mouse models. Treatment of human tumor cells with appropriate phosphorothioate antisense oligodeoxynucleotides led to specific inhibition of C-raf kinase gene expression in cell culture and in vivo at well-tolerated doses. Moreover, oligodeoxynucleotide treatment resulted in potent antiproliferative effects in cell culture and potent antitumor effects in vivo against a variety of tumor types that were highly consistent with an antisense mechanism of action for these compounds. These studies strongly suggest that antisense inhibitors targeted against C-raf-1 kinase may be of considerable value as antineoplastic agents that display activity against a wide spectrum of tumor types at well-tolerated doses.

Selective inhibition of mutant Ha-ras mRNA expression by antisense oligonucleotides.

A biological reporter gene assay was employed to determine the crucial parameters for maximizing selective targeting of a Ha-ras codon 12 point mutation (G----T) using phosphorothioate antisense oligonucleotides. We have tested a series of oligonucleotides ranging in length between 5 and 25 bases, each centered around the codon 12 point mutation. Our results indicate that selective targeting of this point mutation can be achieved with phosphorothioate antisense oligonucleotides, but this selectivity is critically dependent upon oligonucleotide length and concentration. The maximum selectivity observed in antisense experiments, 5-fold for a 17-base oligonucleotide, was closely predicted by a simple thermodynamic model that relates the fraction of mutant to wild type target bound as a function of oligonucleotide concentration and affinity. These results suggest thermodynamic analysis of oligonucleotide/target interactions is useful in predicting the specificity that can be achieved by an antisense oligonucleotide targeted to a single base point mutation.

The characterization of atrial natriuretic peptide (ANP) expression by primary cultures of atrial myocytes using an ANP-specific monoclonal antibody and an ANP messenger ribonucleic acid probe.

The biochemical and morphological characteristics of primary neonatal rat atrial myocytes were examined in order to establish a model system for future studies of the biosynthesis and secretion of atrial natriuretic peptide (ANP). Preliminary studies demonstrated that the quantity of immunoactive ANP/microgram protein within rat atria increased as a function of age from 2 ng/micrograms in 19 day prenatal animals to 400 ng/micrograms in the adult. Gel filtration, reversed phase HPLC, and ion exchange HPLC indicated that there were similar quantities of immunoactive ANP in the right and left atria at various ages, and that the major molecular form of the peptide in the heart is chromatographically indistinguishable from ANP(1-126). Cultures of dissociated cells were prepared from pooled left and right atria derived from 1 day postnatal animals. A complete serum-free medium was developed which resulted in the maintenance of high levels of immunoactive ANP in the cultures. As determined by RIA, the cellular content of ANP increased in the cultures as a function of time through 7 days in vitro. The quantity of immunoactive ANP in the cultures increased approximately 2- to 3-fold between days 3 and 7. When the cultures that had been maintained for 7 days were submitted to immunocytochemistry using an ANP-specific monoclonal antibody, distinct colonies of spindle-shaped cells stained positively. In situ hybridization, utilizing an 35S-labeled ANP messenger RNA probe, demonstrated that these colonies of myocytes expressed the ANP message. Using quantitative dot-blot hybridization it was shown that the ANP mRNA level increased approximately 50-fold between days 1 and 7 in culture. These studies indicate that the serum-free culture medium allows continued accumulation of both ANP and the ANP message in culture and will provide a useful model system to characterize factors that regulate the biosynthesis and secretion of this hormone.