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

Abstract

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.Unequivocal proof of the in vivo mechanism of action for any drug has always been one of the greatest challenges facing the molecular pharmacologist. This challenge is mainly due to the difficulty in obtaining sound experimental evidence for drug mechanisms in complex animal models or in humans. Indeed, the mechanisms of action for many drugs that are used routinely in the clinic today with significant therapeutic benefit are poorly characterized. Unfortunately, without a clear understanding of a particular drug's mechanism in vivo, improvement in the drug's pharmacological properties and advances in the discovery of new drug classes based on novel mechanisms of action will be greatly impeded.Antisense oligonucleotides represent a new paradigm for drug discovery that holds great promise for the delivery of potent and specific drugs with fewer undesired side effects (reviewed in refs. 1 and 2). This paradigm offers the opportunity to rapidly identify lead compounds based upon knowledge of the biology of a disease process. With this knowledge, the practitioner of antisense drug discovery can rapidly design, synthesize, and test a series of compounds in animal models and, because of the rational manner by which these compounds are designed to act, can experimentally address their mechanism of action in vivo with a relatively high degree of confidence. The most compelling evidence to support antisense mechanisms in living systems are (i) specific reduction in target gene expression and (ii) gradual reduction in drug potency as a result of incorporating increasing numbers of nonhybridizing bases (mismatches) into the oligonucleotide (i.e., rank order potency based on hybridization affinity). However, despite the fact that oligonucleotides have been shown to exert potent pharmacological activity in a variety of animal models, rigorous analyses providing strong experimental evidence to support an antisense mechanism of action underlying in vivo pharmacological activity have...

Novel cationic cardiolipin analogue-based liposome for efficient DNA and small interfering RNA delivery in vitro and in vivo

“…As a member of the serine/ threonine protein kinase family, c-raf has been shown to play a key role in mitogen-activated protein kinase signaling cascade 30 and involve in tumor progression. 31,32 Inhibiting the expression of c-raf with antisense oligonucleotides has been shown to suppress the tumor growth in vitro and in xenograft tumor models. 32,33 To test siRNAmediated suppression of c-raf expression using CCLAbased liposome, siRNA was prepared to target c-raf in cultured cancer cells and in mice bearing tumor.…”

“…31,32 Inhibiting the expression of c-raf with antisense oligonucleotides has been shown to suppress the tumor growth in vitro and in xenograft tumor models. 32,33 To test siRNAmediated suppression of c-raf expression using CCLAbased liposome, siRNA was prepared to target c-raf in cultured cancer cells and in mice bearing tumor. A549, PC-3, MDA-MB-231 and SK-OV-3 cells were transfected with siRNA alone, or CCLA-based liposome alone, or the combination of both.…”

Raf kinases: Oncogenesis and drug discovery

“…132 The antitumor activity of ISIS 5132, a 20-nucleotide phosphorothioate 2 0 deoxynucleotide targeting the 3 0 untranslated region of C-RAF mRNA, was very promising at the early preclinical stage. 133,134 However, no major clinical benefits were observed in several Phase I [135][136][137] or Phase II clinical trials. [138][139][140][141] In addition, the use of ISIS 13650, a second generation antisense oligonucleotide with further modifications in the sugar moiety targeting the same C-RAF sequence as ISIS 5132, has not been superior in certain preclinical settings.…”

Ras Family Signaling: Therapeutic Targeting

“…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. 79 A liposomal formulation of antisense c-Raf-1 to improve pharmacodynamics has also been evaluated. 80 These agents are currently under assessment in phase II clinical trials; to date, no efficacy has been reported.…”