Metronomic S-1 dosing and thymidylate synthase silencing have synergistic antitumor efficacy in a colorectal cancer xenograft model.

Metronomic chemotherapy is currently considered an emerging therapeutic option in clinical oncology. S-1, an oral formulation of Tegafur (TF), a prodrug of 5-fluorouracil (5-FU), is designed to improve the antitumor activity of 5-FU in tandem with reducing its toxicity. Clinically, metronomic S-1 dosing has been approved for the standard first- and second-line treatment of metastatic or advanced stage of colorectal (CRC). However, expression of intratumor thymidylate synthase (TS), a significant gene in cellular proliferation, is associated with poor outcome to 5-FU-based chemotherapeutic regimens. In this study, therefore, we examined the effect of a combination of TS silencing by an RNA interfering molecule, chemically synthesized short hairpin RNA against TS (shTS), and 5-FU on the growth of human colorectal cancer cell (DLD-1) both in vitro and in vivo. The combined treatment of both shTS with 5-FU substantially inhibited cell proliferation in vitro. For in vivo treatments, the combined treatment of metronomic S-1 dosing with intravenously injected polyethylene glycol (PEG)-coated shTS-lipoplex significantly suppressed tumor growth, compared to a single treatment of either S-1 or PEG-coated shTS-lipoplex. In addition, the combined treatment increased the proportion of apoptotic cells in the DLD-1 tumor tissue. Our results suggest that metronomic S-1 dosing combined with TS silencing might represent an emerging therapeutic strategy for the treatment of patients with advanced CRC.

The co-delivery of oxaliplatin abrogates the immunogenic response to PEGylated siRNA-lipoplex.

PURPOSE: In vivo application of siRNA/PEGylated cationic liposome complex (lipoplex) is impeded by two main obstacles: cytokine responses and anti-PEG IgM responses to PEGylated siRNA-lipoplex. Here, we investigated whether co-administration of oxaliplatin (l-OHP) abrogates the cytokine release and anti-PEG IgM production by PEGylated siRNA-lipoplex. METHODS: Free l-OHP was administered either simultaneously or 30 min prior to PEGylated siRNA-lipoplex administration, and cytokine response and anti-PEG IgM production were evaluated. In addition, the effect of the liposomal encapsulation of l-OHP on the immunogenic response of PEGylated siRNA-lipoplex was investigated. RESULTS: Simultaneous co-administration of free l-OHP with PEGylated siRNA-lipoplex caused a significant reduction in anti-PEG IgM production, along with an increase in the cytokine response. Free l-OHP injected prior to the lipoplex injection, however, successfully reduced cytokine release and anti-PEG IgM response. Platination of siRNA by simultaneously administered free l-OHP might facilitate the dissociation of double-stranded siRNA to single-stranded siRNA, resulting in the inducement of a potent immuno-stimulation of siRNA via endosomal toll-like receptors (TLRs). On the other hand, encapsulation of l-OHP into the siRNA-lipoplex resulted in a reduction of both anti-PEG IgM production and cytokine responses. CONCLUSIONS: Our results suggest that, besides the expected therapeutic efficacy of co-administration, encapsulation of l-OHP into the PEGylated siRNA-lipoplex has great potential for minimizing the immunostimulation of PEGylated siRNA-lipoplex, resulting in a safe, applicable, and compliant treatment regimen for sequential clinical administration.

Anti-PEG IgM production via a PEGylated nano-carrier system for nucleic acid delivery.

For the systemic application of nucleic acids such as plasmid DNA and small interfering RNA, safe and efficient carriers that overcome the poor pharmacokinetic properties of nucleic acids are required. A cationic liposome that can formulate lipoplexes with nucleic acids has significant promise as an efficient delivery system in gene therapy. To achieve in vivo stability and long circulation, most lipoplexes are modified with PEG (PEGylation). However, we reported that PEGylated liposomes lose their long-circulating properties when they are injected repeatedly at certain intervals in the same animal. This unexpected and undesirable phenomenon is referred to as the accelerated blood clearance (ABC) phenomenon. Anti-PEG IgM produced in response to the first dose of PEGylated liposomes has proven to be a major cause of the ABC phenomenon. Therefore, in a repeated dosing schedule, the detection of anti-PEG IgM in an animal treated with PEGylated lipoplex could be essential to predict the occurrence of the ABC phenomenon. This chapter introduces a method for the evaluation of serum anti-PEG IgM by a simple ELISA procedure, and describes some precautions associated with this method.

Anti-angiogenic therapy via cationic liposome-mediated systemic siRNA delivery.

siRNA has been touted as a therapeutic molecule against genetic diseases, which include cancers. But several challenging issues remain in order to achieve efficient systemic siRNA delivery and a sufficient therapeutic effect for siRNA in vivo. Cationic liposome shows promise as a carrier for nucleic acids, as it can selectively bind to angiogenic tumor blood vessels. In this way, anti-angiogenic therapy via cationic liposome-mediated systemic siRNA delivery could be achieved in cancer therapy. In the present study, we proved our assumption by preparing various kinds of polyethylene glycol (PEG)-coated siRNA/cationic liposome complexes (siRNA-lipoplexes) and screening the avidity of these siRNA-lipoplexes upon angiogenic tumor blood vessels by means of a murine dorsal air sac (DAS) model. The lipoplex, having a lipid composition of DC-6-14/POPC/CHOL/DOPE/mPEG(2000)-DSPE=20/30/30/20/5 (molar ratio) and a charge ratio of cationic liposome and siRNA=3.81 (+/-), showed a higher binding index to newly formed blood vessels. Systemic injection with the lipoplex containing siRNA for the Argonaute2 gene (apoptosis-inducible siRNA) resulted in significant anti-tumor effect without severe side effects in mice with Lewis lung carcinoma. Our results indicate that the PEGylated cationic liposome-mediated systemic delivery of cytotoxic siRNA achieves anti-angiogenesis, resulting in the suppression of tumor growth.

Improved intratumoral delivery of PEG-coated siRNA-lipoplexes by combination with metronomic S-1 dosing in a murine solid tumor model.

Efficient systemic siRNA delivery to cells in the target tissue is a current critical challenge in the drug delivery field. Several studies have demonstrated that nanoparticles such as polyethylene glycol (PEG)-coated siRNA-lipoplexes may enhance the systemic delivery of siRNA to tumor. However, the disordered tumor microenvironment still poses a potential impediment with respect to the efficient delivery of PEG-coated siRNA-lipoplexes. Recently, we showed that metronomic S-1 dosing (daily oral administration) enhanced the accumulation of PEG-coated siBcl-2-lipoplex in DLD-1 solid tumor mouse model. In this study, to extend our work, we investigated the effect of metronomic S-1 dosing on the intratumoral accumulation and, thereby, therapeutic efficacy of PEG-coated siAgo2-lipoplex in Lewis lung carcinoma cells (LLCC) solid tumor mouse model. Also, we tried to elucidate the probable mechanism of the enhanced intratumoral accumulation of PEG-coated siRNA-lipoplexes induced by S-1 combination therapy. Results showed that metronomic S-1 dosing improved systemic delivery of intravenously injected PEG-coated siAgo2-lipoplexes into a LLCC solid tumor. In addition, the combined therapy of S-1 and PEG-coated siRNA-lipoplexes resulted in potent tumor growth suppression. These findings offer proof-of-concept for the improved systemic delivery of PEG-coated siRNA-lipoplexes by metronomic S-1 dosing in whatever tumor model used, and this may pose a promising therapeutic strategy to conquer cancer progression.

Anti-PEG IgM production by siRNA encapsulated in a PEGylated lipid nanocarrier is dependent on the sequence of the siRNA.

We recently reported that the prolonged circulation property of PEGylated cationic liposomes containing nucleic acids disappears, if the second dose is injected within a few days later, due to the production of anti-PEG IgM. This accelerated blood clearance is a concern for treating diseases which require repeated treatment with a PEGylated formulation containing nucleic acids. In this study, we investigated the effect of encapsulation of siRNA in a recently introduced PEGylated lipid nanocarrier for which the term "wrapsome" (PEGylated wrapsome, PEG-WS) was proposed as well as the sequence of the encapsulated siRNA on anti-PEG IgM production. siRNA encapsulated in PEG-WS produced little anti-PEG IgM relative to siRNA in conventional PEGylated lipoplexes. The sequence of siRNA in the PEG-WL dramatically affected the anti-PEG IgM production; a potent immune stimulatory siRNA induced a higher anti-PEG IgM production. Such enhanced effect was abrogated by incorporation of 2'-O-methyl (2'-OMe) uridine into the sequence of siRNA, probably via inhibiting cytokine induction such as IL-6 and TNF-α. Our results strongly indicate that the use of an encapsulation-type lipid nanocarrier with a low immuno-stimulatory siRNA may allow repeated dosing of siRNA containing PEGylated formulations without the induction of a strong immune reaction against PEG and thus may advance synthetic siRNA into a broad range of therapeutic applications.