Anticancer activity of the intraperitoneal-delivered DFP-10825, the cationic liposome-conjugated RNAi molecule targeting thymidylate synthase, on peritoneal disseminated ovarian cancer xenograft model

Abstract

Introduction: Peritoneal disseminated ovarian cancer is one of the most difficult cancers to treat with conventional anti-cancer drugs and the treatment options are very limited, although an intraperitoneal (ip) paclitaxel has shown some clinical benefit. Therefore, treatment of peritoneal disseminated ovarian cancer is a highly unmet medical need and it is urgent to develop a new ip delivered drug regulating the fast DNA synthesis.

Methods: We developed a unique RNAi molecule consisting of shRNA against the thymidylate synthase (TS) and a cationic liposome (DFP-10825) and tested its antitumor activity and PK profile in peritoneally disseminated human ovarian cancer ascites models by the luciferase gene-transfected SCID mice. DFP-10825 alone, paclitaxel alone or combination with DFP-10825 and paclitaxel were administered in an ip route to the tumor-bearing mice. The TS expression level was measured by conventional RT-PCR. The anti-tumor activity and host survival benefit by DFP-10825 treatment on tumor-bearing mice were observed as resulting from the specific TS mRNA knock-down in tumors.

Results: DFP-10825 alone significantly suppressed the growth of SKOV3-luc tumore ascites cells and further extended the survival time of these tumor-bearing mice. Combination with the ip paclitaxel augmented the antitumor efficacy of DFP-10825 and significantly prolonged the survival time in the tumor-bearing mice. Short-hairpin RNA for TS (TS shRNA) levels derived from DFP-10825 in the ascetic fluid were maintained at a nM range across 24 hours but not detected in the plasma, suggesting that TS shRNA is relatively stable in the peritoneal cavity, to be able to exert its anti-tumor activity, but not in blood stream, indicating little or no systemic effect.

Conclusion: Collectively, the ip delivery of DFP-10825, TS shRNA conjugated with cationic liposome, shows a favorable antitumor activity without systemic adverse events via the stable localization of TS shRNA for a sufficient time and concentration in the peritoneal cavity of the peritoneally disseminated human ovarian cancer-bearing mice.

Keywords: DFP-10825; cationic liposome; intraperitoneal dissemination; ip delivery; ovarian cancer; short-hairpin RNA; thymidylate synthase.

Figure 1

Newly designed structure of RNAi molecule for thymidylate synthase (TS shRNA) and preparation of DFP-10825. Notes: TS shRNA is composed of 19 mer sense sequence, 15 mer loop sequence and 19 mer anti-sense sequence. The preparation of DFP-10825 from TS shRNA, and shRNA entrapment and retention of the DFP-10825 after the preparation of cationic liposome (lipoplex). As the particle stability for the therapeutic molecules conjugation, the free TS shRNA in the formulation (TS shRNA-lipoplexes, DFP-10825) of 2.0 mg/kg (as shRNA) was checked by agarose gel electrophoresis.

Figure 2

Detection range of standard short-hairpin RNA for thymidylate synthase (TS shRNA) in ascites and plasma in mice bearing intraperitoneally disseminated ovarian cancer cells. Notes: TS shRNA was linearly detected at the range of 15 pM to 228 nM in the ascites and at 0.6 pM to 228 nM in the plasma. ΔCt (dR) was calculated as follows: ΔCt (dR) = Ct (TS shRNA) - Ct (18S rRNA).

Figure 3

Dose-dependent antitumor activity and toxicity of DFP-10825 on peritoneal disseminated model of SKOV3-luc human ovarian cancer xenografted into NOD/SCID mice. Notes: After intraperitoneal inoculation of SKOV3-luc cells (1×10⁷) into 7–8 mice, 0.5, 1 and 2 mg/kg of short-hairpin RNA for thymidylate synthase (TS shRNA; with 2,000-fold of lipoplex as molar basis) were intraperitoneally administered, respectively, by schedules of q3d ×4. On day 7, 14, 21 and 28, bioluminescent signals in mice were monitored and antitumor activity of each dose was evaluated. Abbreviations: BLI, bioluminescence imaging; q3d, every 3 days.

Figure 4

Tumor bioluminescent signals in mice-bearing SKOV3-luc tumor cells treated with DFP-10825, paclitaxel and their combination. Notes: After intraperitoneal inoculation of SKOV3-luc cells (1×10⁷) into 10 mice, DFP-10825 (1 mg/kg of short-hairpin RNA for thymidylate synthase [TS shRNA] with 2,000-fold of lipoplex as molar basis), paclitaxel (15 mg/kg) and their combination were intraperitoneally administered, respectively, by schedules of every 3rd day for four doses. On day 7, 14, 21 and 26, bioluminescent signals in each group were monitored.

Figure 5

Growth inhibitory effect and body weight change of DFP-10825, paclitaxel and their combination on intraperitoneal disseminated SKOV3-luc tumor xenografts in mice. Notes: From day 7 after intraperitoneal (ip) implantation of tumor cells, DFP-10825 (1 mg/kg of short-hairpin RNA for thymidylate synthase [TS shRNA] with 2,000-fold of lipoplex as molar basis), paclitaxel (15 mg/kg) and their combination were intraperitoneally administered by every 3rd day for two doses, and bioluminescent signals in tumor-bearing mice (n=10) were monitored on day 7, 14, 21 and 28, respectively. BLI value and tumor growth inhibition (TGI, %) in tumor-bearing mice treated with each drug are shown in detail in Table 1. Abbreviations: BLI, bioluminescence imaging; q3d, every 3 days.

Figure 6

Short-hairpin RNA for thymidylate synthase (TS shRNA) levels in ascites fluid and plasma of SKOV3-luc-bearing mice following intraperitoneal administration of TS shRNA-lipoplex (DFP-10825). Notes: TS shRNA levels in ascites were maintained at nM range (0.8–4.5 nM) over 24 hours, while those in plasma were extremely low (under 5 pM) at 2 hours after administration of DFP-10825; half-life time (T_1/2) of TS shRNA was 8.8 hours in ascites. The small figure in the upper right is presented as log scale.

Figure 7

Thymidylate synthase (TS) mRNA expression levels in intraperitoneal ascites tumor cells of ovarian cancer-bearing mice after intraperitoneal DFP-10825. Notes: 18S rRNA was used as the normalization control. Relative gene expression level of TS was 2^−ΔCt [−ΔCt = average Ct (TS) - average Ct (18S rRNA)]. TS mRNA expression was significantly suppressed in ascites tumor cells after intraperitoneal administration of DFP-10825 alone or in combination with paclitaxel. In this experiment, paclitaxel alone did not downregulate the expression of TS mRNA in ascites tumor cells.