IntroductionPeritoneal 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.MethodsWe 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.ResultsDFP-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.ConclusionCollectively, 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.

To reduce 5-fluorouracil (5-FU)-induced serious toxicities without loss of antitumor activity, we have developed DFP-11207, a novel fluoropyrimidine, which consists of 1-ethoxymethyl-5-fluorouracil (EM-FU; a precursor form of 5-FU), 5-chloro-2,4-dihydroxypyridine (CDHP; an inhibitor of 5-FU degradation), and citrazinic acid (CTA; an inhibitor of 5-FU phosphorylation). In vitro studies of DFP-11207 indicated that it strongly inhibited the degradation of 5-FU by dihydropyrimidine dehydrogenase (DPD) in homogenates of the rat liver, and also inhibited the phosphorylation of 5-FU by orotate phosphoribosyltransferase (OPRT) in tumor tissues in a similar magnitude of potency by CDHP and CTA, respectively. Especially, DFP-11207 inhibited the intracellular phosphorylation of 5-FU in tumor cells in a dose-dependent manner whereas CTA alone did not protect intracellular 5-FU phosphorylation. These results postulate that DFP-11207 rapidly entered into the cell and the free CTA produced from DFP-11207 inhibited the phosphorylation of 5-FU in the cell. Furthermore, following oral administration of DFP-11207, CTA was found to be highly retained in the gastrointestinal (GI) tract compared to other tissues in rats. Interestingly, EM-FU, the prodrug of 5-FU was found to specifically produce 5-FU by various species of liver microsomes. When DFP-11207 was administered to rats, the plasma level of 5-FU was persisted for a long-time with lower Cmax and longer half-life than that from other 5-FU prodrugs. The antitumor activity of DFP-11207 was evaluated in human tumor xenografts in nude rats and found that DFP-11207 showed an antitumor activity in a dose-dependent fashion and its efficacy is equivalent to reference 5-FU drugs. In striking contrast, DFP-11207 manifested no or less 5-FU-related toxicities, such as a decrease in body weights, GI injury, and myelosuppression, especially thrombocytopenia. Taken together, the preclinical evaluation of DFP-11207 strongly indicates that DFP-11207 be a potential new version of the oral fluoropyrimidine prodrug for further clinical development.

Summary 5-fluorouracil (5-FU) and 5-FU derivatives, such as capecitabine, UFT, and S-1, are the mainstay of chemotherapy treatment for gastrointestinal cancers, and other solid tumors. Compared with other cytotoxic chemotherapies, these drugs generally have a favorable safety profile, but hematologic and gastrointestinal toxicities remain common. DFP-11207 is a novel oral cytotoxic agent that combines a 5-FU pro-drug with a reversible DPD inhibitor and a potent inhibitor of OPRT, resulting in enhanced pharmacological activity of 5-FU with decreased gastrointestinal and myelosuppressive toxicities. In this Phase I study (NCT02171221), DFP-11207 was administered orally daily, in doses escalating from 40 mg/m2/day to 400 mg/m2/day in patients with esophageal, colorectal, gastric, pancreatic or gallbladder cancer (n = 23). It was determined that DFP-11207 at the dose of 330 mg/m2/day administered every 12 hours was well-tolerated with mild myelosuppressive and gastrointestinal toxicities. The pharmacokinetic analysis determined that the 5-FU levels were in the therapeutic range at this dose. In addition, fasted or fed states had no influence on the 5-FU levels (patients serving as their own controls). Among 21 efficacy evaluable patients, 7 patients had stable disease (33.3%), of which two had prolonged stable disease of >6 months duration. DFP-11207 can be explored as monotherapy or easily substitute 5-FU, capecitabine, or S-1 in combination regimens.

Background: DFP-10917 is a nucleoside analog similar to cytarabine with a unique mechanism of action when administered at a low dose. Upon prolonged administration, DFP-10917 is converted to its nucleotide form and incorporated into tumor DNA, causing DNA strand breaks. Resulting G2/M phase-arrest by cell-checkpoint regulators ultimately leads to apoptosis of tumor cells. Methods: In the Phase 1, DFP-10917 was administered by 7-day continuous infusion (CI) followed by 21 days rest (Phase 1 Stage 1) or 14-day CI followed by 14 days rest (Phase 1 Stage 2) in patients (pts) with relapsed or refractory acute leukemia, to determine the maximum tolerated dose (MTD), recommended phase 2 dose (RP2D) and dose-limiting toxicities (DLT). Phase 2 was an open label, single arm, two-stage study of DFP-10917 administration at the RP2D using a 14-day CI in pts with relapsed or refractory acute myeloid leukemia (AML), or newly diagnosed AML pts ≥60 years not fit for intensive chemotherapy. A Simon 2-stage optimal design was used whereby if ≥1/10 pts responded (CR, CRi, CRp and PR) then an additional 19 pts were to be enrolled. Overall, if ≥4/29 pts respond, DFP-10917 warrants further investigation. A total of 39 AML pts were enrolled in Phase 1 with mean age 65 years (yrs), range 26-85 yrs. In Phase 1 Stage 1 (7-day CI, n=27) 26 pts received DFP-10917 at 8 escalating doses ranging from 4 to 35 mg/m2/day. One pt had CRi after the first treatment cycle of DFP-10917 at 6 mg/m2/day x 7 days. At 35 mg/m2, 1 pt experienced a cycle 1 DLT of grade 3 diarrhea. The starting dose for Phase 1 Stage 2 was calculated as 2/3 the cumulative 7-day dose at the MTD of 35 mg/m2/day divided by 14-day resulting in a dose of 10mg/m2/day×14 days. In Phase 1 Stage 2, (14-day CI, n=12), DFP-10917 at 10 mg/m2/day×14 days resulted in DLTs of prolonged hypo-cellularity, and the MTD/RP2D was defined as 6mg/m2/day×14days. Two pts had CR-one pt received 5 cycles of treatment and the second had continuous CR for over 22 cycles of DFP-10917 treatment. A total of 30 pts, all refractory or relapsed AML, were enrolled in Phase 2, mean age 70 yrs, range 45-88 yrs. Phase 2 pts were treated with DFP-10917 at 6 mg/m2/day x 14 days CI. An overall response rate (ORR) of 48% was observed (6 CRs including 2 transitioned to stem cell transplantation (SCT), 7 CRi including 3 transitioned to SCT, and 1 CRp). Among the 14 responding pts, 3 were refractory, 4 were salvage-1 and 7 were salvage-2 or greater. The median Overall Survival was 6.9 months and median Duration of Response was 3.5 months. The main drug-related AEs >grade 3 were: neutropenia (50%), thrombocytopenia (43%) or anemia (37%). Mild to moderate severity drug-related gastrointestinal AEs were frequently observed (43%) as well as fatigue (13%). No Phase 2 pts discontinued DFP-10917 treatment due to unacceptable drug-related toxicity. Conclusions: The RP2D of DFP-10917 was established at 6 mg/m2/day for 14-day CI with 14-day rest. In Phase 2, DFP-10917 demonstrated important anti-leukemia activity. The safety results of DFP-10917 continuous infusion indicated a tolerable safety profile in pts with relapsed or refractory AML. Overall, DFP-10917 shows promise as salvage treatment in pts with refractory or relapsed AML. Clinical trial information: NCT01702155 Disclosures Kantarjian: ARIAD: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding. DiNardo:Novartis: Research Funding; Abbvie: Research Funding; Agios: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Research Funding. Jain:Infinity: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Seattle Genetics: Research Funding; Incyte: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Novartis: Consultancy, Honoraria; Genentech: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Abbvie: Research Funding; BMS: Research Funding; Servier: Consultancy, Honoraria; Novimmune: Consultancy, Honoraria. Iizuka:Delta Fly Pharma, Inc.: Employment. Jin:Delta Fly Pharma, Inc.: Employment. Zhang:Delta-Fly Pharma, Inc.: Employment.

2'-C-cyano-2'-deoxy-1-β-D-arabino-pentofranocyl-cytosine (DFP-10917, CNDAC) is a 2'-deoxycytidine analog with antitumor activity against various tumor cells. However, a clinically available therapeutic regimen for this compound needs to be established and its functional mechanisms in relation to the dosing schedule need to be clarified. In this study, we evaluated the antitumor activity and toxicity of DFP-10917 by varying the dose and administration schedule in human solid tumor and leukemia xenografts in vivo. Compared to a 1-day infusion with a high-dose of DFP-10917 (30 mg/kg/day), a prolonged 14-day infusion with a low-dose (4.5 mg/kg/day) exerted superior tumor growth inhibitory effects without decreasing the body weights of mice in our human tumor xenograft model. In addition, we found that a 14-day infusion of low-dose DFP-10917 markedly prolonged the lifespan of nude mice bearing both acute leukemia and ovarian cancer cell-derived tumors. On the other hand, gemcitabine (GEM) and cytosine arabinoside (Ara-C), which are similar deoxycytidine analogs and are widely used clinically as standard regimens, exerted less potent antitumor effects than DFP-10917 on these tumors. To elucidate the possible functional mechanisms of the prolonged infusion of DFP-10197 compared with that of GEM or Ara-C, the rate of DNA damage in CCRF-CEM and HeLa cells treated with DFP-10917, Ara-C and GEM was detected using a comet assay. DFP-10917, at a range of 0.05 to 1 µM, induced a clear tailed-DNA pattern in both the CCRF-CEM and HeLa cells; Ara-C and GEM did not have any effect. It was thus suggested that a low concentration and long-term exposure to DFP-10917 aggressively introduced the fragmentation of DNA molecules, namely the so-called double-strand breaks in tumor cells, leading to potent cytotoxicity. Moreover, treatment with DFP-10917 at a low-dose with a long-term exposure specifically increased the population of cells in the G2/M phase, while GEM reduced this cell population, suggesting a unique function (G2/M arrest) of DFP-10917. On the whole, our findings indicate that the prolonged infusion of low-dose DFP-10917 mainly displays a novel functional mechanism as a DNA-damaging drug and may thus prove to be useful in the treatment of cancer patients who are resistant to other cytosine nucleosides, or in patients in which these other nucleosides have been shown to be ineffective.