Lipid core-shell nanoparticles co-deliver FOLFOX regimen and siPD-L1 for synergistic targeted cancer treatment.

FOLFOX regimen, composed of folinic acid, 5-fluorouracil (5-FU) and oxaliplatin (OXP), has been used as clinical standard therapeutic regimen in treatments of colorectal cancer (CRC) and esophageal squamous cell carcinoma (ESCC). To further improve its therapeutic outcomes, FOLFOX was combined with anti-PD-1 antibody to form an advanced chemo-immune combination strategy, which has been proven more efficient in controlling cancer progression and prolonging patients' survival in various clinical trials. However, bad tumor accumulation, relative high toxicity, numerous treatment cycles with high fees and low compliance as well as drug resistance seriously limit the prognosis of FOLFOX regimen. The "all-in-one" formulations, which could precisely delivery multidrug regimen into tumor sites and cells, showed a promising application prospect for targeted drug delivery as well as reducing side effects. However, the design and preparation of the "all-in-one" formulation with high drug encapsulation efficiencies for all drugs was still challenging. Herein, a lipid core-shell nanoparticle codelivery platform was designed for simultaneous encapsulation of variant FOLFOX composed of miriplatin (MiPt), 5-Fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), calcium folinate (CF) and PD-L1 siRNA (siPD-L1) with high efficiencies, and their synergistic anti-tumor mechanisms were studied, respectively. MiPt, a precursor of OXP, was validated capable of inducing efficient immunogenic cell death (ICD) in this work. Additionally, ICD-mediated release of damage associated molecular patterns functionalized synergistically with PD-L1 silence by siPD-L1 to overcome chemoresistance, reverse suppressive tumor microenvironment and recruit more CD8+ T cells. FdUMP, as the intracellular active form of 5-FU, could induce large amounts of reactive oxygen species to enhance the ICD. CF worked as the sensitizer of FdUMP. The enhanced long-term anti-tumor effect of the prepared "all-in-one" formulation compared to free drug regimen and other controls, was verified in heterotopic CRC mice models and ESCC mice models, providing new thoughts for researchers and showing a promising prospect of translation into clinical applications.

The novel anti-cancer fluoropyrimidine NUC-3373 is a potent inhibitor of thymidylate synthase and an effective DNA-damaging agent.

INTRODUCTION: Fluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU. METHODS: Human colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC50 doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC-MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage. RESULTS: We demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU. CONCLUSION: Taken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients.

Formulation of two lipid-based membrane-core nanoparticles for FOLFOX combination therapy.

FOLFOX is a combination of folinic acid (FnA), 5-fluorouracil (5-Fu) and oxaliplatin (OxP). It has been used as the standard treatment for colorectal cancer (CRC) and hepatocellular carcinoma (HCC). This treatment is effective, but its high toxicity is dose limiting, and the drugs need to be taken for a long time. To lower the toxicity so that higher doses can be administered with minimal side effects, two lipid-based membrane-core (MC) nanoformulations, Nano-Folox and Nano-FdUMP, have recently been developed by using the nanoprecipitation technique. The combination of Nano-Folox (containing platinum drug and FnA) and Nano-FdUMP (containing fluorine drug) significantly improves the antitumor effect against CRC and HCC relative to FOLFOX (the combination of free drugs), resulting in long-term survival of animals without significant toxic signs. Here, we describe two formulation protocols. First, for Nano-Folox, a Pt(DACH)•FnA nanoprecipitate is formed by [Pt(DACH)(H2O)2]2+ (the active form of OxP) and FnA2-, and the resultant nanoprecipitate is encapsulated inside the lipid nanoparticles (NPs) modified with the PEGylated aminoethyl anisamide (AEAA, a targeting ligand for sigma-1 receptor overexpressing on CRC and HCC). Second, for Nano-FdUMP, FdUMP (the active metabolite of 5-Fu) is entrapped inside the amorphous Ca3(PO4)2 nanoprecipitate, and the resultant Ca3(PO4)2•FdUMP nanoprecipitate is encapsulated into the AEAA-targeted PEGylated lipid NPs. The procedures for Nano-Folox and Nano-FdUMP take ~17 h and ~4 h, respectively (~17 h if they are prepared simultaneously). Procedures for the physicochemical (~30 h) and cytotoxic (~54 h) characterization are also described.

The cytotoxic and pro-apoptotic activities of the novel fluoropyrimidine F10 towards prostate cancer cells are enhanced by Zn(2+) -chelation and inhibiting the serine protease Omi/HtrA2.

BACKGROUND: Intracellular Zn(2+) levels decrease during prostate cancer progression and agents that modulate intracellular Zn(2+) are cytotoxic to prostate cancer cells by an incompletely described mechanism. F10 is a new polymeric fluoropyrimidine drug-candidate that displays strong activity with minimal systemic toxicity in pre-clinical models of prostate cancer and other malignancies. The effects of exogenous Zn(2+) or Zn(2+) chelation for enhancing F10 cytotoxicity are investigated as is the role of Omi/HtrA2, a serine protease that promotes apoptosis in response to cellular stress. METHODS: To test the hypothesis that the pro-apoptotic effects of F10 could be enhanced by modulating intracellular Zn(2+) we investigated cell-permeable and cell-impermeable Zn(2+) chelators and exogenous Zn(2+) and evaluated cell viability and apoptosis in cellular models of castration-resistant prostate cancer (CRPC; PC3, C4-2). The role of Omi/HtrA2 for modulating apoptosis was evaluated by pharmacological inhibition and Western blotting. RESULTS: Exogenous Zn(2+) initially reduced prostate cancer cell viability but these effects were transitory and were ineffective at enhancing F10 cytotoxicity. The cell-permeable Zn(2+) -chelator tetrakis-(2-pyridylmethl) ethylenediamine (TPEN) induced apoptosis in prostate cancer cells and enhanced the pro-apoptotic effects of F10. The pro-apoptotic effects of Zn(2+) -chelation in combination with F10 treatment were enhanced by inhibiting Omi/HtrA2 implicating this serine protease as a novel target for prostate cancer treatment. CONCLUSIONS: Zn(2+) -chelation enhances the pro-apoptotic effects of F10 and may be useful for enhancing the effectiveness of F10 for treatment of advanced prostate cancer. The serine protease Omi/HtrA2 modulates Zn(2+) -dependent apoptosis in prostate cancer cells and represents a new target for treatment of CRPC. Prostate 75:360-369, 2015. © 2014 Wiley Periodicals, Inc.

Unique dual targeting of thymidylate synthase and topoisomerase1 by FdUMP[10] results in high efficacy against AML and low toxicity.

Acute myeloid leukemia (AML) is an aggressive malignancy that leads to marrow failure and death. There is a desperate need for new therapies. The novel fluoropyrimidine, FdUMP[10], was highly active against both human AML cell lines, (IC(50) values, 3.4nM-21.5nM) and murine lines (IC(50) values, 123.8pM-131.4pM). In all cases, the IC(50) of FdUMP[10] was lower than for cytarabine and ∼ 1000 times lower than 5-fluorouracil (5-FU). FdUMP[10] remained effective against cells expressing the Flt3 internal tandem duplication, BCR-ABL, MN1, and an shRNA against p53. It had activity against patient samples at concentrations that did not affect normal hematopoietic cells. FdUMP[10] inhibited thymidylate synthase (TS) and trapped topoisomerase I cleavage complexes (Top1CCs), leading to DNA damage and apoptosis. All cell lines and nearly all primary AML samples examined expressed both TS and Top1. In vivo, FdUMP[10] was active against a syngeneic AML model with a survival advantage equivalent to doxorubicin plus cytarabine. 5-FU treatment was toxic and did not improve survival. FdUMP[10] was better tolerated than 5-FU or cytarabine plus doxorubicin and did not affect normal HSCs, while 5-FU dramatically impaired their ability to engraft. In summary, FdUMP[10] was highly efficacious and better tolerated than standard therapies.

Cytotoxicity of new duplex drugs linking 3'-C-ethynylcytidine and 5-fluor-2'-deoxyuridine against human melanoma cells.

Melanoma is an increasingly common and potentially fatal malignancy of the skin and some mucous membranes. As no cure exists for metastatic disease, there is an urgent need for novel drugs. 2'-Deoxy-5-fluorouridylyl-(3'-5')-3'-C-ethynylcytidine [5-FdU(3'-5')ECyd] and 3'-C-ethynylcytidinylyl-(5' → 1-O)-2-O-octadecyl-sn-glycerylyl-(3-O → 5')-2'-deoxy-5-fluorouridine [ECyd-lipid-5-FdU] represent cytostatic active duplex drugs, which can be metabolized into various active antimetabolites. We evaluated the cytotoxicity of these heterodinucleoside phosphate analogs, their corresponding monomers ECyd and 5-FdU and combinations thereof on six metastatic melanoma cell lines and six ex vivo patient-derived melanoma cells in comparison to current standard cytostatic agents and the BRAF V600E inhibitor Vemurafenib. In vitro (real-time)-proliferation assays demonstrated that 5-FdU(3'-5')ECyd and ECyd-lipid-5-FdU had a high cytotoxic efficacy causing 75% melanoma cell death at concentrations in the nanomolar and micromolar range. Cytotoxicity was conducted by induction of DNA cleavage indicating apoptotic cells. Chicken embryotoxicity demonstrated that the duplex drugs were less toxic than 5-FdU at 0.01 µM. In vivo the duplex drug 5-FdU(3'-5')ECyd was efficacious in the murine LOX IMVI melanoma xenograph model on administration of 11.2 mg/kg/injection every fourth day. Both duplex drugs are promising novel cytostatic agents for the treatment of malignant melanoma meriting clinical evaluation.

Chromosomal imbalances associated with acquired resistance to fluoropyrimidines in human colorectal cancer cells.

The chromosomal aberrations underlying the development of resistance to fluoropyrimidines have not yet been identified. To characterise the genomic changes that induce the development of resistance to fluoropyrimidines, we used comparative genomic hybridisation (CGH) to analyse and compare the parent DLD-1 human colorectal cancer cell line and two cell lines, DLD-1/5-FU and DLD-1/FdUrd, which were resistant to 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FdUrd), respectively. Both resistant cell lines showed a genetic aberration derived from the parental cell line DLD-1. Losses of 3p and 3q were also detected as additional genetic changes in the two resistant cell lines. Both resistant cell lines showed decreased orotate phosphoribosyltransferase (OPRT) activity, which is associated with the activity of the uridine monophosphate (UMP) synthase gene (3q13). These results suggested that the loss of 3q might be a genetic change responsible for the decreased OPRT activity and fluoropyrimidine cytotoxic response in cancer cells. Amplification of 18p11.2-p11.3 containing the thymidine synthase (TS) gene (18p11.32) was observed only in the DLD-1/FdUrd-resistant cell line, which overexpresses TS. These findings suggested that 18p amplification represents a genetic change associated with the overexpression of the TS protein. Our results indicate that chromosomal aberrations identified by CGH could explain, at least in part, acquired fluoropyrimidine resistance.

Efficacy and safety of FdUMP[10] in treatment of HT-29 human colon cancer xenografts.

Thymidylate synthase (TS) is the molecular target of fluoropyrimidine (FP) chemotherapy, and novel anticancer drugs effective against TS-overexpressing tumors are required to treat patients with FP-refractory solid tumors. We have evaluated the inhibition of cell proliferation in vitro and antitumor activity in vivo of FdUMP[10], an oligodeoxynucleotide 10mer in which 5-fluorouracil (5-FU) is the only nucleobase. FdUMP[10] is a pro-drug of FdUMP, the TS inhibitory metabolite of FPs. FdUMP[10] was 338-fold more potent than 5-FU at inhibiting cell proliferation in the NCI 60 cell line screen. The antitumor activity of FdUMP[10] was compared to 5-FU using H-T29 xenografts in female CD-1 athymic (nu+/nu+) mice. Treatment with FdUMP[10] as a single agent (40 mg/kg/daily x 5, i.v.) delayed tumor growth and resulted in a smaller mean tumor size (T/C value = 51%, p<0.001 compared with the control group). Treatment with 5-FU (25 mg/kg/daily x 5, i.p.) had similar results as single agent FdUMP[10] (T/C value = 65%, p=0.238 compared with the FdUMP[10] treated group. Simultaneous treatment of tumor-bearing mice with both drugs (FdUMP[10] plus 5-FU) further delayed tumor growth (T/C value = 36%; p=0.003 relative to 5-FU). The results from the combined treatment group were not, however, statistically significant relative to the group receiving single agent FdUMP[10] treatment (p=0.059). Histological examination revealed systemic damage was limited to the colonic epithelium in all treatment groups and was least extensive with single agent FdUMP[10] compared to the other treatment groups. The data support the concept that FdUMP[10] is a useful prototype of a novel type of FP that is likely to be more efficacious than FPs in clinical use.

Inhibitory effects of fluorinated pyrimidines, 5'-DFUR, UFT and T-506, in a model of hepatic metastasis of mouse colon 26 adenocarcinoma-assessment of inhibitory activity and adverse reactions at the maximum tolerated dose.

The effects of fluorinated pyrimidines, 5'-DFUR, UFT and T-506, on a mouse model of hepatic metastasis were assessed in regard to inhibitory activity and adverse reactions at the maximum tolerated dose. The model was prepared by injecting the mouse colonic cancer cell line, colon 26, into the portal vein of CDF1 mice. At the treatment regimens employed for 5'-DFUR (1.0 mmol/kg/day, p.o., daily from days 1 to 7), UFT (0.1 mmol/kg/day, p.o., daily from days 1 to 7), and T-506 (0.074 mmol/kg/day, i.v., days 1, 4, 7, and 10), complete inhibition of hepatic metastasis was obtained in six out of seven mice (85.7%) with 5'-DFUR, and in five out of six mice (83.3%) with T-506. Significant inhibition of hepatic metastasis was not achieved with UFT (3/7, 42.9%). 5'-DFUR and T-506 showed the highest rate of inhibition of hepatic metastasis, suggesting that these drugs would be effective for the prophylactic treatment of metastatic disease. 5'-DFUR and UFT exhibited mild adverse reactions such as loss of body weight.

[Development of fluorinated pyrimidines in Japan].

Four fluorinated pyrimidines that are in the stage of clinical trials at present in Japan were reviewed: BOF-A2, Ro09-1390, TT-62 and S-1. Both BOF-A 2 and S-1 are a compound of 5-FU derivative combined or mixed with an inhibitor of 5-FU degradation in order to prolong the blood 5-FU level as well as increase selective toxicity to tumor. Furthermore, an inhibitor of 5-FU phosphorylation in G1 tract contained in S-1 reduces G1 toxicity such as diarrhea etc due to prolongation of blood 5-FU level. Ro09-1390 is an improved compound of 5'-DFUR, which intends to reduce diarrhea caused by the latter. TT-62 is a FdUMP derivative and an active metabolite of 5-FU for oral formulation, which is superior to available 5-FU type anticancer agents in efficacy, and doesn't show cross tolerance to 5-FU.

[Antitumor activity of T-506, a novel synthetic FUDR derivative, on murine colon cancer and its hepatic metastasis].

T-506 is a novel synthetic FUDR derivative which releases FUDR slowly in vivo. We studied antitumor activity of T-506 by i.v. injection against mouse colon cancer, colon 26. When T-506 was administrated to mice daily, from day 1 through day 10, or every 3 days, on days 1, 4, 7, and 11, after s.c. inoculation of the tumor, the survival period was expanded significantly. The subcutaneous tumor growth was also inhibited according to the dose levels. Then, we compared the therapeutic effects on the experimental hepatic metastasis of colon 26 between T-506, 5'-DFUR and UFT at each maximal tolerable dose; that is, T-506 (0.074 m mole/kg/day; i.v. on days 1, 4, 7, and 10), 5'-DFUR (1.0 m mole/kg/day; P. O. from day 1 to 7), UFT (0.1 m mole/kg/day; P. O. from day 1 to 7). T-506 and 5'-DFUR suppressed completely the metastases of 5 of 6 (83.3%) mice and 6 of 7 (85.7%), respectively. UFT did not show a significant inhibitory effect. However, since the loss of body weight was more marked in T-506 than in the other two drugs, the side effect was thought to be a serious problem. These data suggested that if the side effect could be overcome, T-506 would be useful clinically for the treatment of gastrointestinal cancers or hepatic metastases.