Raltitrexed Research Articles

Raltitrexed induces apoptosis through activating ROS-mediated ER stress by impeding HSPA8 expression in prostate cancer cells

Prostate cancer is the most common malignant tumor in males, which frequently develops into castration-resistant prostate cancer (CRPC). CRPC metastasis is the main reason for its high mortality rate. At present, it lacks effective treatment for patients with CRPC. Raltitrexed (RTX) has been shown to be effective in the treatment of colorectal cancer. However, the effect of RTX on prostate cancer and the underlying mechanism remain unknown. In the current study, we found that RTX could dose-dependently inhibit proliferation, migration, colony formation and induce apoptosis in DU145 and PC-3 cells. RTX also increased ROS generation in prostate cancer cells. Pretreatment with N-acetyl-L-cysteine (NAC) significantly prevented RTX-induced cell apoptosis and endoplasmic reticulum (ER) stress signaling activation in prostate cancer cells. Additionally, we found RTX-induced ROS generation and ER stress activation depended on the expression of heat shock protein family A member 8 (HSPA8). Over-expression of HSPA8 could alleviate RTX-induced cell apoptosis, ROS generation and ER stress signaling activation. Finally, our study also showed that RTX attenuated the tumor growth of prostate cancer in the DU145 xenograft model and significantly downregulated HSPA8 expression and activated ER stress signaling pathway in tumor tissues. Our study is the first to reveal that RTX induces prostate cancer cells apoptosis through inhibiting the expression of HSPA8 and further inducing ROS-mediated ER stress pathway action. This study suggests that RTX may be a novel promising candidate drug for prostate cancer therapy.

Prognostic Analysis of Prophylactic Hyperthermic Intraperitoneal Chemotherapy for Advanced Gastric Cancer: a Propensity Score-Matched Analysis

PurposeTo investigate the efficacy of prophylactic hyperthermic intraperitoneal chemotherapy (HIPEC) for advanced gastric cancer (AGC). MethodsWe included 198 patients treated from December 2016 to January 2019; of these patients, the 132 who had clinical T4 gastric cancer were divided into a hyperthermic intraperitoneal chemotherapy group (HIPEC group) and a radical gastrectomy and D2 lymph node dissection group (control group). Because this study was retrospective, we used propensity score matching (PSM) to reduce selectivity bias; we then assessed risk factors for recurrence and compared prognosis in terms of survival in the gastrectomy and prophylactic HIPEC groups. ResultsProphylactic HIPEC reduced the risk of postoperative peritoneal metastasis (PM: 27.5% vs. 10.5%, P = 0.015) and did not increase the risk of postoperative complications, but there was no significant difference in the effect on hepatic metastases or other distant metastases. Risk factors for recurrence included pT4 staging and positive lymph node metastases. Both disease-free survival (DFS: HR 0.592; 95% CI 0.354–0.990; P = 0.042) and peritoneal recurrence-free survival (PFS: HR 0.314; 95% CI 0.127–0.774; P = 0.008) were better in the prophylactic HIPEC group than in the gastrectomy-only group. In addition, there was no difference in the prognosis of patients between the two groups of raltitrexed (RT) and paclitaxel (PTX) for perfusion dosing. ConclusionOur study showed that prophylactic HIPEC could prevent postoperative PM in patients with AGC and did not increase the incidence of postoperative complications. However, it was not found to be effective in the prevention of other metastases, such as hepatic metastases.

Unraveling the Mechanisms of Clinical Drugs-Induced Neural Tube Defects Based on Network Pharmacology and Molecular Docking Analysis.

Chemotherapeutic agents such as methotrexate (MTX), raltitrexed (RTX), 5-fluorouracil (5-FU), hydroxyurea (HU), and retinoic acid (RA), and valproic acid (VPA), an antiepileptic drug, all can cause malformations in the developing central nervous system (CNS), such as neural tube defects (NTDs). However, the common pathogenic mechanisms remain unclear. This study aimed to explore the mechanisms of NTDs caused by MTX, RTX, 5-FU, HU, RA, and VPA (MRFHRV), based on network pharmacology and molecular biology experiments. The MRFHRV targets were integrated with disease targets, to find the potential molecules related to MRFHRV-induced NTDs. Protein-protein interaction analysis and molecular docking were performed to analyze these common targets. Utilizing the kyoto encyclopedia of genes and genomes (KEGG) signaling pathways, we analyzed and searched the possible causative pathogenic mechanisms by crucial targets and the signaling pathway. Results showed that MRFHRV induced NTDs through several key targets (including TP53, MAPK1, HSP90AA1, ESR1, GRB2, HDAC1, EGFR, PIK3CA, RXRA, and FYN) and multiple signaling pathways such as PI3K/Akt pathway, suggesting that abnormal proliferation and differentiation could be critical pathogenic contributors in NTDs induced by MRFHRV. These results were further validated by CCK8 assay in mouse embryonic stem cells and GFAP staining in embryonic brain tissue. This study indicated that chemotherapeutic and antiepileptic agents induced NTDs might through predicted targets TP53, MAPK1, GRB2, HDAC1, EGFR, PIK3CA, RXRA, and FYN and multiple signaling pathways. More caution was required for the clinical administration for women with childbearing potential and pregnant.

The structural maintenance of chromosomes 5 is a possible biomarker for individualized treatment of colorectal cancer.

Although the understanding of resistance to oxaliplatin (OXA) chemotherapy in colorectal cancer (CRC) has been sought for many years, drug tolerance remains a major challenge for cancer therapy. Revealing the molecular mechanism of OXA resistance could help to explain the poor prognosis of patients. Gene expression omnibus (GEO) database was searched, GSE83129, which contains RNA profiling in metastatic CRC patients treated first-line with OXA, was chosen for the following analysis. Differential expressed genes (DEGs) between the adenocarcinoma and adjacent_normal team, respectively, in the OXA responders and no-responders were analyzed. The Gene Ontology (GO) and hub genes in the protein-protein interaction (PPI) network were used for the molecular mechanism of OXA resistance. Tumor-related databases were used for the clinical relevance of the structural maintenance of chromosomes 5 (SMC5) in CRC. The in vitro assays were used to detect the molecular function of SMC5 in CRC cells. Quantitative real-time PCR (qRT-PCR) and western blot were used to detect the expression of the structural maintenance of chromosomes 5/6 (SMC5/6) complex components upon OXA and raltitrexed (RTX) treatment. CCK-8 was used to detect the cell viability of cells with different treatment. SMC5 was downregulated in CRC tissues of OXA no-response patients. Lower expression of SMC5 was correlated with a poor prognosis in CRC patients, improved this gene expression, inhibited the CRC cell growth and invasion in vitro. Furthermore, SMC5 was downregulated upon OXA treatment in CRC cells, while RTX would reverse its expression, and the combination of these two drugs restored the SMC5 level to the normal situation. Finally, RTX treatment enhanced the OXA cytotoxicity. SMC5 is a tumor suppressor, that low expression of this gene is benefit for the development of CRC. Combination treatment with RTX and OXA may be more suitable for those OXA no-responders with lower SMC5.

Thymidylate synthase is essential for efficient HIV-1 replication in macrophages

Thymidylate synthase (TS) is a key enzyme in nucleotide biosynthesis. A study performed by our group on human monocyte-derived macrophages (MDMs) infected with HIV-1 showed that many enzymes related to the folate cycle pathway, such as TS, are upregulated in productively infected cells. Here, we suggest that TS is essential for an effective HIV-1 infection in MDMs. Indeed, a TS specific small interfering RNA (siRNA) as well as the TS specific inhibitor Raltitrexed (RTX) caused a reduction in productively infected cells. Quantitative PCR analysis showed that this treatment decreased the efficacy of the early steps of the viral cycle. The RTX inhibitory effect was counteracted by dNTP addition. These results suggest that TS is essential for the early stages of HIV-1 infection by providing optimal dNTP concentrations in MDMs. TS and its related pathway may thus be considered as a potential therapeutic target for HIV-1 treatment.

Inhibition of Human Uracil DNA Glycosylase Sensitizes a Large Fraction of Colorectal Cancer Cells to 5-Fluorodeoxyuridine and Raltitrexed but Not Fluorouracil.

Previous short-hairpin RNA knockdown studies have established that depletion of human uracil DNA glycosylase (hUNG) sensitizes some cell lines to 5-fluorodeoxyuridine (FdU). Here, we selectively inhibit the catalytic activity of hUNG by lentiviral transduction of uracil DNA glycosylase inhibitor protein into a large panel of cancer cell lines under control of a doxycycline-inducible promoter. This induced inhibition strategy better assesses the therapeutic potential of small-molecule targeting of hUNG. In total, 6 of 11 colorectal lines showed 6- to 70-fold increases in FdU potency upon hUNG inhibition ("responsive"). This hUNG-dependent response was not observed with fluorouracil (FU), indicating that FU does not operate through the same DNA repair mechanism as FdU in vitro. Potency of the thymidylate synthase inhibitor raltitrexed (RTX), which elevates deoxyuridine triphosphate levels, was only incrementally enhanced upon hUNG inhibition (<40%), suggesting that responsiveness is associated with incorporation and persistence of FdU in DNA rather than deoxyuridine. The importance of FU/A and FU/G lesions in the toxicity of FdU is supported by the observation that dT supplementation completely rescued the toxic effects of U/A lesions resulting from RTX, but dT only increased the IC50 for FdU, which forms both FU/A and FU/G mismatches. Contrary to previous reports, cellular responsiveness to hUNG inhibition did not correlate with p53 status or thymine DNA glycosylase expression. A model is suggested in which the persistence of FU/A and FU/G base pairs in the absence of hUNG activity elicits an apoptotic DNA damage response in both responsive and nonresponsive colorectal lines. SIGNIFICANCE STATEMENT: The pyrimidine base 5-fluorouracil is a mainstay chemotherapeutic for treatment of advanced colorectal cancer. Here, this study shows that its deoxynucleoside form, 5-fluorodeoxyuridine (FdU), operates by a distinct DNA incorporation mechanism that is strongly potentiated by inhibition of the DNA repair enzyme human uracil DNA glycosylase. The hUNG-dependent mechanism was present in over 50% of colorectal cell lines tested, suggesting that a significant fraction of human cancers may be sensitized to FdU in the presence of a small-molecule hUNG inhibitor.

Exposure-response analysis of Raltitrexed assessing liver toxicity.

Raltitrexed (RTX) is a thymidylate synthase inhibitor with large pharmacokinetics (PK) variability that can be administered in case of 5-fluorouracil (5FU) intolerance or dihydropyrimidine dehydrogenase deficiency. While it is a more potent thymidylate synthase inhibitor than 5FU, RTX failed to replace this drug for colorectal cancer patients, mainly due to its toxicity at the recommended dose of 3 mg/m2 every 3 weeks. However, every 2 weeks administration at 2 mg/m2 demonstrated a favourable toxicity profile. We performed a randomized crossover comparative population PK study between every 2 weeks TOMOX (RTX 2 mg/m2 ) and every 3 weeks TOMOX (RTX 3 mg/m2 ). A three-compartment model and a proportional error model best describe the data. Creatinine clearance and sex, but not body surface area (BSA), were covariates of RTX clearance leading to decrease of its interindividual variability of 28%. Weight and body surface area were covariates of central and peripheral volumes of distribution, respectively, leading to decreases of interindividual variability of 34.6% and 100%, respectively. In contrast to the dose, AUC was a good predictor of liver toxicity (P = 0.006, OR = 3.91, 95%CI = [1.48-10.34]). Using covariates to compute individual clearance and a threshold AUC (1.639, determined in this study), a covariates-based dose was calculated, leading to less variability in AUC than observed with the actual BSA-based or fixed doses. These results advocate for the use of creatinine clearance and sex to determine the RTX dose instead of BSA.

A Peptidic Thymidylate-Synthase Inhibitor Loaded on Pegylated Liposomes Enhances the Antitumour Effect of Chemotherapy Drugs in Human Ovarian Cancer Cells.

There is currently no effective long-term treatment for ovarian cancer (OC) resistant to poly-chemotherapy regimens based on platinum drugs. Preclinical and clinical studies have demonstrated a strong association between development of Pt-drug resistance and increased thymidylate synthase (hTS) expression, and the consequent cross-resistance to the hTS inhibitors 5-fluorouracil (5-FU) and raltitrexed (RTX). In the present work, we propose a new tool to combat drug resistance. We propose to treat OC cell lines, both Pt-sensitive and -resistant, with dual combinations of one of the four chemotherapeutic agents that are widely used in the clinic, and the new peptide, hTS inhibitor, [D-Gln4]LR. This binds hTS allosterically and, unlike classical inhibitors that bind at the catalytic pocket, causes cell growth inhibition without inducing hTS overexpression. The dual drug combinations showed schedule-dependent synergistic antiproliferative and apoptotic effects. We observed that the simultaneous treatment or 24h pre-treatment of OC cells with the peptide followed by either agent produced synergistic effects even in resistant cells. Similar synergistic or antagonistic effects were obtained by delivering the peptide into OC cells either by means of a commercial delivery system (SAINT-PhD) or by pH sensitive PEGylated liposomes. Relative to non-PEGylated liposomes, the latter had been previously characterized and found to allow macrophage escape, thus increasing their chance to reach the tumour tissue. The transition from the SAINT-PhD delivery system to the engineered liposomes represents an advancement towards a more drug-like delivery system and a further step towards the use of peptides for in vivo studies. Overall, the results suggest that the association of standard drugs, such as cDDP and/or 5-FU and/or RTX, with the novel peptidic TS inhibitor encapsulated into PEGylated pH-sensitive liposomes can represent a promising strategy for fighting resistance to cDDP and anti-hTS drugs.

Thymidylate synthase inhibitor raltitrexed can induce high levels of DNA damage in MYCN-amplified neuroblastoma cells.

MYCN gene amplification is consistently associated with poor prognosis in patients with neuroblastoma, a pediatric tumor arising from the sympathetic nervous system. Conventional anticancer drugs, such as alkylating agents and platinum compounds, have been used for the treatment of high‐risk patients with MYCN‐amplified neuroblastoma, whereas molecule‐targeting drugs have not yet been approved. Therefore, the development of a safe and effective therapeutic approach is highly desired. Although thymidylate synthase inhibitors are widely used for colorectal and gastric cancers, their usefulness in neuroblastoma has not been well studied. Here, we investigated the efficacies of approved antifolates, methotrexate, pemetrexed, and raltitrexed (RTX), on MYCN‐amplified and nonamplified neuroblastoma cell lines. Cell growth‐inhibitory assay revealed that RTX showed a superior inhibitory activity against MYCN‐amplified cell lines. We found no significant differences in the protein expression levels of the antifolate transporter or thymidylate synthase, a primary target of RTX, among the cell lines. Because thymidine supplementation could rescue the RTX‐induced cell growth suppression, the effect of RTX was mainly due to the reduction in dTTP synthesis. Interestingly, RTX treatments induced single‐stranded DNA damage response in MYCN‐amplified cells to a greater extent than in the nonamplified cells. We propose that the high DNA replication stress and elevated levels of DNA damage, which are a result of deregulated expression of MYCN target genes, could be the cause of increased sensitivity to RTX.

Hyperthermic intraperitoneal chemotherapy with recombinant mutant human TNF-α and raltitrexed in mice with colorectal-peritoneal carcinomatosis.

Peritoneum is one of the most common metastatic sites of colorectal cancer (CRC). It has been reported that cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) prolongs the lifespan of patients with peritoneal carcinomatosis of colorectal origin (CRC-PC), while the drugs used for HIPEC are limited. We investigated the application of recombinant mutant human tumor necrosis factor-α (rmhTNF) combined with raltitrexed in the HIPEC treatment in a mice model with CRC-PC. In vitro, we detected the cytotoxicity and apoptosis of human colorectal cancer cells by 3–(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, Western blot, and TdT-mediated dUTP Nick End Labeling (TUNEL) assay. In vivo, we established xenograft models of CRC-PC and assessed the antitumor effect by in vivo imaging, peritoneal cancer index scoring, and TUNEL assay. The results showed that the combination of rmhTNF and raltitrexed under hyperthermia with a temperature of 42°C inhibited the growth of colorectal cancer cells significantly in vitro, and after HIPEC treatments with rmhTNF and raltitrexed, peritoneal tumor growth was prohibited in vivo. Our findings about the efficacy of rmhTNF and raltitrexed used for HIPEC to treat CRC-PC will provide experimental data and basis for their potential clinical application. Impact statement Colorectal peritoneal carcinomatosis exhibits poor prognosis and presents a treatment challenge. At present, the main treatment is surgery, supplemented by hyperthermic intraperitoneal chemotherapy (HIPEC), but the drugs used for HIPEC are limited. Our study found that the combination of recombinant mutant human TNF-α (rmhTNF) and raltitrexed (RTX) under hyperthermia with a temperature of 42°C had antitumor effect both in vitro and vivo. The findings will provide experimental data and basis for the potential clinical application of rmhTNF and RTX, which might offer patients a new choice of therapeutic drugs.

Formulation of Folate-Modified Raltitrexed-Loaded Nanoparticles for Colorectal Cancer Theranostics.

Multifunctional nanoparticles (NPs) that enable the imaging of drug delivery and facilitate cancer cell uptake are potentially powerful tools in tailoring oncologic treatments. Here we report the development of a layer-by-layer (LbL) formulation of folic acid (FA) and folate antimetabolites that have been well-established for enhanced tumor uptake and as potent chemotherapeutics, respectively. To investigate the uptake of LbL coated NPs, we deposited raltitrexed (RTX) or combined RTX-FA on fluorescent polystyrene NPs. The performance of these NP formulations was evaluated with CT26 murine colorectal cancer (CRC) cells in vitro and in vivo to examine both uptake and cytotoxicity against CRC. Fluorescence microscopy and flow cytometry indicated an increased accumulation of the coated NP formulations versus bare NPs. Ex vivo near-infrared (NIR) fluorescence imaging of major organs suggested the majority of NPs accumulated in the liver, which is typical of a majority of NP formulations. Imaging of the CRC tumors alone showed a higher average fluorescence from NPs accumulated in animals treated with the coated NPs, with the majority of RTX NP-treated animals showing the consistently-highest mean tumoral accumulation. Overall, these results contribute to the development of LbL formulations in CRC theranostic applications.

MYC predetermines the sensitivity of gastrointestinal cancer to antifolate drugs through regulating TYMS transcription.

BackgroundThymidylate synthase (TYMS) is a successful chemotherapeutic target for anticancer therapy. Numerous TYMS inhibitors have been developed and used for treating gastrointestinal cancer now, but they have limited clinical benefits due to the prevalent unresponsiveness and toxicity. It is urgent to identify a predictive biomarker to guide the precise clinical use of TYMS inhibitors.MethodsGenome-scale CRISPR-Cas9 knockout screening was performed to identify potential therapeutic targets for treating gastrointestinal tumours as well as key regulators of raltitrexed (RTX) sensitivity. Cell-based functional assays were used to investigate how MYC regulates TYMS transcription. Cancer patient data were used to verify the correlation between drug response and MYC and/or TYMS mRNA levels. Finally, the role of NIPBL inactivation in gastrointestinal cancer was evaluated in vitro and in vivo.FindingsTYMS is essential for maintaining the viability of gastrointestinal cancer cells, and is selectively inhibited by RTX. Mechanistically, MYC presets gastrointestinal cancer sensitivity to RTX through upregulating TYMS transcription, supported by TCGA data showing that complete response cases to TYMS inhibitors had significantly higher MYC and TYMS mRNA levels than those of progressive diseases. NIPBL inactivation decreases the therapeutic responses of gastrointestinal cancer to RTX through blocking MYC.InterpretationOur study unveils a mechanism of how TYMS is transcriptionally regulated by MYC, and provides rationales for the precise use of TYMS inhibitors in the clinic.FundingThis work was financially supported by grants of NKRDP (2016YFC1302400), STCSM (16JC1406200), NSFC (81872890, 81322034, 81372346) and CAS (QYZDB-SSW-SMC034, XDA12020210).

Biomolecular study of human thymidylate synthase conformer-selective inhibitors: New chemotherapeutic approach.

Thymidylate synthase (TS) is a well-validated target for the therapy of adult cancers. Propane-1,3-diphosphonic acid (PDPA) has significant inhibitory properties against human thymidylate synthase (hTS) relative to mouse TS which is not predicted to adopt an inactive conformer. The current research aims to identify novel, lead inhibitors of hTS and examine the prediction that they bind selectively to hTS enzymes existing in different conformational equilibria. Conformer-selectivity was evaluated through performing activity inhibition studies, as well as intrinsic fluorescence (IF) studies in comparison to the known orthosteric inhibitor raltitrexed (RTX). Human TS was isolated from recombinant bacteria expressing either native hTS, capable of conformational switching, or an actively stabilized mutant (R163K-hTS). The examined test compounds were rationally or virtually predicted to have inhibitory activity against hTS. Among these compounds, glutarate, N-(4-carboxyphenyl) succinamic acid, and diglycolic anhydride showed higher selectivity towards native hTS as compared to R163K-hTS. The active site inhibitor RTX showed significantly higher inhibition of R163K-hTS relative to hTS. Targeting hTS via conformational selectivity represents a future approach for overcoming reported resistance towards active-state TS analogs.

Clinical study of raltitrexed plus oxaliplatin compared with S1 in treating the patients with advanced primary liver cancer

Objective To evaluate the therapeutic efficacy and adverse reactions of raltitrexed plus oxaliplatin (RALOX project) and S1 in patients with advanced primary liver cancer. Methods Seventy-one patients with advanced primary liver cancer admitted to 6 cancer centers from July 2013 to July 2015 were divided into 2 groups according to the wishes of the patients and their families: RALOX group (34 patients) and S1 group (37 patients). The therapeutic efficacy such as objective remission rate (ORR), disease control rate (DCR), median overall survival (mOS), median progression free survival (mPFS), one year survival rate (SR), and adverse reactions in these patients were evaluated. Results Thirty-one patients could be evaluated in RALOX group, and 6 patients obtained partial response (PR), 10 stable disease (SD) and 15 progressive disease (PD). Thirty-three patients could be evaluated in S1 group, and 3 patients obtained PR, 8 patients SD and 22 PD. The ORR, DCR, and one year SR were 19.4% vs. 9.1%, 51.6% vs. 33.3%, and 22.6% vs. 12.1% respectively, and there were no statistically significant differences in the two groups (χ2=1.393, P=0.238; χ2=2.190, P=0.139; χ2=1.229, P=0.268). The mOS and mPFS were 7.2 months vs. 6.1 months and 3.4 months vs. 2.8 months, and there were statistically significant differences in the two groups (χ2=6.433, P=0.011; χ2=4.078, P=0.043). There was more serious peripheral nerve toxicity (29.0% vs. 3.0%, χ2=6.344, P=0.012) and lighter hand-foot syndrome (9.7% vs. 30.3%, χ2=4.201, P=0.040) in RALOX group than S1 group. But the incidences of other adverse effects were similar in the two groups. Conclusion RALOX project is safe and effective to the patients with advanced primary liver cancer. Compare with S1 project, RALOX project has better curative effects and the majority of adverse reactions are tolerable. The patients have good condition control and survival benefit. Key words: Liver neoplasms; Platinum compounds; Antimetabolites, antineoplastic; S1

Efficacy observation of concurrent radiotherapy of raltitrexed combined with cisplatin for treatment of advanced esophageal cancer

目的 观察雷替曲塞联合顺铂同步放化疗治疗中晚期食管癌的近期疗效及患者不良反应。 方法 回顾性分析58例采取同步放化疗的中晚期食管癌患者临床资料，分为治疗组28例和对照组30例，治疗组给予雷替曲塞联合顺铂方案同步放疗59.4~60.0 Gy；对照组给予顺铂联合5-氟尿嘧啶方案同步放疗59.4~60.0 Gy。治疗后3个月评价患者疗效及不良反应。计量资料采用t检验，计数资料采用χ2检验。 结果 治疗组近期缓解率为92.9%（26/28），高于对照组近期缓解率的83.3%（25/30），但差异无统计学意义（P>0.05）；两组不良反应主要表现为1~2级的骨髓抑制、放射性肺炎、放射性食管炎以及恶心、呕吐，其发生率分别为39.3%、17.9%、57.1%、42.9%（治疗组），23.3%、20.0%、50.0%、43.3%（对照组），差异均无统计学意义（均P>0.05）。 结论 雷替曲塞联合顺铂同步放疗治疗中晚期食管癌近期疗效不差于FP方案同步联合放疗，具有给药方便、依从性好的优势，安全可行。

Clinical observation of raltitrexed containing regimen for advanced colorectal cancer

Objective To observe the clinical efficacy and adverse reactions of raltitrexed containing regimen for advanced colorectal cancer as the second-line and multi-line treatment. Methods A total of 42 patients with advanced colorectal cancer were treated with raltitrexed containing regimen. The objective response rate (ORR), disease control rate (DCR), median progression-free survival (mPFS) and adverse reactions were evaluated. Results The ORR of the 42 patients was 16.67%, DCR was 80.96%, and mPFS was 4.90 months (95%CI: 2.99-6.81 months). The most of adverse reactions were gradeⅠ-Ⅱ, including leucopenia (30.95%), thrombocytopenia (2.38%), anemia (40.48%), nausea and vomiting (2.38%), anorexia (4.76%), diarrhea (2.38%), transaminase elevation (47.62%) and fatigue (11.90%). Grade Ⅲ-Ⅳ transaminase elevation was found in only 4.76% of patients. Conclusion Raltitrexed containing regimen for advanced colorectal cancer as the second-line and multi-line treatment is effective and well tolerated, and further clinical application is recommended. Key words: Colorectal neoplasms; Drug therapy; Raltitrexed

A phase II prospective study on raltitrexed combined with concurrent radiotherapy for elderly esophageal carcinoma

Objective To evaluate the clinical effects and adverse reaction of raltitrexed combined with radiation for esophageal carcinoma in elderly patients. Methods Sixty patients were randomly divided into two groups by the envelope method, 30 patients in experimental group received raltitrexed combined with radiotherapy and 30 patients in control group received radiotherapy only. Patients in both groups received conventional radiotherapy with a total dose of 56-60 Gy/28-30 F. In experimental group, raltitrexed 2.6 mg/m2 was administered concurrently with the radiotherapy on d1 and d22. Two cycles of concurrent chemotherapy were administered during radiotherapy. The short-term effects, survival times and adverse reactions of the two groups were compared. Results The total effective rates of experimental group and control group were 93.3% and 73.3%, respectively, and there was statistically significant difference between the two groups (χ2=4.320, P=0.038). The median survival times of experimental group and control group was 24.0 months and 12.0 months, respectively, and there was statistically significant difference by Log-rank test (χ2=6.048, P=0.014). The major adverse reactions of grade 3-4 in experimental group and control group were radiation-induced esophagitis (10.0% vs. 3.3%; χ2=0.268, P=0.605), leukopenia (13.3% vs. 10.0%; χ2=0.000, P=1.000), thrombocytopenia (3.3% vs. 0; P=1.000), nausea and vomiting (6.7% vs.0; χ2=0.517, P=0.472), and the differences were not statistically significant. Conclusion Raltitrexed combined with radiotherapy can enhance the short-term effect and prolong the survival time for the elderly esophageal carcinoma patients, and the adverse reactions are mild. It is worthy of further clinical study. Key words: Esophageal neoplasms; Radiotherapy; Drug therapy; Radiation tolerance; Raltitrexed

Progression in the treatment of raltitrexed for colorectal cancer

Raltitrexed is a kind of specific thymidylate synthase inhibitor, and as a novel cytotoxic drug, it is used in the treatment of a variety of solid tumors, with the advantages of good effect, light adverse reaction and easy to use.Especially in the treatment of the colorectal cancer, its efficacy has been recognized.And in clinical, raltitrexed can plug with chemotherapy or targeted drug combination, as well as radiosensitizer.This review summarized the raltitrexed research progress on the treatment of colorectal cancer in recent years. Key words: Raltitrexed; 5-Fluorouracil; Tymidylate synthase; Colorectal cancer; Radiotherapy

Clinical observation of raltitrexed combined with irinotecan as first-line chemotherapy for recurrent or metastatic gastric cancer

Objective To observe the efficacy and adverse reaction of raltitrexed combined with irinotecan as first-line chemotherapy for recurrent or metastatic gastric cancer. Methods From January 2014 to March 2015, 39 patients of recurrent or metastatic gastric cancer who received treatment in the First People′s Hospital of Zhangjiagang of Jiangsu Province were collected. All patients received raltitrexed (3.0 mg/m2, 15 min intravenous drip) on the first day and irinotecan (180.0 mg/m2, 90 min intravenous drip) on the first day. One cycle lasted 21 days. The efficacies were evaluated every 2-cycle. Adverse reactions were evaluated every cycle. Results The efficacies and adverse reactions could be evaluated in 39 patients. The study received 0 complete remission, 16 partial remission, 11 stable disease, 12 progression disease. The objective response rate was 41.03% (16/39). The disease control rate was 69.23% (27/39). The median overall survival time was 9.3 months (95%CI: 8.8-11.1 months). The median progression-free survival time was 6.0 months (95%CI: 5.1-6.8 months). The adverse reactions were mainly neutropenia, anemia, liver dysfunction, the incidence of them were 35.90%, 33.33% and 28.21% respectively. Conlusion Raltitrexed combined with irinotecan as first-line chemotherapy for recurrent or metastatic gastric cancer acquires an definite efficacy, and the adverse reactions can be tolerated, which is worthy of further clinical research. Key words: Stomach neoplasms; Drug therapy; Raltitrexed; Irinotecan

Abstract 4836: Targeted antifolates with thienoyl regioisomers in the side chain result in improved selectivity and potency against KB human tumor cells

Abstract Folates function as cofactors in one-carbon transfer reactions including de novo biosynthesis of nucleotides and play a key in DNA and RNA synthesis, epigenetic processes, cellular proliferation and survival. Cellular folate uptake is mediated by three distinct transporters. Among these, the reduced folate carrier (RFC) is the predominant folate transporter and is critical for antitumor efficacy of currently used classical antifolates such as methotrexate (MTX), pemetrexed (PMX), and raltitrexed (RTX). RFC is ubiquitously expressed in tumor cells and in normal tissues, resulting in dose-limiting toxicity with classical antifolates. We previously reported a series of 6-substituted pyrrolo[2,3-d]pyrimidine antifolates that are selectively transported by folate receptors (FR) and/or by the proton-coupled folate transporter (PCFT) over RFC and inhibit FR/PCFT-expressing human tumor cells (KB and IGROV1) at sub-nanomolar IC50 values. These antifolates are comparatively more potent and reduce the toxicity of clinically available antifolates which have non-selective uptake. As an extension of the SAR, we focused on a novel isosteric series of 6-substituted thieno[2,3-d]pyrimidine analogs, with a 3- or 4- carbon bridge between the thieno[2,3-d]pyrimidine and the thienoyl regioisomeric side chain. This simple isosteric replacement of pyrrole with thiophene in the scaffold, combined with varying the positions of attachment of 3- and 4-carbon bridge and the L-glutamic acid on the thiophene ring, provides compounds with different chain length, conformations and extra hydrogen bond donors and/or acceptors compared to the previously reported compounds. All the analogs (AGF 102, AGF131, AGF 271, AGF 275, AGF 276 and AGF 282) are potently inhibitory toward Chinese hamster ovary (CHO) cells (RT16) expressing human FRá (IC50s of 3.1, 0.33, 0.45, 1.1, 0.83 and 5.6 nM, respectively). All the analogs were inactive in the CHO cell line expressing RFC indicating absolute selectivity for FRá. AGF271, AGF275, and AGF282 showed modest (IC50s from 95-460 nM) with PCFT-expressing CHO cells (R2/PCFT4). AGF102, AGF 131 and AGF 271 AGF 275 AGF 276 AGF 282 were all potently inhibitory toward KB tumor cells (IC50 of 2.10, 1.70, 1.48, 1.81, 5.74 and 7.61nM, respectively). Collectively, our results suggest that AGF102, AGF 131, AGF 271, AGF 275, AGF 276 and AGF 282 are targeted antifolates with activity profiles that suggest that further preclinical studies and optimization are warranted. Citation Format: Aleem Gangjee, Nian Tong, Adrianne Wallace Povrik, Zhanjun Hou, Larry H. Matherly. Targeted antifolates with thienoyl regioisomers in the side chain result in improved selectivity and potency against KB human tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4836.