Dihydropyrimidine Dehydrogenase Inhibitor Research Articles

Enhanced Therapeutic Efficacy of the Nanoscale Fluoropyrimidine Polymer CF10 in a Rat Colorectal Cancer Liver Metastasis Model.

Combination chemotherapy regimens that include fluoropyrimidine (FP) drugs, e.g., 5-fluorouracil (5-FU), are central to the treatment of colorectal cancer liver metastases (CRLMs), a major cause of cancer mortality. We tested a second-generation FP polymer, CF10, in a CC531/WAGRij syngeneic orthotopic rat model of liver metastasis to determine if CF10 improved response relative to 5-FU. CF10 displayed increased potency relative to 5-FU in CC531 rat colorectal cancer cells based on clonogenic assay results and caused increased apoptosis, as shown using a live/dead assay. The increased potency of CF10 to CC531 cells was associated with increased replication stress, as assessed by Western blot for biomarkers of ATR/Chk1 and ATM/Chk2 pathway activation. CF10 dosed to deliver equivalent FP content as an established dose of 5-FU in rats (50 mg/kg) did not cause weight loss in WAGRij rats even when combined with ethynyl uracil (EU), an inhibitor of dihydropyrimidine dehydrogenase, the enzyme primarily responsible for 5-FU degradation in the liver. In contrast, 5-FU caused significant weight loss that was exacerbated in combination with EU. Importantly, CF10 was significantly more effective than 5-FU at inhibiting tumor progression (~90% reduction) in the CC531/WAG/Rij CRLM model. Our results reveal strong potential for CF10 to be used for CRLM treatment.

Fluoropyrimidine Toxicity: the Hidden Secrets of DPYD.

Fluoropyrimidine-induced toxicity is a main limitation of therapy. Currently, polymorphisms in the DPYD gene, which encodes the 5-FU activation enzyme dihydropyrimidine dehydrogenase (DPD), are used to adjust the dosage and prevent toxicity. Despite the predictive value of DPYD genotyping, a great proportion of fluoropyrimidine toxicity cannot be solely explained by DPYD variations. We herein summarize additional sources of DPD enzyme activity variability, spanning from epigenetic regulation of DPYD expression, factors potentially inducing protein modifications, as well as drug-enzyme interactions that contribute to fluoropyrimidine toxicity. While seminal in vitro studies provided evidence that DPYD promoter methylation downregulates DPD expression, the association of DPYD methylation with fluoropyrimidine toxicity was not replicated in clinical studies. Different non-coding RNA molecules, such as microRNA, piwi-RNAs, circular-RNAs and long non-coding RNAs, are involved in post-transcriptional DPYD regulation. DPD protein modifications and environmental factors affecting enzyme activity may also add a proportion to the pooled variability of DPD enzyme activity. Lastly, DPD-drug interactions are common in therapeutics, with the most well-characterized paradigm the withdrawal of sorivudine due to fluoropyrimidine toxicity deaths in 5-FU treated cancer patients; a mechanism involving DPD severe inhibition. DPYD polymorphisms are the main source of DPD variability. A study on DPYD epigenetics (both transcriptionally and post-transcriptionally) holds promise to provide insights into molecular pathways of fluoropyrimidine toxicity. Additional post-translational DPD modifications, as well as DPD inhibition by other drugs, may explain a proportion of enzyme activity variability. Therefore, there is still a lot we can learn about the DPYD/DPD fluoropyrimidine-induced toxicity machinery.

Thymidylate Synthase Inhibition with Capped F10 Is Effective in TP53 Null Acute Myeloid Leukemia (AML)

Acute myeloid leukemia (AML) is an aggressive cancer of the myeloid linage characterized by progressive marrow failure and death. Responses to therapy are between 50 and 80% but relapse is common and salvage rates once relapsed are low. The risk of relapse is greatly influenced by specific somatic mutations. One of the highest risk mutations in AML involves loss of the tumor suppressor TP53. Patients with AML harboring loss of TP53 function have a dismal prognosis and there is a clear need for new approaches. To identify novel agents with activity against TP53 null AML, a TP53 null and WT isogenic cell line pair was generated using CRISPR/CAS9 in a genetically defined murine AML cell line. Clonal isolates were generated by serial dilution and loss of detectable TP53 was confirmed by Western blotting. A high throughput screen of currently FDA approved agents was performed on these cell lines. When agents were ranked by equal cytotoxicity against TP53 null and WT AML cells 20% (6/30) of the top 30 agents either directly targeted thymidylate synthase or its substrates via inhibition of the folate cycle. This finding suggests that thymidylate synthase (TS) is a key enzyme in TP53 null AML cells. To confirm and extend these results, an additional murine AML TP53 null/WT cell line pair was generated. Chemotherapy resistance was confirmed in the TP53 null cell lines when treated with doxorubicin (Figure 1). Capped F10 (CF10) is a second generation fluoropyrimidine oligonucleotide that is a highly potent inhibitor of TS. In contrast to doxorubicin, CF10 treatment resulted in significant cell death when dosed in the low picomolar range with minimal differences between the null and WT cell lines (Figure 1). To extend this result TP53 null cells were injected into syngeneic recipients and upon engraftment mice were treated with CF10 at 300mg/kg twice weekly or doxorubicin 3mg/kg and cytarabine at 100 mg/kg daily for 5 days. CF10 provided a significant survival benefit in the TP53 null AML in this highly aggressive model where doxorubicin and cytarabine did not (p=0.0057, Figure 2). To confirm a TP53 independent mechanism, competition assays were performed with CAS9 expressing AML cells partially infected with a vector expressing an sgRNA targeting TP53 and containing a GFP reporter. When the mixed population was treated with a titration of doxorubicin there was a highly significant increase in GFP+ cells indicating the involvement of TP53 in doxorubicin mediated cell death. In contrast when cells were treated with a titration of CF10 there was a no significant increase in GFP+ population consistent with CF10 inducing a TP53 independent mechanism of cell death. For CF10 to of use clinically there must be an acceptable therapeutic window. Fluoropyrimidine based therapy can be myelosuppressive especially in patients with low dihydropyrimidine dehydrogenase (DPD) activity. To assess the tolerability of CF10, healthy WAG/RAJ rats were treated with the DPD inhibitor ethynyl uracil at 2mg/kg to mimic DPD deficiency. Animals were then treated with repeated doses of either 141 mg/kg CF10 or the molar equivalent of 50 mg/kg 5-flurouracil (5-FU) and weight determined. In contrast to 5-FU, CF10 treated animals maintained their weight compared to controls (data not shown). Taken together these data suggest that TS is a viable target in TP53 null AML and CF10 is well tolerated and provides a survival benefit in a highly aggressive, chemotherapy resistant model. Ongoing studies will explore the mechanisms involved in CF10 induced cell death.

Capped F10, a novel DNA directed fluoropyrimidine, in TP53 mutated acute myeloid leukemia (AML).

7046 Background: Acute myeloid leukemia (AML) is an aggressive malignancy characterized by resistance to treatment and poor outcomes. One of the most devastating mutations in AML involves loss of the tumor suppressor TP53. AML harboring this loss carries a dismal prognosis and there is a clear need for new approaches. Methods: To identify new therapeutics, a TP53 null and WT isogenic cell line pair was generated using CRISPR/CAS9 using a murine AML cell line. Clonal isolates were obtained and loss of detectable TP53 was confirmed by Western Blotting. A high throughput screen of currently FDA approved anticancer agents was performed. Results: When agents were ranked by equal cytotoxicity against TP53 null and WT AML cells 20% (6/30) of the top 30 agents targeted the folate/thymidylate synthase axis. This suggests that thymidylate synthase (TS) is a vulnerability in TP53 null AML cells. To confirm TS as a target in TP53 null AML an additional murine AML TP53 null/WT cell line pair was generated using CRISPR/CAS9. When these cell lines were treated with doxorubicin the TP53 null cells were significantly more resistant (p≤0.001 at 10nM). Capped F10 (CF10) is a second generation fluoropyrimidine oligonucleotide that has been shown to be a highly potent inhibitor of TS. In contrast to doxorubicin, CF10 treatment resulted in significant cell death when dosed in the low picomolar range with minimal differences between the null and WT cell lines (p = 0.31 at 50pM). To extend this result TP53 WT or null cells were injected into syngeneic recipients and upon engraftment mice were treated with CF10 at 300mg/kg twice weekly. CF10 provided a significant survival benefit in the TP53 null but not WT injected mice consistent with CF10 having activity against TP53 null AML (p = 0.035 vs 0.13). To confirm a TP53 independent mechanism, competition assays were performed with CAS9 expressing AML cells partially infected with a vector expressing an sgRNA targeting TP53 and a GFP reporter. When the mixed population was treated with the IC90 concentration of 5-fluorouracil there was a highly significant increase in GFP+ cells indicating a TP53 mediated cell death (p = < 0.001). In contrast when cells were treated with the IC90 concentration of CF10 there was a non-significant increase in GFP+ population (p = 0.074) consistent with a mainly TP53 independent mechanism. Fluoropyrimidine based therapy can be myelosuppressive especially in patients with low dihydropyrimidine dehydrogenase (DPD) activity. To assess the tolerability of CF10, healthy WAG/RAJ rats were treated with the DPD inhibitor ethynyl uracil at 2mg/kg alone or in combination with either 141 mg/kg CF10 or the molar equivalent of 50 mg/kg 5-flurouracil (5-FU) and weight determined. In contrast to 5-FU, CF10 treated animals did not have a significant change in weight compared to controls. Conclusions: Taken together these data suggest that CF10 is a promising new agent for TP53 null AML.

A semimechanistic population pharmacokinetic and pharmacodynamic model incorporating autoinduction for the dose justification of TAS-114.

TAS‐114 is a dual deoxyuridine triphosphatase (dUTPase) and dihydropyrimidine dehydrogenase (DPD) inhibitor expected to widen the therapeutic index of capecitabine. Its maximum tolerated dose (MTD) was determined from a safety perspective in a combination study with capecitabine; however, its inhibitory effects on DPD activity were not assessed in the study. The dose justification to select its MTD as the recommended dose in terms of DPD inhibition has been required, but the autoinduction profile of TAS‐114 made it difficult. To this end, an approach using a population pharmacokinetic (PPK)/pharmacodynamic (PD) model incorporating autoinduction was planned; however, the utility of this approach in the dose justification has not been reported. Thus, the aim of this study was to demonstrate the utility of a PPK/PD model incorporating autoinduction in the dose justification via a case study of TAS‐114. Plasma concentrations of TAS‐114 from 185 subjects and those of the endogenous DPD substrate uracil from 24 subjects were used. A two‐compartment model with first‐order absorption with lag time and an enzyme turnover model were selected for the pharmacokinetic (PK) model. Moreover, an indirect response model was selected for the PD model to capture the changes in plasma uracil concentrations. Model‐based simulations provided the dose justification that DPD inhibition by TAS‐114 reached a plateau level at the MTD, whereas exposures of TAS‐114 increased dose dependently. Thus, the utility of a PPK/PD model incorporating autoinduction in the dose justification was demonstrated via this case study of TAS‐114.

Association of renal function with the safety and efficacy of cisplatin plus S-1 therapy and docetaxel plus cisplatin plus S-1 therapy in patients with advanced gastric cancer: an exploratory analysis of JCOG1013.

Although cisplatin and 5-chloro-2,4-dihydropyrimidine (dihydropyrimidine dehydrogenase inhibitor contained in S-1) are excreted into the urine, it remains unknown how creatinine clearance (CrCl) affects the safety and efficacy of cisplatin plus S-1 and docetaxel plus cisplatin plus S-1 in patients with advanced gastric cancer. Among the 741 participants in JCOG1013 comparing cisplatin plus S-1 with docetaxel plus cisplatin plus S-1, 723 with serum creatinine levels ≤1.2mg/dL were categorized into A1 (CrCl ≥80mL/min), A2 (60≤CrCl <80) and A3 (CrCl <60) in the cisplatin plus S-1 arm and similarly B1, B2 and B3 in the docetaxel plus cisplatin plus S-1 arm. The initial dose modification by CrCl was pre-specified in the docetaxel plus cisplatin plus S-1 arm but not in the cisplatin plus S-1 arm. The numbers of patients categorized as A1/A2/A3 and B1/B2/B3 were 169/136/57 and 170/138/53, respectively. In the cisplatin plus S-1 arm, a lower CrCl was associated with higher incidences of grade 4 leukopenia (P=0.006), neutropenia (P=0.002), and grade 3/4 anorexia (P=0.004) and febrile neutropenia (P=0.049), whereas there was no association in the docetaxel plus cisplatin plus S-1 arm. No significant differences were observed according to CrCl in the overall survival [median: 15.4/15.5/15.4months in A1/A2/A3 (P=0.886) and 15.3/13.7/13.7months in B1/B2/B3 (P=0.719)], progression-free survival [median: 7.1/6.8/6.2months in A1/A2/A3 (P=0.884) and 7.5/7.2/7.8months in B1/B2/B3 (P=0.851)] and response rates [58.9/57.8/46.9% in A1/A2/A3 (P=0.311) and 62.0/61.5/51.5% in B1/B2/B3 (P=0.362)]. Renal impairment was associated with severe adverse events in cisplatin plus S-1 therapy but not with the efficacy in cisplatin plus S-1 and docetaxel plus cisplatin plus S-1 therapy.

The association of renal function with safety and efficacy of cisplatin plus S-1 (CS) therapy and docetaxel plus cisplatin plus S-1 (DCS) therapy in patients with advanced gastric cancer (AGC): An additional analysis of JCOG1013.

e16035 Background: A phase III study, JCOG1013, did not show the superiority of docetaxel plus cisplatin plus S-1 (DCS) to cisplatin plus S-1 (CS) in overall survival (OS) (Yamada Y, Lancet GH 2019). It is known that cisplatin and gimeracil (an inhibitor of dihydropyrimidine dehydrogenase contained in S-1) are excreted in urine. We previously reported (abstr 197, ASCO-GI 2021) exploratory analysis of JCOG1013 which showed creatinine clearance (CrCl) was associated with safety (Grade [G]4 hematological toxicity for CS, and G3-4 non-hematological toxicity for CS and DCS), but not with efficacy in either group. Here, we report the additional detail results of this exploratory analysis. Methods: Among 741 participants in JCOG1013, patients with serum creatinine level &lt; 1.2 mg/dL were included in this analysis and categorized by CrCl and treatment into A1 (CrCl ≥ 80 mL/min, CS), A2 (60 &lt; CrCl &lt; 80, CS), A3 (CrCl &lt; 60, CS), B1 (CrCl &gt; 80, DCS), B2 (60 &lt; CrCl &lt; 80, DCS), and B3 (CrCl &lt; 60, DCS). The dose (mg/m2) of C/S was 60/80 regardless renal function in group A (A1, A2 and A3), and that of D/C/S was adjusted in group B as follows: 40/60/80 in B1, 40/50/80 in B2, and 40/40/65 in B3. Adverse events, OS, progression-free survival (PFS), and objective response rate (ORR) were compared by CrCl in group A (A1 vs. A2 vs. A3) and group B (B1 vs B2 vs B3), respectively. Results: Of 723 pts (169/136/57 in A1/A2/A3 and 170/138/53 in B1/B2/B3), the median CrCl (mL/min) was 94.1/71.9/53.4 in A1/A2/A3 and 98.2/70.0/55.6 in B1/B2/B3. The relative dose intensity of C/S was 90.4/75.3%, 87.8/74.9% and 85.7/72.8% in A1, A2 and A3, and that of D/C/S was 87.5/77.7/74.9%, 85.8/61.2/72.7% and 87.8/49.4/58.3% in B1, B2 and B3. The incidence of G4 white blood cell decreased, G4 neutrophil count decreased, and G3-4 anorexia were 1.2/4.4/9.3% (P &lt; 0.01), 4.8/11.1/18.5% (P &lt; 0.01), 14.4/28.1/28.6% (P &lt; 0.01) in A1/A2/A3, and 1.8/3.0/4.0% (P = 0.36), 27.3/24.8/20.0% (P = 0.28), 22.4/29.3/32.0% (P = 0.11) in B1/B2/B3, respectively. No significant association between CrCl and other adverse events was observed either in CS or in DCS group. The median OS was 15.4/15.5/15.4 months in A1/A2/A3 (P = 0.89) and 15.3/13.7/13.7 months in B1/B2/B3 (P = 0.72). The median PFS was comparable among A1/A2/A3 (7.1/6.8/6.2 months, P = 0.88) and B1/B2/B3 groups (7.5/7.2/7.8 months, P = 0.85). ORR showed no significant difference in A1/A2/A3 (58.9/57.8/46.9%, P = 0.31) and B1/B2/B3 groups (62.0/61.5/51.5%, P = 0.36). Conclusions: Dose modification according to renal function in the DCS arm could control the increase of severe toxicities, which were observed frequently in patients with low renal function in patients receiving fixed dose of CS. Clinical trial information: 000007652.

The association of renal function with safety and efficacy of cisplatin plus S-1 (CS) therapy and docetaxel plus cisplatin plus S-1 (DCS) therapy in patients with advanced gastric cancer (AGC): An exploratory analysis of JCOG1013.

197 Background: JCOG1013, a phase III study comparing CS therapy with DCS therapy for AGC, failed to demonstrate the superiority of DCS to CS in overall survival (OS) (Yamada Y, Lancet GH 2019). Because cisplatin and gimeracil (an inhibitor of dihydropyrimidine dehydrogenase contained in S-1) are excreted in urine, it is speculated that renal dysfunction may affect the safety and efficacy of CS and DCS in patients (pts) with AGC. Methods: Among 741 all randomized pts, pts with serum creatinine level &lt; 1.2 mg/dL were included in this study. Pts were categorized by creatinine clearance (CrCl) and treatment into A1 (CrCl ≥ 80 mL/min, CS), A2 (60 &lt; CrCl &lt; 80, CS), A3 (CrCl &lt; 60, CS), B1 (CrCl ≥ 80, DCS), B2 (60 &lt; CrCl &lt; 80, DCS), and B3 (CrCl &lt; 60, DCS). Adverse events, OS, progression-free survival (PFS), objective response rate (ORR) were compared by CrCl in group A (A1 vs. A2 vs. A3) and group B (B1 vs B2 vs B3), respectively, and between the treatment groups according to CrCl (A1 vs. B1, A2 vs. B2, A3 vs. B3). Results: Of 723 pts (169/136/57 in A1/A2/A3 and 170/138/53 in B1/B2/B3), the median CrCl was 94.1/71.9/53.4 mL/min in A1/A2/A3 and 98.2/70.0/55.6 mL/min in B1/B2/B3 group. The incidence of Grade (G) 4 hematological toxicity was 7.8/16.3/25.9% in A1/A2/A3 (P &lt; 0.01), and 29.1/29.3/22.0% in B1/B2/B3 (P = 0.44), and that of G3-4 non-hematological toxicity was 46.1/64.4/64.3% in A1/A2/A3 (P &lt; 0.01) and 54.6/64.7/74.5% in B1/B2/B3 (P &lt; 0.01), respectively. The median OS was 15.4/15.5/15.4 months in A1/A2/A3 (P = 0.89) and 15.3/13.7/13.7 months in B1/B2/B3 (P = 0.72). The median PFS was comparable in A1/A2/A3 (7.1/6.8/6.2 months, P = 0.88) and B1/B2/B3 groups (7.5/7.2/7.8 months, P = 0.85). ORR showed no significant difference in A1/A2/A3 (58.9/57.8/46.9%, P = 0.31) and B1/B2/B3 groups (62.0/61.5/51.5%, P = 0.36). Comparisons between treatment arms according to CrCl (A1 vs. B1, A2 vs. B2, A3 vs. B3) did not show any significant difference in OS (P = 0.88, 0.65, 0.83) and PFS (P = 0.91, 0.63, 0.46). Conclusions: In this exploratory analysis of JCOG1013, CrCl might be associated with safety (G4 hematological toxicity for CS, and G3-4 non-hematological toxicity for CS and DCS), but not with efficacy in either group. Clinical trial information: 000007652.

5-Fluorouracil Conversion Pathway Mutations in Gastric Cancer.

To date, 5-Fluorouracil (5FU) is a major component of several chemotherapy regimens, thus its study is of fundamental importance to better understand all the causes that may lead to chemoresistance and treatment failure. Given the evident differences between prognosis in Asian and Caucasian populations, triggered by clear genetic discordances and given the extreme genetic heterogeneity of gastric cancer (GC), the evaluation of the most frequent mutations in every single member of the 5FU conversion and activation pathway might reveal several important results. Here, we exploited the cBioPortal analysis software to query a large databank of clinical and wide-genome studies to evaluate the components of the three major 5FU transformation pathways. We demonstrated that mutations in such ways were associated with a poor prognosis and reduced overall survival, often caused by a deletion in the TYMP gene and amplification in TYMS. The use of prodrugs and dihydropyrimidine dehydrogenase (DPD) inhibitors, which normally catabolizes 5FU into inactive metabolites, improved such chemotherapies, but several steps forward still need to be taken to select better therapies to target the chemoresistant pools of cells with high anaplastic features and genomic instability.

An in vitro approach to simulate the process of 5-fluorouracil degradation with dihydropyrimidine dehydrogenase: the process in accordance to the first-order kinetic reaction

Partial or complete deficiency in the dihydropyrimidine dehydrogenase (DPD) has been observed in 3%–5% and 0.1% of the general population, respectively. It causes severe toxicity in the context of 5-fluorouracil (5-FU) therapy. However, the current tests for determination of DPD deficiency have limitations in routine clinical usage. Therefore, an in vitro approach for simulating 5-FU degradation was established by mixing 5-FU with blank whole blood matrix in this study. The effects of initial 5-FU concentrations and temperatures on DPD activities were investigated as well. The degradation process followed the first-order kinetic reaction (r2 > 0.98). The degradation rates were determined by temperature and individually different. The DPD inhibitor, gimeracil, could block this degradation, which indicated that DPD was the main factor. The degradation process of 5-FU in patients’ whole blood in vitro was consistent with it after mixing 5-FU with blank whole blood matrix. In conclusion, mixing 5-FU with blank matrix can simulate the process of 5-FU degradation with DPD.

Bevacizumab plus S-1 and raltitrexed for refractory metastatic colorectal cancer.

e16024 Background: There are lack of effective drugs and regimens for refractory metastatic colorectal cancer (mCRC), especially in China. Dihydropyrimidine dehydrogenase (DPD) is the rate limiting enzyme of 5-FU catabolic pathway. Thymidylate synthase (TS) is the target of 5-FU anti-tumor mechanism. Several studies have shown the up-regulation of the two enzymes after the use of 5-FU in colorectal cancer, which may be closely related to the 5-FU resistance. The preliminary research of our center has shown the efficacy and safety of S-1 (containing a DPD inhibitor) plus raltitrexed (a TS inhibitor) in refractory mCRC. The aim of this study is to evaluate the efficacy and safety of bevacizumab plus S-1 and raltitrexed for patients with mCRC after failure to fluoropyrimidine, irinotecan and oxaliplatin. Methods: This study is a one-center, single-arm, prospective phase II trial, being carried out in the Cancer Center, West China Hospital, Sichuan University, China. The patients who have progressed after the treatment of fluoropyrimidine, irinotecanand oxaliplatin, have at least one measurable lesion according to RECIST 1.1 criteria were enrolled. Patients receive bevacizumab 7.5mg/kg and raltitrexed 3 mg/m2 on days 1 plus S-1 80, 100 or 120 mg/d according to body-surface area on days 1 through 14 of a 21-day cycle. Primary endpoint was objective response rate (ORR). Secondary endpoints included progression-free survival (PFS), overall survival (OS) and toxicity. Results: From Sep 2015 to Nov 2019, 44 patients were enrolled. By Feb 5, 2020, eleven patients were alive. Tumor response evaluation was available in 44 patients at the time of the analysis. There was no complete response, ORR was 15.9%(7/14) and disease control rate was 54.5%(24/44). mPFS and mOS were 110 days (95% confidence interval, 65.0-155.0) and 367 days (95% confidence interval, 310.4-423.6). The most common adverse events were bone marrow depression, dysfunction of digestive system abnormality of liver function and bleeding. Most of these adverse events were mild to moderate. Conclusions: Bevacizumab plus S-1 and raltitrexed further showed its moderate effect for refractory mCRC. Most of the adverse effects were mild to moderate, which could be well controlled. This combined regimen is worthy of further study as third or later line therapy in mCRC. Clinical trial information: ChiCTR1900020485 .

Abstract C059: Inhibition of thymidylate synthase by the ProTide NUC-3373: in vitro analysis and clinical validation

Abstract Background: Although 5-FU-based regimens such as FOLFOX and FOLFIRI remain the standard of care for treatment of patients with colorectal cancer (CRC), their clinical utility is limited by resistance mechanisms and the production of toxic by-products. Anti-cancer activity of 5-FU requires its conversion to the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which binds to and inhibits thymidylate synthase (TS), depleting dTMP, resulting in DNA damage. Due to breakdown by dihydropyrimidine dehydrogenase (DPD), a reliance on enzymatic activation and a short plasma half-life; 5-FU is a sub-optimal inhibitor of TS. NUC-3373, a phosphoramidate transformation of FUDR-MP, was designed to bypass the key resistance mechanisms associated with the activation and breakdown of 5-FU. Herein, we characterize the impact of DPD on NUC-3373 and the role of NUC-3373 on TS biology in vitro. The NuTide:302 clinical study (NCT03428958) is investigating NUC-3373 pharmacokinetics and dTMP levels. Methods: Sensitivity of nine CRC cell lines was assessed by sulforhodamine B assay and IC50values established, with two (one sensitive and one less sensitive) selected for further evaluation. Pharmacological inhibition by gimeracil was used to investigate DPD-induced catabolism in cells treated with 5-FU and NUC-3373. Thymidine supplementation was used to indirectly assess the effect of NUC-3373 on the de novopathway of dTMP synthesis. Immunocytochemistry and Western blot analysis were used to assess the localization and expression of TS. NuTide:302 is a three-part, Phase Ib study in patients with advanced CRC who have relapsed after ≥2 prior lines of 5-FU-containing therapies. Pharmacokinetic analyses via LC-MS from patient PBMCs are intended to determine whether leucovorin (LV) augments NUC-3373 inhibition of TS and its depletion of the dTMP pool. Results: Unlike 5-FU-treated cells, inhibition of DPD by gimeracil had no significant effect on survival following NUC-3373 treatment. Supplementation with exogenous thymidine rescued cells from NUC-3373-induced death. No correlation was found between pre-treatment TS protein expression and the IC50for NUC-3373. In selected cell lines treated with NUC-3373, TS ternary complexes were detected for at least 72 hours. Treatment with NUC-3373 was also associated with increased cytoplasmic TS expression. In the clinical setting, 22 patients have received NUC-3373 in NuTide:302. The clinical impact of LV on TS inhibition and dTMP pool depletion in patient samples will be presented. Conclusions: Unlike 5-FU, NUC-3373 was not catabolised by DPD, highlighting the ability of NUC-3373 to bypass a key rate-limiting factor associated with 5-FU. NUC-3373 targets the de novo pathway of dTMP synthesis and its cytotoxicity is not dependent on basal TS expression. Formation of long-lasting ternary complexes and increased cytoplasmic expression suggest that NUC-3373 is a potent inhibitor of TS activity. Data describing whether LV potentiates the impact of NUC-3373 on TS in patients will be presented. Citation Format: Sarah P Blagden, Fiona G McKissock, Janet S Graham, Kristen K Ciombor, Francesca Aroldi, Lisa J Rodgers, Michelle Myers, Jordan Berlin, T.R. Jeffry Evans, David J Harrison. Inhibition of thymidylate synthase by the ProTide NUC-3373: in vitro analysis and clinical validation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C059. doi:10.1158/1535-7163.TARG-19-C059

ABCC11 gene polymorphism as a potential predictive biomarker for an oral 5-fluorouracil derivative drug S-1 treatment in non-small cell lung cancer.

ABCC11/MRP8 (ABCC11) is an ATP-binding cassette transporter that is involved in regulating cellular sensitivity and resistance for many anti-cancer drugs. Since 5-fluorouracil (5-FU) is one of the substrates for ABCC11, we examined whether ABCC11 is a predictive marker for an oral 5-FU derivative drug S-1 treatment in non-small cell lung cancer (NSCLC). Real-time PCR and MTS assay were carried on 21 human NSCLC cell lines. The drug resistance capabilities of ABCC11 are evaluated by analyzing the resistance profiles of a clone of HeLa cell in which the pump was ectopically expressed. Blood samples of 106 NSCLC patients were collected. There was a significant correlation between dihydropyrimidine dehydrogenase (DPD) gene expression and the IC50 for 5-FU. We then classified NSCLC cell lines into two groups based on the phenotype of the SNP538 (G > A) in ABCC11: a combined G/G and G/A group, and an A/A group. The distribution of the IC50 for 5-FU in combination with a potent inhibitor of DPD 5-chloro-2, 4-dihydropyrimidine (CDHP), which is contained in S-1, showed a significant reduction in the A/A group compared with the combined G/G and G/A group. Next, the clinical usefulness of the ABCC11 SNP in treatment containing S-1 was examined in 106 NSCLC patients, and the disease control rate was found to be significantly better in the A/A group than in the combined G/G and G/A group. These results indicate that the SNP538(G > A) in the ABCC11 gene is a potential determinant for S-1 treatment.

Dihydropyrimidine dehydrogenase in the metabolism of the anticancer drugs.

Cancer caused by fundamental defects in cell cycle regulation leads to uncontrolled growth of cells. In spite of the treatment with chemotherapeutic agents of varying nature, multiple resistance mechanisms are identified in cancer cells. Similarly, numerous variations, which decrease the metabolism of chemotherapeutics agents and thereby increasing the toxicity of anticancer drugs have been identified. 5-Fluorouracil (5-FU) is an anticancer drug widely used to treat many cancers in the human body. Its broad targeting range is based upon its capacity to act as a uracil analogue, thereby disrupting RNA and DNA synthesis. Dihydropyrimidine dehydrogenase (DPD) is an enzyme majorly involved in the metabolism of pyrimidines in the human body and has the same metabolising effect on 5-FU, a pyrimidine analogue. Multiple mutations in the DPD gene have been linked to 5-FU toxicity and inadequate dosages. DPD inhibitors have also been used to inhibit excessive degradation of 5-FU for meeting appropriate dosage requirements. This article focusses on the role of dihydropyrimidine dehydrogenase in the metabolism of the anticancer drug 5-FU and other associated drugs.

Phase I study of DFP-11207, a novel oral 5-FU with enhanced PK and improved tolerability, in patients with solid tumors.

3034 Background: DFP-11207 combines a 5-fluorouracil (5-FU) pro-drug with a reversible dihydropyrimidine dehydrogenase inhibitor and a potent inhibitor of orotate phosphoribosyl transferase resulting in enhanced pharmacological activity of 5-FU with decreased gastrointestinal and myelosuppressive toxicity. Methods: Patients with advanced solid tumors were treated in this single arm, dose escalation study. Accelerated dose escalation using single pt cohorts was followed until drug-related Gr2 adverse events occurred; then a 3+3 design was followed to determine maximum tolerated dose (MTD). Pts were dosed daily in 28-day cycles until intolerable toxicity or disease progression. PK sampling was performed for DFP-11207 metabolites on all pts and a 6 pt cohort received DFP-11207 in fed and fasted states to determine drug bioavailability. Results: Primary tumors among the 23 enrolled pts included esophageal, colorectal, gastric, pancreatic and gallbladder. Seventeen pts were treated at 8 dose levels of oral DFP-11207 administered daily, ranging from 40 to 440 mg/m2/d. At 440 mg/m2/d one pt experienced drug-related Gr3 dehydration and mucosal inflammation, and Gr4 febrile neutropenia; a second pt experienced Gr4 febrile neutropenia. One DLT of Gr3 vomiting occurred among 6 pts treated at 330 mg/m2/d dosed q12hrs confirming the MTD. Six pts were administered 300 mg DFP-11207 q12 hrs in a fed or fasted state to determine drug bioavailability. Among all pts, the most frequently reported drug-related TEAEs were fatigue (47.8%), nausea (47.8%), decreased appetite (39.1%), diarrhea (26.1%), vomiting (21.7%), anemia (13.0%), dysgeusia (13.0%), mucosal inflammation (13.0%) and palmar-plantar erythrodysaesthesia syndrome (13.0%). PK analyses of pts treated at 330 and 440 mg/m2/d indicate 5-FU concentrations of ~20 ng/mL throughout the dose cycle. There was no substantial difference in DFP-11207 bioavailability among pts in a fed or fasted state. Out of 21 efficacy evaluable pts, 7 pts had stable disease (33.3%), of which 2 had prolonged stable disease of &gt; 6 months. No pts achieved CR or PR. Conclusions: DFP-11207 at the dose of 330 mg/m2/d q12hrs is well-tolerated in pts with solid tumors with mild myelosuppressive and gastrointestinal side effects, and results in circulating 5-FU levels conducive to an anti-tumor effect. DFP-11207 can be explored as monotherapy or substitute for 5-FU, capecitabine or S-1 in combination treatment regimens. Clinical trial information: NCT02171221.

Histone deacetylase inhibitors sensitize 5-fluorouracil-resistant MDA-MB-468 breast cancer cells to 5-fluorouracil.

Resistance to 5-fluorouracil (5-FU) is a serious problem in cancer therapy and overcoming it is required in order to improve the efficacy of cancer chemotherapy. Histone deacetylase (HDAC) inhibitors are used in cancer treatments and, recently, it has been reported that HDAC inhibitors can overcome resistance to various anti-cancer drugs in vitro. In the present study, a 5-FU-resistant breast cancer cell line was established, and the effects of HDAC inhibitors in these cells were examined. The 5-FU-resistant cell line MDA-MB-468 (MDA468/FU) was established by continuous exposure of the parental cells to 5-FU. This subline was characterized by high resistance to 5-FU, higher mRNA expression levels of thymidylate synthetase and dihydropyrimidine dehydrogenase (DPD), and lower mRNA expression levels of uridine monophosphate synthetase (UMPS) than the parental cells. Gimeracil, a DPD inhibitor, did not affect the sensitivity of MDA468/FU cells to 5-FU. Oteracil, a UMPS inhibitor, decreased the cytotoxicity of 5-FU in MDA468 cells, but not in MDA468/FU cells. The HDAC inhibitors, valproic acid and suberanilohydroxamic acid sensitized the two cell lines to 5-FU in a concentration-dependent manner. In conclusion, the results of the present study revealed that HDAC inhibitors increase the sensitivity to 5-FU in 5-FU-sensitive and -resistant cells.

TAS-114, a First-in-Class Dual dUTPase/DPD Inhibitor, Demonstrates Potential to Improve Therapeutic Efficacy of Fluoropyrimidine-Based Chemotherapy.

5-Fluorouracil (5-FU) is an antimetabolite and exerts antitumor activity via intracellularly and physiologically complicated metabolic pathways. In this study, we designed a novel small molecule inhibitor, TAS-114, which targets the intercellular metabolism of 5-FU to enhance antitumor activity and modulates catabolic pathway to improve the systemic availability of 5-FU. TAS-114 strongly and competitively inhibited deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), a gatekeeper protein preventing aberrant base incorporation into DNA, and enhanced the cytotoxicity of fluoropyrimidines in cancer cells; however, it had little intrinsic activity. In addition, TAS-114 had moderate and reversible inhibitory activity on dihydropyrimidine dehydrogenase (DPD), a catabolizing enzyme of 5-FU. Thus, TAS-114 increased the bioavailability of 5-FU when coadministered with capecitabine in mice, and it significantly improved the therapeutic efficacy of capecitabine by reducing the required dose of the prodrug by dual enzyme inhibition. Enhancement of antitumor efficacy caused by the addition of TAS-114 was retained in the presence of a potent DPD inhibitor containing oral fluoropyrimidine (S-1), indicating that dUTPase inhibition plays a major role in enhancing the antitumor efficacy of fluoropyrimidine-based therapy. In conclusion, TAS-114, a dual dUTPase/DPD inhibitor, demonstrated the potential to improve the therapeutic efficacy of fluoropyrimidine. Dual inhibition of dUTPase and DPD is a novel strategy for the advancement of oral fluoropyrimidine-based chemotherapy for cancer treatment. Mol Cancer Ther; 17(8); 1683-93. ©2018 AACR.

Abstract CT012: Phase I study of TAS-114 in combination with capecitabine in patients with advanced solid tumors

Abstract Background: TAS-114 is a novel dual inhibitor of deoxyuridine triphosphatase and dihydropyrimidine dehydrogenase, and has shown favorable safety and preliminary efficacy in combination with S-1 or capecitabine in patients (pts) with advanced solid tumors.1,2 This Phase I study further evaluated the TAS-114/capecitabine combination in pts with advanced solid tumors (NCT02025803). Methods: Pts (≥18 years) with histologically or cytologically confirmed advanced solid tumors treated with standard anticancer therapy and an ECOG performance status of 0 or 1 were enrolled. Primary objective was safety and maximum tolerated dose (MTD) of TAS-114 combined with capecitabine; secondary objectives were pharmacokinetics (PK) and antitumor activity. In the dose-escalation phase, pts received an increasing dose of TAS-114 twice daily (BID; 10 mg/m2 [dose level 1] to 360 mg/m2 [level 12]) + capecitabine 380 mg/m2 BID (levels 1 to 9, 11, and 12) or capecitabine 450 mg/m2 BID (TAS-114, 240 mg/m2) for 14 days followed by 7 days' rest (21-day treatment cycle); the expansion cohort received the MTD. PK analyses were performed on days 1 and 14 of cycle 1 in the dose-escalation phase. Results: Of 104 pts, 68 (65.4%) were female, median age was 57 years (28-81), 49 (47.2%) were in the dose-escalation phase, and 55 (52.9%) were in the 2 expansion cohorts (mini [n=8] and MTD [n=47]). In the dose-escalation phase, the majority of pts had colorectal cancer (CRC; n=17; 16.3%) and breast cancer (BC; n=23; 22.1%). Dose-limiting toxicities were reported in 5 pts (1 each had grade 2 and 3 Palmar-plantar erythrodysesthesia, 2 had grade 2 rash, and 1 had grade 3 maculopapular rash). MTD was determined to be TAS-114 360 mg/m2 BID + capecitabine 380 mg/m2 BID (dose level 12). PK analysis on day 1 showed a significant correlation between TAS-114 dose and the Cmax (p=0.0013) and AUC0-last (p&amp;lt;0.0001) of 5-fluorouracil (5-FU), with an average maximum 5-FU AUC0-last of 712 ng·h/mL achieved at the MTD. Of 8 pts in the mini expansion cohort, 6 had CRC and 2 had BC; in the MTD expansion cohort, 29 had CRC and 18 had BC. Grade ≥3 treatment-emergent adverse events were reported in 39 pts (72.2%) and 67 (72.2%) in the overall population, with anemia (19.2%), fatigue (7.7%), stomatitis (6.7%), and maculopapular rash (5.8%) as the most common. No treatment-related deaths occurred during the study. In the 2 expansion cohorts, 2 pts (3.6%) had partial response (PR) and 18 (32.7%) had stable disease (SD) for ≥6 weeks. In the dose-escalation cohort, 3 pts (6.1%) had PR and 24 (48.9%) had SD. Conclusions: TAS-114 combined with capecitabine at 30% of its standard dose achieved an equivalently efficacious 5-FU exposure as the standard capecitabine dose alone, with acceptable safety and preliminary efficacy in pts with advanced solid tumors. 1. Aoyama T et al. Eur J Cancer 2016; 69: S117-8. 2. LoRusso P et al. Eur J Cancer 2016; 69: S119. Citation Format: Patricia M. LoRusso, Kathy D. Miller, Anthony F. Shields, Ki Y. Chung, Kunihiro Yoshisue, Takekazu Aoyama, Heinz-Josef Lenz. Phase I study of TAS-114 in combination with capecitabine in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT012.

Dihydropyrimidine dehydrogenase overexpression correlates with potential resistance to 5-fluorouracil-based treatment in head and neck squamous cell carcinoma

Background: Although dihydropyrimidine dehydrogenase (DPD) expression has been reported to correlate with 5-fluorouracil (5-FU) resistance in several cancers, the relationship between DPD expression and chemotherapeutic resistance to 5-FU remains unclear in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to determine the impact of DPD expression on 5-FU sensitivity and survival in HNSCC patients receiving 5-FU-based treatment. Methods: To study the correlation between DPD expression and clinical outcome of HNSCC patients who had undergone concurrent chemoradiotherapy with 5-FU-based regimens, we first examined DPD mRNA expression of these patient samples. We next developed a 5-FU-resistant HNSCC cell line (HSC-3R) to clarify the association between DPD expression and 5-FU resistance. Clonogenic survival assay was performed to determine the sensitivity of HSC-3 cells and HSC-3R cells to 5-FU. DPD expression levels in parental HSC-3 and HSC-3R cell lines were then examined by Western blotting and real-time quantitative polymerase chain reaction analysis. Lastly, we performed WST-8 assay to examine the effects of 5-Chloro-2,4-dihydroxypyridine (CDHP), a 5-FU modulator to competitively inhibit DPD, on 5-FU cytotoxicity in the HSC-3 and HSC-3R cells, to evaluate if inhibition of DPD can restore the sensitivity of HNSCC cells to 5-FU. Results: Nineteen HNSCC patients who had undergone concurrent chemoradiotherapy with 5-FU-based regimens were enrolled in this study. The cut-off value for DPD mRNA expression calculated from the ROC curve was 8.53 against cancer-specific survival of these patients. The high-level DPD expression group showed significantly shorter overall and cancer-specific survival compared to the low-level DPD expression group (P=0.0018 and 0.0004, respectively). Both of protein and mRNA expression levels were greater in the HSC-3R cells than that in the HSC-3 cells. While HSC-3R cells showed 12.64-fold greater resistance to 5-FU compared with HSC-3 cells, the combination of 5-FU with CDHP had a significant inhibitory effect on 5-FU cytotoxicity in HSC-3R cells in CDHP exposure. Conclusions: In this study, we clarified that the high-level DPD expression group of HNSCC patients undergoing concurrent chemoradiotherapy with 5-FU-based regimens showed significantly shorter overall survival. The 5-FU-resistant cells established from HNSCC cells showed increased DPD expression and attenuated 5-FU resistance induced by CDHP. Our results suggested that a high level of DPD expression was correlated with 5-FU resistance and that DPD expression level might be a predictive biomarker in 5-FU-based chemoradiotherapy for HNSCC patients.

Multicenter, randomized, controlled trial of S-1 monotherapy versus S-1 and interferon-α combination therapy for hepatocellular carcinoma with extrahepatic metastases.

No effective therapies for extrahepatic metastases from hepatocellular carcinoma (HCC) have yet been identified. Previous studies suggested a potentially promising antitumor effect of combination therapy of S-1, a novel oral dihydropyrimidine dehydrogenase inhibitor, and interferon (IFN)-α. The present study aimed to investigate the clinical efficacy of single agent S-1 and S-1/IFN-α for HCC patients with extrahepatic metastases in a randomized, open-label, multicenter trial. A total of 103 patients with HCC with extrahepatic metastases were randomly assigned to the S-1/IFN-α group, receiving the combination of S-1 and IFN-α, or the S-1 group, receiving the single agent of S-1. Clinical efficacy and adverse events were compared between the two groups. A total of 49 patients in the S-1/IFN-α group and 51 patients in the S-1 group were included in the efficacy analysis. The response rate was 22.4% (11/49) in the S-1/IFN-α group and 13.7% (7/51) in the S-1 group; there was no significant difference. Overall and progression-free survival in the two groups were also not significantly different (1-year overall survival 50.8% vs. 72.4%, median progression-free survival 127days vs. 157days). The incidence of grade ≥3 adverse events in the S-1/IFN-α group was 62.7% (32/51), which tended to be higher than in the S-1 group (43.1% [22/51]). Oncological outcomes in both treatment groups were favorable compared with previous reports, though there was no significant beneficial effect of adding IFN-α to S-1 for the treatment of HCC patients with extrahepatic metastases.