Remdesivir for COVID-19 in Hospitalized Children: A Phase 2/3 Study.

OBJECTIVES: Remdesivir decreases the risk of SARS-CoV-2 infection progressing to severe disease in adults. This study evaluated remdesivir safety and pharmacokinetics in infants and children. METHODS: This was a phase 2/3, open-label trial in children aged 28 days to 17 years hospitalized for polymerase chain reaction-confirmed SARS-CoV-2 infection. Participants received for ≤10 days once-daily intravenous remdesivir doses defined using physiologically based pharmacokinetic modeling (for ≥40 kg, 200 mg day 1, then 100 mg/day; for age ≥28 days and ≥3 to <40 kg, 5 mg/kg day 1, then 2.5 mg/kg/day). Sparse pharmacokinetic samples were analyzed using population-pharmacokinetic approaches for remdesivir and metabolites GS-704277 and GS-441524. RESULTS: Among 53 participants, at enrollment the median (Q1, Q3) number of days of COVID-19 symptoms was 5 (3, 7) and hospitalization was 1 (1, 3). Underlying conditions included obesity in 19 (37%), asthma in 11 (21%), and cardiac disorders in 11 (21%). Median duration of remdesivir treatment was 5 days (range, 1-10). Remdesivir treatment had no new apparent safety trends. Two participants discontinued treatment because of adverse events including elevated transaminases; both had elevated transaminases at baseline. Three deaths occurred during treatment (and 1 after). When compared with phase 3 adult data, estimated mean pediatric parameters (area under the concentration-time curve over 1 dosing interval, AUCτ, Cmax, and Cτ) were largely overlapping but modestly increased (remdesivir, 33%-129%; GS-704277, 37%-124%; GS-441524, 0%-60%). Recovery occurred for 62% of participants on day 10 and 83% at last assessment. CONCLUSIONS: In infants and children with COVID-19, the doses of remdesivir evaluated provided drug exposure similar to adult dosing. In this study with a small sample size, no new safety concerns were observed.

Pharmacokinetics, safety, and tolerability of inhaled remdesivir in healthy participants.

Intravenous remdesivir (RDV) is US Food and Drug Administration-approved for hospitalized and nonhospitalized individuals with coronavirus disease 2019. RDV undergoes intracellular metabolic activation to form the active triphosphate, GS-443902, and other metabolites. Alternative administration routes, including localized pulmonary delivery, can lower systemic exposure and maximize exposure at the site of action. This study evaluated the pharmacokinetics (PK) and safety of inhaled RDV in healthy adults. This phase Ia, randomized, placebo-controlled study evaluated inhaled RDV in healthy participants randomized 4:1 to receive RDV or placebo as single doses (4 cohorts) or multiple once-daily doses (3 cohorts). Doses in cohorts 1-6 were administered as an aerosolized solution for inhalation through a sealed facemask; doses in cohort 7 were administered as an aerosolized solution for inhalation through a mouthpiece. Safety was assessed throughout the study. Seventy-two participants were enrolled (inhaled RDV, n = 58 and placebo, n = 14). Following single RDV doses, RDV, GS-704277, and GS-441524 plasma PK parameters indicated dose-proportional increases in area under the concentration-time curve (AUC) extrapolated to infinite time, AUC from time zero to last quantifiable concentration, and maximum observed concentration. Analyte plasma concentrations after multiple RDV doses were consistent with those for single-dose RDV. Analyte plasma exposures were lower when RDV was administered with a mouthpiece versus a sealed facemask. The most common adverse events included nausea, dizziness, and cough. Single- and multiple-dose inhaled RDV exhibited linear and dose-proportional plasma PK. Administration of RDV via inhalation was generally safe and well-tolerated.

Plasma Protein Binding Determination for Unstable Ester Prodrugs: Remdesivir and Tenofovir Alafenamide.

Remdesivir (RDV) and tenofovir alafenamide (TAF) are prodrugs designed to be converted to their respective active metabolites. Plasma protein binding (PPB) determination of these prodrugs is important for patients with possible alteration of free fraction of the drugs due to plasma protein changes in renal impairment, hepatic impairment, or pregnancy. However, the prodrugs' instability in human plasma presents a challenge for accurate PPB determination. In this research work, two approaches were used in the method development and qualification for PPB assessment of RDV and TAF. For RDV, dichlorvos was used to inhibit esterase activity to stabilize the prodrug in plasma during equilibrium dialysis (ED). The impact of dichlorvos on protein binding was evaluated and determined to be insignificant by comparing the unbound fraction (fu) determined by the ED method with dichlorvos present and the fu determined by an ultrafiltration method without dichlorvos. In contrast to RDV, TAF degradation in plasma is ∼3-fold slower, and TAF stability cannot be improved by dichlorvos. Fit-for-purpose acceptance criteria for the TAF PPB method were chosen, and an ED method was developed based on these criteria. These two methods were then qualified and applied for PPB determinations in clinical studies.

The determination of Sulfobutylether ß-Cyclodextrin Sodium (SBECD) by LC-MS/MS and its application in remdesivir pharmacokinetics study for pediatric patients.

SBECD (Captisol®) with an average degree of substitution of 6.5 sulfobutylether functional groups (SBE = 6.5), is a solubility enhancer for remdesivir (RDV) and a major component in Veklury, which was approved by FDA for the treatment of patients with COVID-19 over 12 years old and weighing over 40 kg who require hospitalization. SBECD is cleared mainly by renal filtration, thus, potential accumulation of SBECD in the human body is a concern for patients dosed with Veklury with compromised renal function. An LC-MS/MS method was developed and validated for specific, accurate, and precise determination of SBECD concentrations in human plasma. In this method, the hexa-substituted species, SBE6, was selected for SBECD quantification, and the mass transition from its dicharged molecular ion [(M-2H)/2]2-, Molecular (parent) Ion (Q1)/Molecular (parent) Ion (Q3) of m/z 974.7/974.7, was selected for quantitative analysis of SBECD. Captisol-G (SBE-γ-CD, SBE = 3) was chosen as the internal standard. With 25 µL of formic-acid-treated sample and with a calibration range of 10.0-1000 µg/mL, the method was validated with respect to pre-established criteria based on regulatory guidelines and was applied to determine SBECD levels in plasma samples collected from pediatric patients during RDV clinical studies.

Semi-Mechanistic PK/PD Modeling and Simulation of Irreversible BTK Inhibition to Support Dose Selection of Tirabrutinib in Subjects with RA.

Tirabrutinib is an irreversible, small-molecule Bruton's tyrosine kinase (BTK) inhibitor, which was approved in Japan (VELEXBRU) to treat B-cell malignancies and is in clinical development for inflammatory diseases. As an application of model-informed drug development, a semimechanistic pharmacokinetic/pharmacodynamic (PK/PD) model for irreversible BTK inhibition of tirabrutinib was developed to support dose selection in clinical development, based on clinical PK and BTK occupancy data from two phase I studies with a wide range of PK exposures in healthy volunteers and in subjects with rheumatoid arthritis. The developed model adequately described and predicted the PK and PD data. Overall, the model-based simulation supported a total daily dose of at least 40 mg, either q.d. or b.i.d., with adequate BTK occupancy (> 90%) for further development in inflammatory diseases. Following the PK/PD modeling and simulation, the relationship between model-predicted BTK occupancy and preliminary clinical efficacy data was also explored and a positive trend was identified between the increasing time above adequate BTK occupancy and better efficacy in treatment for RA by linear regression.

Pharmacokinetic, Pharmacodynamic, and Drug-Interaction Profile of Remdesivir, a SARS-CoV-2 Replication Inhibitor.

Remdesivir (RDV, Veklury®) is a once-daily, nucleoside ribonucleic acid polymerase inhibitor of severe acute respiratory syndrome coronavirus 2 replication. Remdesivir has been granted approvals in several countries for use in adults and children hospitalized with severe coronavirus disease 2019 (COVID-19). Inside the cell, remdesivir undergoes metabolic activation to form the intracellular active triphosphate metabolite, GS-443902 (detected in peripheral blood mononuclear cells), and ultimately, the renally eliminated plasma metabolite GS-441524. This review discusses the pre-clinical pharmacology of RDV, clinical pharmacokinetics, pharmacodynamics/concentration-QT analysis, rationale for dose selection for treatment of patients with COVID-19, and drug-drug interaction potential based on available in vitro and clinical data in healthy volunteers. Following single-dose intravenous administration over 2 h of an RDV solution formulation across the dose range of 3-225 mg in healthy participants, RDV and its metabolites (GS-704277and GS-441524) exhibit linear pharmacokinetics. Following multiple doses of RDV 150 mg once daily for 7 or 14 days, major metabolite GS-441524 accumulates approximately 1.9-fold in plasma. Based on pharmacokinetic bridging from animal data and available human data in healthy volunteers, the RDV clinical dose regimen of a 200-mg loading dose on day 1 followed by 100-mg maintenance doses for 4 or 9 days was selected for further evaluation of pharmacokinetics and safety. Results showed high intracellular concentrations of GS-443902 suggestive of efficient conversion from RDV into the triphosphate form, and further supporting this clinical dosing regimen for the treatment of COVID-19. Mathematical drug-drug interaction liability predictions, based on in vitro and phase I data, suggest RDV has low potential for drug-drug interactions, as the impact of inducers or inhibitors on RDV disposition is minimized by the parenteral route of administration and extensive extraction. Using physiologically based pharmacokinetic modeling, RDV is not predicted to be a clinically significant inhibitor of drug-metabolizing enzymes or transporters in patients infected with COVID-19 at therapeutic RDV doses.

Validation of LC-MS/MS methods for determination of remdesivir and its metabolites GS-441524 and GS-704277 in acidified human plasma and their application in COVID-19 related clinical studies.

Remdesivir (RDV) is a phosphoramidate prodrug designed to have activity against a broad spectrum of viruses. Following IV administration, RDV is rapidly distributed into cells and tissues and simultaneously metabolized into GS-441524 and GS-704277 in plasma. LC-MS/MS methods were validated for determination of the 3 analytes in human plasma that involved two key aspects to guarantee their precision, accuracy and robustness. First, instability issues of the analytes were overcome by diluted formic acid (FA) treatment of the plasma samples. Secondly, a separate injection for each analyte was performed with different ESI modes and organic gradients to achieve sensitivity and minimize carryover. Chromatographic separation was achieved on an Acquity UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) with a run time of 3.4 min. The calibration ranges were 4-4000, 2-2000, and 2-2000 ng/mL, respectively for RDV, GS-441524 and GS-704277. The intraday and interday precision (%CV) across validation runs at 3 QC levels for all 3 analytes was less than 6.6%, and the accuracy was within ±11.5%. The long-term storage stability in FA-treated plasma was established to be 392, 392 and 257 days at -70 °C, respectively for RDV, GS-441524 and GS-704277. The validated method was successfully applied in COVID-19 related clinical studies.

Phase 1b study to investigate the safety and tolerability of idelalisib in Japanese patients with relapsed/refractory follicular lymphoma and chronic lymphocytic leukemia.

OBJECTIVE: Idelalisib is an orally administered, highly selective inhibitor of phosphatidylinositol 3-kinase-δ. In this phase 1b study, the safety, tolerability and pharmacokinetics of idelalisib, an oral inhibitor of phosphatidylinositol 3-kinase-δ, were evaluated in Japanese patients with relapsed or refractory indolent B-cell non-Hodgkin lymphoma. METHODS: In total, six patients (follicular lymphoma: n = 3, chronic lymphocytic leukemia: n = 3) were enrolled to receive idelalisib 150 mg twice daily. RESULTS: No dose-limiting toxicities were reported. The most common adverse events were diarrhea (n = 5), gastritis (n = 3), insomnia (n = 3) and pyrexia (n = 3). The most common ≥grade 3 adverse events were diarrhea (n = 2), increased transaminase levels (n = 2) and decreased appetite (n = 2). The maximum idelalisib plasma concentrations (Cmax) were achieved at 2.50 h (range: 1.50-4.00 h). The mean idelalisib plasma concentrations decreased over time but remained detectable in most patients at 12 h. All enrolled patients underwent efficacy evaluation by investigators, and five patients (follicular lymphoma: n = 2, chronic lymphocytic leukemia: n = 3) achieved partial response. The median duration of partial response was 14.5 months (range: 3.7-31.3 months). CONCLUSION: Idelalisib 150 mg twice daily was considered tolerable in Japanese patients with follicular lymphoma or chronic lymphocytic leukemia.(Clinical trial registration: NCT02242045).

Safety, Tolerability, and Pharmacokinetics of Remdesivir, An Antiviral for Treatment of COVID-19, in Healthy Subjects.

Remdesivir (RDV), a single diastereomeric monophosphoramidate prodrug that inhibits viral RNA polymerases, has potent in vitro antiviral activity against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). RDV received the US Food and Drug Administration (FDA)'s emergency use authorization in the United States and approval in Japan for treatment of patients with severe coronavirus disease 2019 (COVID-19). This report describes two phase I studies that evaluated the safety and pharmacokinetics (PKs) of single escalating and multiple i.v. doses of RDV (solution or lyophilized formulation) in healthy subjects. Lyophilized formulation was evaluated for potential future use in clinical trials due to its storage stability in resource-limited settings. All adverse events were grade 1 or 2 in severity. Overall, RDV exhibited a linear profile following single-dose i.v. administration over 2 hours of RDV solution formulation across the dose range of 3-225 mg. Both lyophilized and solution formulations provided comparable PK parameters. High intracellular concentrations of the active triphosphate (~ 220-fold to 370-fold higher than the in vitro half-maximal effective concentration against SARS-CoV-2 clinical isolate) were achieved following infusion of 75 mg or 150 mg lyophilized formulation over 30 minutes or 2 hours. Following multiple-doses of RDV 150 mg once daily for 7 or 14 days, RDV exhibited a PK profile similar to single-dose administration. Metabolite GS-441524 accumulated ~ 1.9-fold after daily dosing. Overall, RDV exhibited favorable safety and PK profiles that supported once-daily dosing.

Phase Ib Study of Tirabrutinib in Combination with Idelalisib or Entospletinib in Previously Treated Chronic Lymphocytic Leukemia.

PURPOSE: Bruton tyrosine kinase (BTK) inhibition alone leads to incomplete responses in chronic lymphocytic leukemia (CLL). Combination therapy may reduce activation of escape pathways and deepen responses. This open-label, phase Ib, sequential dose-escalation and dose-expansion study evaluated the safety, tolerability, pharmacokinetics, and preliminary efficacy of the selective BTK inhibitor tirabrutinib alone, in combination with the PI3K delta (PI3Kδ) inhibitor idelalisib, or with the spleen tyrosine kinase (SYK) inhibitor entospletinib in patients with relapsed/refractory CLL. PATIENTS AND METHODS: Patients received either tirabrutinib monotherapy (80 mg every day) or tirabrutinib 20-150 mg every day in combination with either idelalisib (50 mg twice a day or 100 mg every day) or entospletinib (200 mg or 400 mg every day). RESULTS: Fifty-three patients were included. Systemic tirabrutinib exposure was comparable between monotherapy and combination therapy. No MTD was identified. Across all treatment groups, the most common adverse event was diarrhea (43%, 1 patient grade ≥3); discontinuation due to adverse events was uncommon (13%). Objective response rates were 83%, 93%, and 100%, and complete responses were 7%, 7%, and 10% in patients receiving tirabrutinib, tirabrutinib/idelalisib, and tirabrutinib/entospletinib, respectively. As of February 21, 2019, 46 of 53 patients continue to receive treatment on study. CONCLUSIONS: Tirabrutinib in combination with idelalisib or entospletinib was well tolerated in patients with CLL, establishing an acceptable safety profile for concurrent selective inhibition of BTK with either PI3Kδ or SYK. This small study did not establish a superior efficacy of the combinations over tirabrutinib alone. This trial is registered at www.clinicaltrials.gov (NCT02457598).

Phase 1B Study of Momelotinib Combined With Trametinib in Metastatic, Kirsten Rat Sarcoma Viral Oncogene Homolog-Mutated Non-Small-Cell Lung Cancer After Platinum-Based Chemotherapy Treatment Failure.

INTRODUCTION: Specific treatment options are lacking for Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated non-small-cell lung cancer (NSCLC) despite treatment advances in other mutation-driven subgroups. PATIENTS AND METHODS: In this study we evaluated the multitargeted Janus kinase/TANK-binding kinase 1 (TBK1) inhibitor momelotinib combined with the mitogen/extracellular signal-related kinase (MEK)1/MEK2 inhibitor trametinib in patients with platinum-treated, refractory, metastatic, KRAS-mutated NSCLC. Dose escalations (3 + 3 design) were conducted with momelotinib in combination with trametinib 1.0 mg once daily, then with trametinib in combination with the maximum tolerated dose (MTD) of momelotinib. MTD was determined from dose-limiting toxicity (DLT) during patients' first 28-day cycle. Safety was the primary end point, and efficacy parameters, including disease control rate (DCR) at 8 weeks, were secondary end points. RESULTS: Twenty-one patients were enrolled (median age: 68 years; 14 [66.7%] female). The MTD was momelotinib 150 mg twice daily in combination with trametinib 1.0 mg once daily. DLTs that determined the MTD were increased alanine aminotransferase and fatigue. The most common adverse events of any grade were nausea (n = 14 [66.7%]), diarrhea (n = 11 [52.4%]), and fatigue (n = 11 [52.4%]). The most common Grade ≥3 event was hypoxia (n = 3 [14.3%]). No patients achieved objective response. DCR at 8 weeks was 12 patients (57.1%) (90% confidence interval [CI], 37.2%-75.5%). Median progression-free and overall survival were 3.6 months (90% CI, 2.2-5.6 months) and 7.4 months (90% CI, 4.0-15.3 months), respectively. Maximum momelotinib plasma concentrations were reached 1 to 2 hours after dosing, but were insufficient to achieve significant TBK1 inhibition. CONCLUSION: The additional use of momelotinib with trametinib does not improve on the activity of single-agent trametinib in KRAS-mutated NSCLC on the basis of historic data.

Brief report: Loss of p15Ink4b accelerates development of myeloid neoplasms in Nup98-HoxD13 transgenic mice.

Homeostasis of hematopoietic stem and progenitor cells is a tightly regulated process. The disturbance of the balance in the hematopoietic progenitor pool can result in favorable conditions for development of diseases such as myelodysplastic syndromes and leukemia. It has been shown recently that mice lacking p15Ink4b have skewed differentiation of common myeloid progenitors toward the myeloid lineage at the expense of erythroid progenitors. The lack of p15INK4B expression in human leukemic blasts has been linked to poor prognosis and increased risk of myelodysplastic syndromes transformation to acute myeloid leukemia. However, the role of p15Ink4b in disease development is just beginning to be elucidated. This study examines the collaboration of the loss of p15Ink4b with Nup98-HoxD13 translocation in the development of hematological malignancies in a mouse model. Here, we report that loss of p15Ink4b collaborates with Nup98-HoxD13 transgene in the development of predominantly myeloid neoplasms, namely acute myeloid leukemia, myeloproliferative disease, and myelodysplastic syndromes. This mouse model could be a very valuable tool for studying p15Ink4b function in tumorigenesis as well as preclinical drug testing.

Concise review: erythroid versus myeloid lineage commitment: regulating the master regulators.

Developmental processes, like blood formation, are orchestrated by transcriptional networks. Those transcriptional networks are highly responsive to various environmental stimuli and affect common precursors resulting in increased production of cells of the erythroid lineage or myeloid lineage (granulocytes, neutrophils, and macrophages). A significant body of knowledge has accumulated describing transcription factors that drive differentiation of these two major cellular pathways, in particular the antagonistic master regulators such as GATA-1 and PU.1. However, little is known about factors that work upstream of master regulators to enhance differentiation toward one lineage. These functions become especially important under various stress conditions like sudden loss of red blood cells or pathogen infection. This review describes recent studies that begin to provide evidence for such factors. An increased understanding of factors regulating cellular commitment will advance our understanding of the etiology of diseases like anemia, cancer, and possibly other blood related disorders.

The tumor suppressor p15Ink4b regulates the differentiation and maturation of conventional dendritic cells.

The tumor suppressor p15Ink4b is frequently inactivated by methylation in acute myeloid leukemia and premalignant myeloid disorders. Dendritic cells (DCs) as potent APCs play critical regulatory roles in antileukemic immune responses. In the present study, we investigated whether p15Ink4b can function as modulator of DC development. The expression of p15Ink4b is induced strongly during differentiation and activation of DCs, and its loss resulted in significant quantitative and qualitative impairments of conventional DC (cDC) development. Accordingly, ex vivo-generated BM-derived DCs from p15Ink4b-knockout mice express significantly decreased levels of the antigen-presenting (MHC II) and costimulatory (CD80 and CD86) molecules and have impaired immunostimulatory functions, such as antigen uptake and T-cell stimulation. Reexpression of p15Ink4b in progenitors restored these defects, and confirmed a positive role for p15Ink4b during cDC differentiation and maturation. Furthermore, we have shown herein that p15Ink4b expression increases phosphorylation of Erk1/Erk2 kinases, which leads to an elevated activity of the PU.1 transcription factor. In conclusion, our results establish p15Ink4b as an important modulator of cDC development and implicate a novel function for this tumor suppressor in the regulation of adaptive immune responses.

MiR-24 tumor suppressor activity is regulated independent of p53 and through a target site polymorphism.

MicroRNAs (miRNAs) are predicted to regulate approximately 30% of all human genes; however, only a few miRNAs have been assigned their targets and specific functions. Here we demonstrate that miR-24, a ubiquitously expressed miRNA, has an anti-proliferative effect independent of p53 function. Cell lines with differential p53 status were used as a model to study the effects of miR-24 on cell proliferation, cell cycle control, gene regulation and cellular transformation. Overexpression of miR-24 in six different cell lines, independent of p53 function, inhibited cell proliferation and resulted in G2/S cell cycle arrest. MiR-24 over expression in cells with wt-p53 upregulated TP53 and p21 protein; however, in p53-null cells miR-24 still induced cell cycle arrest without the involvement of p21. We show that miR-24 regulates p53-independent cellular proliferation by regulating an S-phase enzyme, dihydrofolate reductase (DHFR) a target of the chemotherapeutic drug methotrexate (MTX). Of interest, we found that a miR-24 target site polymorphism in DHFR 3' UTR that results in loss of miR-24-function and high DHFR levels in the cell imparts a growth advantage to immortalized cells and induces neoplastic transformation. Of clinical significance, we found that miR-24 is deregulated in human colorectal cancer tumors and a subset of tumors has reduced levels of miR-24. A novel function for miR-24 as a p53-independent cell cycle inhibitory miRNA is proposed.

Epigenetic reversal of acquired resistance to 5-fluorouracil treatment.

Acquired and intrinsic resistance still remains a limitation to the clinical use of 5-fluorouracil (5-FU). The contribution of epigenetic changes to the development of drug resistance remains to be elucidated. Several genes that are hypermethylated and silenced have been identified in colorectal cancer. Based on the findings described in the accompanying article, we hypothesized that acquired resistance to "pulse" 5-FU has an epigenetic origin and might be reversed. Here, we present a novel therapeutic approach to circumvent clinical resistance to bolus 5-FU, that is, treatment of bolus 5-FU-resistant colorectal cancer cells with low-dose 5-azadeoxycytidine (DAC), an inhibitor of DNA hypermethylation, restored sensitivity to 5-FU as well as 5-fluorouridine. Moreover, treatment of nude mice bearing a 5-FU-resistant tumor, characterized by decreased levels of UMP kinase (UMPK), with DAC overcame resistance to bolus 5-FU. DAC-mediated restoration of 5-FU sensitivity was associated with increases in UMPK levels. An increase in UMPK protein and mRNA levels following treatment with low-dose DAC was observed in cultured bolus 5-FU-resistant colorectal cancer cells (HCT-8) and in mice bearing these tumors. We conclude that DAC-mediated restoration of sensitivity to bolus 5-FU is mediated at least in part by increased UMPK levels and clinical resistance to 5-FU due to decreased UMPK in colorectal cancer may be overcome by including methylation inhibitors such as DAC.

Decreased levels of UMP kinase as a mechanism of fluoropyrimidine resistance.

5-Fluorouracil (5-FU) continues to be widely used for treatment of gastrointestinal cancers. Because many tumors show primary or acquired resistance, it is important to understand the molecular basis underlying the mechanism of resistance to 5-FU. In addition to its effect on thymidylate synthase inhibition and DNA synthesis, 5-FU may also influence RNA metabolism. Our previous studies revealed that colorectal cancer cells resistant to bolus 5-FU (HCT-8/4hFU) showed significantly decreased incorporation of the drug into RNA. Resistance to bolus 5-FU was associated with lower expression of UMP kinase (UMPK), an enzyme that plays an important role in the activation of 5-FU to 5-FUTP and its incorporation into RNA. Activities of other 5-FU-metabolizing enzymes (e.g., thymidine kinase, uridine phosphorylase, thymidine phosphorylase, and orotate phosphoribosyltransferase) remained unchanged between sensitive and resistant cell lines. Herein, we show that UMPK down-regulation in 5-FU-sensitive cells (HCT-8/P) induces resistance to bolus 5-FU treatment. Moreover, HCT-8/4hFU cells are even more cross-resistant to treatment with 5-fluorouridine, consistent with the current understanding of 5-fluorouridine as a RNA-directed drug. Importantly, colorectal cancer hepatic metastases isolated from patients clinically resistant to weekly bolus 5-FU/leucovorin treatment exhibited decreased mRNA expression of UMPK but not thymidylate synthase or dihydropyrimidine dehydrogenase compared with tumor samples of patients not previously exposed to 5-FU. Our findings provide new insights into the mechanisms of acquired resistance to 5-FU in colorectal cancer and implicate UMPK as an important mechanism of clinical resistance to pulse 5-FU treatment in some patients.