Pharmacokinetics and pharmacodynamics of an oral formulation of decitabine and tetrahydrouridine.

BACKGROUND: Sickle cell disease (SCD) is caused by an inherited structural abnormality of adult hemoglobin causing polymerization. Fetal hemoglobin interferes with polymerization but is epigenetically silenced by DNA methyltransferase 1 (DNMT1) in adult erythropoiesis. Decitabine depletes DNMT1 and increases fetal and total hemoglobin in SCD patients, but is rapidly catabolized by cytidine deaminase (CDA) in vivo. Tetrahydrouridine (THU) inhibits CDA, safeguarding decitabine. METHODS: The pharmacokinetics and pharmacodynamics of three oral combination formulations of THU and decitabine, with different coatings producing different delays in decitabine release, were investigated in healthy participants. RESULTS: Tetrahydrouridine and decitabine were rapidly absorbed into the systemic circulation after a single combination oral dose, with relative bioavailability of decitabine ≥74% in fasted males compared with separate oral administration of THU followed by decitabine 1 h later. THU and decitabine Cmax and area under the plasma concentration versus time curve were higher in females versus males, and fasted versus fed states. Despite sex and food effect on pharmacokinetics, the pharmacodynamic effect of DNMT1 downregulation was comparable in males and females and fasted and fed states. Treatments were well tolerated. CONCLUSION: Combination oral formulations of THU with decitabine produced pharmacokinetics and pharmacodynamics suitable for oral DNMT1-targeted therapy.

Mycoplasma infection of cancer cells enhances anti-tumor effect of oxidized methylcytidines.

Oxidized methylcytidines 5-hydroxymethyl-2'deoxycytidine (5hmdC) and 5-formy-2'deoxycytidine (5fdC) are deaminated by cytidine deaminase (CDA) into genome-toxic variants of uridine, triggering DNA damage and cell death. These compounds are promising chemotherapeutic agents for cancer cells that are resistant to pyrimidine derivative drugs, such as decitabine and cytarabine, which are inactivated by CDA. In our study, we found that cancer cells infected with mycoplasma exhibited a markedly increased sensitivity to 5hmdC and 5fdC, which was independent of CDA expression of cancer cells. In vitro biochemical assay showed that the homologous CDA protein from mycoplasma was capable of deaminating 5hmdC and 5fdC into their uridine form. Moreover, mycoplasma infection increased the sensitivity of cancer cells to 5hmdC and 5fdC, whereas administration of Tetrahydrouridine (THU) attenuated this effect, suggesting that mycoplasma CDA confers a similar effect as human CDA. As mycoplasma infection occurs in many primary tumors, our findings suggest that intratumoral microbes could enhance the tumor-killing effect and expand the utility of oxidized methylcytidines in cancer treatment.

In Vitro Interaction of Tetrahydrouridine with Key Human Nucleoside Transporters.

NDec is a novel combination of oral decitabine and tetrahydrouridine that is currently under clinical development for the treatment of sickle cell disease (SCD). Here, we investigate the potential for the tetrahydrouridine component of NDec to act as an inhibitor or substrate of key concentrative nucleoside transporters (CNT1-3) and equilibrative nucleoside transporters (ENT1-2). Nucleoside transporter inhibition and tetrahydrouridine accumulation assays were performed using Madin-Darby canine kidney strain II (MDCKII) cells overexpressing human CNT1, CNT2, CNT3, ENT1, and ENT2 transporters. Results showed that tetrahydrouridine did not influence CNT- or ENT-mediated uridine/adenosine accumulation in MDCKII cells at the concentrations tested (25 and 250 µM). Accumulation of tetrahydrouridine in MDCKII cells was initially shown to be mediated by CNT3 and ENT2. However, while time- and concentration-dependence experiments showed active accumulation of tetrahydrouridine in CNT3-expressing cells, allowing for estimation of Km (3,140 µM) and Vmax (1,600 pmol/mg protein/min), accumulation of tetrahydrouridine was not observed in ENT2-expressing cells. Potent CNT3 inhibitors are a class of drugs not generally prescribed to patients with SCD, except in certain specific circumstances. These data suggest that NDec can be administered safely with drugs that act as substrates and inhibitors of the nucleoside transporters included in this study.

Combined inhibition of histone deacetylase and cytidine deaminase improves epigenetic potency of decitabine in colorectal adenocarcinomas.

BACKGROUND: Targeting the epigenome of cancerous diseases represents an innovative approach, and the DNA methylation inhibitor decitabine is recommended for the treatment of hematological malignancies. Although epigenetic alterations are also common to solid tumors, the therapeutic efficacy of decitabine in colorectal adenocarcinomas (COAD) is unfavorable. Current research focuses on an identification of combination therapies either with chemotherapeutics or checkpoint inhibitors in modulating the tumor microenvironment. Here we report a series of molecular investigations to evaluate potency of decitabine, the histone deacetylase inhibitor PBA and the cytidine deaminase (CDA) inhibitor tetrahydrouridine (THU) in patient derived functional and p53 null colon cancer cell lines (CCCL). We focused on the inhibition of cell proliferation, the recovery of tumor suppressors and programmed cell death, and established clinical relevance by evaluating drug responsive genes among 270 COAD patients. Furthermore, we evaluated treatment responses based on CpG island density. RESULTS: Decitabine caused marked repression of the DNMT1 protein. Conversely, PBA treatment of CCCL recovered acetylation of histone 3 lysine residues, and this enabled an open chromatin state. Unlike single decitabine treatment, the combined decitabine/PBA treatment caused > 95% inhibition of cell proliferation, prevented cell cycle progression especially in the S and G2-phase and induced programmed cell death. Decitabine and PBA differed in their ability to facilitate re-expression of genes localized on different chromosomes, and the combined decitabine/PBA treatment was most effective in the re-expression of 40 tumor suppressors and 13 genes typically silenced in cancer-associated genomic regions of COAD patients. Furthermore, this treatment repressed expression of 11 survival (anti-apoptotic) genes and augmented expression of X-chromosome inactivated genes, especially the lncRNA Xist to facilitate p53-mediated apoptosis. Pharmacological inhibition of CDA by THU or its gene knockdown prevented decitabine inactivation. Strikingly, PBA treatment recovered the expression of the decitabine drug-uptake transporter SLC15A1, thus enabling high tumor drug-loads. Finally, for 26 drug responsive genes we demonstrated improved survival in COAD patients. CONCLUSION: The combined decitabine/PBA/THU drug treatment improved drug potency considerably, and given their existing regulatory approval, our findings merit prospective clinical trials for the triple combination in COAD patients.

Upregulation of cytidine deaminase in NAT1 knockout breast cancer cells.

PURPOSE: Arylamine N-acetyltransferase 1 (NAT1), a phase II metabolic enzyme, is frequently upregulated in breast cancer. Inhibition or depletion of NAT1 leads to growth retardation in breast cancer cells in vitro and in vivo. A previous metabolomics study of MDA-MB-231 breast cancer cells suggests that NAT1 deletion leads to a defect in de novo pyrimidine biosynthesis. In the present study, we observed that NAT1 deletion results in upregulation of cytidine deaminase (CDA), which is involved in the pyrimidine salvage pathway, in multiple breast cancer cell lines (MDA-MB-231, MCF-7 and ZR-75-1). We hypothesized that NAT1 KO MDA-MB-231 cells show differential sensitivity to drugs that either inhibit cellular pyrimidine homeostasis or are metabolized by CDA. METHODS: The cells were treated with (1) inhibitors of dihydroorotate dehydrogenase or CDA (e.g., teriflunomide and tetrahydrouridine); (2) pyrimidine/nucleoside analogs (e.g., gemcitabine and 5-azacytidine); and (3) naturally occurring, modified cytidines (e.g., 5-formyl-2'-deoxycytidine; 5fdC). RESULTS: Although NAT1 KO cells failed to show differential sensitivity to nucleoside analogs that are metabolized by CDA, they were markedly more sensitive to 5fdC which induces DNA damage in the presence of high CDA activity. Co-treatment with 5fdC and a CDA inhibitor, tetrahydrouridine, abrogated the increase in 5fdC cytotoxicity in NAT1 KO cells, suggesting that the increased sensitivity of NAT1 KO cells to 5fdC is dependent on their increased CDA activity. CONCLUSIONS: The present findings suggest a novel therapeutic strategy to treat breast cancer with elevated NAT1 expression. For instance, NAT1 inhibition may be combined with cytotoxic nucleosides (e.g., 5fdC) for breast cancer treatment.

In vitro drug-drug interactions of decitabine and tetrahydrouridine involving drug transporters and drug metabolising enzymes.

1. NDec is a novel, oral, fixed-dose formulation of decitabine and tetrahydrouridine that is currently being developed for the treatment of patients with sickle cell disease. Here, we examine the potential for both components of NDec to interact with key drug metabolising enzymes (tetrahydrouridine only) and drug transporters (decitabine and tetrahydrouridine).2. This study assessed the inhibition and induction of cytochrome P450 (CYP) enzymes by tetrahydrouridine, as well as the involvement of specific drug metabolising enzymes in tetrahydrouridine metabolism. Inhibition of efflux and uptake transporters by both decitabine and tetrahydrouridine was also studied.3. Tetrahydrouridine did not inhibit or induce relevant CYP enzymes at concentrations ranging from 0.1 to 100 µM. Metabolism of tetrahydrouridine did not occur in the presence of the human drug metabolising enzymes tested. Tetrahydrouridine showed weak inhibition towards the MATE2-K transporter (∼30% inhibition at 5 and 50 µM), which was not deemed clinically relevant. Tetrahydrouridine did not inhibit any of the remaining uptake or efflux transporters. Decitabine (0.5 and 5 µM) did not inhibit any of the evaluated uptake or efflux drug transporters.4. Data presented confirm that tetrahydrouridine and decitabine are unlikely to be involved in metabolism- or transporter-based drug-drug interactions.

A pilot clinical trial of oral tetrahydrouridine/decitabine for noncytotoxic epigenetic therapy of chemoresistant lymphoid malignancies.

One mechanism by which lymphoid malignancies resist standard apoptosis-intending (cytotoxic) treatments is genetic attenuation of the p53/p16-CDKN2A apoptosis axis. Depletion of the epigenetic protein DNA methyltransferase 1 (DNMT1) using the deoxycytidine analog decitabine is a validated approach to cytoreduce malignancy independent of p53/p16. In vivo decitabine activity, however, is restricted by rapid catabolism by cytidine deaminase (CDA). We, therefore, combined decitabine with the CDA-inhibitor tetrahydrouridine and conducted a pilot clinical trial in patients with relapsed lymphoid malignancies: the doses of tetrahydrouridine/decitabine used (∼10/0.2 mg/kg orally (PO) 2×/week) were selected for the molecular pharmacodynamic objective of non-cytotoxic, S-phase dependent, DNMT1-depletion, guided by previous Phase 1 studies. Patients with relapsed/refractory B- or T-cell malignancies (n = 7) were treated for up to 18 weeks. Neutropenia without concurrent thrombocytopenia is an expected toxicity of DNMT1-depletion and occurred in all patients (Grade 3/4). Subjective and objective clinical improvements occurred in 4 of 7 patients, but these responses were lost upon treatment interruptions and reductions to manage neutropenia. We thus performed parallel experiments in a preclinical in vivo model of lymphoma to identify regimen refinements that might sustain DNMT1-targeting in malignant cells but limit neutropenia. We found that timed-alternation of decitabine with the related molecule 5-azacytidine, and combination with inhibitors of CDA and de novo pyrimidine synthesis could leverage feedback responses of pyrimidine metabolism to substantially increase lymphoma cytoreduction but with less neutropenia. In sum, regimen innovations beyond incorporation of a CDA-inhibitor are needed to sustain decitabine DNMT1-targeting and efficacy against chemo-resistant lymphoid malignancy. Such potential solutions were explored in preclinical in vivo studies.

Intravenous 5-fluoro-2'-deoxycytidine administered with tetrahydrouridine increases the proportion of p16-expressing circulating tumor cells in patients with advanced solid tumors.

PURPOSE: Following promising responses to the DNA methyltransferase (DNMT) inhibitor 5-fluoro-2'-deoxycytidine (FdCyd) combined with tetrahydrouridine (THU) in phase 1 testing, we initiated a non-randomized phase 2 study to assess response to this combination in patients with advanced solid tumor types for which tumor suppressor gene methylation is potentially prognostic. To obtain pharmacodynamic evidence for DNMT inhibition by FdCyd, we developed a novel method for detecting expression of tumor suppressor protein p16/INK4A in circulating tumor cells (CTCs). METHODS: Patients in histology-specific strata (breast, head and neck [H&N], or non-small cell lung cancers [NSCLC] or urothelial transitional cell carcinoma) were administered FdCyd (100 mg/m2) and THU (350 mg/m2) intravenously 5 days/week for 2 weeks, in 28-day cycles, and progression-free survival (PFS) rate and objective response rate (ORR) were evaluated. Blood specimens were collected for CTC analysis. RESULTS: Ninety-three eligible patients were enrolled (29 breast, 21 H&N, 25 NSCLC, and 18 urothelial). There were three partial responses. All strata were terminated early due to insufficient responses (H&N, NSCLC) or slow accrual (breast, urothelial). However, the preliminary 4-month PFS rate (42%) in the urothelial stratum exceeded the predefined goal-though the ORR (5.6%) did not. An increase in the proportion of p16-expressing cytokeratin-positive CTCs was detected in 69% of patients evaluable for clinical and CTC response, but was not significantly associated with clinical response. CONCLUSION: Further study of FdCyd + THU is potentially warranted in urothelial carcinoma but not NSCLC or breast or H&N cancer. Increase in the proportion of p16-expressing cytokeratin-positive CTCs is a pharmacodynamic marker of FdCyd target engagement.

A plant pentatricopeptide repeat protein with a DYW-deaminase domain is sufficient for catalyzing C-to-U RNA editing in vitro.

Pentatricopeptide repeat (PPR) proteins with C-terminal DYW domains are present in organisms that undergo C-to-U editing of organelle RNA transcripts. PPR domains act as specificity factors through electrostatic interactions between a pair of polar residues and the nitrogenous bases of an RNA target. DYW-deaminase domains act as the editing enzyme. Two moss (Physcomitrella patens) PPR proteins containing DYW-deaminase domains, PPR65 and PPR56, can convert Cs to Us in cognate, exogenous RNA targets co-expressed in Escherichia coli We show here that purified, recombinant PPR65 exhibits robust editase activity on synthetic RNAs containing cognate, mitochondrial PpccmFC sequences in vitro, indicating that a PPR protein with a DYW domain is solely sufficient for catalyzing C-to-U RNA editing in vitro Monomeric fractions possessed the highest conversion efficiency, and oligomeric fractions had reduced activity. Inductively coupled plasma (ICP)-MS analysis indicated a stoichiometry of two zinc ions per highly active PPR65 monomer. Editing activity was sensitive to addition of zinc acetate or the zinc chelators 1,10-o-phenanthroline and EDTA. Addition of ATP or nonhydrolyzable nucleotide analogs stimulated PPR65-catalyzed RNA-editing activity on PpccmFC substrates, indicating potential allosteric regulation of PPR65 by ATP. Unlike for bacterial cytidine deaminase, addition of two putative transition-state analogs, zebularine and tetrahydrouridine, failed to disrupt RNA-editing activity. RNA oligonucleotides with a single incorporated zebularine also did not disrupt editing in vitro, suggesting that PPR65 cannot bind modified bases due to differences in the structure of the active site compared with other zinc-dependent nucleotide deaminases.

Biodistribution, Tumor Detection, and Radiation Dosimetry of 18F-5-Fluoro-2'-Deoxycytidine with Tetrahydrouridine in Solid Tumors.

In preclinical studies, 5-fluoro-2'-deoxycytidine (FdCyd), an inhibitor of DNA methyltransferase and DNA hypermethylation, has shown treatment efficacy against multiple malignancies by suppressing epigenetic hypermethylation in tumor cells. Several ongoing clinical trials are using FdCyd, and although some patients may respond to this drug, in most patients it is ineffective. Thus, establishing a noninvasive imaging modality to evaluate the distribution of the drug may provide insight into the variable responses. A novel experimental radiopharmaceutical, 18F-labeled FdCyd, was developed as a companion imaging agent to the nonradioactive form of the drug, FdCyd. We present the first-in-humans radiation dosimetry results and biodistribution of 18F-FdCyd, administered along with tetrahydrouridine, an inhibitor of cytidine/deoxycytidine deaminase, in patients with a variety of solid tumors undergoing FdCyd therapy. Methods: This phase 0 imaging trial examined the 18F-FdCyd biodistribution and radiation dosimetry in 5 human subjects enrolled in companion therapy trials. In each subject, 4 sequential PET scans were acquired to estimate whole-body and individual organ effective dose, using OLINDA/EXM, version 1.0. Tumor-to-background ratios were also calculated for the tumor sites visualized on PET/CT imaging. Results: The average whole-body effective dose for the experimental radiopharmaceutical 18F-FdCyd administered in conjunction with tetrahydrouridine was 2.12E-02 ± 4.15E-03 mSv/MBq. This is similar to the radiation dose estimates for 18F-FDG PET. The critical organ, with the highest absorbed radiation dose, was the urinary bladder wall at 7.96E-02 mSv/MBq. Other organ doses of note were the liver (6.02E-02mSv/MBq), kidneys (5.26E-02 mSv/MBq), and gallbladder (4.05E-02 mSv/MBq). Tumor target-to-background ratios ranged from 2.4 to 1.4, which potentially enable tumor visualization in static PET images. Conclusion: This phase 0 imaging clinical trial provides evidence that 18F-FdCyd administered in conjunction with tetrahydrouridine yields acceptable individual organ and whole-body effective doses, as well as modest tumor-to-background ratios that potentially enable tumor visualization. Dose estimates for 18F-FdCyd are comparable to those for other PET radiopharmaceuticals, such as 18F-FDG. Further studies with larger study populations are warranted to assess 18F-FdCyd imaging as a predictor of FdCyd treatment effectiveness.

Oral tetrahydrouridine and decitabine for non-cytotoxic epigenetic gene regulation in sickle cell disease: A randomized phase 1 study.

BACKGROUND: Sickle cell disease (SCD), a congenital hemolytic anemia that exacts terrible global morbidity and mortality, is driven by polymerization of mutated sickle hemoglobin (HbS) in red blood cells (RBCs). Fetal hemoglobin (HbF) interferes with this polymerization, but HbF is epigenetically silenced from infancy onward by DNA methyltransferase 1 (DNMT1). METHODS AND FINDINGS: To pharmacologically re-induce HbF by DNMT1 inhibition, this first-in-human clinical trial (NCT01685515) combined 2 small molecules-decitabine to deplete DNMT1 and tetrahydrouridine (THU) to inhibit cytidine deaminase (CDA), the enzyme that otherwise rapidly deaminates/inactivates decitabine, severely limiting its half-life, tissue distribution, and oral bioavailability. Oral decitabine doses, administered after oral THU 10 mg/kg, were escalated from a very low starting level (0.01, 0.02, 0.04, 0.08, or 0.16 mg/kg) to identify minimal doses active in depleting DNMT1 without cytotoxicity. Patients were SCD adults at risk of early death despite standard-of-care, randomized 3:2 to THU-decitabine versus placebo in 5 cohorts of 5 patients treated 2X/week for 8 weeks, with 4 weeks of follow-up. The primary endpoint was ≥ grade 3 non-hematologic toxicity. This endpoint was not triggered, and adverse events (AEs) were not significantly different in THU-decitabine-versus placebo-treated patients. At the decitabine 0.16 mg/kg dose, plasma concentrations peaked at approximately 50 nM (Cmax) and remained elevated for several hours. This dose decreased DNMT1 protein in peripheral blood mononuclear cells by >75% and repetitive element CpG methylation by approximately 10%, and increased HbF by 4%-9% (P < 0.001), doubling fetal hemoglobin-enriched red blood cells (F-cells) up to approximately 80% of total RBCs. Total hemoglobin increased by 1.2-1.9 g/dL (P = 0.01) as reticulocytes simultaneously decreased; that is, better quality and efficiency of HbF-enriched erythropoiesis elevated hemoglobin using fewer reticulocytes. Also indicating better RBC quality, biomarkers of hemolysis, thrombophilia, and inflammation (LDH, bilirubin, D-dimer, C-reactive protein [CRP]) improved. As expected with non-cytotoxic DNMT1-depletion, platelets increased and neutrophils concurrently decreased, but not to an extent requiring treatment holds. As an early phase study, limitations include small patient numbers at each dose level and narrow capacity to evaluate clinical benefits. CONCLUSION: Administration of oral THU-decitabine to patients with SCD was safe in this study and, by targeting DNMT1, upregulated HbF in RBCs. Further studies should investigate clinical benefits and potential harms not identified to date. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01685515.

Deoxycytidine and Deoxythymidine Treatment for Thymidine Kinase 2 Deficiency.

OBJECTIVE: Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the nuclear gene, TK2, cause TK2 deficiency, which manifests predominantly in children as myopathy with mtDNA depletion. Molecular bypass therapy with the TK2 products, deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP), prolongs the life span of Tk2-deficient (Tk2-/- ) mice by 2- to 3-fold. Because we observed rapid catabolism of the deoxynucleoside monophosphates to deoxythymidine (dT) and deoxycytidine (dC), we hypothesized that: (1) deoxynucleosides might be the major active agents and (2) inhibition of deoxycytidine deamination might enhance dTMP+dCMP therapy. METHODS: To test these hypotheses, we assessed two therapies in Tk2-/- mice: (1) dT+dC and (2) coadministration of the deaminase inhibitor, tetrahydrouridine (THU), with dTMP+dCMP. RESULTS: We observed that dC+dT delayed disease onset, prolonged life span of Tk2-deficient mice and restored mtDNA copy number as well as respiratory chain enzyme activities and levels. In contrast, dCMP+dTMP+THU therapy decreased life span of Tk2-/- animals compared to dCMP+dTMP. INTERPRETATION: Our studies demonstrate that deoxynucleoside substrate enhancement is a novel therapy, which may ameliorate TK2 deficiency in patients. Ann Neurol 2017;81:641-652.

LC-MS/MS assay for the quantitation of FdCyd and its metabolites FdUrd and FU in human plasma.

The hypomethylating agent 5-fluoro-2'-deoxycytidine (FdCyd, NSC 48006) is being evaluated clinically both via the intravenous route and via the oral route in combination with 3,4,5,6-tetrahydrouridine (THU), a potent inhibitor of FdCyd catabolism. To determine the pharmacokinetics of FdCyd and downstream metabolites, we developed and validated an LC-MS/MS assay for the quantitation of FdCyd, 5-fluoro-2'-deoxyuridine (FdUrd), and 5-fluorouracil (FU) in 0.2mL human plasma. After acetonitrile protein precipitation, the sample was split and separate chromatography was achieved for FdCyd with a Synergi Polar-RP column and for FdUrd and FU with a Shodex Asahipak NH2P-50 2D column. Gradients of 0.1% acetic acid in acetonitrile and water were used. Detection with a Quattromicro quadrupole mass spectrometer with electrospray ionization in positive-ion (FdCyd) or negative-ion (FdUrd and FU) multiple reaction monitoring (MRM) mode. The assay was linear from 5 to 3000ng/mL for all three analytes and proved to be accurate (96.7-105.5%) and precise (<8.1%CV), and fulfilled FDA criteria for bioanalytical method validation. We demonstrated the suitability of this assay for measuring FdCyd and metabolites FdUrd and FU in plasma from a patient who was administered 120mg PO FdCyd 30min after 3000mg THU. Our LC-MS/MS assay will be an essential tool to further define the pharmacology of FdCyd in ongoing and future studies.

Preclinical studies of 5-fluoro-2'-deoxycytidine and tetrahydrouridine in pediatric brain tumors.

Chemotherapies active in preclinical studies frequently fail in the clinic due to lack of efficacy, which limits progress for rare cancers since only small numbers of patients are available for clinical trials. Thus, a preclinical drug development pipeline was developed to prioritize potentially active regimens for pediatric brain tumors spanning from in vitro drug screening, through intracranial and intra-tumoral pharmacokinetics to in vivo efficacy studies. Here, as an example of the pipeline, data are presented for the combination of 5-fluoro-2'-deoxycytidine and tetrahydrouridine in three pediatric brain tumor models. The in vitro activity of nine novel therapies was tested against tumor spheres derived from faithful mouse models of Group 3 medulloblastoma, ependymoma, and choroid plexus carcinoma. Agents with the greatest in vitro potency were then subjected to a comprehensive series of in vivo pharmacokinetic (PK) and pharmacodynamic (PD) studies culminating in preclinical efficacy trials in mice harboring brain tumors. The nucleoside analog 5-fluoro-2'-deoxycytidine (FdCyd) markedly reduced the proliferation in vitro of all three brain tumor cell types at nanomolar concentrations. Detailed intracranial PK studies confirmed that systemically administered FdCyd exceeded concentrations in brain tumors necessary to inhibit tumor cell proliferation, but no tumor displayed a significant in vivo therapeutic response. Despite promising in vitro activity and in vivo PK properties, FdCyd is unlikely to be an effective treatment of pediatric brain tumors, and therefore was deprioritized for the clinic. Our comprehensive and integrated preclinical drug development pipeline should reduce the attrition of drugs in clinical trials.

Oral and intravenous pharmacokinetics of 5-fluoro-2'-deoxycytidine and THU in cynomolgus monkeys and humans.

INTRODUCTION: 5-Fluoro-2'-deoxycytidine (FdCyd; NSC48006), a fluoropyrimidine nucleoside inhibitor of DNA methylation, is degraded by cytidine deaminase (CD). Pharmacokinetic evaluation was carried out in cynomolgus monkeys in support of an ongoing phase I study of the PO combination of FdCyd and the CD inhibitor tetrahydrouridine (THU; NSC112907). METHODS: Animals were dosed intravenously (IV) or per os (PO). Plasma samples were analyzed by LC-MS/MS for FdCyd, metabolites, and THU. Clinical chemistry and hematology were performed at various times after dosing. A pilot pharmacokinetic study was performed in humans to assess FdCyd bioavailability. RESULTS: After IV FdCyd and THU administration, FdCyd C(max) and AUC increased with dose. FdCyd half-life ranged between 22 and 56 min, and clearance was approximately 15 mL/min/kg. FdCyd PO bioavailability after THU ranged between 9 and 25 % and increased with increasing THU dose. PO bioavailability of THU was less than 5 %, but did result in plasma concentrations associated with inhibition of its target CD. Human pilot studies showed comparable bioavailability for FdCyd (10 %) and THU (4.1 %). CONCLUSION: Administration of THU with FdCyd increased the exposure to FdCyd and improved PO FdCyd bioavailability from <1 to 24 %. Concentrations of THU and FdCyd achieved after PO administration are associated with CD inhibition and hypomethylation, respectively. The schedule currently studied in phase I studies of PO FdCyd and THU is daily times three at the beginning of the first and second weeks of a 28-day cycle.

Validation of a hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of gemcitabine in human plasma with tetrahydrouridine.

A simple and reproducible bioanalytical method for the determination of gemcitabine in human plasma treated with tetrahydrouridine (THU) was developed and validated using a hydrophilic interaction ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). To prevent deamination of gemcitabine, blood was treated with THU, and the plasma samples obtained after centrifugation were used in this study. Gemcitabine and gemcitabine-(13)C, (15)N2 used as an internal standard, were extracted from human plasma treated with THU using a 96-well Hybrid SPE-Precipitation plate. Extracts were chromatographed on a hydrophilic interaction chromatography column with isocratic elution. Detection was performed using Quattro Premier with positive electrospray ionization multiple reaction monitoring mode. The standard curve ranged from 10 to 10,000 ng/mL without carryover. No significant interferences were detected in blank plasma and no interferences by 2'-2'-difluoro-2'-deoxyuridine, a metabolite of gemcitabine. Accuracy and precision in the intra-batch reproducibility study using quality control samples with three THU levels did not exceed ±5.4 and 7.3%, respectively, and the inter-batch reproducibility results also met the criteria. Stability of gemcitabine was ensured in whole blood and plasma as well as stability of THU in solutions. The UPLC-MS/MS method developed was successfully validated and can be applied for gemcitabine bioanalysis in clinical studies.

A phase I, pharmacokinetic, and pharmacodynamic evaluation of the DNA methyltransferase inhibitor 5-fluoro-2'-deoxycytidine, administered with tetrahydrouridine.

PURPOSE: Inhibitors of DNA (cytosine-5)-methyltransferases (DNMT) are active antineoplastic agents. We conducted the first-in-human phase I trial of 5-fluoro-2'-deoxycytidine (FdCyd), a DNMT inhibitor stable in aqueous solution, in patients with advanced solid tumors. Objectives were to establish the safety, maximum tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of FdCyd + tetrahydrouridine (THU). METHODS: FdCyd + THU were administered by 3 h IV infusion on days 1-5 every 3 weeks, or days 1-5 and 8-12 every 4 weeks. FdCyd was administered IV with a fixed 350 mg/m(2)/day dose of THU to inhibit deamination of FdCyd. Pharmacokinetics of FdCyd, downstream metabolites and THU were assessed by LC-MS/MS. RBC γ-globin expression was evaluated as a pharmacodynamics biomarker. RESULTS: Patients were enrolled on the 3-week schedule at doses up to 80 mg/m(2)/day without dose-limiting toxicity (DLT) prior to transitioning to the 4-week schedule, which resulted in an MTD of 134 mg/m(2)/day; one of six patients had a first-cycle DLT (grade 3 colitis). FdCyd ≥40 mg/m(2)/day produced peak plasma concentrations >1 µM. Although there was inter-patient variability, γ-globin mRNA increased during the first two treatment cycles. One refractory breast cancer patient experienced a partial response (PR) of >90 % decrease in tumor size, lasting over a year. CONCLUSIONS: The MTD was established at 134 mg/m(2) FdCyd + 350 mg/m(2) THU days 1-5 and 8-12 every 4 weeks. Based on toxicities observed over multiple cycles, good plasma exposures, and the sustained PR observed at 67 mg/m(2)/day, the phase II dose for our ongoing multi-histology trial is 100 mg/m(2)/day FdCyd with 350 mg/m(2)/day THU.

Subchronic oral toxicity study of decitabine in combination with tetrahydrouridine in CD-1 mice.

Decitabine (5-aza-2'-deoxycytidine; DAC) in combination with tetrahydrouridine (THU) is a potential oral therapy for sickle cell disease and ß-thalassemia. A study was conducted in mice to assess safety of this combination therapy using oral gavage of DAC and THU administered 1 hour prior to DAC on 2 consecutive days/week for up to 9 weeks followed by a 28-day recovery to support its clinical trials up to 9-week duration. Tetrahydrouridine, a competitive inhibitor of cytidine deaminase, was used in the combination to improve oral bioavailability of DAC. Doses were 167 mg/kg THU followed by 0, 0.2, 0.4, or 1.0 mg/kg DAC; THU vehicle followed by 1.0 mg/kg DAC; or vehicle alone. End points evaluated were clinical observations, body weights, food consumption, clinical pathology, gross/histopathology, bone marrow micronuclei, and toxicokinetics. There were no treatment-related effects noticed on body weight, food consumption, serum chemistry, or urinalysis parameters. Dose- and gender-dependent changes in plasma DAC levels were observed with a Cmax within 1 hour. At the 1 mg/kg dose tested, THU increased DAC plasma concentration (∼ 10-fold) as compared to DAC alone. Severe toxicity occurred in females receiving high-dose 1 mg/kg DAC + THU, requiring treatment discontinuation at week 5. Severity and incidence of microscopic findings increased in a dose-dependent fashion; findings included bone marrow hypocellularity (with corresponding hematologic changes and decreases in white blood cells, red blood cells, hemoglobin, hematocrit, reticulocytes, neutrophils, and lymphocytes), thymic/lymphoid depletion, intestinal epithelial apoptosis, and testicular degeneration. Bone marrow micronucleus analysis confirmed bone marrow cytotoxicity, suppression of erythropoiesis, and genotoxicity. Following the recovery period, a complete or trend toward resolution of these effects was observed. In conclusion, the combination therapy resulted in an increased sensitivity to DAC toxicity correlating with DAC plasma levels, and females are more sensitive compared to their male counterparts.

Nucleoside-catabolizing enzymes in mycoplasma-infected tumor cell cultures compromise the cytostatic activity of the anticancer drug gemcitabine.

The intracellular metabolism and cytostatic activity of the anticancer drug gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdC) was severely compromised in Mycoplasma hyorhinis-infected tumor cell cultures. Pronounced deamination of dFdC to its less cytostatic metabolite 2',2'-difluoro-2'-deoxyuridine was observed, both in cell extracts and spent culture medium (i.e. tumor cell-free but mycoplasma-containing) of mycoplasma-infected tumor cells. This indicates that the decreased antiproliferative activity of dFdC in such cells is attributed to a mycoplasma cytidine deaminase causing rapid drug catabolism. Indeed, the cytostatic activity of gemcitabine could be restored by the co-administration of tetrahydrouridine (a potent cytidine deaminase inhibitor). Additionally, mycoplasma-derived pyrimidine nucleoside phosphorylase (PyNP) activity indirectly potentiated deamination of dFdC: the natural pyrimidine nucleosides uridine, 2'-deoxyuridine and thymidine inhibited mycoplasma-associated dFdC deamination but were efficiently catabolized (removed) by mycoplasma PyNP. The markedly lower anabolism and related cytostatic activity of dFdC in mycoplasma-infected tumor cells was therefore also (partially) restored by a specific TP/PyNP inhibitor (TPI), or by exogenous thymidine. Consequently, no effect on the cytostatic activity of dFdC was observed in tumor cell cultures infected with a PyNP-deficient Mycoplasma pneumoniae strain. Because it has been reported that some commensal mycoplasma species (including M. hyorhinis) preferentially colonize tumor tissue in cancer patients, our findings suggest that the presence of mycoplasmas in the tumor microenvironment could be a limiting factor for the anticancer efficiency of dFdC-based chemotherapy. Accordingly, a significantly decreased antitumor effect of dFdC was observed in mice bearing M. hyorhinis-infected murine mammary FM3A tumors compared with uninfected tumors.

Design, synthesis, and pharmacological evaluation of fluorinated tetrahydrouridine derivatives as inhibitors of cytidine deaminase.

Several 2'-fluorinated tetrahydrouridine derivatives were synthesized as inhibitors of cytidine deaminase (CDA). (4R)-2'-Deoxy-2',2'-difluoro-3,4,5,6-tetrahydrouridine (7a) showed enhanced acid stability over tetrahydrouridine (THU) 5 at its N-glycosyl bond. As a result, compound 7a showed an improved oral pharmacokinetic profile with a higher and more reproducible plasma exposure in rhesus monkeys compared to 5. Co-administration of 7a with decitabine, a CDA substrate, boosted the plasma levels of decitabine in rhesus monkeys. These results demonstrate that compound 7a can serve as an acid-stable alternative to 5 as a pharmacoenhancer of drugs subject to CDA-mediated metabolism.