Cytidine Deaminase Inhibitor Research Articles

ChemInform Abstract: A Facile Synthetic Route to Diazepinone Derivatives via Ring Closing Metathesis and Its Application for Human Cytidine Deaminase Inhibitors.

AbstractA variety of novel diazepinone derivatives are synthesized from corresponding α‐amino acids and amino alcohols by a new and efficient methodology involving a ring closing metathesis as a key step.

Site Directed Mutagenesis as a Tool to Understand the Catalytic Mechanism of Human Cytidine Deaminase

Cytidine deaminase (CDA), is one of the enzymes involved in the pyrimidine salvage pathways, which catalyzes the formation of uridine and deoxyuridine by the hydrolytic deamination of cytidine and deoxycytidine, respectively. Human CDA is a tetrameric enzyme of identical 15 kDa subunits, each containing an essential zinc atom in the active site. The substrate binds to each active site independently and the cooperativity between subunits has not been reported. CDA is able to recognize as substrates some antitumor and antiviral cytidine analogs rendering them pharmacologically inactive. In light of the role played by this enzyme, a deep knowledge of CDA active site and mechanism of catalysis is required. Site-directed mutagenesis, associated with molecular modeling studies, may be an important tool to discover the active site structure of an enzyme and consequently its mechanism of action. In this review are summarized the site-directed mutagenesis experiments performed on human CDA: through these studies it was possible to understand the role exerted by specific amino acid residues in CDA active site and in the contacts between subunits. The obtained results may open a way for designing new cytidine based drugs or more potent CDA inhibitors.

Pharmacokinetic and pharmacodynamic analysis of 5-aza-2’-deoxycytidine (decitabine) in the design of its dose-schedule for cancer therapy

5-Aza-2′-deoxycytidine (5-AZA-CdR, decitabine), an epigenetic drug that inhibits DNA methylation, is currently used to treat myelodysplastic syndrome (MDS), and is under investigation for treating acute myeloid leukemia (AML) and other malignancies. 5-AZA-CdR can reactivate tumor suppressor genes silenced by aberrant DNA methylation, a frequent event in all types of cancer. Because this epigenetic change is reversible, it is a good target for 5-AZA-CdR therapy. We have reviewed the preclinical data of 5-AZA-CdR to analyze the concentrations and exposure times required to eradicate cancer stem cells. We analyzed the dose-schedules used in animal models that show potent antineoplastic activity of 5-AZA-CdR. We attempted to correlate the preclinical data with the responses obtained in clinical trials of 5-AZA-CdR in patients with cancer. The pharmacokinetics and drug distribution of 5-AZA-CdR are key parameters because adequate therapeutic drug levels are required to eliminate cancer stem cells in all anatomic compartments. The plasma half-life of 5-AZA-CdR in humans is approximately 20 minutes due to the high levels in the liver of cytidine deaminase, the enzyme that inactivates this analogue. This provides a rationale to use an inhibitor of cytidine deaminase in combination with 5-AZA-CdR. Low-dose 5-AZA-CdR is effective for MDS and AML and can induce complete remissions (CR). However, maintenance of CR with low-dose 5-AZA-CdR is difficult. Based on analyses of preclinical and clinical data, low dose 5-AZA-CdR has the potential to be an effective form of therapy in some patients with cancer. For patients who do not respond to low dose therapy we recommend dose-intensive treatment with 5-AZA-CdR. Patients who are candidates for intensive dose 5-AZA-CdR should have a good bone marrow status so as to permit adequate recovery from myelosuppression, the major toxicity of 5-AZA-CdR. Solid tumors are also interesting targets for therapy with 5-AZA-CdR. Both low dose and intensive therapy with 5-AZA-CdR can reduce the proliferative potential of tumor stem cells in animal models. We propose novel dose schedules of 5-AZA-CdR for investigation in patients with cancer. The full chemotherapeutic potential of 5-AZA-CdR to treat cancer merits further clinical investigation and can only be realized when its optimal dose-schedule is determined.

Very Low to Low Doses of Continuous Azacitidine in Combination with Standard Doses of Lenalidomide and Low-Dose Dexamethasone (Rd) in Patients with Relapsed or Refractory Multiple Myeloma (RRMM): Interim Results of a Phase I/II Study

AbstractAbstract 5031 Background:Myeloma inevitably becomes refractory to available agents primarily due to accumulation of genetic and epigenetic mutations in the aberrant clone. The s-phase dependent DNA methylation inhibitor azacitidine may help overcome epigenetically mediated resistance in myeloma cells that cycle despite treatment and reactivate alternate growth control programs in cells with genetic apoptosis resistance. Here we present interim results of an ongoing phase I/II study that explores the safety, antimyeloma activity, and epigenetic effects of continuous azacitidine at predicted non-cytotoxic doses in combination with Rd in RRMM Methods:Entry criteria included measurable disease, relapsed or refractory state, and glomerular filtration rate ≥ 60ml/min (Cockroft-Gault). Lenalidomide 25mg d1–21 every 28d and dexamethasone 40mg weekly were fixed. In phase I (3×3 design) azacitidine was escalated from 30 to 40mg/m2 SC weekly (DL 1 and 2) to 30, 40, and maximally 50mg/m2 SC twice a week (DL 3–5). Dose limiting toxicity (DLT) was assessed during cycle 1 and response every 28 days according to IMWG uniform response criteria (partial response, PR, or better) and adapted EBMT criteria (minor response, MR). Plasma activity of cytidine deaminase (CDA) was measured in dose levels 3–5, using HPLC to quantify deamination of added cytidine (Wilcoxon exact test). Phase II enrolls 10 additional patients at the highest tolerated dose and includes global gene expression and DNA methylation arrays before and after cycle 1 in CD138+, CD34+, and remaining bone marrow cells. Results:Twenty patients with relapsed (n=1) or refractory (n=19) MM were enrolled after they had received a median of 4 prior regimens (range 1–10). Their disease was refractory to lenalidomide (n=17), bortezomib and/or carfilzomib (n=16), or both lenalidomide and proteasome inhibitors (n=14). 18 patients had received alkylators, 8 high dose chemotherapy, and 6 anthracyclines. One DLT was observed in 1 out of 6 patients treated at DL 4; neutropenic fever without other evidence for infection. No DLT occurred in 4 patients treated so far at DL 5. Grade 3 and 4 toxicities were seen in 11 patients, neutropenia in 9 (lowest ANC 0. 24K/mm3), thrombocytopenia in 4 (lowest 16K/mm3, transfusions used in 1 to avoid treatment delay), neutropenic fever in 2 (1 after cycle 1), and diarrhea, depression, deep venous thrombosis, and a possibly related pleural effusion with hypoxemia in 1 patient each. DL 1 and 2 (n=6) yielded only one MR, but as azacitidine was increased to twice a week (DL 3–5, n=14) the clinical benefit response rate (CBRR, ≥MR) and response rate (RR, ≥PR) increased to 35. 7% and 28. 6%, respectively with 1 very good partial response (VGPR), 3 PR and 1 MR. The patient with MR had secondary plasma cell leukemia (sPCL) after 7 prior regimens and had to be taken off trial due to cytopenias but was continued on reduced azacitidine (20mg/m2), lenalidomide (5–10mg), and prednisone (50mg TIW) reaching PR off trial and achieving a 10 month time to progression (TTP). Of all other responding patients, only the one treated with weekly azacitidine progressed while receiving azacitidine (TTP 3 months), one patient with sPCL progressed during azacitidine interruption for neutropenic fever, another patient was taken off trial due to pleural effusion with hypoxemia, 2 are still on study, currently for 8 and 3 months. The median time to response was 1 cycle (range 1–7). Responders in DL 3–5 had similar treatment histories as the entire cohort, refractory to last Rx: 80%, median previous regimens: 4 (range 1–7), refractory to lenalidomide: 60%, to proteasome inhibitors: 80%, and to both: 60%; but their plasma CDA activity was lower than in non-responders (Figure 1). Women treated at DL 3–5 (n=6) responded better than men (n=8), likely related to lower plasma CDA activity (Figure 2). Conclusions:Azacitidine was generally well tolerated up to the target maximal dose of 50mg/m2SC twice a week (BIW) in combination with Rd given to 4 out of 6 patients required for establishing the phase 2 dose. Encouraging response rates in refractory MM correlated with low plasma activity of the azacitidine inactivating enzyme CDA found predominantly in women. Results support development of azacitidine in RRMM and argue for testing combination with the competitive inhibitor of CDA, tetrahydrouridine. Until then, dose escalation in non-responding but tolerating patients will be explored. [Display omitted] [Display omitted] Disclosures:Reu:Celgene: Research Funding. Off Label Use: Azacitidine in multiple myeloma. Kindwall-Keller:Celgene: Speakers Bureau; TEVA: Speakers Bureau. Duong:Celgene: Research Funding.

High cytidine deaminase expression in the liver provides sanctuary for cancer cells from decitabine treatment effects

We document for the first time that sanctuary in an organ which expresses high levels of the enzyme cytidine deaminase (CDA) is a mechanism of cancer cell resistance to cytidine analogues. This mechanism could explain why historically, cytidine analogues have not been successful chemotherapeutics against hepatotropic cancers, despite efficacy in vitro. Importantly, this mechanism of resistance can be readily reversed, without increasing toxicity to sensitive organs, by combining a cytidine analogue with an inhibitor of cytidine deaminase (tetrahydrouridine). Specifically, CDA rapidly metabolizes cytidine analogues into inactive uridine counterparts. Hence, to determine if sheltering/protection of cancer cells in organs which express high levels of CDA (e.g., liver) is a mechanism of resistance, we utilized a murine xenotransplant model of myeloid cancer that is sensitive to epigenetic therapeutic effects of the cytidine analogue decitabine in vitro and hepato-tropic in vivo. Treatment of tumor-bearing mice with decitabine (subcutaneous 0.2mg/kg 2X/week) doubled median survival and significantly decreased extra-hepatic tumor burden, but hepatic tumor burden remained substantial, to which the animals eventually succumbed. Combining a clinically-relevant inhibitor of CDA (tetrahydrouridine) with a lower dose of decitabine (subcutaneous 0.1mg/kg 2X/week) markedly decreased liver tumor burden without blood count or bone marrow evidence of myelotoxicity, and with further improvement in survival. In conclusion, sanctuary in a CDA-rich organ is a mechanism by which otherwise susceptible cancer cells can resist the effects of decitabine epigenetic therapy. This protection can be reversed without increasing myelotoxicity by combining tetrahydrouridine with a lower dose of decitabine.

Tetrahydrouridine inhibits cell proliferation through cell cycle regulation regardless of cytidine deaminase expression levels.

Tetrahydrouridine (THU) is a well characterized and potent inhibitor of cytidine deaminase (CDA). Highly expressed CDA catalyzes and inactivates cytidine analogues, ultimately contributing to increased gemcitabine resistance. Therefore, a combination therapy of THU and gemcitabine is considered to be a potential and promising treatment for tumors with highly expressed CDA. In this study, we found that THU has an alternative mechanism for inhibiting cell growth which is independent of CDA expression. Three different carcinoma cell lines (MIAPaCa-2, H441, and H1299) exhibited decreased cell proliferation after sole administration of THU, while being unaffected by knocking down CDA. To investigate the mechanism of THU-induced cell growth inhibition, cell cycle analysis using flow cytometry was performed. This analysis revealed that THU caused an increased rate of G1-phase occurrence while S-phase occurrence was diminished. Similarly, Ki-67 staining further supported that THU reduces cell proliferation. We also found that THU regulates cell cycle progression at the G1/S checkpoint by suppressing E2F1. As a result, a combination regimen of THU and gemcitabine might be a more effective therapy than previously believed for pancreatic carcinoma since THU works as a CDA inhibitor, as well as an inhibitor of cell growth in some types of pancreatic carcinoma cells.

Effects of tetrahydrouridine on pharmacokinetics and pharmacodynamics of oral decitabine

AbstractThe deoxycytidine analog decitabine (DAC) can deplete DNA methyl-transferase 1 (DNMT1) and thereby modify cellular epigenetics, gene expression, and differentiation. However, a barrier to efficacious and accessible DNMT1-targeted therapy is cytidine deaminase, an enzyme highly expressed in the intestine and liver that rapidly metabolizes DAC into inactive uridine counterparts, severely limiting exposure time and oral bioavailability. In the present study, the effects of tetrahydrouridine (THU), a competitive inhibitor of cytidine deaminase, on the pharmacokinetics and pharmacodynamics of oral DAC were evaluated in mice and nonhuman primates. Oral administration of THU before oral DAC extended DAC absorption time and widened the concentration-time profile, increasing the exposure time for S-phase–specific depletion of DNMT1 without the high peak DAC levels that can cause DNA damage and cytotoxicity. THU also decreased interindividual variability in pharmacokinetics seen with DAC alone. One potential clinical application of DNMT1-targeted therapy is to increase fetal hemoglobin and treat hemoglobinopathy. Oral THU-DAC at a dose that would produce peak DAC concentrations of less than 0.2μM administered 2×/wk for 8 weeks to nonhuman primates was not myelotoxic, hypomethylated DNA in the γ-globin gene promoter, and produced large cumulative increases in fetal hemoglobin. Combining oral THU with oral DAC changes DAC pharmacology in a manner that may facilitate accessible noncytotoxic DNMT1-targeted therapy.

DNA Methyltransferase Inhibitor, Zebularine, Delays Tumor Growth and Induces Apoptosis in a Genetically Engineered Mouse Model of Breast Cancer

Zebularine is a novel potent inhibitor of both cytidine deaminase and DNA methylation. We examined the effect of zebularine on mammary tumor growth in genetically engineered MMTV-PyMT transgenic mice that develop mammary tumors at 60 days of age with 100% penetrance. The MMTV-PyMT transgenic mice were randomized at 46 days of age into control (n = 25) and zebularine (n = 25) treatment groups and monitored for parameters of tumor growth. Zebularine was administered at 5 mg/mL in drinking water. We observed a significant delay in the growth of mammary tumors in zebularine-treated mice with a statistically significant reduction (P = 0.0135) in total tumor burden at 94 days of age when the mice were sacrificed. After 48 days of zebularine treatment, the tumors were predominantly necrotic compared with untreated animals. In addition, a high apoptotic index by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was observed as early as 13 days following treatment. Immunoblot analysis showed depletion of DNMT1 and partial depletion of DNMT3b after zebularine treatment. Microarray analyses of global gene expression identified upregulation of twelve methylation-regulated genes as well as a set of candidate cancer genes that participate in cell growth and apoptosis. In summary, zebularine inhibits the growth of spontaneous mammary tumors and causes early onset of tumor cell necrosis and apoptosis in a genetically engineered mouse model of breast cancer. Defining the parameters of zebularine-mediated tumor inhibition may advance the future development of DNA methyltransferase inhibitors as an effective cancer treatment.

A facile synthetic route to diazepinone derivatives via ring closing metathesis and its application for human cytidine deaminase inhibitors

A variety of diazepinone derivatives were prepared from α-amino acids and amino alcohols by a new synthetic methodology based on ring closing metathesis as a key step. The diazepinones were coupled with ribose derivatives to afford novel diazepinone nucleosides. Among them, (4R)-1-ribosyl-4-methyl-3,4-dihydro-1H-1,3-diazepin-2(7H)-one (3) showed a potent inhibitory effect (K(i) = 145.97 ± 4.87 nM) against human cytidine deaminase.

Combining Tetrahydrouridine with Decitabine Addresses Malignant Cell Sanctuary in the Liver, a Tissue That Expresses High Levels of Cytidine Deaminase,

Abstract 3509Non-cytotoxic DNA methyl-transferase 1 (DNMT1) depletion by decitabine induces p53-independent differentiation mediated cell cycle exit in acute myeloid leukemia (AML) cells and spares normal hematopoietic stem cells, providing a potentially important alternative to conventional apoptosis-based therapy. However, there are pharmacologic barriers that impede optimal clinical translation of pre-clinical observations. One major barrier is the enzyme cytidine deaminase (CDA), which drastically reduces the half-life of decitabine from many hours in vitro to a few minutes in vivo. This problem is significant since decitabine is S-phase specific in its mechanism of action, and for effective DNMT1 depletion, low peak levels but extended half-life are optimal. Furthermore, CDA is highly expressed in the liver and intestines, generating potential sanctuary sites from the effects of decitabine. Finally, upregulation of CDA expression is a potential mechanism by which AML cells may resist decitabine at the cellular level. To overcome the multiple CDA-mediated barriers to effective therapy, we evaluated the combination of very low dose decitabine with the CDA inhibitor tetrahydrouridine (THU) in a murine xenotransplantation model of aggressive human AML (THP1 AML cells – p53 and p16-null). Treatment groups (n=5 per group) received equal volume vehicle (PBS), low dose cytarabine (0.1 mg/kg subcutaneous [SC]) with (THU) (4mg/kg intraperitoneal [IP]), decitabine (0.1 mg/kg SC) with THU (4mg/kg IP), and decitabine alone (0.2 mg/kg SC). Median survival in decitabine (61 days) and THU-decitabine (70 days) treated mice was significantly extended compared to the PBS (38 days) and THU-cytarabine (50 days) treatment groups (Log Rank p=0.00156 (figure 1). In decitabine treated mice, eventual emergence of disease was concentrated in the liver, consistent with a sanctuary function of this organ. Combination of THU with decitabine largely eliminated liver infiltration by AML, reducing liver tumor load from average 5.2g in PBS treated mice to 0.8g in THU-Decitabine treated mice (t-test p<0.001, livers were harvested at different time-points corresponding to the Kaplan-Meier curve). In separate experiments without AML cell xenotransplantation, the non-cytotoxic, DNMT1 depleting character of the decitabine therapy was confirmed by hematologic and bone marrow evaluation for DNA damage by phospho-H2AX staining. In conclusion, combining THU with decitabine can address malignant cell sanctuary in the liver, an organ that expresses high levels of cytidine deaminase. The ability of this combination to address other aspects of CDA-mediated resistance to therapy is also being evaluated. Disclosures:No relevant conflicts of interest to declare.

Oral Administration of Low-Dose Decitabine and Tetrahydrouridine In Combination Increases Fetal Hemoglobin to Therapeutic Levels In the Absence of Cytotoxicity and Reduces Inter-Individual Drug Bioavailability In Baboons

Abstract 2147Increased fetal hemoglobin (HbF) levels alleviate the symptoms and improve survival of patients with sickle cell disease and β-thalassemia. Because therapeutic options remain limited for a large group of patients refractory to hydroxyurea (HU) therapy, alternative therapeutic agents to increase HbF are urgently needed. Decitabine (DAC) increases HbF to therapeutic levels in sickle cell disease patients refractory to HU. Oral administration of DAC would ease administration, increase compliance, and lower costs. Previous studies showed that oral administration of high doses of DAC increased HbF levels in baboons (Lavelle et al Am J Hematol 82:981, 2007). The oral bioavailability of DAC is limited by the degradative action of cytidine deaminase (CDA), and patients with CDA polymorphisms associated with reduced CDA activity may exhibit increased risk of cytotocity. We hypothesized that oral administration of low-dose DAC in combination with the CDA inhibitor THU would improve the DAC concentration-time profile while avoiding high peak drug levels likely to cause DNA damage and cytotoxicity and reduce risks of cytotoxicity in patients with CDA polymorphisms. Pharmacokinetics (PK) analysis in six baboons following oral administration of high-dose DAC alone showed that a dose (10mg/kg) previously shown sufficient to induce maximal (62.3–72%) HbF levels in 3 of 5 baboons when administered daily for ten days was associated with a mean AUC value of 355min*ng/ml. PK analysis following administration of varying doses of DAC in combination with THU showed that this AUC of 355min*ng/ml was attained with a 0.5 mg/kg DAC dose administered orally in combination with THU. The effect of oral low-dose DAC in combination with THU on HbF was then evaluated in four baboons. Initially, a single baboon (7482) was treated at 0.5mg/kg/d 3X/week for 3 weeks followed by 2X/week for an additional 5 weeks. Three additional baboons (7484, 7472, and 7470) were treated with either 0.25 or 0.5mg/kg/d 2X/week for eight weeks. Results (Table 1) showed significant increases in HbF associated with modest decreases in ANC and increases in platelets in all animals.AnimalDecitabine dose (mg/kg/d)SchedulePre-treatment HbF (%)Maximal HbF (%)ΔHbFANC nadirPlt max(x103)74820.53Xwk/3wks 2Xwk/5wks3.523.820.32580100474840.52Xwk/8wks4.223.118.91680102974720.252Xwk/8wks12.830.117.3146060074700.252Xwk/8wks9.929.319.42130895Decreased DNA methylation in the 5’ γ-globin gene region was observed in post-treatment BM samples by quantitative MassARRAY DNA methylation analysis. PK analysis showed that DAC bioavailability following oral administration of high-dose DAC alone differed 33 fold between 7470 and 7484 but only 1.3 fold following oral administration of low-dose DAC in combination with THU. Thus co-administration of low-dose DAC with THU reduced inter-individual variability of drug bioavailability and this was consistent with the similar HbF responses observed in these animals. Importantly, the ability of oral low dose DAC-THU to increase HbF to similar levels in the absence of cytotoxicity in two baboons differing in DAC bioavailability following oral administration of high-dose DAC alone predicts that oral low-dose DAC-THU in combination will reduce risks of cytotoxicity in patients with differences in DAC bioavailability due to CDA polymorphisms. These experiments have determined a dose and schedule of oral low-dose DAC in combination with THU that effectively increases HbF to therapeutic levels in the absence of cytotoxic effects in baboons and thus provide critical information important for the design of a future clinical trial of this drug combination in patients with sickle cell disease refractory to hydroxyurea therapy. Disclosures:No relevant conflicts of interest to declare.

Delineation of the Molecular Mechanisms of Nucleoside Recognition by Cytidine Deaminase through Virtual Screening

Cytidine deaminase (EC 3.5.4.5, CDA), an enzyme of the pyrimidine salvage pathways, is responsible for the degradation and inactivation of several cytidine-based antitumor drugs such as cytarabine, gemcitabine, decitabine, and azacytidine. Thus, CDA inhibitors are highly sought after as compounds to be co-administered with said drugs to improve their effectiveness. Alternatively, the design of antitumor drugs not susceptible to the action of CDA is also regarded as an attractive solution. Herein we describe a virtual screen for CDA ligands based on chemical similarity and molecular docking. The campaign led to the identification of three novel inhibitors and one novel substrate, with a 19 % hit rate, and allowed a significant extension of the structure-activity relationships, also in light of the compounds that resulted inactive. The most active compound identified through the screen is the inhibitor pseudoisocytidine, which has the potential to serve as a lead for highly stable compounds. The study also delineated the detrimental effect of 5-aza and 6-aza substitutions, the incompatibility of the presence of an amino group at the 3'-position, as well as the presence of very strict steric requirements around the 2'-arabino position and, even more, the N4-position. Importantly, these features can be exploited for the design of novel anti-neoplastic agents resistant to the action of CDA.

Abstract 1283: Phase I and pharmacokinetic evaluation of the DNA methyltransferase inhibitor 5-fluoro-2′-deoxycytidine: A California Cancer Consortium study

Abstract Background: The California Cancer Consortium conducted the first-in-human clinical trial of the investigational new drug 5-fluoro-2′-deoxycytidine (FdC), a DNA methyltransferase inhibitor stable in aqueous solution, as a 3-hour infusion concurrent with 350 mg/m2 of the cytidine deaminase inhibitor tetrahydrouridine (THU), included to prolong the half-life of FdC in vivo. Methods: A 3+3 design was used; initial FdC dose doubling ended when two moderate toxicities or one severe toxicity were observed. Pharmacokinetics of FdC and THU were obtained. Results: On the initial schedule, daily (QD) × 5 every 21 days (d), FdC was escalated from 2.5 to 80 mg/m2/d without treatment-related grade 3 toxicity other than anemia and lymphopenia. At FdC doses ≥20 mg/m2/d, peak plasma FdC concentrations were above those that inhibit DNA methylation in vitro (Beumer, et al., Cancer Chemother Pharmacol 62:363-8, 2008). The schedule was amended to QD × 5 for 2 consecutive weeks every 28 d, and patients (pts) were enrolled at 40, 67, 100, 134, and 180 mg/m2/d. Overall, 51 pts were treated [28 M; 23 F; median (range): age 63 (30-85), KPS 80 (60-100)]; 44 pts were evaluable for toxicity (1 cycle). 1/6 pts at 134 mg/m2/d had dose-limiting toxicity (DLT) (grade 3 colitis) and 2/2 pts at 180 mg/m2/d had DLT (1-grade 3 fatigue + LFT; 1-grade 4 mucositis + ANC + platelets). Of the 31 pts on 28-d cycles, 8 had clinical progression prior to the first scheduled evaluation at 8 weeks and 5 did not complete 2 cycles for other reasons; 7 pts had progressive disease at 8 weeks; and 10 pts had stable disease for ≥8 weeks. In addition, 1 heavily-pretreated pt with breast cancer, treated at 67 mg/m2/d, had a partial response lasting 1 year. Conclusions: The Maximum Tolerated Dose was 134 mg/m2/d FdC + 350 mg/m2/d THU QD × 5 for 2 consecutive weeks every 28 d. An expansion cohort is being accrued to determine the pharmacokinetics of oral FdC and THU. Based on the nature of the toxicities and the observed response at 67 mg/m2/d, a multi-histology Phase II trial has been initiated at 100 mg/m2/d QD × 5 for 2 consecutive weeks every 28 d. Supported by U01 CA62505, P30 CA33572, N01-CM-52202, and P30CA47904. The authors acknowledge the invaluable contribution of Dr. M. J. Egorin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1283. doi:10.1158/1538-7445.AM2011-1283

Deoxyuridine analog nucleotides in deoxycytidine analog treatment: secondary active metabolites?

Deoxycytidine analogs (dCa's) are nucleosides widely used in anticancer and anti (retro) viral therapies. Intracellularly phosphorylated dCa anabolites are considered to be their main active metabolites. This article reviews the literature on the formation and pharmacological activity of deaminated dCa nucleotides. Most dCa's are rapidly deaminated into deoxyuridine analogs (dUa's) which are only slowly phosphorylated and therefore relatively inactive. dUa nucleotides are, however, also formed via deamination of dCa monophosphates by deoxycytidine monophosphate deaminase (dCMPD). dUa-monophosphates can interact with thymidylate synthase (TS), whereas dUa-triphosphates are incorporated into nucleic acids and interfere with polymerases. Administration of dCa's as monophosphate prodrugs or co-administration of the cytidine deaminase inhibitor tetrahydrouridine (THU) does not prevent dUa nucleotide formation which is, on the other hand, influenced by the dose and dCMPD activity. Taken together, these observations show that the formation of dUa nucleotides is a common phenomenon in treatment with dCa's and these compounds may play a role in treatment outcome. We conclude that more attention should be given to these relatively unknown, but potentially important metabolites.

Comparison of in vitro metabolic conversion of capecitabine to 5-FU in rats, mice, monkeys and humans - toxicological implications

Capecitabine is an oral anticancer prodrug which is converted to 5-fluorouracil (5-FU) via 3 enzymatic steps, these being 5'-deoxy-5-fluorocytidine (5'-DFCR), 5'-deoxy-5-fluorouridine (5'-DFUR), and finally 5-FU by carboxylesterase (CES), cytidine deaminase (CDA), and thymidine phosphorylase (TP), respectively. Because rats, mice and monkeys are used for preclinical safety studies, we investigated the in vitro conversion from capecitabine to 5-FU by hepatic and intestinal mucosal microsomes and cytosols, to compare their metabolic activity to that of humans. Capecitabine was hydrolyzed to 5'-DFCR in hepatic and intestinal mucosal microsomes in these animal species. In humans and monkeys, CL(int) (V(max)/K(m)) for the hydrolysis of capecitabine in intestine (expressed as µl/min/g tissue) was much lower than that in hepatic microsomes but, in rats and mice, CL(int) was higher in intestine than in liver. In humans and monkeys, similar K(m) values and inhibition patterns by tetrahydrouridine (THU) a CDA inhibitor, were observed in CDA activity of hepatic and intestinal cytosols. However, rats showed very low CDA activity and mice showed non-Michaelis-Menten kinetics and a different inhibition pattern by THU. K(m) values for TP activity were almost similar in rats, mice, monkeys and humans. In conclusion, it was confirmed that monkeys are a suitable animal model for the safety assessment of capecitabine in terms of metabolic enzymes and it was suggested that higher toxic incidences in mouse small intestine were related to high hydrolytic activity of capecitabine in the small intestine.

Tetrahydrouridine Co-Administration Improves Oral Bioavailability and Dampens Inter-Individual Variability of Decitabine Pharmacokinetics In Baboons

AbstractAbstract 2081The cytosine analogue decitabine binds and depletes the chromatin modifying enzyme DNA methyl-transferase 1 (DNMT1), thereby modifying cellular epigenetics and differentiation. An oral formulation of decitabine would potentially increase the time-above-threshold concentration for depleting DNMT1 while avoiding high peak drug levels that cause DNA damage/cytotoxicity, facilitating outpatient epigenetic-differentiation therapy. The enzyme cytidine deaminase (CDA) is the major barrier to oral absorption and furthermore, causes significant inter-individual variability in decitabine pharmacokinetics. Since there is a narrow therapeutic window for depleting DNMT1 without causing cytotoxicity, decreasing inter-individual variability and improving the predictability of dose-pharmacokinetic relationships is important to the objective of non-cytotoxic epigenetic/differentiation therapy. Therefore, we examined if combining the CDA inhibitor tetrahydrouridine (THU) with decitabine could facilitate oral administration and furthermore, decrease inter-individual variability in decitabine pharmacokinetics. Drugs were administered under ketamine/xylazine anesthesia to normal female baboons by intravenous or subcutaneous injection or oral gavage. Blood samples for pharmacokinetic (PK) analysis were drawn at 7 time-points during anesthesia. Decitabine levels were measured using LC-MS/MS. The AUClast (the area under the curve from the time of dosing to the last measurable concentration) was calculated by the linear trapezoidal method. Oral bioavailability was estimated by the ratio of AUCoral to AUCIV, following adjustment of the AUC values for the different doses administered. Minimum doses of THU (400 mg/m2, oral gavage) and decitabine (100 mg/m2, oral gavage), and optimal timing between THU and decitabine administration (60 minutes), that achieved peak levels of decitabine associated with depletion of DNMT1 without cytotoxicity in normal hematopoietic cells (0.2 – 0.5 μM) were determined. Even with AUClast being calculated over a longer period of time for the decitabine alone group (240 versus 180 minutes), the increase in AUClast in the THU group was significant per mg/kg of decitabine administered (p=0.02, Wilcoxon test) (Table 1). Oral decitabine administered 60 minutes after THU produced area under the curve (AUClast) similar to that produced by decitabine alone at double the dose, with a decrease in the inter-individual variability in pharmacokinetics observed with decitabine alone (Table 1).Table 1:The largest increases in AUClast produced by co-administration of THU occurred in baboons which had the lowest AUClast with decitabine alone, thereby decreasing inter-individual variability in decitabine pharmacokineticsAUClast (min*ng/mL)Baboon NumberWeight (kg)Decitabine 10 mg/kg aloneDecitabine 5 mg/kg aloneTHU 20 mg/kg 60 mins before Decitabine 5 mg/kgPA748212.349.98Not doneNot donePA748411.5190.35252.735621.05PA725619.8299.02Not done760.27PA747210.7327.25Not done444.825PA725414.4463Not done2587PA725519.9807.8Not done533.47PA725812.62863.6Not done2284.48PA74709.66278.781219.987515.32Mean±SD1604 ± 22622821 ± 2749Median±IQR463 ± 25652284 ± 5088Median±IQR per mg of decitabine160 ± 226457 ± 1017Fold-variation~30-fold~14-foldCoefficient of Variation14197This is the first report of oral THU in combination with oral decitabine. The significant increase in decitabine bioavailability produced by preceding THU administration may actually be larger than reported here, since AUClast estimates for THU-decitabine were calculated over 180 minutes while AUClast estimates for decitabine alone were calculated over 240 minutes to accommodate the period of allowable anaesthesia. Furthermore, the concentration-time profiles suggested that decitabine levels may have continued to increase beyond the last sampling time in some animals, moreso in the THU-decitabine group. Preceding administration of THU could facilitate oral administration of decitabine and concurrently address other limitations of decitabine therapy, including the problem of inter-individual variability, to advance towards the objective of accessible non-cytotoxic epigenetic-differentiation therapy. Disclosures:Off Label Use: FDA approved decitabine for use in MDS, therefore, its use in thalassemia is off-label. Furthermore, the dose, schedule and route of administration used is also not on the label. Decitabine was used in a study to augment production of fetal hemoglobin in an NIH sponsored trial.

Stability of 5-fluoro-2'-deoxycytidine and tetrahydrouridine in combination.

In vivo, the DNA methyltransferase inhibitor, 5-fluoro-2'-deoxycytidine (FdCyd, NSC-48006), is rapidly converted to its unwanted metabolites. Tetrahydrouridine (THU, NSC-112907), a cytidine deaminase inhibitor can block the first metabolic step in FdCyd catabolism. Clinical studies have shown that co-administration with THU can inhibit the metabolism of FdCyd. The National Cancer Institute is particularly interested in a 1:5 FdCyd/THU formulation. The purpose of this study was to investigate the in vitro pH stability of FdCyd and THU individually and in combination. A stability-indicating high-performance liquid chromatography method for the quantification of both compounds and their degradants was developed using a ZIC(R)-HILIC column. The effect of THU and FdCyd on the in vitro degradation of each other was studied as a function of pH from 1.0 to 7.4 in aqueous solutions at 37 degrees C. The degradation of FdCyd appears to be first-order and acid-catalyzed. THU equilibrates with at least one of its degradants. The combination of FdCyd and THU in solution does not affect the stability of either compound. The stability and compatibility of FdCyd and THU in the solid state at increased relative humidity and at various temperatures are also evaluated.

SP174 Gene signatures: Are we ready to change clinical management in cancer patient treatment?

Gemcitabine is an inhibitor of ribonucleotide reductase (RR) and DNA polymerization with promising activity in hematologic malignancies. Gemcitabine enters the cell mostly via the human equilibrative nucleoside transporter-1 (hENT1), while drug metabolism occurs by phosphorylation by deoxycytidine kinase (dCK), 5′-nucleotidase (cN-II) and cytidine deaminase (CDA) are the main inactivating enzymes. The aim of this study was to investigate the role of these determinants in gemcitabine cytotoxicity and analyze their expression in lymphoid cells. Cytotoxicity was assessed by MTT, and modulated by simultaneous addition of 2′-deoxycytidine (dCK natural substrate), tetrahydrouridine (CDA competitive inhibitor) and diethylpyrocarbonate (cN-II non-competitive inhibitor), while the expression of hENT1, dCK, cN-II, CDA and RR in WIL2-S, Jurkat and CCRF-CEM cells as well as in lymphoid cells from 25 chronic lymphocytic B-leukemia (B-CLL) patients was studied with quantitative-PCR. Cell cycle modulation and induction of apoptosis were analyzed by cytofluorimetry and bisbenzimide staining. Gemcitabine was highly cytotoxic, increased the cells in S-phase and significantly enhanced apoptosis. The crucial role of metabolism in gemcitabine activity was confirmed by the significant modulation of cytotoxicity by inhibitors of dCK, CDA and cN-II. Furthermore, PCR demonstrated a correlation between gemcitabine sensitivity and expression of its determinants, and that their values were within those observed in patients. These data indicate that gemcitabine is cytotoxic against lymphoid cells, affecting cell cycle and apoptosis. Furthermore, chemosensitivity may be predicted on the basis of gene expression profile of critical determinants involved in gemcitabine mechanism of action, suggesting the use of pharmacogenetic profiling for treatment optimization.

DNA mismatch repair (MMR)‐dependent 5‐fluorouracil cytotoxicity and the potential for new therapeutic targets

The metabolism and efficacy of 5-fluorouracil (FUra) and other fluorinated pyrimidine (FP) derivatives have been intensively investigated for over fifty years. FUra and its antimetabolites can be incorporated at RNA- and DNA-levels, with RNA level incorporation provoking toxic responses in human normal tissue, and DNA-level antimetabolite formation and incorporation believed primarily responsible for tumour-selective responses. Attempts to direct FUra into DNA-level antimetabolites, based on mechanism-of-action studies, have led to gradual improvements in tumour therapy. These include the use of leukovorin to stabilize the inhibitory thymidylate synthase-5-fluoro-2'-deoxyuridine 5' monophoshate (FdUMP)-5,10-methylene tetrahydrofolate (5,10-CH(2)FH(4)) trimeric complex. FUra incorporated into DNA also contributes to antitumour activity in preclinical and clinical studies. This review examines our current state of knowledge regarding the mechanistic aspects of FUra:Gua lesion detection by DNA mismatch repair (MMR) machinery that ultimately results in lethality. MMR-dependent direct cell death signalling or futile cycle responses will be discussed. As 10-30% of sporadic colon and endometrial tumours display MMR defects as a result of human MutL homologue-1 (hMLH1) promoter hypermethylation, we discuss the use and manipulation of the hypomethylating agent, 5-fluorodeoxycytidine (FdCyd), and our ability to manipulate its metabolism using the cytidine or deoxycytidylate (dCMP) deaminase inhibitors, tetrahydrouridine or deoxytetrahydrouridine, respectively, as a method for re-expression of hMLH1 and re-sensitization of tumours to FP therapy.

Methylation of Zebularine: A Quantum Mechanical Study Incorporating Interactive 3D pdf Graphs

Methylation of a cytidine deaminase inhibitor, 1-(beta-D-ribofuranosyl)-2-pyrimidone (i.e., zebularine (zeb)), which produces 1-(beta-D-ribofuranosyl)-5-methyl-2-pyrimidinone (d5), has been investigated using density functional theory models. The optimized structures of zeb and d5 and the valence orbitals primarily responsible for the methylation in d5 are presented using state-of-the-art interactive (on a computer or online) three-dimensional (3D) graphics in a portable document format (pdf) file, 3D-PDF (http://www.web3d.org/x3d/vrml/ ). The facility to embed 3D molecular structures into pdf documents has been developed jointly at Swinburne University of Technology and the National Computational Infrastructure, the Australian National University. The methyl fragment in the base moiety shows little effect on the sugar puckering but apparently affects anisotropic properties, such as condensed Fukui functions. Binding energy spectra, both valence space and core space, are noticeably affected; in particular, in the outer-valence space (e.g., IP < 20 eV). The methyl fragment delocalizes and diffuses into almost all valence space, but orbitals 8 (57a, IP = 12.57 eV), 18 (47a, IP = 14.70 eV), and 37 (28a, IP = 22.15 eV) are identified as fingerprint for the methyl fragment. In the inner shell, however, the impact of the methyl can be localized and identified by chemical shift. A small, global, red shift is found for the O-K, N-K and sugar C-K spectra, whereas the base C-K spectrum exhibits apparent methyl-related changes.