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.

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.

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.

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.

Plasma pharmacokinetics and oral bioavailability of the 3,4,5,6-tetrahydrouridine (THU) prodrug, triacetyl-THU (taTHU), in mice.

PURPOSE: Cytidine drugs, such as gemcitabine, undergo rapid catabolism and inactivation by cytidine deaminase (CD). 3,4,5,6-tetrahydrouridine (THU), a potent CD inhibitor, has been applied preclinically and clinically as a modulator of cytidine analogue metabolism. However, THU is only 20% orally bioavailable, which limits its preclinical evaluation and clinical use. Therefore, we characterized THU pharmacokinetics after the administration to mice of the more lipophilic pro-drug triacetyl-THU (taTHU). METHODS: Mice were dosed with 150 mg/kg taTHU i.v. or p.o. Plasma and urine THU concentrations were quantitated with a validated LC-MS/MS assay. Plasma and urine pharmacokinetic parameters were calculated non-compartmentally and compartmentally. RESULTS: taTHU did not inhibit CD. THU, after 150 mg/kg taTHU i.v., had a 235-min terminal half-life and produced plasma THU concentrations >1 µg/mL, the concentration shown to inhibit CD, for 10 h. Renal excretion accounted for 40-55% of the i.v. taTHU dose, 6-12% of the p.o. taTHU dose. A two-compartment model of taTHU generating THU fitted the i.v. taTHU data best. taTHU, at 150 mg/kg p.o., produced a concentration versus time profile with a plateau of approximately 10 µg/mL from 0.5-2 h, followed by a decline with a 122-min half-life. Approximately 68% of i.v. taTHU is converted to THU. Approximately 30% of p.o. taTHU reaches the systemic circulation as THU. CONCLUSIONS: The availability of THU after p.o. taTHU is 30%, when compared to the 20% achieved with p.o. THU. These data will support the clinical studies of taTHU.

Modulation of gemcitabine (2',2'-difluoro-2'-deoxycytidine) pharmacokinetics, metabolism, and bioavailability in mice by 3,4,5,6-tetrahydrouridine.

PURPOSE: In vivo, 2',2'-difluoro-2'-deoxycytidine (dFdC) is rapidly inactivated by gut and liver cytidine deaminase (CD) to 2',2'-difluoro-2'-deoxyuridine (dFdU). Consequently, dFdC has poor oral bioavailability and is administered i.v., with associated costs and limitations in administration schedules. 3,4,5,6-Tetrahydrouridine (THU) is a potent CD inhibitor with a 20% oral bioavailability. We investigated the ability of THU to decrease elimination and first-pass effect by CD, thereby enabling oral dosing of dFdC. EXPERIMENTAL DESIGN: A liquid chromatography-tandem mass spectrometry assay was developed for plasma dFdC and dFdU. Mice were dosed with 100 mg/kg dFdC i.v. or orally with or without 100 mg/kg THU i.v. or orally. At specified times between 5 and 1,440 min, mice (n = 3) were euthanized. dFdC, dFdU, and THU concentrations were quantitated in plasma and urine. RESULTS: THU i.v. and orally produced concentrations >4 microg/mL for 3 and 2 h, respectively, whereas concentrations of >1 microg/mL have been associated with near-complete inhibition of CD in vitro. THU i.v. decreased plasma dFdU concentrations but had no effect on dFdC plasma area under the plasma concentration versus time curve after i.v. dFdC dosing. Both THU i.v. and orally substantially increased oral bioavailability of dFdC. Absorption of dFdC orally was 59%, but only 10% passed liver and gut CD and eventually reached the systemic circulation. Coadministration of THU orally increased dFdC oral bioavailability from 10% to 40%. CONCLUSIONS: Coadministration of THU enables oral dosing of dFdC and warrants clinical testing. Oral dFdC treatment would be easier and cheaper, potentially prolong dFdC exposure, and enable exploration of administration schedules considered impractical by the i.v. route.

Evaluación de alteraciones motoras en modelos animales de enfermedad de Parkinson / Evaluation of motor disorder in animal models of parkinson’s disease

El diagnóstico de la enfermedad de Parkinson es básicamente clínico, es decir, se basa en la observaciónde las alteraciones motoras de los pacientes, por este motivo se considera sencillo; sin embargo, sólo un 75% de los diagnósticos realizados clínicamente se confirma en la autopsia. Los modelos animales generados mediante la aplicación sistémica o intracerebral de neurotoxinas como 6-hidroxidopamina (6-OHDA) para ratas o 1-metil-4-fenil-1,2,3,6-tetrahidropiridina(MPTP) para ratones y primates no humanos, induce un daño en el sistema dopaminérgico nigroestriatal. Esto da como resultado una variedad de síntomas motores como acinesia, bradicinesia, rigidez, temblor, alteraciones en la marcha y posturas anormales; por este motivo es un reto evaluar los cambios de los signos parkinsonianos en los modelos animales.Desarrollo. Se revisa la variedad de paradigmas para valorar estos síntomas en los modelos de ratones, ratas y primates no humanos, los cuales se han utilizado para medir las diferencias que se generan con la aplicación de las neurotoxinas y, en algunos casos, las mejorías de los diferentes tratamientos para los síndromes parkinsonianos inducidos. Conclusiones. Se comentanlos resultados generales de estos trabajos y se discuten los factores que influyen en las pruebas, y los potenciales problemas y beneficios que pueden tener los procedimientos experimentales

The diagnosis of Parkinson’s disease is essentially clinical, that is to say, it is based on theobservation of the motor alterations displayed by patients, and for this reason it is considered to be a simple matter. Yet, only 75% of the diagnoses that are carried out clinically are later confirmed in the autopsy. Animal models can be generated bysystemic or intracerebral application of neurotoxins, like 6-hydroxydopamine (6-OHDA) for rats or 1-methyl-4-phenyl-1,2, 3,6-tetrahydropyridine (MPTP) for mice and non-human primates, which induce damage in the nigrostriatal dopaminergic system. This gives rise to a variety of motor symptoms such as akinesia, bradykinesia, rigidity, tremor, gait disorders andabnormal postures, which is what makes the evaluation of the changes in the signs of Parkinsonism in animal models such a challenge to researchers today. Development. The paper reviews the variety of paradigms available for evaluating these symptoms in mouse, rat and non-human primate models, which have been used to measure the differences brought about byapplying neurotoxins and, in some cases, the improvements produced by different treatments for the Parkinsonian syndromes that were induced. Conclusions. Both the general findings of these works and the factors that influence the trials are discussed, together with the potential problems and benefits that the experimental procedures may have

Plasma pharmacokinetics and oral bioavailability of 3,4,5,6-tetrahydrouridine, a cytidine deaminase inhibitor, in mice.

Cytidine analogues such as cytosine arabinoside, gemcitabine, decitabine, 5-azacytidine, 5-fluoro-2'-deoxycytidine and 5-chloro-2'-deoxycytidine undergo rapid catabolism by cytidine deaminase (CD). 3,4,5,6-tetrahydrouridine (THU) is a potent CD inhibitor that has been applied preclinically and clinically as a modulator of cytidine analogue metabolism. However, THU pharmacokinetics has not been fully characterized, which has impaired the optimal preclinical evaluation and clinical use of THU. Therefore, we characterized the THU pharmacokinetics and bioavailability in mice. Mice were dosed with THU iv (100 mg/kg) or po (30, 100, or 300 mg/kg). Plasma and urine THU concentrations were quantitated with a validated LC-MS/MS assay. Plasma pharmacokinetic parameters were calculated compartmentally and non-compartmentally. THU, at 100 mg/kg iv had a 73 min terminal half-life and produced plasma THU concentrations >1 microg/ml, the concentration shown to effectively block deamination, for 4 h. Clearance was 9.1 ml/min/kg, and the distribution volume was 0.95 l/kg. Renal excretion accounted for 36-55% of the THU dose. A three-compartment model fit the iv THU data best. THU, at 100 mg/kg po, produced a concentration versus time profile with a plateau of approximately 10 mug/ml from 0.5-3 h, followed by a decline with an 85 min half-life. The oral bioavailability of THU was approximately 20%. The 20% oral bioavailability of THU is sufficient to produce and sustain, for several hours, plasma concentrations that inhibit CD. This suggests the feasibility of using THU to decrease elimination and first-pass metabolism of cytidine analogues by CD. THU pharmacokinetics are now being evaluated in humans.

Analytical and pharmacokinetic studies with 5-chloro-2'-deoxycytidine.

5-Chloro-2'-deoxycytidine (NSC 371331, CDC) is in development as a possible radiosensitizing agent for cancer treatment. Previous studies have been done to demonstrate the in vivo efficacy of CDC with various modulators of its metabolism. This paper describes our preclinical studies to determine the pharmacokinetic properties of CDC and the disposition of the drug, both alone and in the presence of the metabolic modulator tetrahydrouridine (THU), a cytidine deaminase inhibitor. Detection of the drug in biological fluids was performed by HPLC analysis using a C-18 column, gradient elution with solvents composed of aqueous trifluoroacetic acid and acetonitrile, and ultraviolet absorbance at 290 nm. Samples were processed by treatment with ammonium sulfate prior to injection into the HPLC system. CDC was stable in aqueous solution and in mouse plasma. High doses of CDC (100mg/kg) were given i.v. or i.p. to mice for the determination of CDC plasma half-life (10 min). CDC was not detectable in plasma after oral administration. It was converted rapidly to 5-chloro-2'-deoxyuridine (CDU) by cytidine deaminase, and CDU was readily discernable in plasma and urine samples collected after i.v. and i.p. administration of CDC. When CDC in doses ranging from 5 to 100mg/kg was given with 100mg/kg of THU, increased plasma levels of CDC were seen. CDC was eliminated through the kidneys, as well as by enzymatic deamination, and did not bind to plasma proteins. The initial steps of the CDC metabolic pathway were determined in vitro with isolated enzymes. Cytidine deaminase from mouse kidney converted CDC into CDU; thymidine phosphorylase converted CDU into 5-chlorouracil (5-CU). The conclusions of these studies are: (a) CDC is a drug with a short half-life and (b) it is excreted through the kidney, mainly in metabolite form. Administration of THU substantially increased the concentrations of CDC in mouse plasma, supporting proposals that the combination of THU with CDC should be evaluated in clinical trials.