Clinical activity, pharmacokinetics, and pharmacodynamics of oral hypomethylating agents for myelodysplastic syndromes/neoplasms and acute myeloid leukemia: A multidisciplinary review.

OBJECTIVE: To review the pharmacokinetic (PK)-pharmacodynamic (PD) profiles, disease setting, dosing, and safety of oral and parenteral hypomethylating agents (HMAs) for the treatment of myelodysplastic syndromes/neoplasms (MDS) and acute myeloid leukemia (AML), and to provide a multidisciplinary perspective on treatment selection and educational needs relating to HMA use. DATA SOURCES: Clinical and real-world data for parenteral decitabine and azacitidine and two oral HMAs: decitabine-cedazuridine (DEC-C) for MDS and azacitidine (CC-486) for AML maintenance therapy. DATA SUMMARY: Differences in the PK-PD profiles of oral and parenteral HMA formulations have implications for their potential toxicities and planned use. Oral DEC-C (decitabine 35 mg and cedazuridine 100 mg) has demonstrated equivalent systemic area under the concentration-time curve (AUC) exposure to a 5-day regimen of intravenous (IV) decitabine 20 mg/m2 and showed no significant difference in PD. The AUC equivalence of oral DEC-C and IV decitabine means that these regimens can be treated interchangeably (but must not be substituted within a cycle). Oral azacitidine has a distinct PK-PD profile versus IV or subcutaneous azacitidine, and the formulations are not bioequivalent or interchangeable owing to differences in plasma time-course kinetics and exposures. Clinical trials are ongoing to evaluate oral HMA combinations and novel oral HMAs, such as NTX-301 and ASTX030. CONCLUSIONS: Treatment with oral HMAs has the potential to improve quality of life, treatment adherence, and disease outcomes versus parenteral HMAs. Better education of multidisciplinary teams on the factors affecting HMA treatment selection may help to improve treatment outcomes in patients with MDS or AML.

Oral hypomethylating agents: beyond convenience in MDS.

Oral hypomethylating agents (HMAs) represent a substantial potential boon for patients with myelodysplastic syndrome (MDS) who have previously required between 5 and 7 visits per month to an infusion clinic to receive therapy. For patients who respond to treatment, ongoing monthly maintenance visits represent a considerable burden to quality of life, and for those who are early in therapy, these sequential visits may tax transportation and financial resources that would be optimally distributed over the treatment cycle to facilitate transfusion support. The availability of oral HMAs may support the optimal application of these agents by contributing to adherence and lessening the burden of therapy, potentially encouraging patients to stay on longer-term treatment. Distinct pharmacokinetic profiles for the recently approved oral HMAs (oral azacitidine and decitabine-cedazuridine) result in differential toxicity profiles and have prompted their clinical trial development in lower- and higher-risk MDS, respectively.

The path to approval for oral hypomethylating agents in acute myeloid leukemia and myelodysplastic syndromes.

Two oral hypomethylating agents, oral azacitidine (CC-486) and decitabine/cedazuridine (ASTX727), have recently entered the clinical domain. CC-486 has been shown to improve overall survival as maintenance therapy for older patients with acute myeloid leukemia in complete remission, whereas the combination of decitabine with cedazuridine, a cytidine deaminase inhibitor, is indicated for the treatment of adult patients with myelodysplastic syndromes and chronic myelomonocytic leukemia with intermediate-1, or higher, International Prognostic Scoring System risk. This article briefly summarizes the clinical development of both drugs, the pivotal studies that led to their approval and some of the issues faced in extending the use of these drugs to other indications.

Lay abstract One of the key challenges in treating acute myeloid leukemia is to prevent relapse after remission has been achieved. This means that developing an effective maintenance treatment is very important. Maintenance treatment is given for a prolonged period and so it needs to be easy to give and well tolerated. Oral azacitidine is an example of this type of treatment and is the first drug that has been shown to improve survival as maintenance therapy for acute myeloid leukemia patients. This article describes the key studies that led to the approval of this important therapy.

Oral cedazuridine/decitabine for MDS and CMML: a phase 2 pharmacokinetic/pharmacodynamic randomized crossover study.

This phase 2 study was designed to compare systemic decitabine exposure, demethylation activity, and safety in the first 2 cycles with cedazuridine 100 mg/decitabine 35 mg vs standard decitabine 20 mg/m2 IV. Adults with International Prognostic Scoring System intermediate-1/2- or high-risk myelodysplastic syndromes (MDS) or chronic myelomonocytic leukemia (CMML) were randomized 1:1 to receive oral cedazuridine/decitabine or IV decitabine in cycle 1, followed by crossover to the other treatment in cycle 2. All patients received oral cedazuridine/decitabine in subsequent cycles. Cedazuridine and decitabine were given initially as separate capsules in a dose-confirmation stage and then as a single fixed-dose combination (FDC) tablet. Primary end points: mean decitabine systemic exposure (geometric least-squares mean [LSM]) of oral/IV 5-day area under curve from time 0 to last measurable concentration (AUClast), percentage long interspersed nuclear element 1 (LINE-1) DNA demethylation for oral cedazuridine/decitabine vs IV decitabine, and clinical response. Eighty patients were randomized and treated. Oral/IV ratios of geometric LSM 5-day AUClast (80% confidence interval) were 93.5% (82.1-106.5) and 97.6% (80.5-118.3) for the dose-confirmation and FDC stages, respectively. Differences in mean %LINE-1 demethylation between oral and IV were ≤1%. Clinical responses were observed in 48 patients (60%), including 17 (21%) with complete response. The most common grade ≥3 adverse events regardless of causality were neutropenia (46%), thrombocytopenia (38%), and febrile neutropenia (29%). Oral cedazuridine/decitabine (100/35 mg) produced similar systemic decitabine exposure, DNA demethylation, and safety vs decitabine 20 mg/m2 IV in the first 2 cycles, with similar efficacy. This study is registered at www.clinicaltrials.gov as #NCT02103478.

Evaluation of BBB permeable nucleolipid (NLDPU): A di-C15-ketalised palmitone appended uridine as neuro-tracer for SPECT.

Despite being in routine for onco-diagnostics for years, the applicability of nucleosidic molecular imaging probes is severely restricted in neurological applications due to their low permeability across blood-brain-barrier (BBB). For extending nucleoside tracers utility for neuro-onco early diagnostics, suitable modification which enhances their BBB permeation needs investigation. Among various modifications, lipidization of nucleosides has been reported to enhance cellular permeability. Extending the concept, the aim was to exemplify the possibility of lipidized nucleosides as potential brain tracer with capability to cross intact BBB and evaluate as metal based neuro-imaging SPECT agent. Uridine based non-lipidic (NSDAU) and di-C15-ketal appended lipidic (NLDPU) ligands were conjugated to chelator, DTPA (DTPA-NSDAU and DTPA-NLDPU) using multi-step chemistry. The ligands were evaluated in parallel for comparative physical and biological parameters. Additionally, effects of enhanced lipophilicity on UV-absorption, acid strength, fluorescence and non-specific protein binding were evaluated. Fluorescence quenching of BSA indicated appreciable interaction of DTPA-NLDPU with protein only above 10 mM without inducing conformational changes. In addition, DTPA-NLDPU was found to be haematocompatible and cytocompatible with low dose-dependent toxicity in HEK-cells. The chelator DTPA was used for 99mTc-complexation for SPECT imaging. Optimized 99mTc-radiolabeling parameters resulted in quantitative (≥97%) labeling with good stability parameters in in-vitro serum and cysteine challenge studies. We demonstrate that the nucleolipid radiotracer (99mTc-DTPA-NLDPU) was successfully able to permeate the BBB with brain uptake of 0.2% ID/g in normal mice as compared to 0.06% ID/g uptake of 99mTc-DTPA-NSDAU at 5 min. Blood kinetics indicate biphasic profile and t1/2(distribution) 46 min for 99mTc-DTPA-NLDPU. The preferential accumulation of 99mTc-DTPA-NLDPU in brain tumor intracranial xenograft indicate the targeting capability of the nucleoside. We conclude that as first-of-its-kind, this work presents the potential of the biocompatible nucleolipidic system for brain targeting and early diagnostics.

Pharmacokinetic analysis of doxifluridine and its metabolites, 5-fluorouracil and 5-fluorouridine, after oral administration in beagle dogs.

Doxifluridine (5'-deoxy-5-fluorouridine, 5'-dFUR) is a fluoropyrimidine derivative that is activated preferentially in malignant cells by thymidine phosphorylase to form 5-fluorouracil (5-FU). The purpose of this study was to investigate the pharmacokinetic properties of doxifluridine and its two major metabolites, 5-FU, and 5-fluorouridine (5-FUrd), in beagle dogs following a single oral administration of 200 mg doxifluridine capsule (Furtulon(®)). After the administration of 200 mg of Furtulon to 23 beagle dogs, the plasma concentrations of doxifluridine, 5-FU, and 5-FUrd were measured simultaneously, using LC-MS/MS. The parent-metabolite compartment model with first-order absorption and Michaelis-Menten kinetics described the pharmacokinetics of doxifluridine, 5-FU, and 5-FUrd. Michaelis-Menten kinetics sufficiently explained the generation and elimination processes of 5-FU and 5-FUrd. The studies described here are the first to evaluate the relationship between pharmacokinetics of doxifluridine and its metabolites in dogs, and these findings will help in understanding the toxicity mechanism of doxifluridine.

Long term effects of denufosol tetrasodium in patients with cystic fibrosis.

RATIONALE: Denufosol stimulates chloride secretion independent of the chloride channel which is dysfunctional in cystic fibrosis (CF) and therefore has the potential to benefit CF patients regardless of genotype. OBJECTIVES: To assess the efficacy of denufosol in CF patients with mild lung function impairment age 5 years and older. METHODS: This multicenter, randomized, parallel group double-blind placebo-controlled trial was conducted at 102 CF care centers in Australia, Canada and the United States (NCT00625612) The active group (n=233) received 60 mg denufosol via inhalation three times daily The primary efficacy endpoint was change in FEV(1) in liters from Day 0 to week 48. MEASUREMENTS AND MAIN RESULTS: 685 patients were screened for the study and 466 patients (233 in each group) were randomized to study treatment. The adjusted mean change in FEV(1)was 40 mL for denufosol and 32 mL for placebo with a resulting treatment effect of 8 mL (95% CI -0.040, 0.056). The average rate of change in FEV(1) percent of predicted over 0 to 48 weeks was -3.04% for placebo vs. -2.30 for denufosol (a difference of 24% relative to placebo) among all patients. The incidence of pulmonary exacerbation was 26% vs. 21% for the placebo and denufosol groups with no differences in the time to first event. The study treatments were well tolerated and there was no evidence of systemic effects in any safety parameter assessed. CONCLUSIONS: In patients with CF treatment with denufosol for 48 weeks did not improve pulmonary function or reduce the incidence of pulmonary exacerbations.

Study of [18F]FLT and [123I]IaraU for cellular imaging in HSV1 tk-transfected murine fibrosarcoma cells: evaluation of the tracer uptake using 5-fluoro, 5-iodo and 5-iodovinyl arabinosyl uridines as competitive probes.

As one of the most intensively studied probes for imaging of the cellular proliferation, [(18)F]FLT was investigated whether the targeting specificity of thymidine kinase 1 (TK1) dependency could be enhanced through a synergistic effect mediated by herpes simplex type 1 virus (HSV1) tk gene in terms of the TK1 or TK2 expression. 5-[(123)I]Iodo arabinosyl uridine ([(123)I]IaraU) was prepared in a radiochemical yield of 8% and specific activity of 21 GBq/µmol, respectively. Inhibition of the cellular uptake of these two tracers was compared by using the arabinosyl uridine analogs such as 5-iodo, 5-fluoro and 5-(E)-iodovinyl arabinosyl uridine along with 2'-fluoro-5-iodo arabinosyl uridine (FIAU). Due to potential instability of the iodo group, accumulation index of 1.6 for [(123)I]IaraU by HSV1-TK vs. control cells could virtually be achieved at 1.5 h, but dropped to 0.2 compared to 2.0 for [(18)F]FLT at 5 h. The results from competitive inhibition by these nucleosides against the accumulation of [(18)F]FLT implied that FLT exerted a mixed TK1- and TK2-dependent inhibition with HSV1-tk gene transfection because of the shifting of thymidine kinase status. Taken together, the combination of [(18)F]FLT and HSV1-TK provides a synergistic imaging potency.

Denufosol: a review of studies with inhaled P2Y(2) agonists that led to Phase 3.

Among the most promising of the new therapies being developed for the treatment of Cystic Fibrosis (CF) are those targeted at increasing mucosal hydration on the surface of the airways. One of these therapies, P2Y(2) receptor agonists, bypasses the defective CFTR chloride channel, and activates an alternative chloride channel. This activation results in an increase in airway surface epithelial hydration, and through these actions and effects on cilia beat frequency, increases mucociliary clearance. The pharmacology of P2Y(2) agonists has been confirmed in several preclinical and clinical studies. Denufosol tetrasodium is a novel second-generation, metabolically stable, selective P2Y(2) receptor agonist currently in Phase 3 clinical development. In radiolabelled deposition studies of P2Y(2) agonists in healthy non-smokers and smokers, approximately 7mg of a 40-mg nebulizer (PARI LC Star) load was deposited in the lungs. In a pharmacokinetic study in healthy volunteers, very limited systemic exposure was observed when doses of 200mg of denufosol were nebulized. Thus, it appears that high concentrations of denufosol can be achieved in the airways with very low systemic absorption. Denufosol has been generally well-tolerated in healthy volunteers and patients with CF. The most common adverse events were in the respiratory system, with cough having the highest frequency. Doses of 20-60mg have been evaluated in Phase 2 trials of up to 28 days duration, and superiority relative to placebo on FEV1 has been observed in patients with relatively normal lung function (FEV1 greater than or equal to 75% of predicted). The first Phase 3 trial is a comparison of denufosol 60mg and placebo in 350 patients with CF with FEV1 at study entry greater than or equal to 75% of predicted.

In vivo evaluation of the uptake of [(123)I]FIAU, [(123)I]IVFRU and [(123)I]IVFAU by normal mouse brain: potential for noninvasive assessment of HSV-1 thymidine kinase gene expression in gliomas.

Radioiodinated 5-iodo-1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)uracil (F *IAU) is most commonly used for noninvasive assessment of herpes simplex virus type 1 thymidine kinase (HSV-1-tk) gene expression. However, it does not permeate the intact blood-brain barrier (BBB) because of its moderate lipophilicity. In this work, three iodo-nucleosides, FIAU, IVFRU, and IVFAU, were radiolabeled with iodine-123 and tested for permeation of the BBB in mice and for potential measurement of HSV-1-tk gene expression in gliomas. The results demonstrate that brain uptake and retention of these nucleosides is not directly related to their lipophilicity. The low brain uptake of IVFAU, in conjunction with its higher and constant brain/blood ratio, may reflect greater stability against hydrolysis of the N-glycosidic bond. In vivo PET evaluations of [(124)I]IVFRU and [(124)I]IVFAU in tumor-bearing mice are warranted.

Synthesis and evaluation of cis-1-[4-(hydroxymethyl)-2-cyclopenten-1-yl]-5-[(124)I]iodouracil: a new potential PET imaging agent for HSV1-tk expression.

In our pursuit to find an appropriate reporter probe for herpes simplex virus type-1 thymidine kinase (HSV1-tk), a carbocyclic nucleoside analogue, cis-1-[4-(hydroxymethyl)-2-cyclopenten-1-yl]-5-[124I]iodouracil, has been efficiently synthesized. A Pd(0)-catalyzed coupling reaction together with organotin and exchange reactions for radiolabeling gave more than 80% radiochemical yield with greater than 95% radiochemical purity and 1.15 GBq/mumol specific activity. Biological data reveal that the analogue is stable in vitro, less toxic than ganciclovir (GCV), and selective to HSV1-tk-expressed cells based on micro positron emission tomography (microPET) image analyses. Thus, this new carbocyclic nucleoside, referred to in this paper as carbocyclic 2',3'-didehydro-2',3'-dideoxy-5-iodouridine (carbocyclic d4IU) is a potential imaging probe for HSV1-tk.

Synergistic activity of troxacitabine (Troxatyl) and gemcitabine in pancreatic cancer.

BACKGROUND: Gemcitabine, a deoxycytidine nucleoside analog, is the current standard chemotherapy used as first-line treatment for patients with locally advanced or metastatic cancer of the pancreas, and extends life survival by 5.7 months. Advanced pancreatic cancer thus remains a highly unmet medical need and new therapeutic agents are required for this patient population. Troxacitabine (Troxatyl) is the first unnatural L-nucleoside analog to show potent preclinical antitumor activity and is currently under clinical investigation. Troxacitabine was recently evaluated as a first-line therapy in 54 patients with advanced adenocarcinoma of the pancreas and gave comparable overall results to those reported with gemcitabine in recently published randomized trials. METHODS: The human pancreatic adenocarcinoma cell lines, AsPC-1, Capan-2, MIA PaCa-2 and Panc-1, were exposed to troxacitabine or gemcitabine alone or in combination, for 72 h, and the effects on cell growth were determined by electronic particle counting. Synergistic efficacy was determined by the isobologram and combination-index methods of Chou and Talalay. Mechanistic studies addressed incorporation of troxacitabine into DNA and intracellular levels of troxacitabine and gemcitabine metabolites. For in vivo studies, we evaluated the effect of both drugs, alone and in combination, on the growth of established human pancreatic (AsPC-1) tumors implanted subcutaneously in nude mice. Statistical analysis was calculated by a one-way ANOVA with Dunnett as a post-test and the two-tailed unpaired t test using GraphPad prism software. RESULTS: Synergy, evaluated using the CalcuSyn Software, was observed in all four cell-lines at multiple drug concentrations resulting in combination indices under 0.7 at Fa of 0.5 (50% reduction of cell growth). The effects of drug exposures on troxacitabine and gemcitabine nucleotide pools were analyzed, and although gemcitabine reduced phosphorylation of troxacitabine when cells were exposed at equal drug concentrations, there was no effect on phosphorylated pools at drug combinations that were synergistic. The amount of troxacitabine incorporated into DNA was also not affected by the presence of gemcitabine. In vivo testing against a human pancreatic (AsPC-1) xenograft mouse tumor model indicated that both drugs were more than additive at well-tolerated doses and schedule. The biological basis for this synergy is unclear as we did not observe changes in apoptosis, DNA repair, troxacitabine incorporation into DNA or troxacitabine metabolism in the presence of gemcitabine. CONCLUSION: These data, together with phase I clinical data showing tolerability of both agents when combined, suggest combination therapy with troxacitabine and gemcitabine warrants further evaluation in advanced pancreatic cancer patients.

Cytidine is a novel substrate for wild-type concentrative nucleoside transporter 2.

Nucleoside transporter (NT) plays key roles in the physiology of nucleosides and the pharmacology of its analogues in mammals. We previously cloned Na+/nucleoside cotransporter CNT2 from mouse M5076 ovarian sarcoma cells, the peptide encoded by it differing from that by the previously reported mouse CNT2 in five substitutions, and observed that the transporter can take up cytidine, like CNT1 and CNT3. In the present study, we examined which of the two aforementioned CNT2 is the normal one, and whether or not cytidine is transported via the previously reported CNT2. The peptide encoded by CNT2 derived from mouse intestine, liver, spleen, and ovary was identical to that previously reported. The uptake of [3H]cytidine, but not [3H]thymidine, by Cos-7 cells transfected with CNT2 cDNA obtained from mouse intestine was much greater than that by mock cells, as in the case of [3H]uridine, a typical substrate of NT. [3H]Cytidine and [3H]uridine were taken up via CNT2, in temperature-, extracellular Na+-, and substrate concentration-dependent manners. The uptake of [3H]cytidine and [3H]uridine mediated by CNT2 was significantly inhibited by the variety of nucleosides used in this study, except for thymidine, and inhibition of the [3H]uridine uptake by cytidine was competitive. The [3H]uridine uptake via CNT2 was significantly decreased by the addition of cytarabin or gemcitabine, antimetabolites of cytidine analogue. These results indicated that the previously reported mouse CNT2 is the wild-type one, and cytidine is transported mediated by the same recognition site on the CNT2 with uridine, and furthermore, cytidine analogues may be substrates for the transporter.

The role of human nucleoside transporters in cellular uptake of 4'-thio-beta-D-arabinofuranosylcytosine and beta-D-arabinosylcytosine.

4'-Thio-beta-D-arabinofuranosyl cytosine (TaraC) is in phase I development for treatment of cancer. In human equilibrative nucleoside transporter (hENT) 1-containing CEM cells, initial rates of uptake (10 microM; picomoles per microliter of cell water per second) of [3H]TaraC and [3H]1-beta-D-arabinofuranosyl cytosine (araC) were low (0.007 +/- 003 and 0.034 +/- 0.003, respectively) compared with that of [3H]uridine (0.317 +/- 0.048), a highactivity hENT1 permeant. In hENT1- and hENT2-containing HeLa cells, initial rates of uptake (10 microM; picomoles per cell per second) of [3H]TaraC, [3H]araC, and [3H]deoxycytidine were low (0.30 +/- 0.003, 0.42 +/- 0.03, and 0.51 +/- 0.11, respectively) and mediated primarily by hENT1 (approximately 74, approximately 65, and approximately 61%, respectively). In HeLa cells with recombinant human concentrative nucleoside transporter (hCNT) 1 or hCNT3 and pharmacologically blocked hENT1 and hENT2, transport of 10 microM[3H]TaraC and [3H]araC was not detected. The apparent affinities of recombinant transporters (produced in yeast) for a panel of cytosine-containing nucleosides yielded results that were consistent with the observed low-permeant activities of TaraC and araC for hENT1/2 and negligible permeant activities for hCNT1/2/3. During prolonged drug exposures of CEM cells with hENT1 activity, araC was more cytotoxic than TaraC, whereas coexposures with nitrobenzylthioinosine (to pharmacologically block hENT1) yielded identical cytotoxicities for araC and TaraC. The introduction by gene transfer of hENT2 and hCNT1 activities, respectively, into nucleoside transport-defective CEM cells increased sensitivity to both drugs moderately and slightly. These results demonstrated that nucleoside transport capacity (primarily via hENT1, to a lesser extent by hENT2 and possibly by hCNT1) is a determinant of pharmacological activity of both drugs.

Functional characterization and haplotype analysis of polymorphisms in the human equilibrative nucleoside transporter, ENT2.

The equilibrative nucleoside transporter 2 (ENT2; SLC29A2) is a bidirectional transporter that is involved in the disposition of naturally occurring nucleosides as well as a variety of anticancer and antiviral nucleoside analogs. The goal of the current study was to evaluate the function of genetic variants in ENT2 in cellular assays and to determine the haplotype structure of the coding and flanking intronic region of the gene. As part of a large study focused on genetic variation in membrane transporters (Leabman et al., 2003), DNA samples from ethnically diverse populations (100 African-Americans, 100 European-Americans, 30 Asians, 10 Mexicans, and 7 Pacific Islanders) were screened for variants in membrane transporters, including SLC29A2. Fourteen polymorphic sites in SLC29A2 were found, including 11 in the coding region. Five protein-altering variants were identified: three nonsynonymous variants, and two deletions. Each of the protein-altering variants was found at a very low frequency, occurring only once in the sample population. The nonsynonymous variants and the deletions were constructed via site-directed mutagenesis and were subsequently characterized in Xenopus laevis oocytes. All variants were able to take up inosine with the exception of ENT2-Delta845-846, which resulted in a frameshift mutation that prematurely truncated the protein. ENT2 showed very infrequent variation compared with most other transporter proteins studied, and it was found that five haplotypes were sufficient to describe the entire sample set. The low overall genetic diversity in SLC29A2 makes it unlikely that variation in the coding region contributes significantly to clinically observed differences in drug response.

Nucleoside transport in primary cultured rabbit tracheal epithelial cells.

The present study aimed at elucidating the mechanisms of nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) grown on a permeable filter support. Uptake of (3)H-uridine, the model nucleoside substrate, from the apical fluid of primary cultured RTEC was examined with respect to its dependence on Na(+), substrate concentration, temperature and its sensitivity to inhibitors, other nucleosides and antiviral nucleoside analogs. Apical (3)H-uridine uptake in primary cultured RTEC was strongly dependent on an inward Na(+) gradient and temperature. Ten micromolar nitro-benzyl-mercapto-purine-ribose (NBMPR) (an inhibitor of es-type nucleoside transport in the nanomolar range) did not further inhibit this process. (3)H-uridine uptake from apical fluid was inhibited by basolateral ouabain (10 microM) and apical phloridzin (100 microM), indicating that uptake may involve a secondary active transport process. Uridine uptake was saturable with a K(m) of 3.4 +/- 1.8 microM and the V(max) of 24.3 +/- 5.2 pmoles/mg protein/30 s. Inhibition studies indicated that nucleoside analogs that have a substitution on the nucleobase competed with uridine uptake from apical fluid, but those with modifications on the ribose sugar including acyclic analogs were ineffective. The pattern of inhibition of apical (3)H-uridine, (3)H-inosine and (3)H-thymidine uptake into RTEC cells by physiological nucleosides was consistent with multiple systems: A pyrimidine-selective transport system (CNT1); a broad nucleoside substrate transport system that excludes inosine (CNT4) and an equilibrative NBMPR-insensitive nucleoside transport system (ei type). These results indicate that the presence of apically located nucleoside transporters in the epithelial cells lining the upper respiratory tract can lead to a high accumulation of nucleosides in the trachea. At least one Na(+)-dependent, secondary, active transport process may mediate the apical absorption of nucleosides or analogous molecules.

Fine tuning of rabbit equilibrative nucleoside transporter activity by an alternatively spliced variant.

The full-length cDNA encoding an equilibrative nucleoside transporter (rbENT2) and its novel C-terminal variant, rbENT2A, were isolated from rabbit trachea. Rabbit ENT2 protein consists of 456 amino acid residues; rbENT2A is shorter by 41 residues. Both rbENT2 and rbENT2A transcripts are found in rabbit tissues including intestine, kidney cortex, kidney, and trachea, at varying levels of expression. When transfected in a heterologous expression system-Madin Darby canine kidney (MDCK) epithelial cell line-both rbENT2 and rbENT2A were expressed. rbENT2 had a molecular mass of 49 kDa; rbENT2A had a molecular mass of 44 kDa. Clones of both transporters yielded functional proteins that were capable of mediating uridine uptake and efflux without the needing to be coupled to a secondary ion (e.g. Na(+)). Remarkably, rbENT2A displayed a higher affinity (K(m) = 41 microM) and a lower capacity (V(max) = 0.6 nmol/mg protein/5 min) towards substrates than rbENT2 (K(m) = 272.8 microM, V(max) = 1.26 nmol/mg protein/5 min). Pharmacological profiles showed that nitro-benzyl-mercapto-purine-ribose (NBMPR) potently inhibited (3)H-uridine uptake mediated by rbENT2A, but not uptake mediated by rbENT2. The constitutive splicing, broad expression, markedly different kinetics, and distinct pharmacological characteristics of rbENT2A appear to act in conjunction with the wild type, rbENT2, to fine-tune basolateral nucleoside transport function in rabbit trachea.

Functional and pharmacological mechanisms of nucleoside transport across the basolateral membrane of rabbit tracheal epithelial cells.

The role of basolateral membrane nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) was studied. Primary cultured RTEC were grown on permeable support at an air-interface. Transport studies were conducted in the uptake, efflux, and transepithelial transport configurations using (3)H-uridine as a model substrate. Time, temperature and concentration dependency of (3)H-uridine transport were evaluated in parallel to the metabolism of this substrate using scintillation counting and thin layer chromatography. Inhibition of (3)H-uridine uptake from basolateral fluid was estimated in presence of all unlabeled natural nucleosides as well as analogs and nucleobases. Functional modulation pathways of (3)H-uridine uptake were studied after treatment of RTEC with pharmacological levels of A23187, forskolin, tamoxifen, H89 and colchicine. The basolateral aspect has a low-affinity and high-capacity transport system that exhibits characteristics of bi-directionality, temperature/concentration dependency, and broad specificity towards purines and pyrimidines without requiring Na(+). Basolateral equilibrative-sensitive/insensitive (es/ei) type transport machinery manifested as a biphasic dose response to nitro-benzyl-mercapto-purine-ribose (NBMPR) inhibition. In addition, a number of therapeutically relevant nucleoside analogs appeared to compete with the uptake of uridine from basolateral fluid. Short-term pre-incubation of primary cultured RTEC with the calcium ionophore A23187 inhibited basolateral uridine uptake without affecting the J(max) and K(m). The inhibitory effect was not reversible with a protein kinase C (PKC) antagonist, tamoxifen. In contrast, basolateral uridine uptake was increased by adenylyl cyclase activator forskolin (reversible with protein kinase A (PKA) inhibitor H89), resulting in a decreased K(m), but a lower J(max). Uridine exit across the basolateral membrane of primary cultured RTEC occurs via a facilitative diffusion carrier, which can be modulated by intracellular Ca(2+) levels and PKA. Information about these carriers will help improve the transportability of antitumor and antiviral nucleoside analogs in the pulmonary setting.

5-(Phenylthio)acyclouridine: a powerful enhancer of oral uridine bioavailability: relevance to chemotherapy with 5-fluorouracil and other uridine rescue regimens.

PURPOSE: The purpose of this investigation was to evaluate the effectiveness of oral 5-(phenylthio)acyclouridine (PTAU) in improving the pharmacokinetics and bioavailability of oral uridine. PTAU is a potent and specific inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3), the enzyme responsible for uridine catabolism. This compound was designed as a lipophilic inhibitor in order to facilitate its access to the liver and intestine, the main organs involved in uridine catabolism. PTAU is fully absorbed after oral administration with 100% oral bioavailability. METHODS: Uridine (330, 660 or 1320 mg/kg) and/or PTAU (30, 45, 60, 120, 240 or 480 mg/kg) were orally administered to mice. The plasma levels of uridine, its catabolite uracil, and PTAU were measured using HPLC, and pharmacokinetic analysis was performed. RESULTS: Oral PTAU up to 480 mg/kg per day is not toxic to mice. Oral PTAU at 30, 45, 60, 120 and 240 mg/kg has a prolonged plasma half-life of 2-3 h, and peak plasma PTAU concentrations (C(max)) of 41, 51, 74, 126 and 161 microM with AUCs of 70, 99, 122, 173 and 225 micromol h/l, respectively. Coadministration of uridine with PTAU did not have a significant effect on the pharmacokinetic parameters of plasma PTAU at any of the doses tested. Coadministration of PTAU (30, 45, 60 and 120 or 240 mg/kg) with uridine (330, 660 or 1320 mg/kg) elevated the concentration of plasma uridine over that following the same dose of uridine alone, a result of reduced metabolic clearance of uridine as evidenced by decreased plasma exposure (C(max) and AUC) to uracil. Plasma uridine was elevated with the increase of uridine dose at each PTAU dose tested and no plateau was reached. Coadministration of PTAU at 30, 45, 60, 120 and 240 mg/kg improved the low oral bioavailability (7.7%) of uridine administered at 1320 mg/kg by 4.3-, 5.9-, 9.9-, 11.7- and 12.5-fold, respectively, and reduced the AUC of plasma uracil (1227.8 micromol h/l) by 5.7-, 6.8-, 8.2-, 6.3-, and 6.9-fold, respectively. Similar results were observed when PTAU was coadministered with lower doses of uridine. Oral PTAU at 30, 45, 60, 120 and 240 mg/kg improved the oral bioavailability of 330 mg/kg uridine by 1.7-, 2.4-, 2.6-, 5.2- and 4.3- fold, and that of 660 mg/kg uridine by 2.3-, 2.7-, 3.3-, 4.6- and 6.7-fold, respectively. CONCLUSION: The excellent pharmacokinetic properties of PTAU, and its extraordinary effectiveness in improving the oral bioavailability of uridine, could be useful to rescue or protect from host toxicities of 5-fluorouracil and various chemotherapeutic pyrimidine analogues used in the treatment of cancer and AIDS, as well as in the management of medical disorders that are remedied by the administration of uridine including CNS disorders (e.g. Huntington's disease, bipolar disorder), liver diseases, diabetic neuropathy, cardiac damage, various autoimmune diseases, and transplant rejection.