Fludarabine Triphosphate Research Articles

Glycodendrimer Nanocarriers for Direct Delivery of Fludarabine Triphosphate to Leukemic Cells: Improved Pharmacokinetics and Pharmacodynamics of Fludarabine.

Fludarabine, a nucleoside analogue antimetabolite, has complicated pharmacokinetics requiring facilitated transmembrane transport and intracellular conversion to triphosphate nucleotide form (Ara-FATP), causing it to be susceptible to emergence of drug resistance. We are testing a promising strategy to improve its clinical efficacy by direct delivery of Ara-FATP utilizing a biocompatible glycodendrimer nanocarrier system. Here, we present results of a proof-of-concept experiment in several in vitro-cultured leukemic cell lines (CCRF, THP-1, U937) using noncovalent complexes of maltose-modified poly(propyleneimine) dendrimer and fludarabine triphosphate. We show that Ara-FATP has limited cytotoxic activity toward investigated cells relative to free nucleoside (Ara-FA), but complexation with the glycodendrimer (which does not otherwise influence cellular metabolism) drastically increases its toxicity. Moreover, we show that transport via hENT1 is a limiting step in Ara-FA toxicity, while complexation with dendrimer allows Ara-FATP to kill cells even in the presence of a hENT1 inhibitor. Thus, the use of glycodendrimers for drug delivery would allow us to circumvent naturally occurring drug resistance due to decreased transporter activity. Finally, we demonstrate that complex formation does not change the advantageous multifactorial intracellular pharmacodynamics of Ara-FATP, preserving its high capability to inhibit DNA and RNA synthesis and induce apoptosis via the intrinsic pathway. In comparison to other nucleoside analogue drugs, fludarabine is hereby demonstrated to be an optimal candidate for maltose glycodendrimer-mediated drug delivery in antileukemic therapy.

Fludarabine resistance mediated by aminoglycoside-3'-phosphotransferase-IIa and the structurally related eukaryotic cAMP-dependent protein kinase.

While working with G418-resistant stably transfected cells, we realized the neomycin resistance (NeoR) gene, which encodes the aminoglycoside-3'-phosphotransferase-IIa [APH(3')-IIa], also confers resistance to the nucleoside analog fludarabine. Fludarabine is a cytostatic drug widely used in the treatment of hematologic and solid tumors, as well as in the conditioning of patients before transplantation of hematopoietic progenitors. We present evidence that NeoR-transfected cells do not incorporate fludarabine, thus avoiding DNA damage caused by the drug, evidenced by a lack of FANCD2 monoubiquitination and impaired apoptosis. A screening of other nucleoside analogs revealed that APH(3')-IIa only protects against ATP purine analogs. Moreover, APH(3')-IIa ATPase activity is inhibited by fludarabine monophosphate, suggesting that APH(3')-IIa blocks fludarabine incorporation into DNA by dephosphorylating its active fludarabine triphosphate form. Furthermore, overexpression of the catalytic subunit of the eukaryotic kinase PKA, which is structurally related to APHs, also provides resistance to fludarabine, anticipating its putative utility as a response marker to the drug. Our results preclude the use of Neo marker plasmids in the study of purine analogs and unveils a new resistance mechanism against these chemotherapeuticals.-Sánchez-Carrera, D., Bravo-Navas, S., Cabezón, E., Arechaga, I., Cabezas, M., Yáñez, L., Pipaón, C. Fludarabine resistance mediated by aminoglycoside-3'-phosphotransferase-IIa and the structurally related eukaryotic cAMP-dependent protein kinase.

Determination and quantification of intracellular fludarabine triphosphate, cladribine triphosphate and clofarabine triphosphate by LC-MS/MS in human cancer cells.

Purine nucleoside analogues are widely used in the treatment of haematological malignancies, and their biological activity is dependent on the intracellular accumulation of their triphosphorylated metabolites. In this context, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to study the formation of 5'-triphosphorylated derivatives of cladribine, fludarabine, clofarabine and 2'-deoxyadenosine in human cancer cells. Br-ATP was used as internal standard. Separation was achieved on a hypercarb column. Analytes were eluted with a mixture of hexylamine (5 mM), DEA (0.4%, v/v, pH 10.5) and acetonitrile, in a gradient mode at a flow rate of 0.3mLmin-1. Multiple reactions monitoring (MRM) and electrospray ionization in negative mode (ESI-) were used for detection. The application of this method to the quantification of these phosphorylated cytotoxic compounds in a human follicular lymphoma cell line, showed that it was suitable for the study of relevant biological samples.

Severe fludarabine neurotoxicity after reduced intensity conditioning regimen to allogeneic hematopoietic stem cell transplantation: a case report

Key Clinical MessageWe present a case of severe, irreversible neurotoxicity in a 55-year-old-patient with myelofibrosis undergoing hematopoietic stem cell transplantation following a reduced intensity conditioning including fludarabine. The patient developed progressive sensory-motor, visual and consciousness disturbances, eventually leading to death. MRI imaging pattern was unique and attributable to fludarabine neurotoxicity.

Association of fludarabine pharmacokinetic/dynamic biomarkers with donor chimerism in nonmyeloablative HCT recipients.

Fludarabine monophosphate (fludarabine) is an integral component of many reduced-intensity conditioning regimens for hematopoietic cell transplantation (HCT). Fludarabine's metabolite, 9-β-D-arabinofuranosyl-2-fluoroadenine (F-ara-A), undergoes cellular uptake and activation to form the active cytotoxic metabolite fludarabine triphosphate (F-ara-ATP), which inhibits cellular DNA synthesis in CD4(+) and CD8(+) cells. In this study, we evaluated whether fludarabine-based pharmacologic biomarkers were associated with clinical outcomes in HCT recipients. Participants with hematologic diseases were conditioned with fludarabine and low-dose total body irradiation (TBI) followed by allogeneic HCT and post-grafting immunosuppression. After fludarabine administration, we evaluated pharmacological biomarkers for fludarabine-F-ara-A area under the curve (AUC) and the ratio of circulating CD4(+) and CD8(+) cells (CD4(+)/CD8(+) ratio) after fludarabine administration-in 102 patients; F-ara-ATP accumulation rate in enriched CD4(+) and CD8(+) cells was evaluated in 36 and 34 patients, respectively. Interpatient variability in the pharmacological biomarkers was high, ranging from 3.7-fold (F-ara-A AUC) to 39-fold (F-ara-ATP in CD8(+) cells). Circulating CD8(+) cells were more sensitive to fludarabine administration. A population pharmacokinetic-based sampling schedule successfully allowed for estimation of F-ara-A AUC in this outpatient population. There was a poor correlation between the F-ara-AUC and the F-ara-ATP accumulation rate in CD4(+) (R (2)=0.01) and CD8(+) cells (R (2)=0.00). No associations were seen between the four biomarkers and clinical outcomes (day +28 donor T cell chimerism, acute graft-versus-host disease (GVHD), neutrophil nadirs, cytomegalovirus reactivation, chronic GVHD, relapse, non-relapse mortality, or overall mortality). Considerable interpatient variability exists in pharmacokinetic and fludarabine-based biomarkers, but these biomarkers are not associated with clinical outcomes in fludarabine/TBI-conditioned patients.

Determination of intracellular fludarabine triphosphate in human peripheral blood mononuclear cells by LC–MS/MS

Fludarabine is a nucleoside analog routinely used in conditioning regimens of pediatric allogeneic stem cell transplantation to promote stem cell engraftment. In children, it remains a challenge to accurately and precisely quantify the active intracellular triphosphate species of fludarabine in vivo, primarily due to limitations on blood volume and inadequate assay sensitivity. Here we report a liquid chromatography tandem mass spectrometry (LC–MS/MS) method for determination of fludarabine triphosphate in human peripheral blood mononuclear cells (PBMC). PBMC (∼5 million cells) were collected and lysed in 1mL 70% methanol containing 1.2mM tris buffer (pH 7.4). The lysate (80μL) was mixed with internal standard (2-chloro-adenosine triphosphate, 150ng/mL, 20μL) and injected onto an API5000 LC–MS/MS system. Separation was achieved on a hypercarb column (100mm×2.1mm, 3μm) eluted with 100mM ammonium acetate (pH 9.8) and acetonitrile in a gradient mode at a flow rate of 0.4mL/min. Multiple reactions monitoring (MRM) and electrospray ionization in negative mode (ESI−) were used for detection. The ion pairs 524.0/158.6 for the drug and 540.0/158.8 for the IS were selected for quantification and 524.0/425.7 used for confirmation. Retention time was 3.0 and 3.4min for fludarabine triphosphate and the IS, respectively. The concentration range for the calibration curve was 1.52–76nM. Our method is simple, fast, and has been successfully applied in a clinical dose–concentration study in children to quantify intracellular fludarabine in low volume clinical samples. The median concentration was 1.03 and 3.19pmole/million PBMC at trough and peak time points, respectively. Fludarabine triphosphate is degraded in water within hours but relatively stable in 70% methanol–tris (1.2mM, pH 7.4). One limitation is that the hypercarb column takes a longer time to equilibrate than conventional reverse phase columns, and peaks become broad and distorted if the column is not washed and stored properly.

Fludarabine, Bendamustine, and Rituximab (FBR) Chemoimmunotherapy Is a Safe and Active Regimen for Relapsed/Refractory CLL with in Vivo Mechanism of Action for Combination Chemotherapy

Abstract 437Chemoimmunotherapy (such as fludarabine, cyclophosphamide, and rituximab) is highly effective CLL therapy; adding novel agents or replacing standard with more effective agents could improve outcomes. Bendamustine (B) is a potent alkylating agent that induces DNA damage and repair response. Marked DNA damage response (H2AX phosphorylation) was seen with activation of p53 protein and PUMA, and cell death when fludarabine was combined with bendamustine in vitro. Base on this, we are conducting a phase I/II trial of escalating doses of bendamustine at 20 (n=6), 30 (n=17), 40 (n=6), or 50 (n=6) mg/m2 on D1, 2, 3 with fludarabine 20 mg/m2 administered prior to bendamustine on D2 & 3. Rituximab 375–500 mg/m2 was given D3. Courses were repeated each 28 days for 6 planned courses to assess the safety and tolerability, clinical efficacy, and pharmacodynamics (PD) in previously treated CLL. Response assessment (IWCLL 2008 criteria) was after 3 courses and end of treatment; bone marrow residual disease was assessed by 4-color flow. We previously reported (ASH 2011) that no MTD was identified in phase I and identified bendamustine 30 mg/m2 as safe for phase II expansion. We now report on efficacy of this FBR regimen in 35 pts treated in phase I & II who have response data available. The median age was 62 yrs; number of prior treatments was 3 (1-6); and number of FBR courses was 3 (1-6). Dose-reduction after course 1 occurred in 10/35 pts. Responses are shown (Table) and were seen across all dose levels. Time-to-event endpoints will be presented. Pts had high-risk features: median b-2 microglobulin was 4.1 (1.8–10.4); 18/35 were Rai stage III-IV; 23/29 were ZAP70+; 23/29 had unmutated IGHV; and FISH identified 3 pts with del17p and 13 with del11q. Myelosuppression was the most common treatment-related toxicity considering all courses given (n=106). Grade(G) 3&G4 neutropenia occurred in 26&29% of courses, respectively; thrombocytopenia G3&G4 occurred in 14&9% of courses, respectively; and anemia G3&G4 occurred in 15&2% of courses, respectively. There were no treatment-related deaths.To test fludarabine triphosphate-mediated mitigation of DNA repair response induced by bendamustine, on D1 bendamustine was infused alone and on D2, fludarabine was administered 2 hours prior to second bendamustine infusion; circulating CLL cells from 11 pts at different bendamustine doses (3 at 20, 3 at 30, 3 at 40, and 2 at 50 mg/m2) were evaluated. Phosphorylation of histone 2A variant × (H2AX) was used as damage response marker. There was heterogeneity in extent of DNA damage response elicited after first bendamustine infusion. Considering basal phosphorylation level in the pretreatment sample on D1 as 1.0, the H2AX phosphorylation at D1-6hr (bendamustine alone) ranged between 0.2–8 (n=11). Median intracellular fludarabine triphosphate level at the start of bendamustine infusion was 12 μM (range 5–21 μM). This was sufficient to increase H2AX phosphorylation in all 11 pts tested. At the end of D2-4hr (bendamustine combined with fludarabine) the range was between 0.9–22 and remained the same on D2-6hr. In 1 pt sample, the phosphorylation persisted at 26 until D3-pretreatement, while in others it ranged between 3–12. Consistent with H2AX data, molecular markers of DNA damage response showed activation of ATM measured in 5 pt samples as ser1981 phosphorylation and phosphorylation of p53 at ser15. In parallel, there was a decrease in anti-apoptotic proteins Mcl-1 and Bcl-2 at the end of D2-6hr; however protein levels were retained on D3.In conclusion, this FBR regimen was tolerated up to the highest evaluated bendamustine dose; efficacy was demonstrated in previously treated pts with CLL. DNA damage and repair response biomarkers validated the hypothesis that fludarabine triphosphate inhibited bendamustine-induced DNA repair resulting in increased or sustained DNA damage. We continue to extend the clinical and PD investigations in phase II.TableCharacteristicn% CR/CRi% OR%MRD NegAll Pts35267111Rai StageIII-IV181767110-II17357612No. Prior Rx>219165851-216388819B2M34 mg/l19115311<4 mg/l16449413FISH17p del333100011q del1323690+127578643None30333313q del301000IGHVUnmutated2326709Mutated63310033ZAP70Positive23227013Negative6178317CD38 (>7%)Positive2623738Negative8387525CR, complete remission; CRi, CR with incomplete recovery of cytopenias; OR, overall response; MRD, minimal residual disease. Disclosures:No relevant conflicts of interest to declare.

A Phase I/II Trial of Fludarabine, Bendamustine, and Rituximab (FBR) Chemoimmunotherapy for Previously Treated Patients with CLL,

Abstract 3901Chemoimmunotherapy (such as fludarabine, cyclophosphamide, and rituximab) has been the most significant advance in treatment for patients with CLL, achieving the highest complete remission rates, longest progression-free and overall survival compared to chemotherapy combinations or monotherapy. Bendamustine (B) is a well-tolerated, alkylating agent that induces a DNA damage and repair response. In vitro data in 30 CLL patient (pt) samples suggested an increased DNA damage response (measured as H2AX phosphorylation), activation of p53 protein and PUMA, and cell death when fludarabine was combined with bendamustine (El-Mabhouh, A, unpublished). To translate this observation to the clinic, we are conducting a phase I/II trial of escalating doses of bendamustine at 20, 30, 40, or 50 mg/m2 on D1,2,3 with fludarabine 20 mg/m2 administered prior to bendamustine on D2&3. Rituximab 375–500 mg/m2 was given on D3. Courses were repeated each 28 days to assess the safety and tolerability, clinical efficacy, and pharmacodynamics (PD) in previously treated pts with CLL. Responses were assessed after 3 courses and end of treatment. We report results of the phase I portion of this study. For phase I, dose-limiting toxicities (DLT) were assessed in course 1 and were Grade (G) ≥3 treatment-related, non-hematologic adverse event (AE), and hematologic toxicity G≥3 that lasted beyond D42 of course 1. MTD was defined as the cohort with ≤1 DLT in 6 treated pts. All pts (n=19) had active CLL and were previously treated; median number of prior treatments was 2 (1–6). Pts had high-risk features, median >102<−2 microglobulin was 4 (2.4–8.7); Rai stage III-IV was 10/19; 13/15 were ZAP70+; 12/15 had unmutated IGHV; and FISH identified 2 with del17p and 7 with del11q. 19 patients were evaluable for course 1 toxicities and DLT. Course 1 toxicities were predominantly G1-2 and most common were nausea, fatigue, and hyperglycemia. One of 6 pts experienced DLT (G3 nausea/vomiting/dehydration) in the B-20 cohort; 0 of 3 pts experienced DLT in the B-30 cohort; 1 of 6 pts experienced DLT (G4 sepsis) in the B-40 cohort; and 1 of 4 pts experienced DLT (G3 neutropenia) in the B-50 cohort. Pts continued on treatment, 5 with dose reduction, (Table) for up to 6 courses. The B-50 cohort continues enrollment and treatment, all other cohorts completed treatment. Among 14 pts evaluable for response, there were 5 complete responders (3 MRD negative by 4-color flow cytometry) and 8 partial responders (2 PRs were CRi by IWCLL 2008 criteria); only 1 pt was a non-responder (Table). Considering all courses given, the most common G3-4 AEs that occurred in more than 10% of courses (n=56) were: neutropenia (30%) and thrombocytopenia (13%). All other AEs were G1-2 and resolved. There were no treatment-related deaths. More frequent AEs with higher doses of bendamustine supports selection of the 30 mg/m2 dose level to move forward in phase II. To test fludarabine triphosphate-mediated mitigation of DNA repair response induced by bendamustine, on D1, bendamustine was infused alone and on D2, the fludarabine dose was given 2 hours prior to bendamustine infusion. Circulating CLL cells from 7 pts (3 B-20 and 3 B-40, and 1 B-50) were evaluated for PD endpoints. Median intracellular fludarabine triphosphate level at the start of bendamustine infusion was 12 μM (range 5–21 μM). This was sufficient to increase by 3–5-fold the H2AX phosphorylation response. Molecular markers of DNA damage response and cell death (ATM, p53, PUMA, Mcl-1) are being evaluated.In conclusion, the FBR regimen was tolerated up to the highest bendamustine dose evaluated, with significant efficacy in previously treated patients with CLL. We are extending the clinical and PD investigations in a phase II study with B-30 dose.TableCohort*nMedian coursesTotal coursesTotal AEs per Cohort (C1)Eval for ResponsePercent RespondersG1-2G3-4nCRORB-2063 (2–6)2222465083B-3034 (3–5)1218130100B-4062.5 (1–4)164514540100B-5041.5 (1–2)**6**318–––AEs, adverse events; G, grade; n, number; CR, complete remission; OR, overall response*Bendamustine dose mg/m2 daily × 3;**Treatment continues Disclosures:No relevant conflicts of interest to declare.

A pilot pharmacologic biomarker study of busulfan and fludarabine in hematopoietic cell transplant recipients

Sixteen patients diagnosed with various hematologic malignancies participated in a phase II study evaluating the addition of rabbit antithymocyte globulin (rATG, Thymoglobulin(®)) to the hematopoietic cell transplant (HCT) conditioning regimen of IV fludarabine monophosphate (fludarabine) and targeted intravenous (IV) busulfan (fludarabine/(T)busulfan). Our goal was to evaluate pharmacologic biomarkers pertinent to both medications in these patients. We characterized the interpatient variability of pharmacologic biomarkers relevant to busulfan, specifically busulfan concentration at steady state, and fludarabine, specifically F-ara-A area under the curve (AUC) and fludarabine triphosphate (F-ara-ATP) intracellular accumulation and concentration in separate CD4(+) and CD8(+) T-lymphocyte populations. Acute and chronic graft versus host disease (GvHD) occurred in 11 patients and one patient, respectively. Four patients died before day +100 of non-relapse causes, which met the protocol stopping guidelines. The cumulative incidence of relapse was 25% at 3year post-HCT. Interpatient variability in the busulfan- and fludarabine-relevant pharmacologic biomarkers was 2.1- to 2.5-fold. F-ara-A AUC and accumulated F-ara-ATP in CD8(+) cells had the highest hazard ratio for non-relapse mortality and overall survival, respectively. However, neither achieved statistical significance. The low rates of GvHD, particularly in its chronic form, were encouraging, and further biomarker studies are warranted to optimize the fludarabine/(T)busulfan/rATG conditioning regimen.

Intracellular disposition of fludarabine triphosphate in human natural killer cells.

Fludarabine is a key component of several reduced-intensity conditioning regimens for hematopoietic cell transplantation (HCT). Shortly after reduced-intensity conditioning, the percent of donor natural killer (NK) cells has been associated with progression-free survival. Insufficient suppression of the recipient's NK cells by fludarabine may lead to lower donor chimerism; however, the effect of fludarabine upon NK cells is poorly understood. Thus, in purified human NK cells we evaluated the uptake and activation of fludarabine to its active metabolite, fludarabine triphosphate (F-ara-ATP), and assessed the degree of interindividual variability in F-ara-ATP accumulation. Intracellular F-ara-ATP was measured in purified NK cells isolated from healthy volunteers (n = 6) after ex vivo exposure to fludarabine. Gene expression levels of the relevant transporters and enzymes involved in fludarabine uptake and activation were also measured in these cells. F-ara-ATP accumulation (mean +/- SD) was 6.00 +/- 3.67 pmol/1 x 10(6) cells/4 h, comparable to average levels previously observed in CD4(+) and CD8(+) T-lymphocytes. We observed considerable variability in F-ara-ATP accumulation and mRNA expression of transporters and enzymes relevant to F-ara-ATP accumulation in NK cells from different healthy volunteers. Human NK cells have the ability to form F-ara-ATP intracellularly and large interindividual variability was observed in healthy volunteers. Further studies are needed to evaluate whether F-ara-ATP accumulation in NK cells are associated with apoptosis and clinical outcomes.

A novel phenotypic method to determine fludarabine triphosphate accumulation in T-lymphocytes from hematopoietic cell transplantation patients

Fludarabine is an integral anticancer agent for patients with chronic lymphocytic leukemia (CLL) and those receiving conditioning regimens prior to allogeneic hematopoietic cell transplantation (HCT). An individual's response to fludarabine may be influenced by the amount of CD4(+) and CD8(+) T-lymphocyte suppression. Fludarabine undergoes cellular uptake and activation to form the cytotoxic metabolite, fludarabine triphosphate (F-ara-ATP). We have previously developed a highly sensitive LC-MS method to quantitate intracellular F-ara-ATP concentrations in a leukemic cell line. However, quantitation of F-ara-ATP concentrations within CD4(+) and CD8(+) T-lymphocytes from pharmacokinetic blood samples obtained from patients receiving fludarabine therapy is not feasible because of the limited number of T-lymphocytes that can be isolated from each blood sample. Thus, we sought to determine F-ara-ATP accumulation after ex vivo exposure of freshly isolated human CD4(+) or CD8(+) T-lymphocytes to fludarabine. The method was optimized in T-lymphocytes obtained from healthy volunteers, and proved to be a feasible method to determine F-ara-ATP accumulation in patients undergoing HCT. Considerable variability was observed in F-ara-ATP accumulation in HCT patients (10.5- and 12.5-fold in CD4(+) and CD8(+) cells, respectively), compared to healthy volunteers (1.6- and 1.9-fold in CD4(+) and CD8(+) cells, respectively). Larger variability was also observed in gene expression of transporters and enzymes involved in F-ara-ATP accumulation in HCT patients; however, F-ara-ATP accumulation was not correlated with gene expression, which is in agreement with previous studies. The quantitation of F-ara-ATP accumulation in T-lymphocytes provides a novel tool to evaluate patient sensitivity to fludarabine. This tool can be used in future studies to evaluate whether intracellular F-ara-ATP accumulation is associated with efficacy and/or toxicity in patients receiving fludarabine.

Imatinib inhibition of fludarabine uptake in T-lymphocytes

We investigated the potential drug-drug interaction between imatinib and fludarabine, which may be concomitantly administered in chronic myeloid leukemia (CML) patients receiving fludarabine-based conditioning for allogeneic hematopoietic cell transplantation (HCT). Imatinib is an inhibitor of human equilibrative transporters (hENTs), which are responsible for the intracellular uptake of fludarabine. Intracellular accumulation of fludarabine triphosphate (F-ara-ATP), the active metabolite of fludarabine, was measured in CD4(+) and CD8(+) T-lymphocytes isolated from healthy volunteers, which were treated in vitro with fludarabine alone, and in the presence of either imatinib or NBMPR, a known hENT inhibitor. Imatinib significantly inhibited F-ara-ATP accumulation in CD4(+) and CD8(+) T-lymphocytes in a concentration-dependent manner. The observed imatinib inhibition was comparable to inhibition observed with NBMPR. The inhibition of F-ara-ATP by imatinib is likely due to inhibition of nucleoside transporters hENT1 and hENT2. There is significant in vitro drug interaction between imatinib and fludarabine. This effect may be of important consideration in patients receiving fludarabine-based conditioning prior to HCT.

Fludarabine-Mediated Circumvention of Cytarabine Resistance Is Associated with Fludarabine Triphosphate Accumulation in Cytarabine-Resistant Leukemic Cells

The combination of cytarabine (ara-C) with fludarabine is a common approach to treating resistant acute myeloid leukemia. Success depends on a fludarabine triphosphate (F-ara-ATP)-mediated increase in the active intracellular metabolite of ara-C, ara-C 5'-triphosphate (ara-CTP). Therapy-resistant leukemia may exhibit ara-C resistance, the mechanisms of which might induce cross-resistance to fludarabine with reduced F-ara-ATP formation. The present study evaluated the effect of combining ara-C and fludarabine on ara-C-resistant leukemic cells in vitro. Two variant cell lines (R1 and R2) were 8-fold and 10-fold more ara-C resistant, respectively, than the parental HL-60 cells. Reduced deoxycytidine kinase activity was demonstrated in R1 and R2 cells, and R2 cells also showed an increase in cytosolic 5'-nucleotidase II activity. Compared with HL-60 cells, R1 and R2 cells produced smaller amounts of ara-CTP. Both variants accumulated less F-ara-ATP than HL-60 cells and showed cross-resistance to fludarabine nucleoside (F-ara-A). R2 cells, however, accumulated much smaller amounts of F-ara-ATP and were more F-ara-A resistant than R1 cells. In HL-60 and R1 cells, F-ara-A pretreatment followed by ara-C incubation produced F-ara-ATP concentrations sufficient for augmenting ara-CTP production, thereby enhancing ara-C cytotoxicity. No potentiation was observed in R2 cells. Nucleotidase might preferentially degrade F-ara-A monophosphate over ara-C monophosphate, leading to reduced F-ara-ATP production and thereby compromising the F-ara-A-mediated potentiation of ara-C cytotoxicity in R2 cells. Thus, F-ara-A-mediated enhancement of ara-C cytotoxicity depended on F-ara-ATP accumulation in ara-C-resistant leukemic cells but ultimately was associated with the mechanism of ara-C resistance.

A highly sensitive high-performance liquid chromatography–mass spectrometry method for quantification of fludarabine triphosphate in leukemic cells

A high-performance liquid chromatography (HPLC)–mass spectrometry (MS) method has been developed for the analysis of 9-β- d-arabinofuranosyl-2-fluoroadenine 5′-triphosphate (F-ara-ATP) from biological samples. Quantification is carried out by selected ion monitoring of the parent ion. Baseline separation of the monophosphate (F-ara-AMP) and diphosphate (F-ara-ADP) is achieved using the volatile ion-pairing reagent dimethylhexylamine. This method is selective and sensitive with an on-column detection limit of ∼50 fmol. It also permits simultaneous monitoring of endogenous adenosine phosphates. The utility of the assay has been demonstrated by the analysis of F-ara-ATP in human leukemic cells after incubation with 9-β- d-arabinosyl-2-fluoroadenine (F-ara-A) at clinically relevant concentrations.

The Histone Deacetylase Inhibitor MS-275 Interacts Synergistically with Fludarabine to Induce Apoptosis in Human Leukemia Cells

Interactions between the novel benzamide histone deacetylase (HDAC) inhibitor MS-275 and fludarabine were examined in lymphoid and myeloid human leukemia cells in relation to mitochondrial injury, signal transduction events, and apoptosis. Prior exposure of Jurkat lymphoblastic leukemia cells to a marginally toxic concentration of MS-275 (e.g., 500 nM) for 24 h sharply increased mitochondrial injury, caspase activation, and apoptosis in response to a minimally toxic concentration of fludarabine (500 nM), resulting in highly synergistic antileukemic interactions and loss of clonogenic survival. Simultaneous exposure to MS-275 and fludarabine also led to synergistic effects, but these were not as pronounced as observed with sequential treatment. Similar interactions were noted in the case of (a) other human leukemia cell lines (e.g., U937, CCRF-CEM); (b) other HDAC inhibitors (e.g., sodium butyrate); and (c) other nucleoside analogues (e.g., 1-beta-D-arabinofuranosylcytosine, gemcitabine). Potentiation of fludarabine lethality by MS-275 was associated with acetylation of histones H3 and H4, down-regulation of the antiapoptotic proteins XIAP and Mcl-1, enhanced cytosolic release of proapoptotic mitochondrial proteins (e.g., cytochrome c, Smac/DIABLO, and apoptosis-inducing factor), and caspase activation. It was also accompanied by the caspase-dependent down-regulation of p27(KIP1), cyclins A, E, and D(1), and cleavage and diminished phosphorylation of retinoblastoma protein. However, increased lethality of the combination was not associated with enhanced fludarabine triphosphate formation or DNA incorporation and occurred despite a slight reduction in the S-phase fraction. Prior exposure to MS-275 attenuated fludarabine-mediated activation of MEK1/2, extracellular signal-regulated kinase, and Akt, and enhanced c-Jun NH(2)-terminal kinase phosphorylation; furthermore, inducible expression of constitutively active MEK1/2 or Akt significantly diminished MS-275/fludarabine-induced lethality. Combined exposure of cells to MS-275 and fludarabine was associated with a significant increase in generation of reactive oxygen species; moreover, both the increase in reactive oxygen species and apoptosis were largely attenuated by coadministration of the free radical scavenger L-N-acetylcysteine. Finally, prior administration of MS-275 markedly potentiated fludarabine-mediated generation of the proapoptotic lipid second messenger ceramide. Taken together, these findings indicate that the HDAC inhibitor MS-275 induces multiple perturbations in signal transduction, survival, and cell cycle regulatory pathways that lower the threshold for fludarabine-mediated mitochondrial injury and apoptosis in human leukemia cells. They also provide insights into possible mechanisms by which novel, clinically relevant HDAC inhibitors might be used to enhance the antileukemic activity of established nucleoside analogues such as fludarabine.

Simple and sensitive method for quantification of fludarabine triphosphate intracellular concentration in leukemic cells using isocratic liquid chromatography

A simple, isocratic HPLC method was newly developed for quantitating intracellular fludarabine triphosphate (F-ara-ATP). Samples (500 μl) were injected onto an anion-exchange column and eluted isocratically with phosphate-acetonitrile buffer (flow rate: 0.7 ml/min) at an ambient temperature. F-ara-ATP was quantitated according to its peak area at the absorbance of 261 nm. The standard curve was linear with minimal within-day and inter-day variability. The low and high quantification limits were 50 pmol and 20 nmol, respectively. The method was capable of measuring F-ara-ATP generated in cultured leukemic cells in vitro. Thus, our method will be useful because of its sensitivity and simplicity as well as applicability to biological materials.

High-performance liquid chromatography method for the determination and quantitation of arabinosylguanine triphosphate and fludarabine triphosphate in human cells

A gradient anion-exchange high-performance liquid chromatographic assay was developed for the simultaneous determination and quantitation of the cytotoxic triphosphates of arabinosylguanine (ara-GTP) and fludarabine (F-ara-ATP). The method was validated with respect to selectivity, recovery, linearity, precision, and accuracy using authentic standards. To test this assay in a more complex biological matrix, perchloric acid extracts of circulating human leukemia cells spiked with known concentrations of ara-GTP and F-ara-ATP were examined. Finally, to assess the clinical utility of our method, perchloric acid extracts of circulating human leukemia cells isolated from patients treated with fludarabine and nelarabine were analyzed. The range of quantitation was 0.0125–10 nmol for the ara- and native NTPs in cellular extracts. This assay should be helpful in establishing the mechanistic rationales for drug scheduling and combinations of nelarabine and fludarabine, and for correlating the therapeutic efficacy and levels of the cytotoxic triphosphates in target cells.

Use of novel plasmid constructs to demonstrate fludarabine triphosphate inhibition of nucleotide excision repair of a site-specific 1,2-d(GpG) intrastrand cisplatin adduct.

We previously showed that fludarabine triphosphate (F-ara-ATP) acts as a potent inhibitor of nucleotide excision repair (NER). To determine how F-ara-ATP inhibits NER, we designed closed circular DNA duplexes, each containing a site-specific d(GpG) cisplatin adduct, as the substrate for an in vitro repair assay. We used the assay to determine the effects of F-ara-ATP on the incision, repair synthesis, and ligation steps in the NER process. A closed circular DNA duplex, pSSA, was first constructed by inserting an 87-bp oligonucleotide into pGEM-7Zf(+), from which a single-stranded plasmid (pTDS) was derived. The 87-bp insert included two potential repair patches; each contained a d(GpG) site flanked by unique sequences 22 nucleotides upstream and 6 nucleotides downstream so that four dAMP sites were strategically placed in patch 1 but were absent from patch 2. Each duplex substrate was then synthesized by annealing a unique primer containing a platinated and 32P-end-labeled oligonucleotide to the pTDS template, which was then converted to a duplex through polymerization and ligation. At 50 microM, F-ara-ATP inhibited repair synthesis; F-ara-ATP also inhibited incision and ligation, but only at concentrations of 200 microM and above. Chemical DNA sequencing of the repair patch revealed that F-ara-ATP induced the formation of a truncated repair patch in which DNA polymerization stopped one nucleotide before the first installed dAMP site - a potential site for F-ara-ATP's incorporation. In contrast, truncation of a repair patch was not detectable when the repair patch contained no dAMP. Taken together, the results suggest that F-ara-ATP induced patch truncation by self-incorporation and the incorporated F-ara-AMP was subsequently excised, presumably by polymerase-associated exonuclease activity. We conclude that F-ara-ATP blocks the NER process by strongly inhibiting DNA repair synthesis as well as by less strongly inhibiting incision and ligation. Our approach of designing plasmid constructs that contain sequence-specific repair patches with a strategically placed 32P label may provide a powerful tool for dissecting the mechanism of NER inhibition not only for F-ara-ATP but also for other NER inhibitors.

Fludarabine-mediated repair inhibition of cisplatin-induced DNA lesions in human chronic myelogenous leukemia-blast crisis K562 cells: induction of synergistic cytotoxicity independent of reversal of apoptosis resistance.

We demonstrated previously that the nucleoside of fludarabine (F-ara-A), a clinically effective agent against chronic lymphocytic leukemia and low-grade lymphoma, produces synergistic cytotoxicity against cisplatin-resistant CP2.0 human colon tumor cells when administered in combination with cisplatin. The purpose of this study was 2-fold: (i) to determine whether the synergy occurs in K562 human chronic myelogenous leukemia cells, which, unlike CP2.0 cells, are relatively resistant to drug-induced apoptosis because they express P210(bcr-abl) and (ii) to study the underlying mechanism for the synergy if the enhancement of cytotoxicity occurs in K562 cells. When K562 cells were treated with fludarabine nucleoside and cisplatin as single agents for 4 hr, IC50 values for fludarabine and cisplatin were 3.33 and 2.28 microM, respectively, as measured by a clonogenic survival assay. The simultaneous treatment of K562 cells with the two agents resulted in synergistic cell killing as determined by median-effect analysis. Such synergistic cell killing by combined cisplatin and fludarabine could not be detected in repair-deficient human xeroderma pigmentosum cell lines. Within the range of cytotoxic concentrations, fludarabine (2.5-15 microM) and cisplatin (3-30 microM) as single agents produced no detectable internucleosomal DNA fragmentation as revealed by gel electrophoresis, nor did the combination of the two drugs induce apoptotic DNA degradation. The effects of fludarabine on the repair of cisplatin-induced DNA adducts and interstrand cross-links in K562 cells were analyzed to determine their correlation with the cytotoxic synergy. The interstrand cross-links were measured by the ethidium bromide binding fluorescence assay and quantitative Southern blotting technique. Repair of the intrastrand adducts was detected with whole-cell extracts using a cisplatin-damaged plasmid as the substrate for the in vitro repair assay. Fludarabine at clinically achievable concentrations (1.5-4.5 microM fludarabine nucleoside; 20-100 microM fludarabine triphosphate) inhibited the repair of the DNA lesions induced by cisplatin in a dose-dependent fashion in K562 cells but not in xeroderma pigmentosum cells. Cotreatment with fludarabine preferentially increased the number of interstrand cross-links induced by cisplatin in actively transcribed genes in K562 cells. These data demonstrate the DNA-repair-inhibitory effect of fludarabine and suggest that this effect may contribute to the synergistic cytotoxicity of the fludarabine/cisplatin combination that resulted in decreased clonogenic survival of apoptosis-resistant K562 cells.