Intracellular ara-CTP Concentration Research Articles

Intracellular cytarabine triphosphate in circulating blasts post-treatment predicts remission status in patients with acute myeloid leukemia

Cytarabine remains the backbone of therapy in acute myeloid leukemia (AML). The ability to assess intracellular cytarabine triphosphate (ara-CTP) levels in patients receiving cytarabine represents a major goal in the prediction of treatment response. This study, conducted within a clinical setting, aimed to assess ara-CTP levels in circulating peripheral blasts from non-M3 AML patients receiving cytarabine at one of three dosing levels, using a novel biosensor assay. Results from the initial 72 hours post-commencement were correlated with day 28 remission status, with feasibility parameters concurrently assessed. Intracellular ara-CTP was detectable in ex vivo blasts post-treatment for standard-dose (SD) and high-dose (HD) patients (p < 0.05), and quantification revealed a 27-fold increase in intracellular steady-state concentration between the two dosing levels. For low-dose cytarabine, high rates of patient discharge and low intracellular concentrations limited analysis; however, assessment of intracellular ara-CTP concentration was achievable in a dwindling population of blasts for SD and HD treatment cohorts, with 4 hours post-treatment commencement potentially being most predictive of clinical response (r = -0.912, p = 0.0113). Concurrent assessment of peripheral leukemia-associated immunophenotype (LAIP)-positive cells revealed a decline in burden (0-72 hours), which correlated with remission status (p < 0.05). Unexpectedly high rates of night sampling led to challenges associated with sampling rates, but did not have an impact on patient compliance. Additional training of night staff improved feasibility substantially. Multiple peripheral sampling during the initial 72 hours of treatment is feasible in newly diagnosed patients, and ara-CTP is detectable over the initial 24 hours, facilitating prediction of chemosensitivity of leukemic blasts to cytarabine.

Comprehensive Ara-C SNP score predicts leukemic cell intracellular ara-CTP levels in pediatric acute myeloid leukemia patients.

Cytarabine (Ara-C), a mainstay of acute myeloid leukemia (AML) treatment, is a prodrug requiring activation to ara-CTP for its antileukemic activity. Aim of this study was to evaluate impact of genetic variants in the key genes involved in ara-C metabolism on the leukemic cell intracellular levels of ara-CTP. We investigated SNPs in 14 ara-C metabolic-pathway genes,for association with intracellular ara-CTP levels, in leukemic cells obtained post-initiation of cytarabine infusion in pediatric AML patients (n = 68). Nine SNPs were significantly associated with leukemic cell intracellular concentration of ara-CTP.A comprehensive ara-CTP-SNP-score (ACSS) was further developed from top four SNPs identified in regression model. Patients were classified into three groupsbased on ACSS: high-ACSS (score>0), intermediate-ACSS (score=0) and low-ACSS (score<0). ACSS designation was significant predictor of intracellular ara-CTP levels (p=0.00012), suggesting a cumulative or synergistic effect of the significant SNPs. ACSS score designation holds promise in definfing ara-C dose. Validation of the clinical utility of ACSS score in other independent cohorts will help identification of patients with potentially lower or higher levels of the ara-CTP in leukemic cells,thereby opening up opportunities for dose management to reduce toxicity and enhance efficacy.

AraU accumulation in patients with renal insufficiency as a potential mechanism for cytarabine neurotoxicity

Neurotoxicity of cytarabine (AraC) is believed to be related to renal insufficiency. We examined the plasma pharmacokinetics of AraC and its deamination product uracil arabinoside (AraU) in four patients with AML and concomitant severe renal insufficiency after treatment with AraC. Additionally, in one of these patients the concentration of intracellular AraCTP, the active metabolite of AraC, was analysed. Patients 2 and 3 were treated with AraC 1.0 g/m(2) infused for 3 h at 12-h intervals on days 1-4. Patient 1 received the same schedule of AraC with 0.5 g/m(2) and patient 4 with 0.25 g/m(2) AraC. Plasma concentrations of AraC, AraU and the intracellular concentration of AraCTP were analysed at different time points using HPLC. AraC pharmacokinetics in patients with severe renal insufficiency was comparable to patients with normal renal function. Peak plasma levels as well as intracellular AraCTP kinetics were also not significantly influenced by renal dysfunction. As expected from the high dose AraC pharmacokinetic parameters, the AraU serum levels accumulated during treatment. Under the conditions of renal impairment, AraU half-life was about 75 h and the AUC was about 12-fold higher than for patients with normal renal function. AraU could be the pathophysiologic cause for the known correlation between the incidence of neurotoxicity and renal insufficiency in high-dose AraC. To avoid AraU accumulation, intermittent hemodialysis during high-dose AraC treatment could be a suitable method considering the low protein-binding and low distribution volume of AraU.

Deoxycytidine Kinase Genotype Influences Leukemia Cell Concentration of Cytarabine 5′-Triphosphate in Pediatric AML Patients.

Cytarabine (Ara-C) is a prodrug requiring conversion to its 5′-triphosphate (ara-CTP) form for antileukemic effect. Alteration of deoxycytidine kinase (DCK), a rate-limiting enzyme in this conversion, is associated with ara-C resistance. We sought to identify and determine the functional and clinical significance of DCK genetic variants. Sixty-four genetic polymorphisms were identified, including 3 coding changes (Ile24Val, Ala119Gly, and Pro122Ser amino acid substitutions), in subjects of European or African ancestry. The activity of recombinant DCK-24Val, DCK-119Gly, and DCK-122Ser was 85±5%, 66±3%, and 43±4%, respectively, that of wild-type (WT) DCK. These 3 DCK mutants also demonstrated altered substrate kinetics. Lymphoblast cell lines derived from subjects heterozygous for these coding changes had DCK activity significantly lower than that of homozygous-WT lymphoblasts (176.7±15 vs. 340.8±19 pmol CdA-MP/min/mg protein; p=0.0004). Moreover, for a 3′UTR 35708T>C SNP, the C allele was significantly associated with lower DCK mRNA expression in lymphoblast cell lines (p=0.02). To determine the clinical implications of DCK SNPs, we screened pediatric AML patients treated on the St Jude AML 97 protocol (n=55) for the 3 coding variants and the 3UTR SNP. Patients were randomly assigned to receive ara-C as either a short daily infusion (500 mg/m2/dose intravenously over 2 hrs daily for 5 days) or a continuous infusion (500 mg/m2/day as a continuous infusion over 5 days). Bone marrow was collected at the end of the ara-C infusion on day1 for patients receiving the short daily infusion (n=27), and at 10 hours after the start of the infusion for those receiving the continuous infusion (n=28). Ara-CTP levels in leukemia cells were analyzed by HPLC. Intracellular accumulation of ara-CTP was significantly higher in patients given a short daily infusion than in those given a continuous infusion (0.56±0.5 vs. 0.33±0.31 nmol ara-CTP/2x107 leukemia cells; p = 0.014). The inter-patient variability of blast ara-CTP concentration was 40-fold (0.06–2.43 nmol/2x107 cells) when ara-C was administered as short infusion and 101-fold (0.012–1.22 nmol/2x107 cells) when given as a continuous infusion. Karyotype, sex, age, and ethnicity were not significantly associated with leukemia-cell ara-CTP level. An Ala119Gly or Pro122Ser non-synonymous polymorphism was present in 1 patient each; the patient heterozygous for Ala119Gly (continuous infusion arm) had the lowest intracellular concentration of ara-CTP (0.012 nmol/2x107 cells; Figure). Both of these patients had relapses, despite the presence of favorable cytogenetic abnormalities [inv16 and t(8;21)]. The 3′UTR 35708 T>C SNP was significantly associated with lower intracellular ara-CTP concentration in patients receiving ara-C as a continuous infusion (35708T allele 0.5±0.3 vs. 35708C allele 0.22±0.17 nmol/2x107 cells; p= 0.04; Figure). These results suggest that genetic polymorphism of DCK influences its activity and expression and may underlie interpatient variability in intracellular ara-CTP concentration, which in turn can influence treatment response. [Display omitted]

A sensitive new method for clinically monitoring cytarabine concentrations at the DNA level in leukemic cells

Cytarabine (ara-C), a major antileukemic agent, is phosphorylated in the cell to cytarabine triphosphate (ara-CTP), which is then partly incorporated into DNA. The drug incorporation into DNA poisons the extending primer against further incorporation of deoxyribonucleotides including dCTP, ultimately inhibiting DNA synthesis. While intracellular ara-CTP concentration has been found to predict clinical outcome, cytotoxicity in vitro is determined primarily by the extent of drug incorporation into DNA. However, clinically appropriate quantitation methods for ara-C at the DNA level have not been available. We developed a sensitive new method for monitoring ara-C incorporated into DNA in vivo. After DNA from leukemic cells was fractionated using the Schmidt-Thannhauser-Schneider method, it was degraded to constituent nucleosides to release ara-C, which was isolated from the nucleosides using HPLC and then measured by radioimmunoassay. Recovery for DNA fractionation, ara-C release by degradation, and ara-C isolation were 92.0 ± 6.4%, 90.7 ± 9.4%, and 98.5 ± 1.4%, respectively. The method was found to determine ara-C incorporation into DNA of ara-C-treated HL 60 cells in vitro with minimal interassay variation. The values determined were compatible with those determined by scintillation counting in parallel experiments using tritiated ara-C. Our method could be used to monitor DNA-incorporated ara-C concentrations during ara-C therapy, together with plasma ara-C and intracellular ara-CTP concentrations. ara-C incorporation into DNA appeared to be associated with intracellular retention of ara-CTP or persistence of plasma ara-C. Thus, the present method is sensitive, accurate, precise, and may permit therapeutic drug monitoring at the DNA level for better individualization of antileukemic regimens.

Plasma and leukemic cell pharmacokinetics of high-dose N4-behenoyl-1-beta-D-arabinofuranosylcytosine in acute leukemia patients.

The pharmacokinetics of N4-behenoyl-1-beta-D-arabinofuranosylcytosine (BHAC), a lipophilic antitumor analog of 1-beta-D-arabinofuranosylcytosine (ara-C), was investigated, by assay of plasma and leukemic cells of ten acute leukemic patients receiving 60-minute intravenous (IV) infusion of 700 mg/m2 BHAC, for BHAC and 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) by high-performance liquid chromatography, ara-C by radioimmunoassay, and 1-beta-D-arabinofuranosyluracil (ara-U) by gas chromatography-mass fragmentography. The plasma concentration of BHAC reached a maximum (173.4 +/- 75.3 micrograms/mL) at the end of the infusion and then declined in a biphasic pattern with an initial-phase half-life (t1/2 alpha) of 1.00 +/- .36 hours and a second-phase half-life (t1/2 beta) of 4.28 +/- 2.35 hours. That of ara-C similarly reached a maximum (102.2 +/- 39.9 mg/mL) at the end of the infusion and then declined with t1/2 alpha of 1.37 +/- 1.11 hours and t1/2 beta of 11.2 +/- 4.31 hours. Intracellular ara-CTP concentration increased in a linear-accumulation manner for the first 4 hours after the infusion, reached a maximum of .081 +/- .112 micrograms/10(7) cells at approximately 7 hours, and then declined very slowly in accordance with a one-compartment model with t1/2 of 13.56 +/- 9.62 hours.

Ara-C in plasma and ara-CTP in leukemic cells after subcutaneous injection and continuous intravenous infusion of ara-C in patients with acute nonlymphoblastic leukemia.

Six patients with acute nonlymphoblastic leukemia (ANLL) were treated sequentially with one subcutaneous (SC) injection (50 mg/m2) and one 156 hour continuous intravenous (IV) infusion (100 mg/m2/d) of 1-beta-D-arabinofuranosylcytosine (ara-C) with an interval of 12 hours. Leukemic cells were isolated from venous blood samples. The intracellular concentration of ara-C 5'-triphosphate (ara-CTP) was determined by high-performance liquid chromatography, and in two of the patients the plasma concentration of ara-C was determined by radioimmunoassay. A rapid biphasic elimination of the drug from plasma with an initial half-life of 18 minutes was seen after SC injection. After about 1.5 hours, the concentration was lower than that obtained at steady-state during continuous IV infusion. In the leukemic cells, the concentration of ara-CTP was higher after SC injection than during continuous IV infusion for about five hours. The mean half-life was 2.1 hours. Judging from the intracellular concentration of ara-CTP, considered to be the active metabolite, the results suggest that SC administration of ara-C can mimic continuous IV infusion if the dose interval is reduced from the traditional 12 hours to four to six hours. The results of this study emphasize the differences between the plasma pharmacokinetics of the parent drug and the concentration of the active metabolite in leukemic cells.