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Chemically modified oligonucleotides are routinely used as diagnostic and therapeutic agents due to their enhanced biological stability relative to natural DNA and RNA. Here, we examine the biological stability of α-l-threofuranosyl nucleic acid (TNA), an artificial genetic polymer composed of repeating units of α-l-threofuranosyl sugars linked by 2',3'-phosphodiester bonds. We show that TNA remains undigested after 7days of incubation in the presence of either 50% human serum or human liver microsomes and is stable against snake venom phosphordiesterase (a highly active 3' exonuclease). We further show that TNA will protect internal DNA residues from nuclease digestion and shield complementary RNA strands from RNA degrading enzymes. Together, these results demonstrate that TNA is an RNA analogue with high biological stability., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cytotoxic nucleosides have proven to be ineffective for the treatment of hepatocellular carcinoma (HCC) due, in part, to their inadequate conversion to their active nucleoside triphosphates (NTP) in the liver tumor and high conversion in other tissues. These characteristics lead to poor efficacy, high toxicity, and a drug class associated with an unacceptable therapeutic index. Cyclic 1-aryl-1,3-propanyl phosphate prodrugs selectively release the monophosphate of a nucleoside (NMP) into CYP3A4-expressing cells, such as hepatocytes, while leaving the prodrug intact in plasma and extrahepatic tissues. This prodrug strategy was applied to the monophosphate of the well-known cytotoxic nucleoside cytosine-1-beta-D-arabinofuranoside (cytarabine, araC). Compound 19S (MB07133), in mice, achieves good liver targeting compared to araC, generating >19-fold higher cytarabine triphosphate (araCTP) levels in the liver than levels of araC in the plasma and >12-fold higher araCTP levels in the liver than in the bone marrow, representing a >120-fold and >28-fold improvement, respectively, over araC administration.

Cytosine arabinoside (AraC) is rapidly inactivated via systemic deamination with half-lives ranging from 1 h (i.v.) to 4 h (s.c.) -- and cannot be applied orally due to its hydrophilic properties. These limitations might be overcome by YNK01 -- a lipophilic prodrug of AraC -- that is resistant to deoxycytidine deaminase and can be applied orally. In the present study the therapeutic activity, side-effects and pharmacokinetics of YNK01 were evaluated in a phase I/II study including patients with relapsed or refractory acute myeloid leukemia (AML) (n=23) or low-grade non-Hodgkin's lymphoma (NHL) (n=20). YNK01 was given by 14 day cycles with escalating doses starting with a daily dose of 50 mg/m2 (equivalent to 20 mg/m2 AraC on a molar basis). The maximum tolerated dose was reached at the 600 mg/m2 dose level with WHO grade 3-4 diarrhoea as the main toxicity. In the 23 patients with AML two complete remissions, four partial remissions and three patients with stable disease were observed. In the 23 patients with AML two complete remissions, four partial remissions and three patients with NHL two cases reached partial remission and six other patients mainained stable disease. Pharmacokinetic evaluations were performed during 34 treatment cycles in 28 patients. The data suggest that YNK01 was absorbed in the distal part of the small intestine and taken up into hepatocytes. After hepatic psi and subsequent beta-oxydation of YNK01 the released AraC (and its deamination product AraU) appeared in the systemic circulation. Time of maximum concentration (h), half-life (h) and area under the curve (ng x h/ml, at the 1200 mg dose level) were as follows (VC variation coefficient) YNK01: 1.0 (0.58), 10.1 (0.43), 12622 (0.65); AraC: 23.2 (0.57), 22.6 (0.36), 3496 (0.76); AraU: 19.2 (0.58) 22.3 (0.33) 15441 (0.66). Of the total dose of YNK01 15.8% was absorbed and metabolized to AraC and AraU, defining the metabolic bioavailability of this prodrug. A linear relationship was observed between YNK01 dose and YNK01 AUC and AraC AUC over the whole dose range tested. AraC was released from hepatocytes over a prolonged period of time resulting in long lasting plasma levels similar to a continuous i.v. infusion. After administration of YNK01 at a dosage of 100-150 mg/m2 plasma levels of AraC were comparable to those achieved after low-dose AraC treatment (20 mg/m2) while at doses of YNK01 of 450-600 mg/m2 concentrations of standard-dose AraC (100 mg/m2) were obtained. We conclude that YNK01 shows considerable activity against relapsed and refractory AML and NHL and that its pharmacokinetic properties offers advantages in comparison to (standard) i.v. or s.c. AraC in clinical practice.

Cytarabune ocfosfate (SPAC) is rapidly transformed into cytarabine (ara-C) when orally administered. The pharmacokinetics of SPAC was studied in six patients with acute non-lymphocytic leukemia (ANLL) and/or myelodysplastic syndromes (MDS) after oral administration of SPAC at 100 to 400 mg/day for 14 days. Plasma ara-C concentrations reached a plateau in 48 to 96 hours after initiation of SPAC administration, remained at this or a little higher level until one day after its termination and were less than 1 ng/ml 8 days after the termination. From all of pharmacokinetic data, the oral administration of SPAC at 150 to 300 mg/m2/day was pharmacokinetically concluded to be comparable to the continuous infusion of ara-C at 20 mg/m2/day. All of the patients could receive SPAC for 14 days. SPAC is considered to be useful for consolidation or maintenance chemotherapy of ANLL or MDS outpatients who are unable to undergo intensive chemotherapy.

Purine nucleoside phosphorylase deficiency leads to a dGTP-mediated T-lymphopenia, suggesting that an analogue of deoxyguanosine would be selectively effective in T-cell disease. 9-beta-D-Arabinofuranosylguanine (ara-G) is relatively resistant to hydrolysis by purine nucleoside phosphorylase and selectively toxic to T cells, but its low solubility has prevented its use in the clinic. 2-Amino-6-methoxy-arabinofuranosylpurine (GW506U) serves as the water-soluble prodrug for ara-G. A Phase I trial in patients with refractory hematological malignancies demonstrated that the clinical responses to this agent were directly related to the peak levels of ara-G 5'-triphosphate (ara-GTP) in target cells. The aim of the present study was to develop and test strategies to increase intracellular accumulation of ara-GTP in primary human leukemia cells of myeloid and B-lymphoid origin. Three strategies were tested. First, incubations with 100 microM ara-G for 4 h produced a linear median accumulation rate of 19 microM/h (range, 2-45 microM/h; n = 15) in lymphoid leukemia cells and 16 microM/h (range, 0.5-41 microM/h; n = 11) in myeloid leukemia cells. Saturation of ara-GTP accumulation was achieved only after 6-8 h exposure in both lymphoid and myeloid leukemia cells, suggesting a rationale for prolonged infusion. Second, a dose-dependent increase in ara-GTP accumulation was observed with incubations of 10-300 microM ara-G for 3 h. Hence, dosing regimens that achieve high plasma levels of ara-G during therapy may increase cellular levels of ara-GTP. Finally, a biochemical modulation approach using in vitro incubation of leukemia cells with 10 microM 9-beta-D-arabinofuranosyl-2-fluoroadenine for 3 h, followed by either 50 or 100 microM ara-G for 4 h, resulted in a statistically significant median 1.3-fold (range, 1.1-9.0-fold; P = 0.034) and 1. 8-fold (range, 0.9-10.6 fold; P = 0.018) increase in ara-GTP compared to cells incubated with ara-G alone. Extension of these studies to ex vivo incubations confirmed our in vitro findings. These strategies will be used in the design of clinical protocols to increase ara-GTP accumulation in leukemia cells during therapy.

1-beta-d-Arabinofuranosylcytosine (ara-C), an effective drug for acute leukemias, must be phosphorylated to its 5'-triphosphate, ara-CTP, for activity. Our previous studies during therapy of acute myelogenous leukemia (AML) patients demonstrated that the accumulation of ara-CTP in circulating leukemia blasts was increased by a median of 2-fold when fludarabine (30 mg/m2/day over 30 min) was infused 4 h prior to intermediate dose ara-C. The augmentation was dependent on the cellular concentration of fludarabine triphosphate (F-ara-ATP). To determine the lowest dose of fludarabine needed for modulation of ara-C metabolism, the present study administered fludarabine at a test dose (15 mg/m2 over 30 min) followed by 2 g/m2 ara-C infused over 4 h. The next day, the fludarabine/ara-C couplet was repeated but with a standard dose (30 mg/m2) of fludarabine. There was a dose-dependent accumulation of F-ara-ATP in circulating leukemia blasts; the median peak concentrations were 33 and 41 microM with 15 and 30 mg/m2 of fludarabine, respectively. These intracellular levels of F-ara-ATP effectively increased ara-CTP accumulation to similar levels. To further titrate the dose of fludarabine, the next cohort of patients (n = 4) initially received fludarabine test doses of 7.5 or 5 mg/m2, followed by the 30 mg/m2 dose of fludarabine on the next day; each dose was infused 4 h prior to 2 g/m2 of ara-C. The peak levels of F-ara-ATP at 7.5 and 5 mg/m2 fludarabine were between 3 and 39 microM. The AML blasts that achieved >/=10 microM intracellular F-ara-ATP accumulated ara-CTP similar to the levels achieved after 30 mg/m2 of fludarabine. However, <10 microM intracellular F-ara-ATP resulted in less ara-CTP accumulation compared to that observed after the conventional dose of fludarabine. These data suggest that the modulation of the ara-CTP accumulation by fludarabine is dependent on the cellular concentration of F-ara-ATP, and that 15 mg/m2 fludarabine infused over 30 min consistently produces cellular F-ara-ATP levels that maximize ara-CTP accumulation in AML blasts. These findings point to the feasibility of intensifying the fludarabine-ara-C regimen by using fludarabine as a 15 mg/m2/dose twice daily with intermediate-dose ara-C.

Fludarabine and 1-beta-D-arabinofuranosylcytosine (ara-C) are effective nucleoside analogues for the treatment of leukemias when used as single agents or together. Recent trials of the fludarabine and ara-C therapy with or without growth factors suggested an improved clinical response by combining fludarabine and ara-C. The activity of these antimetabolites depends on their phosphorylation to the respective triphosphates, F-ara-ATP and ara-CTP. The principal mechanism through which these triphosphates cause cytotoxicity is incorporation into DNA and inhibition of further DNA synthesis. A model system of DNA primer extension on a defined template sequence was used to quantitate the consequences of incorporation of one or two analogues by human DNA polymerase alpha (pol alpha). The template (31-mer) was designed so that DNA pol alpha incorporated six deoxynucleotides (alternately G and T) on the 17-mer primer, followed by insertion of an A and then a C. The primer was then elongated with G and T to the full-length product. The apparent Kms of DNA pol alpha to incorporate these analogues (0. 053 and 0.077 microM, respectively) were similar to the Km for dCTP (0.037 microM) and dATP (0.044 microM), suggesting that the enzyme recognized these analogues and incorporated them efficiently on the growing DNA primer. The velocity of extension (Vmax) of these primers ranged between 0.53 and 0.77%/min when normal nucleotides were present. Once inserted at the 3'-terminus, F-ara-AMP or ara-CMP were poor substrates for extension. However, in reactions lacking dCTP and dATP and with high concentrations of ara-CTP, ara-CMP was inserted by pol alpha after incorporation of the F-ara-AMP residue. This tandem incorporation of the two analogues resulted in almost complete inhibition (99.3%) of further extension of the primer. In the presence of competing deoxynucleotides, each analogue resulted in a dose-dependent inhibition of DNA synthesis. When present together, inhibition of the primer elongation was more than additive at low concentrations of analogue triphosphates. Based on these results and the intracellular pharmacokinetics of ara-CTP and F-ara-ATP in leukemia blasts, we propose a pharmacodynamic model to explain interactions between these analogues during combination chemotherapy.

Cytarabine ocfosfate (YNK01) is a novel orally applicable prodrug of cytosine arabinoside. Recent pharmacokinetic studies have revealed a prolonged release of the cytotoxic agent cytosine arabinoside (araC) from hepatocytes into the systemic circulation, resulting in a half-life of approximately 24 h for araC. The specific pharmacokinetic characteristics of cytarabine ocfosfate lead to a prolonged exposure of leukemic cells to this antineoplasstic agent during the 14-day cycle. the oral applicability during outpatient treatment and the sustained antineoplastic activity of araC against slowly proliferating leukemic B-cells suggest that cytarabine ocfosfate might be a useful drug in the treatment of chronic lymphocytic leukemia. Four years after diagnosis of B-CLL, a 50-year-old patient was started on cytarabine ocfosfate. Sequentially, the patient's disease had proved refractory to treatment with chlorambucil/prednisone (31 months), fludarabine (5 months), and prednimustine/mitoxantrone (3 months). These established regimens were discontinued because of increasing lymphocytosis, significant thrombocytopenia, and progressive B-symptoms. Following three cycles of cytarabine ocfosfate B-symptoms resolved, lymphadenopathy disappeared, and thrombocytopenia was significantly reduced. The patient has been free of these symptoms on a dosage of 1500 mg cytarabine ocfosfate/day (cycle of 14 days with intervals of 14-21 days) for 24 months and remains in an ongoing partial remission.

Ara-CMP-Stearate (1-beta-D-arabinofuranosylcytosine-5'-stearylphosphate, YNK 01, Fosteabine) is the orally applicable prodrug of cytosine-arabinoside (Ara-C). During a phase I study in patients with advanced low-grade non-Hodgkin lymphomas or acute myeloid leukemia, the pharmacokinetic parameters of Ara-CMP-Stearate (kindly provided by ASTA Medica, Frankfurt, Germany) were determined by HPLC analysis. Seventy-two hours after a first starting dose which served for the determination of baseline pharmacokinetic parameters, Ara-CMP-Stearate was administered over 14 days by daily oral application. Ara-CMP-Stearate was started at a dose of 100 mg/day and was escalated in subsequent patients to 200 mg/day and 300 mg/day. Plasma and urine concentrations of Ara-CMP-Stearate, Ara-C and Ara-U were measured during the initial treatment phase and within 72 h after the end of the 14-day treatment cycle. So far six patients have been treated with 100 mg/day, three with 200 mg/day and another six with 300 mg/day. One patient was treated consecutively with 100 mg, 300 mg and 600 mg. Fitting the results of the plasma concentration measurements of Ara-CMP-Stearate to a one-compartment model, the following pharmacokinetic parameters were obtained (average and variation coefficient VC). Ara-CMP-Stearate dose-independent parameters: lag time = 1.04 h (0.57); tmax = 5.72 h (0.30); t1/2 = 9.4 h (0.36). Dose-dependent parameters: at 100 mg: AUC = 1099 ng/h/ml (0.31); concentration(max) = 53.8 ng/ml (0.28); at 200 mg: AUC = 2753 ng/h/ml (0.32); concentration(max) = 154.8 ng/ml (0.46); at 300 mg: AUC = 2940 ng/h/ml (0.66); concentration(max) = 160.0 ng/ml (0.59). The long lag time and late tmax can be explained by resorption in the distal part of the small intestine. No Ara-CMP-Stearate was detected in urine samples (limit of detection = 500 pg/ml). Pharmacokinetic parameters of Ara-C following Ara-CMP-Stearate application showed the following characteristics: t1/2 = 24.3 h (0.39); AUC (100 mg) = 262 ng/h/ml (0.93); AUC (200 mg) = 502 ng/h/ml (0.87); AUC (300 mg) = 898 ng/h/ml (1.07). Since Ara-CMP-Stearate causes intravascular hemolysis after intravenous administration, it was not possible to determine its bioavailability by comparing the AUC after oral and i.v. application. Instead, the renal elimination of Ara-U, as the main metabolite of Ara-C was measured during the first 72-h period and after the last application.(ABSTRACT TRUNCATED AT 400 WORDS)

Plasma concentration of cytosine arabinoside (Ara-C) was determined in elderly patients with myelodysplastic syndromes or acute myelocytic leukemia who were treated with subcutaneous injection of Ara-C (Ara-C s.c.; 10 mg/m2/12 hr, 14-21 days), continuous drip infusion of Ara-C (Ara-C d.i.v.; 20 mg/m2/day, 24 hr 14 days) and/or oral administration of cytarabine ocfosfate (SPAC) (SPAC p.o.; 100 mg-300 mg/body/day, 14 days) by radioimmunoassay. In the Ara-C s.c. patients, the peak plasma level (Cmax) of Ara-C was 103 ng/ml and the time to reach Cmax was 15 min. The elimination half-like (t1/2) was 25 min and no accumulation was detected after 14 days of consecutive Ara-C s.c. administrations. In the SPAC p.o. patients, Cmax of Ara-C was 3-8 ng/ml and it took 3-5 days to reach Cmax. The plasma concentration level of Ara-C remains almost at the Cmax level during the SPAC p.o. administration and it remained higher than 0.32 ng/ml for as long as 15 days after the end of administration. In a Ara-C d.i.v. patient, plasma level of Ara-C was detected 4-7 ng/ml during the administration (day 7 through day 14). In all patients bone marrow suppression was observed after chemotherapy regardless of regimen, and there was no significant difference between nadir peripheral cell blood counts of Ara-C s.c. patients and SPAC p.o. patients.

1-beta-D-Arabinofuranosylcytosine-5'-stearylphosphate (cytarabine ocfosfate, stearyl-ara-CMP) is a newly synthesized 5'-alkylphosphate derivative of 1-beta-D-arabinofuranosylcytosine (ara-C), which is lipophilic, resistant to inactivation by deamination, and orally active. Pharmacology of this drug was studied in patients with hematological malignancies. The concentrations of stearyl-ara-CMP, ara-C (its active metabolite), and 1-beta-D-arabinofuranosyluracil (ara-U, its inactive metabolite) were determined by radioimmunoassay. When six patients received a single p.o. dose of the drug (500 mg/m2), stearyl-ara-CMP, ara-C, and ara-U could be detected in the plasma for at least 72 h afterwards. The plasma disappearance curve of stearyl-ara-CMP corresponded to a one-compartment open model with first-order absorption kinetics. The peak plasma level (Cmax) was 322 +/- 218 nM, and the predicted time to reach Cmax (Tmax) was 6.5 +/- 4.5 h, while the elimination half-life (t1/2) was very long (32.0 +/- 8.4 h). The plasma ara-C level increased slowly to a Cmax of 26.3 +/- 12.7 nM (Tmax, 13.3 +/- 4.7 h) after stearyl-ara-CMP administration. This level was quite low compared with that achieved by low-dose s.c. ara-C therapy, but ara-C persisted longer in the plasma in the former case, and the area under the curve was similar for both regimens. For ara-U, the Cmax, Tmax, and t1/2 were 483 +/- 315 nM, 23.6 +/- 4.0 h, and 19.6 +/- 5.3 h, respectively. No stearyl-ara-CMP was detected in the urine, and only 8.0% of the administered dose was excreted as ara-C and ara-U within 72 h. The stearyl-ara-CMP concentration in the cerebrospinal fluid was below the limit of detection in three patients without meningeal involvement at 6 h. During clinical use of stearyl-ara-CMP, macrocytic anemia was observed, and some patients also developed megaloblastic change of their erythroblasts, suggesting a mild and persistent cytostatic effect. In conclusion, p.o. therapy with stearyl-ara-CMP achieved prolonged maintenance of the plasma drug level. Thus, the drug released a very low dose of ara-C over a long period in plasma and tissues and had a prolonged mild antineoplastic effect in patients with hematological malignancies.

Cytarabine ocfosfate (commercial name: Starasid) is a prodrug having stearyl group attached to phosphoric acid at 5' position of arabinose moiety of cytosine arabinoside (Ara-C). This drug is given orally. The mode of action is in the inhibition of DNA synthesis after conversion to Ara-CTP as in Ara-C. The drug is metabolized in the liver, producing the intermediate metabolite, C-C3PCA which is converted to Ara-C gradually. This property results in the maintenance of relatively long time the blood Ara-C levels. This was proved to be active clinically against acute leukemia and MDS.

The pharmacology of fludarabine triphosphate (F-ara-ATP) in leukemic lymphocytes was studied during a phase II trial of fludarabine in 24 patients with chronic lymphocytic leukemia (CLL). Fludarabine was given as a 30-min i.v. infusion at a dose of 25 or 30 mg/m2 daily for 5 days. The concentrations of F-ara-ATP, the active metabolite of fludarabine, were determined in leukemic lymphocytes at intervals up to 24 hr after the first infusion. A median peak concentration of 19 microM (range, 6-52 microM) was generally reached 4 hr after the beginning of the infusion. No significant relationship was observed between clinical response and the median peak level of F-ara-ATP or the retention of F-ara-ATP in leukemic lymphocytes. In vitro incubation of CLL cells with the parent nucleoside of fludarabine, arabinosyl-2-fluoroadenine (F-ara-A), indicated that F-ara-ATP accumulated in a linear fashion in response to the product of the F-ara-A concentration times the duration of incubation. Exposing cells longer with lower F-ara-A concentrations or shorter with higher F-ara-A concentrations resulted in similar intracellular levels of F-ara-ATP as long as the products of fludarabine concentration and time of exposure were equal. These results and the fact that the fludarabine dose rate currently administered is well tolerated suggest that it may be the optimal dose rate for F-ara-ATP accumulation in CLL cells.

The active 5'-triphosphate of arabinosyl-2-fluoroadenine (F-ara-ATP) increases the anabolism of arabinosylcytosine (ara-C), whereas ara-C 5'-triphosphate inhibits the phosphorylation of arabinosyl-2-fluoroadenine (F-ara-A) in human leukemia cells in vitro. These interactions have a potential impact on drug scheduling. Clinical trials of relapsed leukemia in which fludarabine (F-ara-A 5'-monophosphate) and ara-C were given in sequence provided the opportunity to evaluate the effects of ara-C infusion on two sequelae: the pharmacokinetics of F-ara-A in plasma and that of F-ara-ATP in leukemia cells. First, F-ara-A pharmacokinetics were altered by ara-C infusion. This was visualized as a transient increase in F-ara-A plasma levels during the ara-C infusion that was given 4 h after fludarabine. The perturbation in F-ara-A plasma levels was dependent on the dose ara-C. Second, peak F-ara-ATP concentrations were lower in leukemia cells of patients who received ara-C in addition to fludarabine as compared with those who received fludarabine alone. The terminal half-life of F-ara-A in plasma and the half-life of intracellular F-ara-ATP were reduced after the ara-C infusion in a concentration-dependent manner. Studies using purified deoxycytidine kinase support the conclusion that the increase in plasma levels of F-ara-A is in part the result of an effective competition by ara-C for phosphorylation by this enzyme, leading to a perturbation of the pharmacokinetics of intracellular F-ara-ATP.

Exponentially growing K562 cells incubated with 1-beta-D-arabinofuranosylcytosine (ara-C) accumulate ara-C triphosphate (ara-CTP) at a higher rate and to a greater concentration after pretreatment with 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) than do cells treated with ara-C alone. Potentiation of ara-C metabolism is due in part to an indirect effect of F-ara-A triphosphate (F-ara-ATP)-mediated reduction in deoxynucleotide pools and consequent activation of deoxycytidine kinase. Because the levels of deoxynucleotide pools and the activity of deoxycytidine kinase are cell cycle-specific, we investigated the effect of cell cycle phases on the accumulation of ara-CTP and the influence of F-ara-A pretreatment on such accumulation. Exponentially growing K562 cells were fractionated into G1, S, and G2+M phase-enriched subpopulations (each enriched by > 60%) by centrifugal elutriation. The rate of ara-CTP accumulation was 22, 25, and 14 microM/h and the rate of F-ara-ATP accumulation was 38, 47, and 33 microM/h in the G1, S, and G2+M subpopulations, respectively. The rate of elimination of arabinosyl triphosphates was similar among the different phases of the cell cycle. After pretreatment with F-ara-A, the rate of ara-CTP accumulation in the G1, S, and G2+M phase-enriched subpopulations was 43, 37, and 26 microM/h, indicating a 1.7-, 1.5-, and 1.9-fold increase, respectively. These results suggest that a combination of F-ara-A and ara-C may effectively potentiate ara-CTP accumulation in all phases of the cell cycle. This observation is consistent with the results of studies on the modulation of ara-C metabolism by F-ara-A in lymphocytes and leukemia blasts obtained from patients with chronic lymphocytic leukemia and acute myelogenous leukemia, respectively.

YNK01 (a daily oral dose of 450 mg) was administered for 22 days to a 58-year-old-female with Ph1-positive acute unclassified leukemia. Leukemia cells were negative for peroxidase and esterase, but were positive for CD19, CD13, CD34, CD9, HAL-DR, CD25, and TdT. Complete remission was obtained and continued for at least a month. The main side effects noted were diarrhea and melena. The administration of YNK01 resulted in plasma ara C levels that ranged between 15.4 to 23.0 ng/ml, which appear to be nearly equivalent to dose achieved during continuous IV infusion of a low dose (20 mg/m2/day) of ara C.

We evaluated relationships between ara-CTP pharmacokinetics in myeloblasts, response, and karyotype in 147 patients with newly diagnosed AML treated on three protocols each initiated by a 3 g/m2 ara-C dose given over 2 h. Area under the curve of ara-CTP concentration times time (AUC) or peak ara-CTP concentration after this dose did not predict response or response duration, suggesting that inability to form ara-CTP is an unlikely mechanism of ara-C resistance. Following high-dose bolus ara-C therapy patients with INV [16] or del [16q] had long remissions despite low AUC and peak values while patients with -5, del [5q], -7, or del [7q] were frequently resistant despite average AUC and peak values. In 55 patients treated with high-dose continuous infusion ara-C as a single agent, steady-state ara-CTP concentrations were significantly lower in resistant patients (who again were disproportionately those with -5, del [5q], -7, or del [7q]) although there was no correlation with CR duration.

Accumulation of cytosine arabinoside triphosphate (araCTP) from a range of cytosine arabinoside (araC) concentrations (1-50 microM) was measured during incubations of leukemic cells freshly isolated from patients with acute leukemia. In all but one patient, increments in extracellular araC above 10 microM did not increase intracellular araCTP levels. This maximal level of araCTP accumulation ranged from 254 to 1607 pmol/10(7) cells attained after 1 h incubation and did not correlate with either the number of nucleoside transporters on the cell membrane or the Vmax of araC phosphorylation in cell free extracts. Extremely low araCTP accumulation (103 pmol/10(7) cells/h at 50 microM araC) was observed in an AML patient with the unusual finding of micromyeloblasts. These cells also had very low numbers of nucleoside transport sites (less than 500 sites/cell) and were mitotically inactive. The unique feature of the myeloblasts from this patient was that intracellular araCTP accumulation showed a linear dependence on extracellular araC up to 50 microM with no evidence of saturation.

The cellular metabolism of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'monophosphate (F-araAMP), a soluble nucleoside analog with proven antileukemic activity in animal and human tumors, has been studied in mice bearing P388 leukemia. Earlier studies showed markedly less in vivo accumulation of F-ara ATP the principal active metabolite, in gastrointestinal mucosa (GI) and bone marrow (BM) compared with P388 after F-araA or F-araAMP administration. To elucidate the mechanism of toxicity this work has examined the pharmacodynamics of F-araAMP anabolites, F-araATP and F-ATP, in P388 cells, BM and GI mucosa tissues after nontoxic (LD1) and toxic (LD50) doses of F-araAMP. F-araATP was the major triphosphate metabolite in acid-soluble extracts from P388 cells, BM, and GI mucosa tissues. F-araATP accumulated to approximately 1 mM in P388 cells after either LD1 or LD50 treatment of F-araAMP and was eliminated with a t1/2,el of approximately 5 h. The ratio of the area under the concentration-time curve (AUC 0----infinity) of F-araATP was 1.01 after the LD50 over the LD1 doses of F-araAMP. BM and GI mucosa tissues accumulated 40-fold less F-araATP than the concentration in P388 cells. 2-Fluoro-ATP, a second toxic anabolite, accumulated in P388 cells to 156 +/- 39 microM and 447 +/- 79 microM after the two doses of the drug, respectively. The ratio of area under the curve (AUC) of F-ATP in P388 cells after the two doses of F-araAMP was 38.77, which approaches the ratio of % lethality (LD50/LD1 = 50). F-ATP was also quantitated in BM and GI mucosa reaching one-fifth to one-half the concentration of F-araATP after the LD50 dose of F-araAMP. The AUC values of F-ATP (0----24 h) were 9.5- to 12.5-fold higher after the LD50 than after the LD1 dose of F-araAMP. These results suggest that there is a selective therapeutic action of F-araAMP against P388 and that the increased cellular concentration of F-ATP in both the tumor cells and the host tissues (BM and GI mucosa) could explain the mode of toxicity of F-araAMP.

The antitumor activity of 1-beta-D-arabinofuranosylcytosine-5'-alkylphosphates (CnPCAs) against L1210 leukemia in mice after oral administration was demonstrated. The optimum length of the alkyl group on the phosphate moiety of CnPCA for exhibiting a high antitumor activity was found to be between tetradecyl (C14) and tricosyl (C23). The most active alkyl derivative in this system was found to be 1-beta-D-arabinofuranosylcytosine-5'-stearylphosphate (C18PCA). The optimum and minimum effective doses of C18PCA were 100 and 6.25 mg/kg/day (q1d, day 1 to day 5), respectively. The maximum T/C% of C18PCA was approximately 220. The antitumor activity of C18PCA was not greatly dependent on the treatment schedule and route. Plasma concentration of 1-beta-D-arabinofuranosylcytosine (ara-C) remained in the range of 0.4 to 0.75 nmol/ml [corrected] for 24 h after oral administration of 100 mg/kg (170 mumol/kg) of C18PCA. These results indicate that C18PCA administered per orally is absorbed intact through the gastrointestinal tract and area-C is released of long period of time. C18PCA is regarded as an orally active depot form of ara-C.

The pharmacokinetics of 1-beta-D-arabinofuranosylcytosine (ara-C) and 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) and their respective 5'-triphosphates, ara-CTP and F-ara-ATP, were compared in human plasma and circulating leukemic blasts (CLB) since initial phosphorylation of ara-C and F-ara-A is catalyzed by the same enzyme, deoxycytidine kinase. These investigations were conducted in 4 patients after the first infusion of high-dose ara-C therapy (3 g/m2 i.v. infused over 2 hr) and, following the failure of each to respond, after the initial bolus of F-ara-A monophosphate (50-100 mg/m2 i.v. over 30 min) in a subsequent treatment regimen. The median terminal rate of elimination (t1/2) of F-ara-A was 8.4 hr compared to 2.2 hr for ara-C. The median t1/2 for F-ara-ATP in CLB was 12.2 hr relative to 1.9 hr for ara-CTP. To evaluate the possibility that diminished deoxycytidine kinase was a mechanism of drug resistance, the relative area under the concentration X time curve (AUC) of the active triphosphate of each prodrug in the CLB of individuals was compared. The intracellular nucleotide AUC was normalized by dividing it by the AUC of the respective nucleoside in plasma. A value of 1.0 for the resulting ratio would be expected if the accumulation and retention of F-ara-ATP and ara-CTP were identical. In these patients, however, this ratio ranged between 0.2 and 68.2. When a similar analysis was performed in vitro using four established human leukemia cell lines, a 150-fold variation was found in the normalized nucleotide AUC ratio. Thus, the metabolic characteristics of ara-CTP in CLB of patients who fail to respond to high-dose ara-C may not predict the cellular metabolism of F-ara-ATP in the same patient at a later disease stage.