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BACKGROUND The objectives of this study were to assess the incidence of clinical allergy and end-induction antiasparaginase (anti-ASNase) antibodies in children with high-risk acute lymphoblastic leukemia treated with pegylated (PEG) Escherichia coli ASNase and to determine whether they carry any prognostic significance. METHODS Of 2057 eligible patients, 1155 were allocated to augmented arms in which PEG ASNase replaced native ASNase postinduction. Erwinia chrysanthemi (Erwinia) ASNase could be used to replace native ASNase after allergy, if available. Allergy and survival data were complete for 990 patients. End-induction antibody titers were available for 600 patients. RESULTS During the consolidation phase, 289 of 990 patients (29.2%) had an allergic reaction. There were fewer allergic reactions to Erwinia ASNase than to native ASNase (odds ratio, 4.33; P

L-Asparaginase (ASNase) is a front-line chemotherapy for acute lymphoblastic leukemia (ALL), which acts by deaminating asparagine and glutamine. To evaluate the importance of glutaminase activity, we exploited a recently developed mutant of Helicobacter pylori ASNase (dm HpA), with amino acid substitutions M121C/T169M. The mutant form has the same asparaginase activity as wild-type but lacks glutaminase activity. Wild-type and dm HpA were compared with the clinically used ASNases from Escherichia coli (l-ASP) and Erwinia chrysanthemi (ERWase). Asparaginase activity was similar for all isoforms, while glutaminase activity followed the rank order: ERWase>l-ASP>wild-type HpA>dm HpA. Cytotoxic efficacy of ASNases was tested on 11 human leukemia cell lines and two patient-derived ALL samples. Two cell lines which we had previously shown to be asparagine-dependent were equally sensitive to the asparaginase isoforms. The other nine lines and the two patient-derived samples were more sensitive to isoforms with higher glutaminase activities. ERWase was overall the most effective ASNase on all cell lines tested whereas dm HpA, having the lowest glutaminase activity, was the least effective. These data demonstrate that asparaginase activity alone may not be sufficient for ASNase cytotoxicity, and that glutaminase activity may be required for full anti-leukemic efficacy.

Obesity is associated with increased cancer incidence and mortality. We have previously found that obesity in children is associated with a 50% increased recurrence of acute lymphoblastic leukemia (ALL) in high-risk patients. We have therefore developed novel in vivo and in vitro preclinical models to study the mechanism(s) of this association. Obesity increased relapse after monotherapy with vincristine (P = 0.03) in obese mice injected with syngeneic ALL cells. This occurred although the drug was dosed proportionally to body weight, equalizing blood and tissue drug levels. In coculture, 3T3-L1 adipocytes significantly impaired the antileukemia efficacy of vincristine, as well as three other chemotherapies (P < 0.05). Interestingly, this protection was independent of cell-cell contact, and it extended to human leukemia cell lines as well. Adipocytes prevented chemotherapy-induced apoptosis, and this was associated with increased expression of the two prosurvival signals Bcl-2 and Pim-2. These findings highlight the role of the adipocyte in fostering leukemia chemotherapy resistance, and may help explain the increased leukemia relapse rate in obese children and adults. Given the growing prevalence of obesity worldwide, these effects are likely to have increasing importance to cancer treatment. [Cancer Res 2009;69(19):7867–74]

In the past 25 years, effective new drugs along with better treatment decisions based on disease factors have resulted in significantly improved clinical outcomes in acute lymphoblastic leukemia. Despite these successes in the last 2 decades, 15% to 25% of acute lymphoblastic leukemia patients relapse. Therefore, better dosing therapies are still needed. Insights in the pharmacokinetic and pharmacodynamic (PK-PD) contributions of licensed drugs may guide us into better protocol design and optimal use of existing combination drug regimens. Currently, 3 asparaginase formulations are available in the United States, Escherichia coli native asparaginase (ASNase), Pegaspargase, and Erwinase. On the basis of these clinical studies, PK and PD population modeling (NONMEM) have been used to delve into new insights as to the optimal dose, formulation, and time intervals of ASNases that may be used in future clinical trials. Pegaspargase 2500 IU/m2 Q2week dosing seems to be the "golden standard" as far as being safe and effective. Lower doses of this formulation Qweek may achieve better PK "steady-state" profiles in serum. Native E. coli or Erwinia ASNase at 6000 IU/m2 showed inferior PK parameters (peak, trough, and area under the curve) than Pegaspargase. Assuming linear handling of ASNase modeling, simulations of higher doses of these ASNase formulations on a daily or Q48 hours regimen are showing bioequivalency with Pegaspargase PK-PD parameters. Future clinical trial designs may prove these efforts useful.

Purpose: Many molecular pathways, including cell cycle control, angiogenesis, and drug resistance, mediate tumor growth and survival. Vascular endothelial growth factor-A (VEGF-A) serum levels 100 pg/mL have been associated with good and poor prognoses, respectively. Experimental Design: The hypothesis was that serum VEGF-A levels in standard-risk acute lymphoblastic leukemia pediatric patients at induction are predictive of event-free survival (EFS). One hundred seventeen patients were entered in CCG-1962 study and randomized into the native and polyethylene glycolated asparaginase arms. VEGF-A levels were quantified by an ELISA assay. Results: All patients had a decrease in VEGF-A levels by day 14 of induction, but they later dichotomized; EFS group levels remained low and event group levels increased. A correlation exists between high VEGF-A levels at entry to induction and time to event. Moreover, 6-year EFS patients have lower end of induction VEGF-A levels (28 ± 6 pg/mL) than event patients (>100 pg/mL; P < 0.01). Kaplan-Meier curves using various VEGF-A values were produced; with ≤30 at entry into induction (day 0) and ≤60 pg/mL at the end of induction (day 28), patients with low VEGF-A levels had superior EFS (P < 1e−4). Furthermore, patients who had an increase in VEGF-A during induction (ΔVEGF-positive, days 0-28) were more likely to have an event (P < 1e−4). Bifurcation by asparaginase treatment arm did not alter these results. Conclusions: These observations strongly support that high VEGF-A levels in induction are an asparaginase treatment–independent predictive marker for EFS. Hence, an anti-VEGF-A therapy should be tested in acute lymphoblastic leukemia.

In contrast to that in children, pharmacokinetic, pharmacodynamic, and safety information on pegaspargase in adults is very limited. We administered a single intravenous dose of pegaspargase (2000 IU/m2) as part of a standard frontline induction regimen to 25 adults with newly diagnosed acute lymphoblastic leukemia (ALL), and obtained serum samples on several time points. The population mean peak serum concentration of asparaginase enzymatic activity was 1 IU/mL, the elimination half-life was 7 days, and the volume of distribution was 2.43 L/m2. After the single dose, asparagine deamination was complete in all patients after 2 hours, and in 100%, 81%, and 44% on days 14, 21, and 28, respectively. A pharmocodynamic correlation model showed minimal enzymatic activity of 0.2 IU/mL for optimal asparagine depletion. The kinetic posthoc analyses demonstrated enzymatic activity for 3 weeks or more. One patient developed neutralizing antiasparaginase antibodies on day 22 after administration. Pegaspargase was well tolerated, with few grade 3/4 side effects. No allergic reactions or pancreatitis were observed. In adults aged 55 years or younger, pegaspargase produces a long duration of asparagine depletion and can be given intravenously, with a safety profile that is similar to equivalent multiple doses of intramuscular Escherichia coli asparaginase.

We investigated the anti-asparaginase antibody (Ab) and asparaginase enzymatic activity in the sera of 1,001 patients (CCG-1961) with high-risk acute lymphoblastic leukemia (HR-ALL). Patients received nine doses of native Escherichia coli asparaginase during induction. Half of rapid early r

Background Determination of the frequency of antibody formation during first and second exposure to Erwinia asparaginase after i.v. and i.m. administration. Procedure Thirty-nine children with newly diagnosed acute lymphoblastic leukemia (ALL) were included in this prospective study. Antibodies were determined (ELISA method) in plasma from these patients on specific days during and after therapy with 30,000 IU/m2 i.v. or i.m. every day for ten days during the induction phase (first exposure). For 19 children, antibodies were measured in plasma during and after the re-induction phase (second exposure) following treatment with 30,000 IU/m2 i.v. or i.m. twice a week for two weeks (Mondays and Thursdays). On the same days of therapy, enzyme activity (spectrophotometric method) and the concentration of asparagine (HPLC) was determined. Results During the first exposure, none of the patients developed anti-Erwinia asparaginase antibodies. During the second exposure, one patient (1 of 8 patients) treated intravenously developed antibodies, which were associated with disappearance of enzyme activity and reappearance of asparagine. Three of eleven patients developed antibodies of pharmacokinetic importance after i.m. therapy. None of the children had any clinical symptoms of hypersensitivity. Conclusions The formation of antibodies and subsequently altered pharmacokinetics of Erwinia asparaginase seemed to be of importance only during a second period of asparaginase therapy. Med. Pediatr. Oncol. 2002;38:310–316. © 2002 Wiley-Liss, Inc.

Purpose Asparaginase treatment is standard in all pediatric acute lymphoblastic leukemia (ALL) regimens, whereas in adults, it is either excluded or administered for a shorter duration. Several adult ALL protocols are adapting pediatric regimens, but the optimal implementation of asparaginase is not well studied, considering its potential higher toxicity. We studied a pegaspargase dosing strategy based on its pharmacokinetic characteristics in adults. Patients and Methods Between 2004 and 2009, 51 adults age 18 to 57 years with newly diagnosed ALL were treated with a regimen adapted from a pediatric trial that included six doses of intravenous pegaspargase at 2,000 IU/m2 per dose. Intervals between doses were longer than 4 weeks and rationally synchronized with other chemotherapy drugs to prevent overlapping toxicities. Pegaspargase was administered with steroids to reduce hypersensitivity. Asparaginase-related toxicities were monitored after 173 pegaspargase doses. Results The most common grade 3/4 asparaginase-related toxicities were lengthy hyperbilirubinemia and transaminitis, occasionally resulting in subsequent treatment delays. All toxicities resolved spontaneously. Forty-five percent of patients were able to receive all six doses of pegaspargase, and 61% received ≥ three doses. In only 20% of patients, the drug was discontinued after pegaspargase-related serious toxicity. Ninety-six percent achieved complete remission, almost all within 4 weeks, and a low induction death rate was seen. Seven-year disease-free and overall survival were 58% and 51%, respectively. Conclusion Our dose and schedule of pegaspargase, based on its pharmacokinetics, and our detailed toxicity profile could be applied for safer adaptation of pediatric ALL protocols in adults.

Combinations of nucleoside analog drugs such as 6-MP. ara-C. or F-araA are synergistic against human leukemic T-cells and induce apoptotic cell death. Addition of Taxotere or PEG-ASNase to the synergistic combination of nucleoside analog drugs augments the synergism several fold by enhancing cellular apoptosis.

The cure rate for children with acute lymphoblastic leukaemia (ALL) has increased to approximately 70%, in part related to the use of the protein synthesis inhibitor drug asparaginase in multiagent chemotherapy regimens. Its lack of haematological toxicity allows its incorporation into phases of therapy in which myelosuppression would be expected either from the disease itself (induction therapy) or secondary to other chemotherapeutic agents (consolidation, intensification or reinduction phases of therapy). Its antileukaemic effect is related to the degree and duration of asparagine depletion. The 2 native forms of L-asparaginase are derived from Escherichia coli and Ewinia chrysantherni. The half-lives (t½) of these forms are approximately 1.2 and 0.6 days, respectively. In order to increase the biological t½, pegaspargase was synthesised by the covalent attachment of monomethoxypolyethylene glycol (PEG) to native E. coli L-asparaginase: it has a t½, of approximately 5.7 days. The duration of asparagine depletion, the substrate amino acid of the drug, is directly related to asparaginase t½. Asparaginase is associated with several unique toxicities, including hyperglycaemia, hypolipoproteinaemia, hypoalbuminaemia, coagulation factor deficiencies, hepatotoxicity and pancreatitis. Since asparaginase is a protein, it may induce hypersensitivity reactions. The incidence of these reactions increases with use. In addition, silent hypersensitivity, i.e. the development of IgG antibodies without clinical reactions, results in a decreased t½ of asparaginase, shortened duration of asparagine depletion, and probably decreased efficacy. The use of pegaspargase allows continued treatment with asparaginase in patients with clinical hypersensitivity reactions. In addition, its use in patients with silent hypersensitivity may maintain the efficacy of asparaginase. So far, the optimal use of the 3 forms of asparaginase has not been determined in children with ALL, partly due to the lack of appropriate pharmacokinetic monitoring methods. As the technology has become available, it has been demonstrated that there is little rationale for the dosage and administration schedules presently in use. Studies are required to determine appropriate dosages and administration methods (intravenous or intramuscular) and schedules for each form of asparaginase, based upon pharmacokinetic parameters. The incidence and time to onset of hypersensitivity (clinical or silent) reactions and the appropriate means of continuing asparaginase therapy with therapeutic effect needs to be evaluated. Pharmacokinetic studies are now available as a research tool. These will allow further investigation to determine if failure to maintain asparagine depletion is a remediable cause of treatment failure.

A phase I clinical trial of 13-cis-retinoic acid (cis-RA) was undertaken to determine the maximally tolerated dose (MTD) and pharmacokinetics (PK) of cis-RA following bone marrow transplantation (BMT) in children with high-risk neuroblastoma. Mean peak serum levels of cis-RA in 31 pediatric patients ranged from 4.9 to 8.9 microM following doses of 100-200 mg/m2 per day, divided into two doses every 12 h administered orally. The PK of cis-RA obeyed a single-compartment model following first-order absorption in the majority of patients. A linear increase in the mean peak serum levels and area under the time-concentration curve (AUC) with increasing dose was observed. The average half-lives of absorption and elimination were 1.0 and 5.8 h, respectively. At the MTD of 160 mg/m2 per day, the mean cis-RA peak serum concentration was 7.2 +/- 5.3 microM. AUC values were not altered significantly during a 2-week course of treatment or over a long period of multiple courses. Levels of trans-retinoic acid, a metabolite of cis-RA, remained low but were similar on days 1 and 14, whereas the 4-oxo-13-cis-RA metabolite had increased in 64% of patients by day 14. Peak serum cis-RA concentrations correlated with clinical toxicity as grade 3 to 4 toxicity was seen in 44% of patient-courses (8/18) with peak serum levels10 microM, but only 13% (12/96) with peak serum levels10 microM. These results show that cis-RA given at 160 mg/m2 to children achieved serum concentrations known to be effective against neuroblastoma in vitro, and the PK for cis-RA differs from that reported for trans-retinoic acid in children.

We examined the population pharmacokinetics (PPK) of cytosine arabinoside (ara-C) after high-dose ara-C (HDara-C) (3 g/m2 every 12 h) and after a loading bolus (LB) plus continuous infusion (C1) of ara-C for 72 h in 52 pediatric patients with leukemias, enrolled in four clinical trials. The PPK analyses of the drug were performed using the NONMEM program. The patients' ages ranged from 2 months to 19 years. The ara-C data were analyzed using a two-compartment open model. Interindividual variability was described by the constant coefficient of variation (CCV) model, while the intraindividual variability was described by a combined additive and CCV error model. The covariates age (AGE) and surface area (SA) were tested to examine their influence on the estimation of the ara-C PPK parameters. In the absence of model covariates, the data fit was characterized by considerable bias, as indicated by the plot of measured vs predicted ara-C concentrations. The fit of the data was greatly improved when the parameters total body clearance (CL), intercompartmental clearance (Q), and volumes of distribution of central (Vd1) and peripheral (Vd2) compartments were expressed as linear functions of the covariate product, AGE x SA. The final parameter estimates were: CL = 2.59 x AGE x SA 1/h, Q = 2.01 x AGE x SA 1/h, Vd1 = 0.48 x AGE x SA1, and Vd2 = 38.1 x AGE x SA1. The coefficients of variation of CL, Q, Vd1 and Vd2 were 83.79%, 12.08%, 40.0%, and 52.54%, respectively, indicating substantial interindividual variability. In separate NONMEM analyses, the PK of ara-C and its metabolite uracil arabinoside (ara-U) were modeled simultaneously in order to investigate whether the dependence of ara-C on patient age was due to increased deamination of ara-C to ara-U. The PK of ara-C were described by the two-compartment open model while the PK of ara-U were simultaneously described by the one-compartment open model. The conversion of ara-C to ara-U was modeled as a first-order kinetic process due to the relatively low concentrations of ara-C in plasma. These PPK analyses indicated that elimination of ara-C from the central compartment occurs primarily by its metabolic conversion to ara-U and that the rate of conversion of ara-C to ara-U increases with increasing patient age, which explains the higher ratios of ara-U to ara-C and, hence, the increased ara-C clearance observed in older children as compared to infants. We conclude that the NONMEM PPK methodology allowed the simultaneous analyses of data from different doses and dose regimens and explained phenomena that prior standard two-stage analyses could not.

PURPOSE Treatment of neuroblastoma cell lines with 13-cis-retinoic acid (cis-RA) can cause sustained inhibition of proliferation. Since cis-RA has demonstrated clinical responses in neuroblastoma patients, it may be effective in preventing relapse after cytotoxic therapy. This phase I trial was designed to determine the maximal-tolerated dosage (MTD), toxicities, and pharmacokinetics of cis-RA administered on an intermittent schedule in children with neuroblastoma following bone marrow transplantation (BMT). PATIENTS AND METHODS Fifty-one assessable patients, 2 to 12 years of age, were treated with oral cis-RA administered in two equally divided doses daily for 2 weeks, followed by a 2-week rest period, for up to 12 courses. The dose was escalated from 100 to 200 mg/m2/d until dose-limiting toxicity (DLT) was observed. A single intrapatient dose escalation was permitted. RESULTS The MTD of cis-RA was 160 mg/m2/d. Dose-limiting toxicities in six of nine patients at 200 mg/m2/d included hypercalcemia (n = 3), rash (n = 2), and anemia/thrombocytopenia/emesis/rash (n = 1). All toxicities resolved after cis-RA was discontinued. Three complete responses were observed in marrow metastases. Serum levels of 7.4 +/- 3.0 mumol/L (peak) and 4.0 +/- 2.8 mumol/L (trough) at the MTD were maintained during 14 days of therapy. The DLT correlated with serum levels > or = 10 mumol/L. CONCLUSION The MTD of cis-RA given on this intermittent schedule was 160 mg/m2/d. Serum levels known to be effective against neuroblastoma in vitro were achieved at this dose. The DLT included hypercalcemia, and may be predicted by serum cis-RA levels. Monitoring of serum calcium and cis-RA levels is indicated in future trials.

The 2-hydroxy-1H-isoindole-1,3-dione (HISD) derivatives are inhibitors of ribonucleotide reductase (RR). Among the seven new compounds of the PL series, three were found to be active in cell lines sensitive (CEM/0) or resistant to ara-C (CEM/ara-C/7A; CEM/dCk[-]). The compounds PL4, PL7 and PL8 exhibited IC50 values in micromolar range against the three CEM cell lines. The 3 compounds showed 100- to 1000-fold higher cytotoxicity compared to HU against all three CEM cell lines. Combination of each of the three active PL compounds with ara-C showed high degree of synergism in comparison to the combinations of HU with ara-C against both CEM/0 and CEM/ara-C/7A cell lines. The DNA synthetic capacity of CEM/0 cells treated with 10 μM PL4 for 24 or 48 h was inhibited ≥ 99.8% simultaneously the cellular NTP pools were severely depleted. Treatment of CEM/0 cells with 10 μM PL4 showed steady depletion in all the dNTP pools at 1 or 2 h and complete depletion by 4 h. The enzyme activity did not recover up to 48 h in ...

A series of eight new N-hydroxy-N′-aminoguanidine (HAG) Schiff bases [ArCH = NNHC( = NH)NHOH · tosylate] was synthesized as potential antitumor agents through the inhibition of the enzyme ribonucleotide reductase (EC 1.17.4.1). Five of the HAG derivatives (LK02 through LK06) were designed to contain an orthohydroxy group on the phenyl ring of ArCH = to increase the stability of the Schiff base formed. In addition, two compounds with a substituted quinoline [LK10; ArCH = (4-hydroxy-7-trifluoromethylquinolin-3-yl)methylene] or isoquinoline (LK11; ArCH = (5-nitroisoquinolin-l-yl)methylene] moiety were synthesized through multiple-step reactions involving reduction and/or oxidation. The IC50 values of the newly synthesized HAG Schiff bases were determined against human leukemic CCRF-CEM/0 cells in culture. The IC50 values of two previously reported HAG derivatives [ATL25; ArCH = (5-nitro-isoquinolin-l-yl)methylene] and [LW02; ArCH = 2-hydroxy-3-allyl-benzylidene)] were also determined for the first time against CCRF-CEM/0 cells. Among the compounds tested, LK11 was found to be the most potent (IC50, 2.95 ± 0.1 µM) and the 4-methoxy-2-hydroxy-phenyl derivative (LK02) to be the least potent (IC50, 121 ± 16 µM). LK11 was about 15-fold more potent against CCRF-CEM/0 cells compared to the parent compound hydroxyguanidine sulfate (IC50, 46 ± 7.1 µM) and was about 32 times more potent than LK10 (IC50, 97.6 ± 0.9 µM). LK11 in combination was incubated in sequence with cytarabine (ara-C) at various molar ratios against CCRF-CEM/0 cells for 48 hr. The results were analyzed using both the isobologram and the median-effect methods. This combination at a 1:1 molar ratio was about 6-fold more potent (IC50, 0.16 µM) compared to ara-C alone (IC50, 1.05 µM) and about 18-fold more potent compared to LK11 alone (IC50, 2.95 ± 0.1 µM). In summary, the antileukemic potency of certain HAG derivatives can be improved by the presence of orthohydroxy groups of the phenyl ring, and a 1:1 molar combination of an isoquinoline HAG compound and ara-C leads to significant synergistic antileukemic activity against CCRF-CEM/0 cells in vitro.

Purpose Re-induction outcomes vary for children with acute lymphoblastic leukemia (ALL) and marrow relapse. We explored possible relationships among asparaginase (ASNase) activity levels, asparagine (ASN) depletion, anti-ASNase antibody titers, and response to re-induction therapy in children and adolescents with ALL and an ‘early’ first marrow relapse. Patients and methods After appropriate informed consent, we enrolled children and adolescents 1–21 years old with ALL and first marrow relapse within 12 months of completion of primary therapy. Induction therapy included intramuscular pegylated ASNase on Days 2 and 16. We assessed ASNase activity, anti-ASNase antibody titers against native and pegylated (E. coli) ASNase, and amino acid levels of asparagine (ASN) and glutamine (GLN) on Days 0, 14, and 35 of re-induction. Results Ninety-three patients were at least partially assessable. Among 21 patients with M1 marrow status at Day 35, the median Day 14 ASN level was

The discovery of the tumour-inhibitory properties of asparaginase began 50 years ago with the observation that guinea-pig serum-treated lymphoma-bearing mice underwent rapid and often complete regression. Soon afterwards, the asparaginase of bacterial origin was isolated. The asparaginases of bacterial origin induce anti-asparaginase neutralising antibodies in a large proportion of patients (44-60%), thus negating the specific enzymatic activity and resulting in failure of the target amino acid deamination in serum. There is immunological cross-reaction between the antibodies against various formulations of native Escherichia coli-asparaginase and polyethylene glycol (PEG)-asparaginases, but not to Erwinia asparaginase, as suggested by laboratory preclinical findings. This evidence was strongly inferred from the interim analyses in the Children's Cancer Group (CCG)-1961 study. Thus, anti-E. coli or PEG-asparaginase antibodies seropositive patients may benefit from the Erwinia asparaginase. The inter-relationships between asparaginase activity, asparagine (ASN) and glutamine deamination remain largely unexplored in patients. Studies have shown that ASN depletion is insufficient to induce apoptosis in T lymphoblasts in vitro and that the inhibitory concentration of CEM T-cell line is correlated with the asparaginase concentration responsible for 50% glutamine deamination. The optimal catalysis of ASN and glutamine deamination in serum by asparaginase induces apoptosis of leukaemic lymphoblasts. The percentage of ASN and glutamine deamination was predicted by asparaginase activity. Asparaginase activity of 0.1 IU/mL provided insufficient depletion of both amino acids in high-risk acute lymphoblastic leukaemia (ALL) patients. With increasing glutamine deamination, mean asparaginase activities and percentages of post-treatment samples with effective ASN depletion (3 micromol/L) increase. Both glutamine and ASN deamination are predicted by asparaginase activity. Further population analyses resulted in identification of sigmoid relationships between asparaginase levels and post-treatment glutamine and ASN deamination.Furthermore, pharmacodynamic analyses strongly suggested that/=90% deamination of glutamine must occur before optimal ASN deamination takes place, due to the de novo ASN biosynthesis by the liver. These pharmacodynamic results from the best-fit population pharmacokinetic/pharmacodynamic model obtained from nonlinear mixed effects model pharmacodynamic analyses for standard-risk ALL patients are similar. These analyses produced the following results: (i) asparaginase activity/=0.4 IU/mL provided insufficient deamination of ASN, whereas0.4-0.7 IU/mL was required for optimal (90%) ASN and glutamine deamination; and (ii) deamination of glutamine is dependent on asparaginase activity and it correlates with enhanced serum ASN deamination. Thus, glutamine deamination enhances asparaginase efficacy in ALL patients. Deamination of ASN/=90% of control or ASN concentration3 micromol/L may be associated with improved survival in this subset of patients. Our findings support the pharmacodynamic mechanism of PEG-asparaginase for disease control in ALL patients. These results taken together strongly support new experimental approaches for application of population pharmacokinetic/pharmacodynamic analyses to further enhance survival of leukaemia patients.

Purpose: Asparaginase therapy is an important component in the treatment of children with acute lymphoblastic leukemia. Polyethylene glycol-conjugated asparaginase (PEG-ASNase) has significant pharmacological advantages over native Escherichia coli asparaginase. We investigated the pharmacokinetics of PEG-ASNase, presence of antibodies to PEG-ASNase, and concentrations of asparagine in serum and cerebrospinal fluid (CSF) in combination chemotherapy for relapsed pediatric acute lymphoblastic leukemia. Experimental Design: Twenty-eight pediatric patients with relapsed medullary (n = 16) and extramedullary (n = 11) acute lymphoblastic leukemia were enrolled at three pediatric institutions and had at least two serum and CSF samples obtained for analysis. Patients received induction therapy (including PEG-ASNase 2500 IU/m2 intramuscularly weekly on days 2, 9, 16, and 23) and intensification therapy (including PEG-ASNase 2500 IU/m2 intramuscularly once on day 7). Serum samples were obtained weekly during induction and intensification. CSF samples were obtained during therapeutic lumbar punctures during induction and intensification. Results: Weekly PEG-ASNase therapy resulted in PEG-ASNase activity of >0.1 IU/ml in 91–100% of patients throughout induction. During intensification, PEG-ASNase on day 7 resulted in PEG-ASNase activity >0.1 IU/ml in 94% and 80% of patients on days 14 and 21, respectively. Serum and CSF asparagine depletion was observed and maintained during induction and intensification in the majority of samples. PEG-ASNase antibody was observed in only 3 patients. Conclusions: Intensive PEG-ASNase therapy in the treatment of relapsed acute lymphoblastic leukemia reliably results in high-level serum PEG-ASNase activity, and asparagine depletion in serum and CSF is usually achieved. Incorporation of intensive PEG-ASNase in future trials for recurrent acute lymphoblastic leukemia is warranted.

For this study, 118 children with standard-risk acute lymphoblastic leukemia (ALL) were given randomized assignments to receive native or pegylated Escherichia coli asparaginase as part of induction and 2 delayed intensification phases. Patients treated with pegaspargase had more rapid clearance of lymphoblasts from day 7 and day 14 bone marrow aspirates and more prolonged asparaginase activity than those treated with native asparaginase. In the first delayed intensification phase, 26% of native asparaginase patients had high-titer antibodies, whereas 2% of pegaspargase patients had those levels. High-titer antibodies were associated with low asparaginase activity in the native arm, but not in the pegaspargase arm. Adverse events, infections, and hospitalization were similar between arms. Event-free survival at 3 years was 82%. A population pharmacodynamic model using the nonlinear mixed effects model (NONMEM) program was developed that closely fit the measured enzyme activity and asparagine concentrations. Half-lives of asparaginase were 5.5 days and 26 hours for pegaspargase and native asparaginase, respectively. There was correlation between asparaginase enzymatic activity and depletion of asparagine or glutamine in serum. In cerebrospinal fluid asparagine, depletion was similar with both enzyme preparations. Intensive pegaspargase for newly diagnosed ALL should be tested further in a larger population.

L-Asparaginase (ASNase) is a front-line chemotherapy for acute lymphoblastic leukemia (ALL), which acts by deaminating asparagine (Asn) and glutamine (Gln). We and others have shown that its cytotoxicity depends more on Gln than Asn depletion. Novel ASNases with different activities have been developed in an attempt to decrease side effects and allergic reaction. To evaluate the importance of glutaminase activity, we developed a novel form of Helicobacter pylori ASNase (dm HpA) using site-directed mutagenesis, with amino acid substitutions M121C/T169M. This mutant form was designed to have the same asparaginase activity as wild type (wt), but lack glutaminase activity. WT and dm HpA were compared with the clinically used ASNases from E. coli (EcA-II) and Erwinia chrysanthemi (ErA). Asparaginase and glutaminase activities of each enzyme were measured with Nessler's assay at pH 8.6. The ratio of glutaminase/asparaginase activity was 1.4% for dm HPase, 7.5% for wt HPase, 41% for EcA-II, and 99% for ErA (at 0.6 IU), respectively. L-Asparaginases (0.01 to 3 IU/ml over 72 hrs) were tested on 8 human leukemia cell lines: pre B ALL (BV173, Nalm-6, RCH-ACV, RS4;11, SEM, SupB15), T cell ALL (Molt-4), and acute promyelocytic leukemia (HL-60). Viable cells were determined by trypan blue exclusion. Two cell lines which we had previously shown to be Asn-dependent (RS4;11 and SupB15) were equally sensitive to the asparaginase isoforms (>90% kill at 0.03 IU/mL). The other 6 lines were more sensitive to isoforms with higher glutaminase activities (Table). ErA was the most effective on all three cell lines on which it was tested. These data show that asparaginase activity alone may not be sufficient for ASNase cytotoxicity, and that glutaminase activity may be required for full efficacy. These sensitivities to ASNases match previous data on Asn- and Gln-dependent cell proliferation (Ehsanipour et al., Cancer Research, 2013). % cell growth inhibition at 3 IU/ml ASNase (n=1-2 for each cell line; nd: not done)Cell linedm HpAwt HpAEcA-IIErARS4;11919291ndSupB15919193ndRCH-ACV13362866SEM22484165BV17324326ndNalm-61621574HL-60204033ndMolt4325963nd Citation Format: Jean-Hugues Parmentier, Maristella Maggi, Erika Tarasco, Claudia Scotti, Vassilios Avramis, Steven D. Mittelman. Glutaminase activity determines cytotoxicity of L-asparaginases on leukemia cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3378. doi:10.1158/1538-7445.AM2014-3378

PURPOSE: A case-control study was performed to determine whether patients who had been treated with Erwinia asparaginase as part of their treatment for childhood acute lymphoblastic leukemia (ALL) and who showed relapsed of their disease more often developed anti-asparaginase antibodies than patients who remained in remission.METHODS: A group of 13 patients who showed relapsed of their disease (median follow-up 35 months) were randomly matched with control patients of the same risk group (two control patients to each case), who had received therapy of the same intensity during the same period (median follow-up 70 months). Anti- Erwinia asparaginase antibodies were measured (ELISA method) during maintenance therapy after asparaginase treatment (30,000 IU/m(2) daily for 10 days in all patients plus twice weekly for 2 weeks in intermediate-risk and high-risk ALL patients).RESULTS: The overall incidence of anti- Erwinia asparaginase antibodies was 8% (3 of 39 patients). There was no statistically significant difference in the incidence of antibody formation between patients who had suffered relapse (1 of 13) and those who had not (2 of 26). In two of the three patients who developed antibodies, the antibodies disappeared after some time, whereas one patient had measurable antibody levels for more than a year after asparaginase therapy.CONCLUSIONS: In this study, the development of anti-Erwinia asparaginase antibodies was rare and was unrelated to the risk of relapse.

Background: Sequence-specific combinations of purine analogs, such as fludarabine or 6-mercaptopurine (6-MP), administered prior to cytosine arabinoside (ara-C) have been shown to abrogate ara-C resistance in human leukemia cells in vitro and in patients with relapsed acute myeloid or lymphoblastic leukemias. The two-drug combination of 6-MP plus ara-C results in greater cytotoxicity than that achieved with either ara-C or 6-MP alone. Further preclinical investigations have shown that the addition of PEG-asparaginase (PEG-ASNase) to the combination of 6-MP plus ara-C (6-MP + ara-C + PEG-ASNase) results in 15.6-fold synergism over that achieved with the two-drug regimen. This is due to increased DNA damage leading to apoptotic cell death. Purpose: Since the intravenous preparation of 6-MP is no longer available and since oral 6-thioguanine (6-TG) provides higher levels of intracellular thioguanine nucleotides than an isotoxic dose of oral 6-MP, we investigated the potential drug synergism of 6-TG plus ara-C plus PEG-ASNase (TGAP) in myeloid (HL60/S, HL60/SN3, U937) and lymphoblastic (CEM/0, CEM/ara-C/B, CEM/ara-C/I, MOLT-4) leukemia cell lines. The CEM clones, MOLT-4 and HL60/SN3 cell lines expressed functional or measurable p53 protein, while the other cell lines did not. Methods: The MTT and trypan blue dye exclusion assays were used to determine drug cytotoxicity. In addition, cellular apoptosis and cellular p53, p21/waf-1 and bcl-2 protein concentrations were determined by FACS analysis and ELISA assays. Results: Sequential exposure to 6-TG (24 h) plus ara-C (24 h) plus PEG-ASNase (24 h) produced 1.3- to 18.3-fold drug synergism over the two-drug combination of 6-TG plus ara-C. The molecular mechanism of synergism was due to the fact that the three-drug combination was capable of downregulating bcl-2 oncoprotein levels in these cell lines even when p53 was absent. Conclusion: These studies strongly demonstrate that the TGAP regimen is highly synergistic in p53-null and p53-expressing leukemia cell lines. We conclude that this combination regimen is collaterally sensitive with ara-C and further evaluation in an investigational phase I trial in relapsed leukemia patients is warranted.

Introduction Cure rates of pediatric acute lymphoblastic leukemia (ALL) have markedly improved to approximately 80%, while in adult ALL the rates remain 40-50%. Pediatric ALL regimens contain higher doses of non-myelosuppressive chemotherapy, e.g., vincristine, corticosteroids, and, particularly, higher cumulative doses of asparaginase. Asparaginase use in adults was previously limited due to toxicity concerns. However, several recent studies, using pediatric regimens in adults, contain higher cumulative doses of asparaginase and are showing promising preliminary results. In these studies it was also noted that the long-acting pegaspargase (PEG-ASN) was much more commonly associated with hepatotoxicity in adults than in children. Although hepatotoxicity appears to be the commonest adverse effect of PEG-ASN in adults, it has not been well defined. We report the frequency and characteristics of PEG-ASN-related high-grade hepatotoxicity after multiple doses in adults treated by a pediatric regimen. Methods Between July 2004 and July 2009, 51 adults aged 18 to 57 years were enrolled on a phase II trial with a pediatric ALL regimen that included six planned PEG-ASN doses. PEG-ASN-related toxicities were carefully monitored on a weekly basis after each dose and reported using NCI CTCAE v3.0 for 185 doses delivered. The PEG-ASN dosing schedule was: two doses in induction phases I and II, and four during post-induction cycles (ASH Abstract 1495, 2012). Each PEG-ASN dose was 2000 IU/m2/dose IV, given at intervals of four weeks or greater. Pegaspargase was not discontinued and subsequent doses were not reduced after hepatotoxicity. Results A total of 192 pegaspargase doses were delivered (3.8 doses/patient), with 23 patients receiving all six doses. Of the 28 patients who received fewer than six doses, only 10 (20%) discontinued due non-hepatic toxicity (pancreatitis, allergy, and DVT). Eight (16%) patients discontinued due to allogeneic HSCT while in CR1, while nine (18%) discontinued for other reasons (death post-induction, induction failure, and relapse). Grade 3/4 hyperbilirubinemia occurred in 16 patients (31%) and in 23 doses (12%); grade 3/4 transaminitis occurred in 33 patients (65%) and in 62 doses (34%). Patients with grade 3/4 hyperbilirubinemia tended to be older than those without hepatotoxicity (median age 39 vs 31 years), but all other baseline characteristics were similar. Results of different parameters related to high-grade liver toxicity are detailed in Table 1. Patients with grade 3/4 hyperbilirubinemia and transaminitis received a mean of 4.0 and 4.3 PEG-ASN doses, while the mean number of PEG-ASN doses causing hyperbilirubinemia and transaminitis was only 1.4 and 1.9 doses per patient, respectively. Those without hepatotoxicity received 2.8 PEG-ASN doses per patient. Induction I had the highest incidence (20% of doses delivered) of grade 3/4 hyperbilirubinemia. High-grade transaminitis was spread more evenly among cycles. Grade 3/4 hepatotoxicity was long, with a median duration of 34 days to return to grade 1 for bilirubin and 38 days to return to grade 2 for transaminitis. Of the 16 patients with grade 3/4 hyperbilirubinemia, five did not receive a subsequent PEG-ASN dose for other reasons; of the 11 other patients who received subsequent doses, five (45%) did not re-experience the same toxicity. Of the 33 patients with grade 3/4 transaminitis, eight did not receive a subsequent PEG-ASN dose due to other reasons; of the 25 other patients who received subsequent doses, 10 (40%) did not have this toxicity recur. Summary Our study shows in adults with ALL treated with multiple doses of PEG-ASN that: (1) high-grade hepatoxicity (grade 3/4 hyperbilirubinemia and transaminitis) is a common adverse effect of PEG-ASN; (2) recovery from hepatotoxicity is often long and can delay subsequent chemotherapy; (3) high-grade hepatotoxicity did not necessarily recur after subsequent doses and did not lead to PEG-ASN discontinuation; (4) the dose and schedule of other hepatically cleared or hepatotoxic drugs should be adjusted during periods of PEG-ASN-related hepatotoxicity. In conclusion, although PEG-ASN at this dose and interval is associated in adults with a high rate of hepatotoxicity, it is tolerable and can be given again despite earlier PEG-ASN-related hepatotoxicity. Disclosures: Douer: Sigma Tau Pharmaceuticals : Research Funding.

1-beta-D-arabinofuranosyl-5-azacytosine (ara-AC) and 5,6-dihydro-5-azacytidine (DHAC) are two new antitumor agents under clinical investigations, which exhibit the chemical similarities found in the tumoricidal drug cytosine arabinoside (ara-C) and the nitrogen substitution in the 5 position of the pyrimidine ring found in 5-azacytidine (5-aza-C). The cellular anabolism of ara-AC and DHAC and their effect on DNA methylation have been examined in two new human leukemia cell lines, which are sensitive (PER-145) and resistant (PER-163) to ara-C. The triphos-phate anabolite of ara-AC, ara-ACTP, was the major cellular anabolite in the cellular extracts of the PER-145 cells, reaching a cellular saturation concentration of 64.1 +/- 3.2 microM using 25 microM of the drug. Only trace levels of ara-ACTP were detected in the PER-163 cell line, which lacks deoxycytidine kinase, after exposure to a similar concentration. Notably, after 1 mM, the ara-ACTP concentration averaged 12 +/- 3 microM. DHAC was anabolized by both cell lines to a similar degree but required much higher nucleoside concentrations (100 microM or higher) to achieve similar cellular concentrations of its triphosphate, DHACTP. Although the deoxy-derivative, DHAdCTP, was detected in both cell lines, it was detected at 1-2 log10 lower concentrations than DHACTP. DNA methylation studies showed that DHAC had a profound effect in inducing DNA hypomethylation in both cell lines, with nadir values of 27.3 and 29.2% of control.(ABSTRACT TRUNCATED AT 250 WORDS)

Obesity is associated with increased cancer incidence and mortality. Adipose tissue has been demonstrated to play a role in several types of cancer, including acute lymphoblastic leukemia (ALL). We have shown that obesity impairs the survival of mice transplanted with ALL and treated with the front-line chemotherapy, L-Asparaginase (ASNase), which acts through the deamination of asparagine (ASN) and glutamine (GLN). We have also shown that adipocytes protect ALL cells from ASNase in vitro. We hypothesized that adipocytes protect ALL cells from ASNase via production of GLN. Fat pads were excised from control and diet-induced obese mice. Glutamine synthetase, the rate-limiting enzyme for GLN synthesis, was analyzed by western blot. Amino acids secreted by fat pads were measured using HPLC. ALL cell sensitivity to ASN and/or GLN depletion, and ASNase, was determined by trypan blue exclusion in culture alone and in transwells over 3T3-L1 adipocytes. Mouse fat pads expressed high levels of glutamine synthetase protein, and this was not affected by obesity or 5 days of L-asparaginase treatment (3,000 IU/kg/day). Fat pad explants secreted GLN (251±50 nmoles/100 mg tissue), but not ASN, over 72 hours. Confirming that GLN secretion could protect ALL from ASNase, twice daily addition of 500 nmoles of GLN to murine ALL cells completely reversed ASNase cytotoxicity (64.0x10^4 vs. 7.9x10^4 viable cells after 72 hours of 1 IU/mL ASNase, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5245. doi:1538-7445.AM2012-5245

O-6-methylguanine (O6-mG), a guanine analog recently shown to be a potent inhibitor of alkylguanine-DNA alkyltransferase, has been found to potentiate the antitumor activity of nitrosoureas, in particular, carmustine (BCNU), in resistant cell lines (HT-29 mer+) and is targeted for development as a modulating agent with chloroethyl nitrosoureas. A high-performance liquid chromatography (HPLC) assay of O6-mG in plasma has been developed using a microC18 reverse-phase column. O6-mG and the internal standard deoxyguanosine (dGuo) were eluted with a linear gradient of from 15% to 35% methanol in 0.5 M ammonium acetate (pH 6.5) at a flow rate of 1 ml/min. The assay was linear over a 4-log concentration range with a detection limit of 0.1 microgram/ml. The within-run and between-run coefficients of variation (CV) were found to be 8.1% and 9.3%, respectively. The pharmacokinetics (PK) of O6-mG were investigated in healthy CDF1 mice following separate i.v. and i.p. administrations. At 20 mg/kg i.v., plasma O6-mG gave a biexponential profile with a terminal half-life (t1/2) of 24 min and a total clearance (CLT) of 23.7 ml min-1 kg-1. Higher doses (40-80 mg/kg) revealed a fluctuating third phase, probably due to enterohepatic cycling. Dose-dependent kinetics as measured by CLT and area under the plasma-concentration curve (AUC) values were also seen. Following i.p. dosing, O6-mG was completely absorbed and available to the circulation. No acute toxicity was observed in the animals, except for mild sedation, a possible side effect of the 10% ethanol used in the formulation. Studies on the cellular metabolism of highly purified [3H]-O6-mG have shown that the compound is not anabolized by a human lymphoblastoid cell line (CEM). Biochemistry studies have shown that the parent molecule is inactivating the alkylguanine-DNA alkyltransferase (AGT), thus exerting its pharmacological effect.

Obesity increases cancer incidence and mortality. This is also true for acute lymphoblastic leukemia (ALL). L-Asparaginase (ASNase), an important front-line drug in leukemia treatment, acts through the hydrolysis of asparagine (ASN) and glutamine (GLN) into aspartic acid and glutamic acid. As lymphoblastic leukemia cells are highly dependent on these two non-essential amino acids, ASNase leads to decreased protein synthesis, cell cycle arrest, and apoptosis. We have previously shown that obesity impairs ASNase treatment in C57BL/J mice transplanted with syngeneic leukemia cells. The present study was designed to elucidate how obesity protects ALL cells from this drug. The effects of adipocytes on ALL ASNase resistance were examined in vitro using co-cultures with 3T3-L1 adipocytes and ALL cells, separated by TransWells. Differentiated 3T3-L1 adipocytes significantly protected ALL cells from a 3-day treatment with ASNase, compared to undifferentiated 3T3-L1 fibroblasts or no feeder layer (viable cells after ASNase treatment: 3.1 ± 1.2 fold greater than no feeder layer, 2.1 ± 0.6 greater than fibroblast feeder layer, p The current data demonstrate that adipocytes directly protect leukemia cells from ASNase treatment. We have demonstrated that adipocytes increase leukemia survival in media starved of ASN and/or GLN, secrete ASN and GLN, and enhance mTOR signaling in amino acid starved leukemia cells. Thus, obesity may impair the effects of ASNase on leukemia cells through adipose tissue secretion of both ASN and GLN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1419.

Abstract 3077 Poster Board III-14 Asparaginase is an essential component of combination therapy for ALL and is derived from either Escherichia coli (Native ASNase) or Erwinia chrysanthemi (Erwinia ASNase). Allergic reaction is the most common toxicity of ASNase therapy. Some studies have shown no adverse impact of allergy to native ASNase on outcome. However, compared with native ASNase, in equal doses (“unit for unit” substitution), Erwinia ASNase has been associated with less toxicity and inferior survival in two large multi-institutional cooperative group trials. Pegylated Escherichia coli asparaginase (PEG) has a longer half-life compared to native ASNase and is presumed to be less immunogenic with equivalent efficacy at an appropriate dose and schedule. Between November 1996 and May 2002, 2057 eligible patients with newly diagnosed high-risk ALL were enrolled on Children's Oncology Group study, CCG-1961, and treated with a standard four-drug induction that included native ASNase. Rapid early responders (RER) randomized to stronger post-induction intensification (arms C &D) and all slow early responders (SER) were assigned 6 or 10 doses of PEG (2500 IU/m2/dose) post-induction (n=1157). Patients received native ASNase if PEG was not available. Those with adverse reactions to PEG received Erwinia ASNase (10,000 IU/m2 qod x 6 during consolidation and delayed intensification and 25000 IU/m2 during interim maintenance per single dose of PEG). Antibody titers were collected on a subset of patients pre-consolidation and through all phases. Reaction incidence and event-free survival (EFS) in patients who received PEG versus Erwinia ASNase and in patients with negative versus positive antibody titer were compared among patients assigned to PEG containing regimens. Likelihood of allergic reactions was similar with PEG, native ASNase, and Erwinia during all phases with the exception of Interim Maintenance 1 (IM1). Reactions were less likely with Erwinia (n=235) than with Native ASNase (n=87; odds ratio [OR] = 0.23, p 1.1) and 243 (71.5%) had a negative antibody (titer ≤1.1). Patients with a positive antibody pre-consolidation were more likely to have an allergic reaction to PEG during consolidation or a subsequent phase than patients who had a negative antibody (OR = 2.41, p Disclosures Gaynon: Enzon: Speakers Bureau.

The better outcome in pediatric ALL may not be solely related to the biology of the disease. Adult regimens are less intensive than used in children and include less, shorter or no use of asparaginase; the drug is often considered more toxic than in children. However, adequate serum asparagine depletion was shown to be associated with improved overall outcome. Further in several, multi-agent, large pediatric randomized trials, longer administration of asparaginase in the post remission phase and more sustained asparagine depletion resulted in better outcome. PEG-asparaginase (PEG-ASP) is a modified E.coli ASP, with less hypersensitivity reactions and longer half-life. In adults a single IV dose of PEG-ASP during induction produces long duration of asparagine depletion of up to 4 weeks, t½ = 7 days, with similar toxicity to equivalent multiple doses of E.coli ASP (Douer et al Blood 19:2744,2007). We now report the use of multiple doses of PEGASP, given during induction and throughout the post-remission phases in order to produce prolonged and sustained asparagine depletion in the context of an intensified pediatric protocol, in newly diagnosed, treatment naïve, adult ALL. Sustained asparaginase activity after the short acting E.coli ASP, can be impaired by development of neutralizing anti – asparaginase antibodies. We therefore measured the asparaginase enzymatic activity in a cohort of our patients who received PEG-ASP which in children was shown to be associated with less antibody formation (Avramis et al Blood2002;99:1986). Methods: The backbone of our protocol is an augmented BFM pediatric ALL regimen consisting of 8 cycles of multi-agent chemotherapy, followed by maintenance. PEG-ASP (2000 U/m2/dose) is given IV once on day 15 of the following cycles (total 6 doses): induction phase I (cycle 1), induction phase II (cycle 2), two cycles of consolidation (cycles 3 and 6), and two cycles of delayed re-induction (cycles 5 and 8). Since December 2006, Ph+ patients receive imatinib 600mg/daily. Asparaginase enzymatic activity was measured in samples obtained before and after PEG-ASP doses in cycles 1, 5 and 8. Results: 41 patients, aged 19–57 (median 33) years, with precursor B cell – 36, T cell-5, Ph+ 9, were studied. Median WBC at diagnosis - 15,900/cumm (range 1,900–512,000). CR rate: 37 (95%) pts. all after induction phase I. To date 15 patients received all 6 doses of PEG-ASP, having completed all consolidation cycles. The other patients are still being treated. So far the number of PEG-ASP doses given is: 6(15pts), 3(6 pts), 2(7 pts), 1(13 pts) Fifteen patients could not continue the protocol for: allogeneic stem-cell transplantation (8), refusal (1), pancreatitis (5), grade 3 DVT(1). Two additional patients died in CR from neutropenic sepsis. Patients with elevated liver enzymes, high bilirubin, or hyperglycemia continued on the study. Total number of PEG-ASP doses was 135. No pt had an allergic reaction. The number of pts with grade 3/4 toxicities during PEG-ASP cycles were: elevated liver enzymes 24 (59%), hyperbilrubinema 8 (20%), hyperglycemia 12 (29 %), pancreatitis 6(15%), fatigue 4(10%), hypertriglyceridemia 3 (7%), catheter thrombosis- 4 (10%), neuropathy-1. All toxicities were reversible. With a median follow up of 18 months, EFS at 3 and 4 yrs is 68 % (Ph- patients 74%) and CIR is 23%. Asparaginase enzymatic activity after each dose was very long in a cohort of 19 pts with a population average of t½ = 10.99 days (peak concentration 0.88 IU/ml). All 74 specimens analyzed from this cohort of patients had significant asparaginase activity after each PEG-ASP dose, highly suggesting that none of the patients analyzed had developed neutralizing anti asparaginase antibodies. Additional patients with enzymatic measurements will be presented Conclusions: Multiple doses of PEG-ASP IV in adults (ages 19–57 years) provide prolonged and sustained asparagine depletion and no apparent drug inactivating by antibody formation. In combination with an intensified BFM-based protocol this pediatriclike strategy is feasible, without allergic reactions, and with acceptable toxicity. Although the follow up is relatively short the EFS appears to be high. Such approach may benefit adults with ALL

Introduction: The mechanisms of treatment failure in ALL are poorly understood. Attention has focused on the intrinsic resistance of the leukemic blast. ASNase contributes importantly to response and outcome in ALL. In about 1/3 of 1st relapse patients, we found inadequate ASN depletion, i.e., ASN > 3 mmoles on Day 14 of Induction (11 days following a single dose of pegylated ASNase) and an inferior re-induction rate (Pediatr Blood Cancer2006; 47:141). Patients with relapse have a superior re-induction rate with qwk rather than conventional qow pegylated ASNase (Blood2000; 96:1709). In the present study, we hypothesized that inadequate ASN depletion with standard ASNase dosing leads to relapse in newly diagnosed children with ALL. Methods: Between 1997 and 1998, 118 children with NCI standard risk ALL were enrolled on Children’s Cancer Group CCG-1962 study after informed consent (Blood2002; 99:1986). By random allocation, patients received either native ASNase 6,000 IU/m2 tiw × 9 in Induction and × 6 in each of 2 Delayed Intensification (DI) phases (n=59, 15 relapses, 1 2nd malignancy) or a single dose of pegylated ASNase 2500 IU/m2 (n=59, 10 relapses) in Induction and each of 2 DI phases. All ASNase was administered intramuscularly and begun on Day 3–5 of Induction and each DI phase. Multiple samples were obtained for each patient and cooled promptly to prevent ex-vivo deamination, which may lead to falsely low, not falsely high ASN values. We examined the incidence of relapse by 8 years in patients ranked by ASN concentration and ASNase activity. Results: The overall 4-year EFS is 84%. Overall, 17/ 31 patients (55%) in the highest Day 14 ASN tertile relapsed compared to 7/ 75 (9%) in the lower two tertiles (p Conclusion: In vivo, asparagine (ASN) depletion reflects a balance between ASNase activity and ASN production, which may vary for an individual over time and among individuals. ASN depletion on Induction Day 14, 9–11days after ASNase administration, predicts outcome in children with SR ALL receiving either native or pegylated ASNase. ASNase activity is not predictive. Failure to achieve adequate ASN depletion in Induction may contribute to relapse in childhood ALL. ASN Concentration Induction Day Pegylated ASNase Native ASNase Day 7 ASN μmoles Relapses/ total μmoles Relapses/ total 1st tertile > 1.14 3/ 16 > 0.99 6/ 16 2nd tertile 0.03–1.12 4/ 16 0.04–0.73 3/17 3rd tertile 0.01 2/ 19 0.01 4/23 DAy 14 ASN 1st tertile > 2.78 9/ 17 > 2.62 8/ 14 2nd tertile 0.18–2.68 0/ 17 0.23–0.27 3/ 15 3rd tertile 0.01–0.07 1/ 17 0.01 3/ 26 ASNase Activity Induction Day Pegylated ASNase Native ASNase Day 7 Activity IU/ml Relapses/ total IU/ml Relapses/ total 1st tertile ≤0.64 3/17 ≤0.23 5/19 2nd tertile 0.65–0.92 2/18 0.24–0.51 3/18 3rd tertile 0.93–1.63 4/17 0.52–1.13 5/19 DAy 14 Activity 1st tertile ≤ 0.31 2/17 ≤ 0.23 5/18 2nd tertile 0.34–0.66 4/18 0.24–0.42 4/19 3rd tertile 0.68–1.25 4/17 0.42–1.24 5/18

13010 Background: Using the linear PK first order (FO) compartmental approach, the methodical and computational uniformity in modeling various linear systems (ERW) is the dominant characteristic of the population PK analyses. However, saturation of the enzymatic reaction results in nonlinear kinetics based on the MM equation, i.e., the deamination of ASN by ERW, which complicates the PK-PD model. The PK with FO & the MM PK-PD model is theoretically better when single dose of ERW is given. To simulate the PK-PD data and to project patient (pt) data using this model, we used simultaneous integration of the FO+MM equations. Methods: In the 1990’s, ERW was used in pediatric ALL pt (n=23, 1st cohort evaluated for PK-PD). The current study was started to assess if the parallel MM+FO (MM+FO) PK-PD model fit the limited ERW (25K) data better than the MM model, and to validate the MM+FO model and its population parameter estimates. Results: The population PK-PD model best-fitted serum ERW & ASN-time pairs obtained in 23 and from 5 pediatric pt with ALL, using nonlinear mixed-effects modeling (NONMEM). The validity of the MM+FO population PK-PD model and the estimated parameters were tested using the naive prediction method. Patients were administered ERW 6K or 25 K IU/m2 when allergy to E. coli formulations appeared. High correlation between ERW peak serum levels calculated from limited individual pt’ KM (900 μM) & Vmax values in 5 pediatric pt (2nd cohort) and the observed ERW trough levels & its substrate (ASN) were found. The T1/2 averaged 16 hr (25K ERW), and the trough level of 0.1–0.2 IU/ml was correlated with ASN st cohort. When simulations on population parameters were conducted, the MM+FO predicted the multiple dose steady-state serum ERW & ASN levels nicely. Therefore, the MM+FO model was clearly superior to either the FO or the MM PD models. Moreover, simulations compared favorably ERW 25K Q2 days x3 doses & x2 weeks vs. Pegaspargase 2,5K Q2 weeks. Conclusions: The NONMEM PK-PD model for ERW fitted the simultaneous analyses of data from different doses and regimens better than either standard Two-stage or MM could. No significant financial relationships to disclose.

Background: ASNase is a critical treatment component in childhood acute lymphoblastic leukemia (ALL). As ASNase is a bacterial protein, production of anti-ASNase Ab, often associated with clinical allergy, poses a frequent problem during ASNase therapy. Sixty percent of higher risk ALL patients, who received native ASNase in induction, were Ab(+) by 5 months into therapy (Panosyan et al., 2004). Moreover, 22%–29% of these patients had no clinical allergy (silent hypersensitivity) and were at increased risk for relapse. Patients and Methods: Physicians from 13 institutions submitted samples for 48 children in first remission, aged 14 to 252 months, on various SR or HR ALL protocols with suspected ASNase allergy. The male/female ratio was 1.4. The serum samples were assessed for ASNase activity and anti-ASNase Ab as reported earlier (Avramis, et al., Blood 2002). Ab titer is expressed as the numeric ratio relative to an Ab negative control. Results: Allergic symptoms ranged from localized skin rash and swelling to respiratory difficulties and/or anaphylaxis. Neutralizing anti-PEG-ASNase Ab’s were seen in 41 patients (85%). The results were examined with respect to age, gender, ASNase formulation received, time of post-ASNase administration, and the physician’s clinical observations. Higher Ab ratios > 1.1 correlated with low or no ASNase activity post-PEG-ASNase (46/48 patients) or post-Native ASNase dosing (2 patients). Fourteen patients had cutaneous manifestations (rash, hives, urticaria). All had neutralizing Ab and no enzymatic activity. Nine had higher Ab ratios and 5 had lower Ab ratios. Eight additional patients had localized or generalized swelling; all had higher Ab ratios. Similarly, over the entire CCG-1961 study, 526/1100 patients had a clinical reaction; 476/526 had higher Ab ratios (90%). Conclusions: We found neutralizing Ab in 85% of patients with apparent clinical ASNase allergy. Neutralizing Ab was correlated with lower or absent ASNase activity. Lower Ab ratios may be associated with earlier time points in the anamnestic response. In the remaining 15% of patients with an apparent clinical reaction, we found no Ab and substantial ASNase activity. Monitoring ASNase activity and Ab may be useful for guiding ASNase therapy. Patients with no Ab and substantial activity, might be rechallenged with the same product despite an apparent allergic reaction.

Background: We found enhanced cytotoxicity against human leukemia cell lines resistant to cytarabine with the sequence specific three-drug combination, 6-thioguanine (6TG) + cytarabine + PEG-asparaginase (“TGAP”) [Fu et al, Cancer Chemother Pharmacol 48:123–133, 2001] and developed a Phase I study to evaluate TGAP’s feasibility in children with relapsed/refractory leukemia or non-Hodgkin’s lymphoma with marrow involvement. Patients and Methods: Three days of twice daily 6TG was followed by cytarabine @ 487 mg/m2 loading dose and 126 mg/m2/hr by 72-hour continuous infusion. Cytarabine was followed by pegylated asparaginase @ 2,500 units/m2. Complete response (CR) required fewer than 5% marrow blasts with recovery of neutrophils and platelets. Partial response required marrow blasts between 5% and 25% with no peripheral blasts. Results: Between April 1999-January 2006, 11 patients, age 1 to 18 years, were enrolled after local IRB approval and individual or parental informed consent. Six patients had relapsed/refractory ALL, 4 patients had relapsed ANLL (2/4 had Down syndrome) and 1 patient had relapsed Burkitt’s lymphoma/leukemia. Six patients were enrolled at dose level #1 (6TG @ 100mg/m2/dose) and 5 patients were enrolled at dose level #2 (6-TG @ 150mg/m2/dose). The last patient enrolled was later found ineligible, but was included for toxicity. Chemotherapy was well tolerated in 9 patients. All patients developed grade 3–4 neutropenic fevers or bacteremia. Other grade 3–4 toxicities included transient elevations in transaminases. Asparaginase related pancreatitis occurred in two patients, in one with symptoms. Dose limiting acute vascular leak syndrome occurred in two patients at dose level #2 during the cytarabine infusion and just after the completion of therapy. Both required mechanical ventilation but recovered fully. Complete responses (CR) occurred in 2 of 10 evaluable patients and partial response (PR) occurred in 2 for total response rate (CR + PR) of 40%. All four responders tolerated a second course of TGAP. Both CR patients subsequently underwent matched unrelated BMT with no veno-occlusive disease or other excessive toxicity. In one CR patient, ara-CTP incorporation in the patients leukemic blasts ex vivo studied at baseline, pre chemotherapy, and repeated after 6TG and a 24-hour infusion of cytarabine showed a four-fold increase in intracellular ara-CTP, confirming our previous work in cell lines. Conclusions: TGAP is a feasible combination for patients with relapsed leukemias. A phase II study is currently under development using the maximum tolerated dose for 6TG of 100mg/m2/dose in combination with CI ara-C and IM PEG-asparaginase.

9027 Background: Asparaginase (ASNase) induces apoptosis in ALL lymphoblasts by depleting cells of extracellular asparagine (ASN) and possibly glutamine (GLN). On CCG-1961 [Seibel NL et al. Blood 2003; 102(11): 224a (abstract #787)], all patients (pts) received 9 doses of native E.coli-ASNase in induction (IND). Pts with rapid early response (marrow blasts < 25% on Day 7, RER) were randomized to standard intensity therapy and received native ASNase thereafter or to stronger intensity therapy and received PEG-ASNase post induction. All pts with slow early response (marrow blasts ≥ 25% on Day 7, SER) received PEG-ASNase after induction. Pts with clinical allergy to E.coli-ASNase were switched to Erwinase. Methods: Anti-ASNase Antibody (Ab) titers were assayed at the end of IND, prior to Delayed Intensification #1 and prior to starting Maintenance. Amino acid levels were measured in 430 sera from 187 patients with measurable ASNase activity. Results: Population pharmacokinetics (PK) and pharmacodynamics (PD) of the ASNase formulations demonstrated half-lives of native ASNase (6000 IU/m2): 30 hours, PEG-ASNase (2500 IU/m2): 156 hours, and Erwinase (6000 IU/m2): 18 hours. Subsequent exposures to either formulation resulted in increased activity with moderate prolongation of the elimination half-lives. One-hundred forty-two sera from 70 patients with ASNase activity between 0.03 and 1.11 IU/ml, had a mean ± SDEV ASNase activity, ASN levels, and GLN levels of 0.21 ± 0.23 IU/ml, 14.6 ± 17.7 μM, and 300.4 ± 233.8 μM, respectively; with median values of 0.11 IU/ml (range: 0.03–1.114 IU/ml), 4.7 μM (range: 0.38 - 81.34 μΜ), and 270.2 μM (range: 5.3 - 1189.1 μM), respectively. TTEST analyses linked higher ASNase activity with lower ASN levels and greater % ASN deamination. Higher ASNase activity did not assure greater GLN depletion. Conclusions: A sigmoid relationship between ASNase levels and % ASN deamination showed that activity of 0.41 ± 0.24 IU/ml (mean ± SDEV) provided 93% ± 13% ASN deamination. Therefore, >90% ASN deamination required ASNase >0.4 IU/ml. Kaplan-Meier analyses are pending. [Table: see text]

9025 Background: ASNase is an important agent in the treatment of childhood acute lymphoblastic leukemia. Most other chemotherapeutic agents require entry into cells and some variety of activation. ASNase acts unmodulated and totally outside of cells, targeting extracellular ASN and glutamine. Several investigators have described a relationship between ASNase activity and ASN. In vivo, the equilibrium asparagine level depends on the input rate of ASN derived from the nutrients plus the de novo biosynthesis minus its deamination from the serum ASNase activity. Imax is a pharmacodynamic (PD) parameter developed in 1980’s representing the cumulative input of ASN from nutrients and de novo biosynthesis. Of note, glutamine is the amino group source for the synthesis of ASN from aspartate. In Enzyme-substrate relationships a sigmoid relationship exists that reaches a maximum product formation (Imax). We reported a link between Day 14 ASN depletion - not ASNase activity - and response for children with ALL in first marrow relapse (Jarrar et al, 2006). Methods: On CCG-1962, 117 children with standard risk ALL were randomly allocated to native or pegylated ASNase. Relevant PK-PD parameters have been reported (Avramis et al, Blood 2002). ASNase activity was not predictive of EFS. Induction (IND) Day 3 - 24 serum ASN levels were fitted into a PD model and the Imax values were evaluated for 112 patients. Results: The median Imax values were 1E-6 nmoles/ml/min in all 112 (range 2.0E-2 to 1.0E-7) and in the native or PEG-ASNase randomized patients. The mean values were 1.1E-3.1±3E-3 in 112 patients and 1.5E-3±4.0E-3 & 1.1E-3±3.0E-3 nmoles/ml/min in the native or PEG-ASNase randomized patients. We examined EFS by Imax values in lifetable analyses. High Imax values predicted statistically poorer outcome (p−3 or Imax ≥5 × 10−4 nmoles/ml/min. The effect was similar in the native and pegylated ASNase subsets. ASNase activity was not prognostic. Conclusions: Hence, ASN Imax in serum is a treatment-independent PD prognostic factor in a subset of children with standard risk ALL. Strategies to provide uniform therapeutic effect among patients with differing Imax are under consideration. No significant financial relationships to disclose.

9026 Background: Many molecular pathways including cell-cycle control, angiogenesis & drug resistance mediate tumor growth and survival. Activation of Akt, PI3K and Ras can be induced by the activation of receptor tyrosine kinases, such as the VEGF-R and related growth factor receptors. VEGF-A serum levels 100 pg/ml have been associated with good and poor prognoses, respectively. The higher the VEGF-A levels the greater the chance of MRD blasts surviving chemotherapy. ALL blasts secrete VEGF-A and express VEGF-R. The hypothesis was that serum VEGF-A levels in SR ALL pediatric patients at entry of Induction (IND) are predictive of Event-Free Survival (EFS). Methods: 117 patients were entered in CCG-1962 study and were randomized into the Native & PEG-ASNase arms. VEGF-A serum levels in Induction were quantified by an ELISA assay. Results: . All patients had a decrease in VEGF-A levels by Day 14 of IND, but they later dichotomized; with EFS group levels remained low and Event group increasing. The VEGF-A levels at the end of IND remained unchanged in all patients in the DI1 and DI2 treatment phases. A linear relationship exists between high VEGF-A levels at entry to IND and time to Event. Moreover, 7 yr EFS patients have lower VEGF-A average levels of 28±2 pg/ml than Event patients, >100 pg/ml (P No significant financial relationships to disclose.

Deamination of asparagine (Asn) and glutamine (Gln) by asparaginase (ASNase) induces apoptosis of ALL lymphoblasts, which lack ability to synthesize Asn de-novo. Early treatment intensification after Induction, including PEG-ASNase instead of native E.Coli-ASNase, in addition to more vincristine and intravenous methotrexate, improves outcome in pediatric ALL patients presenting with unfavorable features, who show rapid early response to Induction chemotherapy (Seibel NL, et al., Blood, 2003;102:224a). Erwinase was used for patients with overt clinical allergy to E.Coli-ASNase. We evaluated the population pharmacokinetics and pharmacodynamics (PK-PD) of the three ASNase formulations employed on CCG-1961. The PK-PD parameters of native and PEG-ASNase in this group of HR ALL patients were similar to those reported from SR ALL patients from CCG-1962 (Avramis VI, et al., Blood, 2002;99:1986–1994). Population PK (NONMEM) showed the expected half-lives of native E.Coli-ASNase (6000 IU/m2): 1.3 days; PEG-ASNase (2500 IU/m2): 6.5 days; and Erwinase (6000 IU/m2): 0.8 days. Repeated doses resulted in an accumulation of activity with moderate prolongation of elimination half-lives for all formulations. PEG-ASNase provided > 0.3 IU/ml activity for > 21 days for most patients in Delayed Intensification. We measured simultaneous amino acid levels in 430 sera samples obtained from various phases of CCG-1961 from these 187 fully evaluable patients with detectable post-treatment ASNase activity. The averages of pre-treatment control serum levels for Asn and Gln were 44.8 ± 3.0 μM (mean ± SE) and 363.8 ± 23.9 μM, respectively. There were no statistical differences between the PD effects of ASNase formulations on serum Asn deamination, although the lowest Asn levels averaging 2.6 ± 0.4 μM were obtained on 11–14 days post-PEG-ASNase samples (n=14). Glutamine deamination enhanced Asn depletion, and deamination correlated with ASNase activity. At levels >0.4 IU/ml, PEG-ASNase and native ASNase provided 95 ± 8% and 85 ± 26% Gln deamination, respectively (p=0.04). Eleven to 14 days after PEG-ASNase, Gln levels averaged 13 ± 5 μM (n=14). In general, Erwinase provided greater Gln deamination (94 ± 5%, n=19) 2–3 days post administration, than either native or PEG-ASNases at comparable activity levels, which were 70–80% (p

8556 Background. Recent reports demonstrated the incomplete Asn depletion in CSF (Appel et al., Leukemia, 2003), which verified our earlier findings (Avramis et al., Blood 2002). Methods. We examined the measured by HPLC amino acid assay CSF and concomitant serum Asn deamination in 20 out of 24 relapsed plus 28 randomly selected event-free (CCR) patients (pts) from CCG-1962 SR ALL study. Results. These analyses are shown below. Asn levels in CSF or serum are lower significantly in both arms after ASNase treatment, however neither ASNase formulation resulted in complete Asn depletion in these biological fluids. In addition, specific PD analyses were conducted to evaluate the impact of the Input of Asn in the PEG-ASNase arm pts, characterized by the biochemical parameters of Imax plus additive error from the host, i.e., nutrients & the de-novo biosynthesis by the liver. The Imax of Asn in serum was estimated to be statistically higher in pts with events (8.9E-3 ±6E-3 nmoles/ml/min), than in the randomly sel...

Angiogenesis is involved in many physiological and pathological conditions including embryonic development, wound healing, menstrual cycle, chronic inflammation and the development of tumors. Vascular endothelial growth factor (VEGF) is a major initiator and regulator of angiogenic processes and is associated with tumor growth, invasion and metastasis. The VEGF family of proteins (VEGF-A, VEGF-B, VEGF-C, VEGF-D and the placenta growth factor [PGF]) share tyrosine kinase receptors (VEGFR-1, VEGFR-2, VEGFR-3) which are expressed on many cell types including endothelial cells, hematopoietic stem cells and many solid tumor and leukemia blast cells. The VEGF receptor signaling pathway activates a tyrosine kinase cascade involving various intracellular proteins (particularly Pl-3 and RAS/MAP kinases) and is a potential antiangiogenic target that can be inhibited at various levels. The work presented here deals primarily with the small molecule tyrosine kinase inhibitors such as adaphostin. Adaphostin and congeners have the potential to specifically inhibit tyrosine kinases of the VEGF receptors with an acceptable toxicity profile to the host. These compounds have shown good inhibition of VEGFR-1 and relatively good inhibition of VEGFR expressing human leukemia cells. Furthermore, combination regimens of this class of compounds with cytotoxic drugs can downregulate both angiogenesis and its associated effects in the bone marrow of mammalian systems and provide synergistic combination drug regimens for the treatment of refractory malignancies.

Development of Resistance to Gamma Radiation in T-Lymphoblastic Cell Lines after Treatment with Chemotherapeutic Agents

Murine P388 cells incubated in vitro with the anticancer drug arabinosyl 2-fluoroadenine accumulate its 5′-triphosphate. F-araATP, as the major phosphorylated metabolite. A new chromatographically separate metabolite that accumulated to levels 10% of that of F-araATP was identified as 2-fluoro-ATP, by the following criteria. 1. The metabolite coeluted with the authentic compound on anio-exchange HPLC. 2. Dephosphorylation of the metabolite yielded a compound that was chromatographically identical to 2-fluoroadenosine. 3. The compound was sensitive to NaIO4 oxidation. Cellular incubation experiments indicated that 2-fluoroadenine, but not arabinosyl 2-fluorohydroxanthine, was the likely intermediate in the formation of 2-fluoro-ATP.

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.

Deamination of cytosine arabinoside (ara-C) by cytidine deaminase (Cyt DA) is the main mode of the inactivation of this drug in vivo. Tetrahydrouridine (THU) and the deamination product, uracil arabinoside (ara-U) are potent inhibitors of Cyt DA. We investigated whether ara-U or THU pretreatments can protect ara-C from excessive deamination in tumor- (L1210) bearing mice. In order to determine this, plasma concentrations of ara-C, ara-U, and the intracellular levels of ara-CTP, the active anabolite of ara-C, were assayed. The control peak plasma levels of ara-C and ara-U were 3.3 and 0.78 mM and they were eliminated with a half life (t 1/2) of 1.26 and 1.43 hours, respectively. One hour pretreatment with a nontoxic dose of ara-U (single dose of 300 mg/kg intraperitoneally), resulted in increased ara-C levels by 5.9-fold, while ara-U increased 14.3 fold in comparison with controls. A 24-hour (every 8 hours) pretreatment with ara-U increased ara-C plasma levels by 3.0-fold and it was eliminated with a t 1/2 of 1.21 hours. One hour pretreatment with THU (single dose 25 mg/kg intraperitoneally) enhanced ara-C plasma levels by 5.3-fold. In control L1210/0 acid extracts, ara-CTP peaked at 2 hours and reached 2030 +/- 85 microM; ara-CTP was eliminated with a t 1/2 1.47 hours. The ara-CTP cellular concentrations after 1- and 24-hour pretreatments were 1875 +/- 534 and 2624 +/- 429 microM at 4 hours; the t 1/2 were 2.20 and 1.44 hours, respectively. The THU pretreatment resulted in a peak concentration of ara-CTP of 2208 +/- 366 microM at 2 hours and was eliminated with a t 1/2 of 2.54 hours. We concluded that all pretreatments increased both the peak plasma ara-C concentrations and the area under the plasma concentration-time curve (AUC). One hour ara-U pretreatment did not enhance the peak ara-CTP cellular concentration, but did extend the t 1/2. The 24-hour ara-U and the 1-hour THU pretreatments increased, to some extent, the cellular ara-CTP concentrations, but these differences were not statistically significant. THU pretreatment increased the time the peak occurred and the t 1/2 of ara-CTP. The area under the cellular ara-CTP concentration-time curve (AUC) in L1210 cells was either the same or increased by a small amount after the pretreatments.(ABSTRACT TRUNCATED AT 400 WORDS)

5,6-Dihydro-5-azacytidine (DHAC) is a hydrolytically stable analog of 5-azacytidine (5-aza-C) that has antileukemic activity against experimental leukemias and, like 5-aza-C, causes DNA hypomethylation. We report the cellular metabolism of DHAC and its incorporation into nucleic acids in the CCRF/CEM/O and deoxycytidine kinase mutant CCRF/CEM/dCk(-) human lymphoid cell lines. The cells were incubated with their respective IC50 concentrations for 24 h, then aliquot samples were removed at predetermined intervals and extracted for nucleotides. The acid-soluble extracts of the cells were assayed on HPLC for nucleotides of DHAC. The major anabolite of [3H]DHAC, [3H]DHACTP, peaked at 110.3 +/- 30.7 microM in CEM/O and at 96.3 +/- 41.9 microM in CEM/dCk(-) cells at 9 and 12 h, respectively. The intracellular concentrations of the deoxyribonucleoside triphosphate, [3H]DHAdCTP, peaked at 13.5 +/- 7.7 microM at 4 h in CEM/O and at 80.8 +/- 13.8 microM at 12 h, a 6-fold greater cellular concentration, in the dCk mutant cell line. The amount of DHAC anabolites incorporated into CEM/O nucleic acids reached a plateau in RNA at 552.6 +/- 7.8 pmol/10(7) cells and in DNA at 64.55 +/- 10.0 pmol/10(7) cells. In CEM/dCk(-) cells, DHAC anabolites reached a plateau in RNA and DNA at 4,256.3 +/- 631.0 and 395.5 +/- 145.4 pmol/10(7) cells, respectively. Thus, with equitoxic treatments of DHAC, the incorporation of its analog anabolites into RNA and DNA was 8- and 6-fold greater in CEM/dCk(-) cells. DNA methylation levels were depressed equally despite a 6-fold greater incorporation of the analog in DNA in the CEM/dCk(-) cells indicating that hypomethylation may be saturated after DHAC treatment. The DNA methylation levels reached a nadir of 0.19% and 0.20% methyl-C (percentage of methylation) in the two cell lines at 6 and 12 h after the beginning of drug treatment and remained relatively constant for the duration of the 24-h treatment. A curve-linear relationship was obtained between the DNA methylation levels in both cell lines and the amounts of DHAC anabolite incorporated into DNA.

The development of acquired resistance to antitumor agents is a major impediment to the cure of pediatric cancers and leukemias. The causes of drug resistance include: (1) defective drug transport, (2) defective drug metabolism to active species, (3) altered intracellular pools of nucleotides, (4) increased drug inactivation, (5) altered DNA repair, (6) gene amplification and (7) altered target proteins [1–3]. These mechanisms may be due to enhanced expression of specific genes as seen in cells that have amplification of dihyrofolate dehydrogenase (DHFR), adenosine deaminase, ribonucleotide reductase, and possibly pleotropic drug resistance, a condition where cells are resistant to anthracyclines, vinca alkaloids and dactinomycin [4–6]. Alternatively, resistant cells may have altered genes or reduced expression of the genes that code for transport proteins, drug receptors and enzymes that activate certain antitumor agents. In most cases of drug resistance, we do not know the mechanisms for this altered gene expression.

1-beta-D-arabinosyl-5-azacytosine (ara-AC) is a relatively new antitumor agent under clinical investigation, which has the 2'-beta arabinosyl configuration found in the tumoricidal drug ara-C and the nitrogen substitution in the 5-position of the pyrimidine ring found in 5-azacytidine (5-aza-C). The present study examined the cellular metabolism and the effect on DNA methylation of ara-AC in human CCRF/CEM cells sensitive and resistant to ara-C. The triphosphate anabolite of the drug, ara-ACTP, was the major anabolite in the CEM cellular extracts, peaking at 50.6 +/- 23 microM 4 h after incubation with IC50 concentrations (0.25 microM) of [3H]ara-AC. The mono- and diphosphate anabolites accumulated 10-fold lower cellular concentrations than ara-ACTP. The nucleoside triphosphate (NTP) pools and, especially, cellular ATP declined significantly by 9 h after the initiation of drug treatment and remained depleted for the 24-h treatment. The drug anabolite was gradually incorporated into both RNA and DNA, peaking in CEM/0 at 3.44 and 0.14 nmol/10(7) cells, respectively. The DNA methylation levels in these cells declined rapidly after treatment with ara-AC, attaining a nadir plateau at 29% of control methylation value. The deoxycytidine kinase (dCK) mutant CEM cell line [CEM/dCk(-)] neither activated ara-AC at appreciable levels nor induced DNA hypomethylation at low concentrations (0.25-1 microM). However, the drug was activated at 0.2-1 microM extracellular concentrations of ara-AC, probably by an as yet unknown nucleoside kinase at approximately 10% of the amount in CEM/0 cells. Ara-AC appears to mediate its cytotoxic action through the accumulation of its triphosphate anabolite, ara-ACTP, and the subsequent incorporation into nucleic acids. DNA methylation may also contribute to its cytotoxicity.

HIV is the causative agent of AIDS. The purpose of this study was to examine the biochemical pharmacology of the anti-viral agent zidovudine (AZT) in the T-cell origin line (CEM). We have shown that zidovudine is activated by thymidine kinase (TK) in CEM cells to the triphosphate anabolite, which is incorporated into DNA. One microM zidovudine is sufficient for saturation of activation by TK and also of zidovudine monophosphate, by thymidylate kinase, to the diphosphate. Zidovudine triphosphate peaked 4 h after initiation of drug administration in CEM cells and then declined biexponentially. Nucleoside triphosphate (NTP) cellular concentrations declined rapidly in the cells after exposure to zidovudine. Concomitantly phosphorylation of zidovudine to zidovudine monophosphate and zidovudine monophosphate to zidovudine diphosphate declined in a similar manner in the CEM cells. The amount of zidovudine anabolite incorporated into purified DNA peaked 1 h after zidovudine treatment and declined thereafter with first order elimination kinetics. These studies elucidate the cellular activation of zidovudine in a T-cell line, CEM, and enhance our understanding of this important anti-HIV drug.

Dihydro-5-azacytidine (DHAC) is a hydrolytically stable congener of 5-azacytidine, which retains antileukemic activity against experimental leukemias. The biochemical pharmacology of DHAC was studied in tumor-bearing mice in order to elucidate the mode of action of this drug. We found that after an LD10 dose of DHAC, the plasma peak concentration achieved was 317 microM and was eliminated biexponentially, with a t1/2 alpha of 1.03 h and a t1/2 beta of 5 h. By 4 h, an unidentified metabolite of [3H]DHAC peaked and was eliminated biexponentially, with a t1/2 alpha of 1.06 h and a t1/2 beta of 10.6 h. [3H]DHACTP was the major anabolite in the L1210/0 cells, and was also eliminated biexponentially, with a t1/2 alpha of 4.3 h and a t1/2 beta of 12.2 h. An unknown anabolite of [3H]DHAC that eluted 5 min after [3H]DHACTP, between UTP and ATP, peaked at 3 h and could possibly be the deoxy-derivative [3H]DHAdCTP. A tissue distribution study revealed that the liver, L1210/0, and lung accumulate the most radioactivity per gram of wet tissue. Methylation studies showed that an LD10 dose of [3H]DHAC resulted in a 25.06% hypomethylation of DNA in L1210/0 cells and a 46.32% hypomethylation in a deoxycytidine kinase mutant cell line L1210/dCK(-), compared with their respective controls.