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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

Background Epidemiologic studies find sex-based differences in incidence, survival, and long-term outcomes for children with cancer. The purpose of this study was to determine whether male and female patients differ with regard to acute treatment-related toxicities. Procedures We reviewed data collected on the Children's cancer group (CCG) high-risk acute lymphoblastic leukemia (ALL-HR) study (CCG-1961), and compared male and female patients' toxicity incidence and related variables in the first four phases of treatment. Similar analyses were performed with standard-risk ALL (ALL-SR) patients enrolled in CCG-1991. Results Among ALL-HR patients, females had significantly more hospital days, delays in therapy, grade 3 or 4 toxicities (e.g., gastrointestinal, liver), and supportive care interventions (e.g., transfusions, intravenous antibiotics) than males. Females were significantly more likely to have died of treatment-related causes than males (Hazard ratio = 2.8, 95%CI = 1.5–5.3, P = 0.002). Five months after beginning the treatment, the cumulative incidence of treatment-related deaths was 2.6% for females and 1.2% for males. Similar disparities were found among ALL-SR patients, with females experiencing significantly more hospital days and treatment-related toxicities than males. Conclusions This study complements cancer survivorship studies that also report an increase in treatment-related late effects among females. Risk profiles appear to be different for male and female patients, with females having greater risk of developing both acute and long-term treatment-related toxicities. The underlying biological mechanisms for these sex differences are poorly understood and warrant further study in order to determine how sex-based outcome disparities can be addressed in future clinical trials and practice. Pediatr Blood Cancer 2015;9999:1–10. © 2015 Wiley Periodicals, Inc.

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 poorer outcome from many cancers, including leukemia. One possible contributor to this could be suboptimal chemotherapy dosing in obese patients. We have previously found that vincristine (VCR) is less effective in obese compared to non-obese mice with leukemia, despite weight-based dosing. In the present study, we administered (3)H-VCR to obese and control mice to determine whether obesity would cause suboptimal VCR exposure. Blood VCR concentrations were fitted with a three-compartment model using pharmacokinetic analysis (two-stage PK) in three subsets of VCR concentrations vs. time method. Tissue and blood VCR concentrations were also analyzed using non-compartmental modeling. Blood VCR concentrations showed a triexponential decay and tended to be slightly higher in the obese mice at all time-points. However, the t(1/2,beta) and t(1/2,gamma) were shorter in the obese mice (9.7 min vs. 44.5 min and 60.3h vs. 85.6h, respectively), resulting in a lower AUC(0-infinity) (13,099 ng/m Lh vs. 15,384 ng/mL h). Had the dose of VCR been "capped", as is done in clinical practice, the AUC(0-infinity) would have been 36% lower in the obese mice than the controls. Tissue disposition of VCR revealed a biexponential decay from spleen, liver, and adipose. Interestingly, VCR slowly accumulated in the bone marrow of control mice, but had a slow decay from the marrow in the obese mice. Thus, obesity alters VCR PK, causing a lower overall exposure in circulation and bone marrow. Given the high prevalence of obesity, additional PK studies should be performed in obese subjects to optimize chemotherapy dosing regimens.

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.

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.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.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 .0001) or PEG ASNase (odds ratio, 3.08; P .0001) only during phase 1 of interim maintenance. There was no significant difference in 5-year event-free survival (EFS) between patients who received PEG ASNase throughout the entire study postinduction versus those who developed an allergic reaction to PEG ASNase during consolidation phase and subsequently received Erwinia ASNase (80.8% ± 2.8% and 81.6% ± 3.8%, respectively; P = .66). Patients who had positive antibody titers postinduction were more likely to have an allergic reaction to PEG ASNase (odds ratio, 2.4; P .001). The 5-year EFS rate between patients who had negative versus positive antibody titers (80% ± 2.6% and 77.7% ± 4.3%, respectively; P = .68) and between patients who did not receive any ASNase postconsolidation and those who received PEG ASNase throughout the study (P = .22) were significantly different.The current results demonstrate differences in the incidence rates of toxicity between ASNase preparations but not in EFS. The presence of anti-ASNase antibodies did not affect EFS.

Epidemiologic studies find sex-based differences in incidence, survival, and long-term outcomes for children with cancer. The purpose of this study was to determine whether male and female patients differ with regard to acute treatment-related toxicities.We reviewed data collected on the Children's cancer group (CCG) high-risk acute lymphoblastic leukemia (ALL-HR) study (CCG-1961), and compared male and female patients' toxicity incidence and related variables in the first four phases of treatment. Similar analyses were performed with standard-risk ALL (ALL-SR) patients enrolled in CCG-1991.Among ALL-HR patients, females had significantly more hospital days, delays in therapy, grade 3 or 4 toxicities (e.g., gastrointestinal, liver), and supportive care interventions (e.g., transfusions, intravenous antibiotics) than males. Females were significantly more likely to have died of treatment-related causes than males (Hazard ratio = 2.8, 95%CI = 1.5-5.3, P = 0.002). Five months after beginning the treatment, the cumulative incidence of treatment-related deaths was 2.6% for females and 1.2% for males. Similar disparities were found among ALL-SR patients, with females experiencing significantly more hospital days and treatment-related toxicities than males.This study complements cancer survivorship studies that also report an increase in treatment-related late effects among females. Risk profiles appear to be different for male and female patients, with females having greater risk of developing both acute and long-term treatment-related toxicities. The underlying biological mechanisms for these sex differences are poorly understood and warrant further study in order to determine how sex-based outcome disparities can be addressed in future clinical trials and practice.

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.

To determine the maximum tolerated dose (MTD) and assess the toxicity profile and pharmacokinetics of weekly gemcitabine infusions in pediatric patients with refractory hematologic malignancies. Fourteen patients under 21 years old were given infusions of gemcitabine for escalating durations at 10 mg/m2/min weekly for three consecutive weeks. Two males and two females were studied at each dose level. Pharmacokinetics of the drug's metabolism were measured by high pressure-liquid chromatography (HPLC) for 24 h after the first dose. Intracellular difluorodeoxycytidine triphosphate formation in leukemic blasts was measured in selected patients. The MTD of gemcitabine in these patients was 3600 mg/m2/week for three consecutive weeks (10 mg/m2/min for 360 min). Hepatotoxicity was the dose limiting toxicity. Thirty to fifty percent of patients exhibited fever, rash, or myalgia. Rare instances of hypotension and pulmonary toxicity were observed. Two of six patients [one acute lymphoblastic leukemia (ALL) and one acute myelogenous leukemia (AML)] treated at the MTD had at least M2 marrows, although peripheral blood counts did not recover sufficiently for the patients to be considered in complete response. Pharmacokinetics of gemcitabine fit a two-compartment open model with terminal half-life and plasma clearance value of 62 min and 2.2 l/min/m2, respectively. No gender differences were observed. In conclusion, the MTD of gemcitabine was 10 mg/m2/min for 360 min every week for 3 weeks. This is the recommended phase II dose schedule for children with leukemia. The activity of the drug at this schedule in heavily pretreated, refractory patients warrants a phase II trial in hematologic malignancies.

Asparaginases develop innovative “tumor starvation” conditions for all antileukemia treatments; however, administrations are limited by the toxicities of this drug. Patients exhibiting moderate toxicity have optimal treatment outcomes. Certain asparagine synthetase polymorphisms may contribute to severe host toxicities in divergent subsets of patients, whereas others do not. Clinical correlations should be evaluated. Clin Cancer Res; 21(2); 230–2. ©2014 AACR. See related article by Tanfous et al., p. 329

In this issue of Blood, Chan et al report on an innovative study using molecular dynamics and mapping to identify and produce a promising bioengineered clone of Escherichia coli asparaginase (ASNase), mutated at Q59L position (Gln59Leu). The Q59L variant ASNase has undetectable glutaminase coactivity, while retaining its ASNase activity.1

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.

Protein phosphatase 1 (PP1) plays important roles in many different aspects of cellular activities including cell cycle control. One important function of PP1 is to activate the retinoblastoma protein pRB. Here we show that pRB is one of PP1's downstream targets during apoptosis. When HL-60 cells synchronized at the G1/S boundary were treated with pro-apoptotic cytosine arabinoside (araC), PP1alpha protein increased twofold and PP1 activity about 30% within 1 h. This was followed by pRB dephosphorylation, pRB cleavage by caspases, DNA fragmentation, the appearance of cells with

PURPOSE The Children's Cancer Group (CCG) undertook a phase I study (CCG-0922) to determine a tolerable dose of idarubicin given with fludarabine and cytarabine in children with relapsed or refractory leukemia. The phase I study was extended to a limited phase II study to assess the activity of this combination in children with acute myelogenous leukemia (AML). PATIENTS AND METHODS This was a multiinstitutional study within the CCG. Eleven patients were entered onto the phase I study: seven with AML, three with acute lymphoblastic leukemia (ALL), and one with chronic myelogenous leukemia (CML). The maximal-tolerated dose (MTD) of fludarabine and cytarabine determined in a previous study was a fludarabine loading dose (LD) of 10.5 mg/m2 followed by a continuous infusion (CI) of 30.5 mg/m2/24 hours for 48 hours, followed by cytarabine LD 390 mg/m2, then CI 101 mg/m2/h for 72 hours. Idarubicin was given at three dose levels: 6, 9, and 12 mg/m2 intravenously (I.V.) on days 0, 1, and 2. The phase II portion of the trial included 10 additional patients with relapsed or refractory AML. RESULTS A dose of idarubicin 12 mg/m2/d for 3 days given in combination with fludarabine and cytarabine was tolerated. The major toxicity encountered was hematologic. Nonhematologic toxicities included transaminase elevations, hyperbilirubinemia, and infections. Eight of 10 patients with AML in the phase II portion (12 mg/m2 idarubicin) achieved a complete remission (CR). CONCLUSION This combination is active in patients with relapsed or refractory AML. The major toxicity encountered is hematologic. This regimen may be useful therapy for AML and should be compared with standard induction therapy in children with newly diagnosed AML.

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.

Obesity is a significant risk factor for cancer. A link between obesity and a childhood cancer has been identified: obese children diagnosed with high-risk acute lymphoblastic leukemia (ALL) had a 50% greater risk of relapse than their lean counterparts. l-asparaginase (ASNase) is a first-line therapy for ALL that breaks down asparagine and glutamine, exploiting the fact that ALL cells are more dependent on these amino acids than other cells. In the present study, we investigated whether adipocytes, which produce significant quantities of glutamine, may counteract the effects of ASNase. In children being treated for high-risk ALL, obesity was not associated with altered plasma levels of asparagine or glutamine. However, glutamine synthetase was markedly increased in bone marrow adipocytes after induction chemotherapy. Obesity substantially impaired ASNase efficacy in mice transplanted with syngeneic ALL cells and, like in humans, without affecting plasma asparagine or glutamine levels. In coculture, adipocytes inhibited leukemic cell cytotoxicity induced by ASNase, and this protection was dependent on glutamine secretion. These findings suggest that adipocytes work in conjunction with other cells of the leukemia microenvironment to protect leukemia cells during ASNase treatment. Cancer Res; 73(10); 2998–3006. ©2013 AACR.

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.

This is an ambitious effort attempting to present as many aspects as possible in a review article on asparaginases (ASNase), and their use against acute lymphoblastic leukemia (ALL) and T-cell lymphomas. In the process, the modes of drug resistance are described both of the host and in the leukemia cells themselves. These modes of drug resistance, developed by the ALL cells, are an attempt to overcome the toxic insult this class of anti-leukemic drugs causes to them. It is expected that by reading this article one would obtain a better understanding of the initial events in the leukemia development, its microenvironment, and the many issues that a leukemia specialist has to deal with, especially in the treatment of refractory and relapsed patient populations. The specific issues addressed in this review deal with the importance of nutrients in tumor growth and progression of malignancies; the cytogenetics of ALL, as well as its chemotherapy, are also briefly presented. The emphasis will turn to ASNase, their mechanisms of action, the immune responses they cause in a significant percentage of the ALL patients, the significance of the up-regulation of glutamine synthetase and asparagine synthetase and the complexity of the elucidation of the mechanisms of action of ASNase. Additional details on the ASNase epitope mapping of anti-ASNase antibodies, the degradation of the protein, and the unmet needs in producing an optimal ASNase protein, will be also presented. Finally, a brief description of the toxicity, as well as the correlative factor of ALL treatment with ASNase is given.

Selective combinations of purine and pyrimidine analogs increase remission rates in pediatric patients with relapsed leukemias. The combination of 6-mercaptopurine (6-MP) and cytosine arabinoside (ara-C) may exhibit synergism similar to that observed for fludarabine and ara-C and may diminish the potential for development of resistance since the two drugs are activated by separate enzymatic pathways. To determine the efficacy of the combination against human leukemia cells, we investigated the time-concentration relationships of the drugs given alone or in combination to the resultant cytotoxicity. To determine whether the combination leads to enhanced activity of deoxycytidine kinase (dCk), the rate-limiting enzyme in ara-C activation, we characterized the cellular dCk in CCRF/CEM/0, CCRF/CEM/ara-C/7A, and CCRF/CEM/ara-C/3A monoclonal cells before and after treatment with 6-MP. CCRF/CEM/0 (wild type), CCRF/CEM/ara-C/7A (approximately 50% ara-C-resistant as determined by ara-C sensitivity assay and dCk characterization), and CCRF/CEM/ara-C/3A (approximately 90% resistant to ara-C) human leukemia cells were incubated with various concentrations of 6-MP and ara-C given alone or in combination. Cell survival, inhibition of DNA synthetic capacity (DSC), ara-CTP anabolism, and dCk enzymatic characteristics were studied. Incubation of CEM/0 cells with 6-MP for 24 h, followed by ara-C for 48 h, increased cell-growth inhibition by approximately 0.5-1 log10, corresponding to 5- to 10-fold synergism, as compared with ara-C alone after identical drug incubation in all cell lines. Simultaneous administration showed no synergism, whereas reversal of the sequence produced an antagonistic effect. The ara-CTP levels were 2- to 3.5-fold and 3- to 5-fold higher in CEM/0 and CEM/ara-C/7A cells, respectively, in cells exposed to 6-MP followed by ara-C than in those exposed to ara-C alone at the same concentrations. Furthermore, a progressive increase in ara-CTP levels was noted in CEM/0 cells exposed to increasing concentrations of 6-MP followed by 10 or 20 microM ara-C. A significant decrease in DSC was observed upon treatment of wild-type and ara-C-resistant cells with 6-MP and ara-C. The combination of 6-MP and ara-C exhibits significant sequence-specific synergism in both wild-type and partially ara-C-resistant leukemia cell lines. The combination also exerts collateral sensitivity in the ara-C-resistant cell lines. 6-MP pretreatment may play a role in enhancing ara-C activation, thus producing drug synergism in sensitive and resistant leukemia cell lines.

Objective. Zidovudine and didanosine are both beneficial for the treatment of human immunodeficiency virus (HIV) infection in children. Because disease progression and toxicity often limit their long-term use as single agents, new approaches to using nucleoside analogues are necessary to improve current antiretroviral therapy. Design. We conducted a phase I-II study to evaluate the tolerance, pharmacokinetics, and antiviral activity of the combination of zidovudine and didanosine in children with HIV infection. Sixty-eight children who were either previously untreated or who had manifested hematologic toxicity on full-dose zidovudine were enrolled. Eight dose combinations were studied in the previously untreated children, with doses of zidovudine ranging from 90 to 180 mg/m2 every 6 hours and doses of didanosine ranging from 90 to 180 mg/m2 every 12 hours. Results. Fifty-four previously untreated HIV-infected children were enrolled in this part of the study, of whom 49 remained in the study for a minimum of 24 weeks. For children with previous zidovudine-related hematologic toxicity, three dose levels with zidovudine at 60 mg/m2 every 6 hours orally and didanosine ranging from 90 to 180 mg/m2 every 12 hours orally were used. A total of 14 children were enrolled in this part of the study, and 12 remained on therapy for at least 24 weeks. No evidence of new or enhanced toxicity was observed in either group. After 24 weeks, the median CD4 cell count for all patients increased from 331 to 556 cells/mm3 (P = .01). For the previously untreated group, the median increase in CD4 counts was from 386 to 726 cells/mm3 (P = .003). The median p24 antigen concentration (in those with a detectable level at baseline) decreased from 95 to Conclusions. The combination of zidovudine and didanosine was well-tolerated at doses as high as those used in single agent therapy. Potent in vivo antiviral activity was observed. Combination therapy with nucleoside analogues may be an important approach to optimizing the use of these agents in the treatment of HIV infection.

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.

Therapeutic uses of asparaginases (ASNase) have been shown to elicit immune responses resulting in the development of potentially life-threatening human anti-bacterial antibodies (Ab). A robust screening enzyme-linked immunosorbent assay (ELISA) to detect binding Ab(+) against ASNase has been developed and validated for therapeutic monitoring to support clinical trials. Recently, a protein chip bioassay (Biacore) was developed for the Ab of these proteins. These methods were compared.A Biacore T-100 analyzer using a protein bioassay and an ELISA assay were used to determine the IgG immmuboglobulin Ab against ASNase in sera from 84 acute lymphoblastic leukemia (ALL) patients plus 6 controls (n=121 samples). These samples were characterized for anti-ASNase Ab neutralizing activity. Human E. coli ASNase, pegaspargase and Erwinase proteins were covalently coupled to the carboxy-methylated dextran matrix of a CM5 sensor chip (surface plasmon resonance, SPR). In the course of a nested experimental design, a wide range of human sera from patients who had obvious clinical allergic reactions after either native or pegaspargase treatments were tested. The data were fitted by a parametric logistic equation (+/-95% confidence interval, CI), which ranged from3.0% to14%.The specificity of Ab(+) was evaluated using "spiked" human IgG antibodies. Both assays provide near excellent linearity and sensitivity of response (0.8 to500 ratio and 1-3000 resonance units [RU]) of anti-ASNase Ab in human sera with low variance. The bioassay method was ten times more sensitive than the ELISA Ab assay. The lowest limit of quantification of Ab(+) ratio for the SPR assay was 0.6 whereas the upper limit of quantification was 3000 RU. The SPR assay results were in excellent accord with both the Ab(-) and the Ab(+). Ab(-) by the ELISA method (1.003 ratio) was related to a mean RU value of 8.1. Despite the narrow range of ambiguity around the 1.1 Ab(+) ratio values, the majority of the specimens (93.2%) were determined to be Ab(+) by either ELISA or SPR determination.The vast majority (81/84 = 96.4%) of the IgG Ab(+) were neutralizing. The SPR Ab determination technique is reliable, accurate and more sensitive than the ELISA method.

Computer software NONMEM for nonlinear mixed-effects modeling was used to determine the population pharmacokinetics (PPK) and pharmacodynamics (PPD) of intravenously administered nucleoside analogs cytosine arabinoside (ara-C) and fludarabine phosphate (F-ara-A) in pediatric patients with leukemias. A description of the major NONMEM tasks as well as the statistical models has been given for these drugs. Two pharmacokineticpharmacodynamic (PK-PD) models have been developed for ara-C and one for F-ara-A. These NONMEM population analyses present a novel approach for determining the PK parameters in many patients; these parameters are superimposable to the ones derived from the classical two-stage PK approach. However, by minimizing errors, population analyses explained the significant variabilities in the elimination of these drugs from the central compartment and the variable high peak plasma concentrations in younger leukemia patients. Moreover, the influence of the covariate product (TR AGE × SA) demonstrated an increase in the PK parameter values with increasing patient age (AGE) and surface area (SA). The evaluation of the intercompartmental clearance of ara-C demonstrated statistically significant linear relationships with the covariate product (AGE × SA). These results explained the lower drug plasma concentration in the older children as compared to the infant and younger patients with leukemias. In addition to the classical population pharmacokinetic and population pharmacodynamic parameters of half-lives of elimination, clearance, and volumes of distribution, these analyses in pediatric patients determined the effects of deamination by cytidine deaminase on the clearance of ara-C from the circulation. Moreover, the K m of activation of these drugs to their respective phosporylated anabolites in patients, and under treatment conditions, was determined by the Michaelis-Menten equation, which was fused into the INPUT PK-PD subroutine. Overall, NONMEM population PK-PD analyses are complicated and time consuming and require a rich database from many patients. However, they produce the best possible PK-PD analyses for a drug and its metabolites, the parameters of which give better understanding of the biochemical pathways in plasma and in leukemic blasts simultaneously, which can then be applied to many other patients in future clinical trials.

The discovery of the tumor-inhibitory properties of asparaginase (ASNase) began in the early 1950s with the observation that guinea pig serum-treated lymphoma-bearing mice underwent rapid and often complete regression. About 4000 cases of acute lymphoblastic leukemia (ALL) are diagnosed very year in the US and many more through out the world. The majority of these cases are in children and young adults, making ALL the most common form of malignancy in these age groups. The treatment protocols of ALL are complex and use 6–12 drugs. Consequently, the improvement in the protocol design has improved significantly the success rate for long-term event-free survival in the past 20–30 years, which is now approximately 75% for patients afflicted with the higher risk ALL features and just above this percentage for patients with standard or good features. Despite this success, approximately 15% of patients die from ALL, making leukemic relapse the most common cause of treatment failure in pediatric oncology. ASNases have been the cornerstone of ALL therapies since the late 1970s. Native or pegylated L-asparaginase (ASNase or PEG-ASNase) are highly specific for the deamination of L-asparagine (Asn) to aspartic acid and ammonia. Depletion of Asn leads to a nutritional deprivation and inhibition of protein biosynthesis, resulting in apoptosis in T-lymphoblastic leukemias, which require Asn from external sources. The reactions of the host exposed to repeated ASNase treatments as well as the up-regulation of the mammalian enzymes to overcome the ASN-depletion toxic condition are of significant importance and may make us relearn the lessons on this important antileukemic drug.

Asparaginases are the cornerstone therapy of many successful combination regimens for the treatment of acute lymphoblastic leukemia (ALL), the most common malignancy in children and adolescents. Currently, two asparaginase formulations are available in the US, native Escherichia coli asparaginase (ASNase) and pegaspargase. A third formulation native Erwinia asparaginase (Erwinase, ERW) has recently been made available under a licensing exception for personal use. We report here the development and validation process of ERW pharmacoanalytical assays and the results in a few patients.We developed and systematically validated the ERW enzyme activity and ERW concentration, anti-ERW antibody and related assays. Pharmacokinetic and pharmacodynamic (PK-PD) studies were performed in a limited number of patients who received 6,000 IU/m2 x 3 per week x 2 courses, and 4 patients who received 25,000 IU/m2 x 3 per week x 2 courses of ERW.The linearity and range of the Erwinase calibration lines for the pharmacoanalytical assays were excellent. The accuracy and precision were better than the FDA limit allows for oncology biological products (30%) coefficient of variation (%CV) and related parameters in the quantification of ERW concentration. The validation of these parameters was equal to or better than during the assay development. PK-PD analyses of ERW in a few patients yielded an average half-life of elimination of 15.8+/-1.64 hours. There was an excellent PD response post ERW administration resulting in an ERW concentration-dependent asparagine (ASN,0.5 microM) and glutamine (GLN,50 microM) deamination. Pharmacodynamic correlations demonstrated that 0.1 to 0.2 IU/ml of ERW in serum were sufficient for 90% GLN and/or ASN deamination for up to 2 weeks. No anti-ERW antibody [Ab(+)] was seen among those few patients. None of the other 5 patients had an adverse event. Based on these post hoc results, simulations on various doses and schedules of this drug have been made.The pharmacoanalytical assays were excellent tools to evaluate the PK and PD data of ERW in pediatric patients with HR ALL. However, this initial PK-PD evidence needs further validation in future clinical trials. Insights into the PD contributions of ERW in anti-E. coli ASNase Ab(+) patients will guide us in optimal design and use of ERW as part of combination chemotherapy regimens in future clinical trials.

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.

Glutamine (Gln) deamination by asparaginase (ASNase) appears to contribute in the decrease of serum asparagine (Asn) levels and enhance leukemic cell apoptosis. The pharmacodynamic (PD) rationale is based on the role of Gln as the main amino group donor for Asn synthesis from aspartate by the enzyme asparagine synthetase (AS).Relationships between ASNase enzymatic activity and Asn or Gln levels were examined in 274 pairs of pre- and post-ASNase serum specimens from 200 high-risk acute lymphoblastic leukemia (ALL) patients from the Children's Cancer Group (CCG-1961). Data were analyzed according to a novel PD model based on previous best-fit projections (NONMEM) from the CCG-1962 standard-risk ALL study.The PD results from high-risk and standard-risk ALL patients were superimposable. The percentages of Asn and Gln deamination were predicted by ASNase activity in patients' sera. Pharmacodynamic analyses strongly suggested that90% deamination of Gln must occur before optimal Asn deamination takes place in vivo. Asparaginase activityor = 0.4 IU/ml yielded mean Gln and Asn % deamination values of 90%. Lower ASNase concentrations yielded lower Gln or Asn % deamination. This ASNase concentration coincides with the in vitro determined IC50 value on CEM/0 human T-lymphoblastic leukemia cells.Asparaginase activity ofor = 0.4 IU/ml provided optimal Asn and Gln deamination in high-risk ALL patients. Deamination of Gln correlates with enhanced serum Asn deamination in vivo. Therefore, deamination of Gln may enhance the antileukemic effect of ASNase.

Deamination of asparagine (Asn) and glutamine (Gln) by asparaginases (ASNase) is associated with good prognosis in acute lymphoblastic leukemia (ALL). Chemotherapy drugs used for ALL may accelerate catabolism of other amino acids (AA).We studied ASNase activity and changes of Asn, Gln, serine (Ser), threonine (Thr), histidine (His), proline (Pro) and arginine (Arg) levels and sought relationships in sera from 73 pediatric ALL patients, who received ASNase-containing chemotherapy.Asparaginase activity averaged 0.4+/-0.34 IU/ml (mean+/-SDEV) in all specimens. All AA decreased after treatment, ranging from 18.6%-82.6% of control. Asparaginase activity of 0.7 IU/ml provided 90% Asn and Gl deamination. The data were dichotomized in subsets of low ASNase (range 0.02-0.39 IU/ml, mean=0.17+/-0.09 IU/ml) and high ASNase (range 0.4-1.69 IU/ml and mean=0.72+/-0.32 IU/ml). Asparagine and Gln % deamination values were correlated with ASNase activity (p=0.0002 and p=0.0001). Similarly, decreases of Arg and Ser levels were also correlated, p =0.0009 and p=0.032, respectively. In the high ASNase subset, a 39% decrease of Arg and 26% of Ser was obtained. Low ASNase activity was correlated with lower Asn and Gln % deamination and with moderate decrease of Ser (14.6%) and Arg (19.6%). Threonine, Pro and His also decreased, but no correlations were obtained with ASNase activity.Asparagine, Gln and five other AA declined during ASNase treatment. Asparagine and Gln % deamination values are highly correlated with serum ASNase activity. Asparaginase may indirectly cause moderate depletion of serum Arg and Ser levels, providing an enhancement in leukemia blasts apoptosis. Toxicity from the ASNase and other drugs could enhance the decrease of AA serum levels. Further studies are needed to verify these findings and their potential clinical importance in the treatment of ALL patients.

The primary growth factor receptors involved in angiogenesis and lymphomagenesis can be grouped into the vascular endothelial growth factor (VEGF) receptors and related families. Inhibition of VEGF and other growth factors, including c-Abl, c-Kit, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and insulin-like growth factor (IGF), or their receptors containing tyrosine kinase domains by antiangiogenesis drugs disrupts cell survival signal transduction pathways and may contribute to the proapoptotic pathways in malignant cells. However, clinical trials suggest that signal transduction inhibitors have considerable antitumor activity when used as single agents only for a short time, most likely due to the development of drug resistance by the host or by the tumor cells. In order to prevent this problem and to augment their antitumor efficacy, these agents could be administered in combination with cytotoxic antineoplastic drugs. We hypothesized that the combination of the antiangiogenesis tyrosine kinase inhibitors with cytotoxic drugs would produce synergistic drug regimens. Two human T-lymphoblastic leukemia cell lines that express VEGF-R1, CEM/0 (wild-type, WT) and the drug-resistant clone CEM/ara-C/I/ASNase-0.5-2, were utilized in the drug combination studies. NSC 680410, a tyrosine kinase inhibitor given at 0.1 to 1 microM for 72 h, inhibited VEGF secretion and leukemic cell growth at 90% of vehicle-treated control cultures with an IC50 value of less than 1 microM. The cytotoxic drugs idarubicin (IDA), fludarabine (Fludara), and cytosine arabinoside (ara-C) were used for the various drug combinations. One-, two-, three-, and four-drug treatments were tested. Cell viability was documented by the MTT assay and photomicrographic estimation of apoptotic cells. Both the combination index (CI) and isobologram evaluations demonstrated strong synergism between these drugs and the tyrosine kinase inhibitor. NSC 680410 was highly synergistic with IDA, IDA + ara-C, and IDA + Fludara + ara-C, over the respective cytotoxic drug regimens at concentrations easily achieved in patient plasma. NSC 680410 potentiated the activity of IDA in both leukemia cell lines by 17.8- and 221.4-fold in the WT and drug-resistant line, respectively. The activity of NSC 680410 + IDA + ara-C was also potentiated by 58.8-fold in the WT line, and the activity of NSC 680410 + IDA + Fludara + ara-C by 2.4- and 6.47x10(6)-fold in the WT and drug-resistant lines, respectively. The results suggest that IDA was not needed for optimal synergistic activity in the CEM/0 cells, but IDA was a necessary component to obtain drug synergism in the drug-resistant clone. Similarly, STI571 (imatinib mesylate, Gleevec), the p210(bcr/abl) tyrosine kinase inhibitor, demonstrated synergism with Fludara + ara-C or IDA + ara-C. Most importantly STI571 showed synergism with NSC 680410, suggesting that these drugs inhibit different tyrosine kinase domains in human leukemia cells. Lastly, pretreatment of leukemic cells with NSC 680410 showed additivity with gamma radiation in comparison to either treatment modality alone. The data, taken together, suggest that by inhibiting the pro-survival signal transduction pathway (VEGF-R1) and DNA replication by cytotoxic drugs, leukemic cells undergo apoptosis in a synergistic manner. In conclusion, the combinations of antiangiogenesis and DNA-damaging cytotoxic drugs are highly synergistic regimens in both WT and drug-resistant leukemic cell lines and they should be examined further.

Determination of the frequency of antibody formation during first and second exposure to Erwinia asparaginase after i.v. and i.m. administration.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/m(2) 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/m(2) 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.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.The formation of antibodies and subsequently altered pharmacokinetics of Erwinia asparaginase seemed to be of importance only during a second period of asparaginase therapy.

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.

Purpose.NSC 680410, the novel adamantyl ester of AG957, an inhibitor of the p210bcr/abl tyrosine kinase (CML, Ph+) and possibly other kinases, was tested for antitumor activity in ten human leukemia and human glioblastoma cell lines. Methods. CEM/0, seven ara-C- and/or ASNase-resistant clones, Jurkat/0, the myelomonocytic line U937 and U87 MG glioblastoma cell lines were used for these studies. The drug-resistant leukemic clones lack p53, express bcl-2 and VEGF-R1, and thus are refractory to apoptosis. Since tyrosine kinases drive many proliferative pathways and these activities are increased in many leukemic cells, we hypothesized that NSC 680410 may induce cytotoxicity in drug-resistant leukemia clones, independently of p210bcr/abl expression. Results. NSC 680410 exhibited significant antileukemic activity in CEM/0, Jurkat E6-1, and in the drug-resistant leukemic cell lines. The IC50 values in nine leukemia lines ranged from 17 to 216 nM. Western blot analyses after NSC 680410 treatment demonstrated caspase-3 cleavage and ELISAs showed a fivefold upregulation of its activity in cellular extracts. In addition, U87 MG human glioblastoma cells, which express VEGF-R1, were treated with the Flt-1/Fc chimera, a specific inhibitor of VEGF, and showed 30–43% cell kill in the MTT assay. Furthermore, the combination of NSC 680410 plus Flt-1/Fc chimera demonstrated an eightfold synergism against U87 MG cells in vitro. To verify this observation in vivo, athymic mice were inoculated orthotopically into the caudate putamen with 106 U87 MG cells. On day 3, five mice per group were treated i.p. with either 8.3 mg/kg NSC 680410 daily for three doses per week for 4 weeks alone or in combination with one dose of Flt-1/Fc chimera 100 mg/kg subcutaneously. Treatment with NSC 680410 alone produced no weight changes and increased the median survival to 133%, whereas treatment with NSC680410 plus Flt-1/Fc chimera increased survival to 142% over control. Control animals had large intra- and extracranial tumors while the NSC 680410-treated mice had small, only intracranial tumors with necrotic centers. The combination treatment resulted in small residual tumors around the needle track, indicating significant inhibition of tumor growth. Conclusions. These studies demonstrate that the tyrosine kinase inhibitor NSC 680410 has significant antileukemic activity in p53-null, drug-resistant human leukemia cell lines, as well as significant antitumor activity in combination with Flt-1/Fc chimera against U87 MG glioblastoma brain tumors implanted in situ in athymic mice.

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