Structure-function relationships and clinical applications of L-asparaginases.

L-asparaginase (L-ASNase, EC 3.5.1.1) catalyzes the hydrolysis of the non-essential amino acid L-Asn to LAsp and ammonia and is widely used for the treatment of haematopoetic diseases such as acute lymphoblastic leukaemia (ALL) and lymphomas. Therapeutic forms of L-ASNase come from different biological sources (primarily E. coli and Erwinia chrysanthemi). It is well established that the various preparations have different biochemical pharmacology properties, and different tendency to induce side-effects. This is due to different structural, physicochemical and kinetic properties of L-ASNases from the various biological sources. Understanding these properties of various L-ASNases would allow a better decipherment of their catalytic and therapeutic features, thus enabling more accurate predictions of the behaviour of these enzymes under a variety of therapeutic conditions. In addition, detailed understanding of the catalytic mechanism of L-ASNases might permit the design of new forms of L-ASNases with optimal biochemical properties for clinical applications. In this paper we review the available biochemical and pharmacokinetic information of the therapeutic forms of bacterial L-ASNases, and focus on a detailed description of structure, function and clinical applications of these enzymes.

Phase I study of gemcitabine (difluorodeoxycytidine) in children with relapsed or refractory leukemia (CCG-0955): a report from the Children's Cancer Group.

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.

Taxotere and vincristine inhibit the secretion of the angiogenesis inducing vascular endothelial growth factor (VEGF) by wild-type and drug-resistant human leukemia T-cell lines.

Recent studies have shown that angiogenesis, which is induced by VEGF, may be involved in the pathogenesis of hematopoietic malignancies. A human leukemia model consisting of T-lymphoblastic CEM/0, 7 monoclonal refractory clones resistant to both cytosine arabinoside (ara-C) and L-asparaginase (ASNase), Jurkat/E6-1 and U937, representing the leukemic blasts from relapsed patients with leukemias was investigated for secretion of VEGF before and after treatment with various agents. The T-lymphoblastic cell line, Jurkat/E6-1, was used as the negative control, which has been characterized as not expressing mRNA nor the VEGF protein, and did not secrete VEGF. With no treatment, U937, the positive control, secreted the highest VEGF concentration of 1612.7 pg/ml. The CEM/O wild type cell line and 5 other drug-resistant clones secreted VEGF at levels ranging from 180.9 to 414.2 pg/ml. Two CEM drug-resistant clones, CEM/ara-C/G/ASNase-0.5-1 and CEM/ara-C/G/ASNase-1-1, lacked VEGF production. Docetaxel (Taxotere, TXR), Vincristine (VCR), ASNase, and the Fit-1/Fc chimera, a specific inhibitor of VEGF-dependent human umbilical vein endothelial cell (HUVEC) proliferation, were tested for inhibition of VEGF secretion. Treatment of the leukemic cell lines with 2 microg/ml Flt-1/Fc chimera for 24 hours completely inhibited VEGF secretion to the detection limit of the assay (<10pg/ml). After 24 hours incubation with Flt-1/Fc chimera, the leukemic cells appeared to be undergoing apoptosis, based on microphotography examination, suggesting that VEGF could be used in an autocrine loop to promote cell survival by the leukemic cells. Treatment with 0.5, 1, and 2 microg/ml Flt-1/FC chimera for 48 hours demonstrated a 15-25% growth inhibition by MTT assay. Strong inhibition of VEGF secretion in the culture media was observed after 10 microM TXR or 0.1 microM VCR for 24 hours in the wild-type and drug-resistant clones, except CEM/ara-C/I, in comparison with controls. In contrast, treatment with 1 IU/ml ASNase, a specific T-cell protein inhibitor, in 5 cell lines for 24 hours demonstrated no inhibition of VEGF in CEM/0 3 drug-resistant clones and the myeloid U937 line. We conclude that the leukemia cell lines actively secrete VEGF, in vitro. TXR and VCR, but not ASNase, strongly inhibit the VEGF production, suggesting that inhibition of this growth factor may be a mechanism of antileukemic activity. Moreover, the leukemic cell lines examined here may constitute a useful model to study antiangiogenic drugs, alone or in combination with established drug regimens used against refractory leukemias.

Protein phosphatase 1alpha-mediated stimulation of apoptosis is associated with dephosphorylation of the retinoblastoma protein.

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 <2n DNA content and finally, dying and dead cells. In vitro, pRB was protected from caspase-3 digestion by prior Cdk-mediated phosphorylation, whereas PP1alpha converted phospho-pRB into an efficient substrate for caspase-3. Introduction of active PP1alpha into HL-60 cells by electroporation was sufficient to induce characteristics of apoptosis. Similarly, araC-resistant cells, normally unable to die in response to araC, initiated apoptosis when electroporated with active PP1alpha. This was also accompanied by pRB cleavage. In contrast, introduction of inhibitor-2 delayed the onset of araC-induced apoptosis, whereas concomitant introduction of PP1alpha and inhibitor-2 completely prevented PP1alpha-induced apoptosis. These results suggest that dephosphorylation of key proteins by PP1alpha may be crucial for the initiation of apoptosis and further support the concept of PP1 serving as a potential target for anti-cancer therapy.

Reversal of cytosine arabinoside (ara-C) resistance by the synergistic combination of 6-thioguanine plus ara-C plus PEG-asparaginase (TGAP) in human leukemia lines lacking or expressing p53 protein.

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.

Synergistic antiviral effect of PEG-asparaginase (ONCASPAR), with protease inhibitor alone and in combination with RT inhibitors against HIV-1 infected T-cells: a model of HIV-1-induced T-cell lymphoma.

We evaluated the anti-HIV-1 activity of the T-cell-specific protein inhibitor PEG-asparaginase (PEG-ASNase) in human HIV-1-infected T-cells. We further examined the drug synergism between PEG-ASNase and the protease inhibitor Saquinavir (SAQ), both alone and in combination with nucleoside analog reverse transcriptase inhibitors (NRTI). Our drug synergism studies served as a model for an HIV-induced T-cell lymphoma. Phytohemagglutinin [PHA(+)] stimulated T-cells were infected with HIV-1 and then treated with one or more drugs 90 minutes from the viral exposure. To measure inhibition of viral replication, we examined HIV-1 RT and HIV-1 RNA in the supernatant and intracellularly on day 7 post-infection and drug treatment. Last, we examined the effect of administering drugs immediately after HIV-1 infection of T-cells to simulate treatment after an accidental exposure to the virus. PEG-ASNase, even when used alone, has anti-HIV-1 activity in PHA(+)-stimulated T-cells due to inhibition of protein synthesis. When the drug was used with SAQ, the combination was synergistic in inhibiting HIV-1 RT and RNA in the supernatant and intracellularly by 2.5 log10 in comparison with controls. PEG-ASNase and SAQ were even more effective in inhibiting HIV-1 replication when combined with the NRTI inhibitors azidothymidine (AZT) and (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC, lamivudine). The addition of ribonucleotide reductase inhibitor, 2-methyl-1H-isoindole-1,3-dione (MISID), further potentiated the antiviral effect of the regimen. HIV-1 RT and RNA analyses showed that the administration of the PEG-ASNase + SAQ drug combination immediately following exposure to HIV-1 completely inhibited the infection of T-cells in our in vitro T-cell model. From these results we conclude that PEG-ASNase is a valuable T-cell-specific protein inhibitor against HIV-1 infection, when used singly or in combination with a protease inhibitor, an RT inhibitor and an RR inhibitor. Since PEG-ASNase is a drug of choice for the treatment of T-cell lymphomas, a combination regimen containing PEG-ASNase could be very effective in the treatment of HIV-1-induced T-cell lymphoma and possibly AIDS. Future studies are needed in HIV-infected and/or HIV-induced T-cell lymphoma patients to investigate these findings.

Development of a double-drug-resistant human leukemia model to cytosine arabinoside and L-asparaginase: evaluation of cross-resistance to other treatment modalities.

We have developed an in vitro model of 38 T-lymphoblastic leukemia lines resistant to cytosine arabinoside (ara-C) and L-asparaginase (ASNase). Of these, 26 cell lines resistant to both drugs, 6 resistant to ara-C, and 6 resistant to ASNase were isolated. In 18 of these cell lines, all randomly selected, resistance to ara-C, ASNase and gamma radiation was documented by the MTT and trypan blue assays, as well as flow cytometry with Annexin V and propidium iodide (PI) staining. In these lines, p53, p21WAF1, and bcl-2 levels were measured by ELISA. Results show that P21WAF1 upregulation following p53 induction did not occur, suggesting that p53 function may be lost. Moreover, the data imply that upregulation of bcl-2 is critical in the development of resistance to ara-C and ASNase in these leukemic lines. In the CEM/0 parent line, p53 maintained its ability to interact with its DNA binding site as documented by the electrophoretic mobility shift assay (EMSA). But in one single- and one double-resistant leukemic cell line examined, p53 was not shown to maintain this ability. We conclude that double-resistant clones to ara-C and ASNase are refractory to both drugs, providing an excellent leukemic model to investigate the multiple-drug resistance.

Comparison of intramuscular therapy with Erwinia asparaginase and asparaginase Medac: pharmacokinetics, pharmacodynamics, formation of antibodies and influence on the coagulation system.

Asparaginase comes from different biological sources and the various preparations have different pharmacokinetic properties, and their tendency to induce side-effects is different. Erwinia asparaginase (ASNase) has a shorter half-life than the Escherichia coli preparations, and it has been reported to be less immunogenic than the E. coli preparations and to induce fewer coagulation disorders. Children with newly diagnosed acute lymphoblastic leukaemia (ALL) were included in this study. Twenty-seven patients were treated with Erwinia ASNase (induction therapy 30.000 IU/m2/d i.m. for 10 d, and re-induction therapy 30.000 IU/m2 twice a week for 2 weeks) and 15 were treated with ASNase Medac (induction therapy 1.000 IU/m2/d i.m. for 10 d, and re-induction therapy 5.000 IU/m2 i.m. twice a week for 2 weeks). Blood samples were drawn to determine enzyme activity, l-asparagine, anti-asparaginase antibodies, and coagulation parameters. After i.m. administration, Erwinia ASNase displayed a protracted absorption phase compared to ASNase Medac. The mean bioavailability after i.m. administration was 27% for Erwinia ASNase and 45% for ASNase Medac respectively. Mean trough enzyme activities during induction therapy were Erwinia ASNase 1748 IU/l and ASNase Medac 272 IU/l, and during re-induction therapy Erwinia ASNase 83 IU/l and ASNase Medac 147 IU/l. We conclude that in this setting, therapy with ASNase Medac resulted in sufficient treatment during both phases of therapy, whereas treatment with Erwinia ASNase resulted in unnecessarily intense therapy during the induction phase and insufficient treatment during the re-induction phase. There was no significant difference in the incidence of antibody formation, and therapy with Erwinia ASNase resulted in a more pronounced influence on the coagulation parameters than therapy with ASNase Medac.

Pharmacodynamic studies (PD) of didanosine (ddI) alone and in combination with azidothymidine (AZT) in human T-cells; a stochastic biochemical approach to antiretroviral nucleoside drug combination in inhibiting HIV-reverse transcriptase (RT).

Didanosine (ddI) is used in the treatment of HIV-1 infection alone and in combination with azidothymidine (AZT). When combined with AZT, patients exhibit improved patterns of surrogate markers after sequential combination regimens of ddI and AZT compared to either drug monotherapy. We have investigated the biochemical mechanism(s) of this synergistic drug combination in human PBMC cells and in human T-cell lines sensitive and resistant to AZT due to lack of thymidine kinase (TK). DdI is preferentially activated to its triphosphate anabolite, ddATP, at 3:1 ratio in human T-lymphocytes compared to monocytes from the same individual. There are no apparent differences in the intracellular concentrations of ddATP in Jurkat/0 and Jurkat/AZT-10, an AZT resistant human T-cell line, when ddI is administered alone or in combination with AZT, hence there appears to be a case of collateral sensitivity. Intracellular increases of AZTTP concentrations in patient's PBMC cells have been determined clinically after AZT alone and in a combination regimen with ddI. A stochastic biochemical model has been developed that estimates the velocity of HIV-RT under uninhibited and inhibited conditions by the active anabolites, AZTTP and ddATP. This model provides a rational explanation for the greater inhibition of HIV-RT in the presence of both inhibitors, AZTTP and ddATP, as compared to the presence of either anabolite triphosphate alone. Expanding this model to describe the inhibition of HIV-RT in the presence of three competitive inhibitors, AZTTP, ddATP and 3TCTP demonstrated that the presence of these HIV-RT inhibitors resulted in an even greater inhibition of this viral enzyme necessary for HIV integration and replication. Hence, a more effective inhibition of HIV-RT enzyme is achieved by the combination of the three drugs, AZT, ddl and 3TC. In an effort to verify this model with experimental data the kinetics of HIV-RT were studied in the absence and after inhibition by AZTTP or ddATP alone, both AZTTP + ddATP or AZTTP + ddATP + 3TCTP. Treatment of HIV-RT with high concentrations of these triphosphate inhibitors, as high as 3Kis, inhibited this enzyme to greater than 90% of untreated control. However, a small percentage of residual HIV-RT, 6%, was uninhibited even after exposure to 3Ki concentrations of each inhibitor. These studies strongly suggested that: 1) AZT plus ddI or AZT plus ddI plus 3TC are synergistic at the active anabolite level against HIV-RT; 2) the combination of the three nucleoside analog drugs (AZT, ddI 3TC) is needed for more effective inhibition of HIV-RT; 3) that the combination of the triphosphates at concentrations much greater than those pharnacologically achieved in T-Cells or PBMC under treatment conditions did not inhibit completely HIV-RT. Hence, the three nucleoside HIV-RT inhibitors must be combined with other classes of antiviral drugs or T-cell specific inhibitor drugs.

Cytosine arabinoside (ara-C) resistance confers cross-resistance or collateral sensitivity to other classes of anti-leukemic drugs.

The major limitation of treatment with antimetabolite drugs is that they produce resistant clones both in vitro and in patients who either do not respond to treatment or relapse soon after response has been documented. To better understand the phenomenon of cross-resistance, we developed seven CEM/ara-C-resistant leukemic clones from the CEM/0 (wt) cell line. These clones ranged from 4- to 3.5 x 10(8)-fold more resistant to ara-C than the wt CEM/0 cell line. Using this model, we determined IC50 concentrations to several chemotherapeutic agents and gamma radiation, and we also studied pro- (p53) and anti-apoptotic (bcl-2) proteins, as well as P-glycoprotein (P-gp) and multidrug resistance related protein (MRP). The cell viability assays showed that these clones were cross-resistant to 6-thioguanine (6-TG) or 6-mercaptoguanosine (6-TGuo) from 1.1- to 8.8-fold with ara-C; cross-resistance to vincristine (VCR) was from 200- to 1 x 10(4)-fold with ara-C. Taxotere (TXR) showed cross-resistance with ara-C from 1.39- to 3.03 x 10(3)-fold; dexamethasone (DEX) also showed a significant degree of cross-resistance from 27.4- to 3.87 x 10(7)-fold. Gamma radiation treatments from 0.77 Gy to 12.3 Gy showed a radiation dose-dependent cross-resistance with ara-C from 1.43- to 2.93-fold. Idarubicin was collaterally sensitive with ara-C from 4.6- to 1 x 10(9)-fold in these cell lines. The CEM/ara-C/G resistant cell line was 3-fold more sensitive to 6-TG or VCR than CEM/0 (wt), and 5-fold more sensitive to 6-TGuo. This cell clone expressed p53 and did not overexpress bcl-2 protein. All of the cell lines studied, CEM/0 (wt) and the ara-C resistant clones, showed functional p53 protein. The cell treatment with 0.1, 1 and 10 microM ara-C for 48 hours showed increased p53 protein expression in most of these lines. No increase in bcl-2 protein expression was seen in the wt cell line after ara-C treatment for 48 hours. Three cell lines resistant to ara-C (CEM/ara-C/B, CEM/ara-C/D and CEM/ara-C/I) showed an important increased expression of bcl-2 protein after treatment with 1 microM ara-C, but not after 10 microM. This alteration may lead to resistance to apoptosis and enhanced cell survival. The ratio of bcl-2 to p53 was increased significantly in these three clones, thus favoring an anti-apoptotic drive. All of the cell lines examined were negative for MRP expression and only two, CEM/ara-C/B and CEM/ara-C/J, were positive for MRP functional activity. However, three ara-C resistant cell clones, CEM/ara-C/7A, CEM/ara-C/B and CEM/ara-C/G, were positive for P-gp expression and functional activity. It is apparent that selection for ara-C resistance confers cross-resistance to many other classes of drugs and gamma radiation, probably due to bcl-2 protein overexpression or P-gp and MRP expression, as independent mechanisms.

Inhibition of ribonucleotide reductase by a new class of isoindole derivatives: drug synergism with cytarabine (Ara-C) and induction of cellular apoptosis.

The hydroxyisoindole dione derivatives ISID and MISID are new compounds with structures resembling purines and possessing a hydroxamic acid moiety which is the pharmacophore of hydroxyurea (HU), an inhibitor of ribonucleotide reductase (RR). ISID and MISID exhibited 100- to 500-fold higher cytotoxicity as compared to HU against cell lines sensitive (CEM/0) or resistant to ara-C (CEM/ara-C/7A; CEM/dCk[-]). Both MISID and ISID showed significant inhibitory activity of ribonucleotide reductase (RR). Treatment of CEM/0 cells with 10 microM ISID showed a linear decrease in all the dNTPs leading to a complete depletion by 4 hours with no recovery of enzymatic activity of RR up to 48 hours in the presence of the drug, suggesting an irreversible inhibition of this enzyme. However, 10 microM MISID caused a significant time dependent, but reversible inhibition of RR in a whole cell assay in CEM/0 cells. Pretreatment of CEM/0 cells with 10 microM MISID for 1 hour increased cellular ara-CTP concentrations approximately 2-fold as compared to untreated controls. However, a reduction in intracellular ara-CTP concentration was observed following a commensurate depletion of ATP in these cells after 4 hrs of ISID pretreatment. Similarly, the ara-CTP concentration was augmented by 1.6-fold following pretreatment of CEM/0 cells with 10 microM MISID for 4 hours. Significant apoptotic cell death was detected in CEM/0 cells treated with ara-C, ISID or MISID alone or in combination. Ara-C treatment induced HMW (high molecular weight) DNA fragmentation at earlier times which subsequently led to oligonucleosomal DNA fragmentation by 48 hrs. The sequential treatment of CEM/0 cells with MISID followed by ara-C resulted in increased DNA fragmentation in the 2.0 to 4.0 Kb range in comparison to those cells treated with either ara-C or MISID alone. The increased apoptotic cell death explained the synergistic cytotoxicity of the combination of ara-C and MISID against CEM/0 cells observed earlier. We conclude that the inhibition of RR by these agents induces leukemic cell apoptosis, a mechanism which is further potentiated when these RR inhibitors are combined with ara-C. Since new compounds do not require activation, as do other clinically useful RR inhibitors, further studies for their potential use against leukemias and solid tumors are warranted.

Pharmacology of cytarabine given as a continuous infusion followed by mitoxantrone with and without amsacrine/etoposide as reinduction chemotherapy for relapsed or refractory pediatric acute myeloid leukemia.

BACKGROUND: The outcome of patients with acute myeloid leukemia (AML) who relapse or fail to achieve an initial remission has been dismal. PROCEDURE: Fifteen pediatric patients with AML, 4 relapsed and 11 primary refractory, were reinduced with a loading bolus of 0.5 g/m2 cytarabine (ara-C) followed immediately by a continuous infusion of ara-C (130 mg/m2/day) for 72 hours, followed with four daily doses (12 mg/m2/day) of mitoxantrone. Eight of 15 patients received an additional course of amsacrine and etoposide. RESULTS: Ten of 15 (66%) achieved complete response (CR) and 3 achieved partial response (PR) (20%), with an objective response rate of 86% after ara-C/mitoxantrone. One patient died before disease assessment, and one had no response after ara-C/mitoxantrone. Pharmacokinetic studies of ara-C and ara-U were performed in 13 of 15 patients. A steady-state (Css) ara-C concentration was achieved at 2 hours after the bolus ara-C dose and was maintained up to 72 hours. The Css plasma concentrations of ara-C and ara-U averaged 10.33 +/- 0.81 microM and 139.14 +/- 17.8 microM, respectively. Also, cellular pharmacokinetic studies of ara-CTP were performed on circulating leukemic cells from 5 patients. Four patients who had a significant increase (P = 0.0041) in their Css ara-CTP concentrations achieved CR, whereas one patient with an insignificant increase achieved PR. CONCLUSIONS: Continuous infusion of ara-C followed by mitoxantrone is an active reinduction regimen in refractory or relapsed pediatric AML patients. The addition of amsacrine and etoposide did not improve the remission induction rate. Further studies are needed in a larger patient population to confirm these observations.

Increased p21/WAF-1 and p53 protein levels following sequential three drug combination regimen of fludarabine, cytarabine and docetaxel induces apoptosis in human leukemia cells.

Combinations of nucleoside analog drugs, such as F-araA and ara-C, combined with Topoisomerase II inhibitors, such as anthracyclines, are synergistic against human leukemic T-cells and induce apoptotic cell death. Similarly, nucleoside analog drugs followed by mitotic inhibitors also have a synergistic effect. Sequence specific combinations of F-araA followed by ara-C and Taxotere (docetaxel) in CEM/0 cells showed a 2- to 3-fold synergism over the two drug (F-araA + ara-C) combinations and 2- to 4-fold synergism over Taxotere alone. This synergism was evident due to enhanced cellular apoptosis. In the CEM/ara-C/7A cell line, which is partially resistant to ara-C, the synergy observed with the triple drug combination was 9-fold greater than the F-araA plus araC combination, and 3-fold greater than Taxotere alone, making this three-drug regimen collaterally sensitive to ara-C. This study describes the mechanisms of the synergistic effect in regards to apoptosis achieved by three-drug regimens comprised of two nucleoside analog drugs and a mitotic inhibitor in comparison with the combination of two nucleotide analog drugs. The study also demonstrates that the possible biochemical mechanism of cellular toxicity and drug synergism is attributed to induction of apoptosis following drug treatment and the onset of the apoptotic cascade is primarily regulated by p21/WAF-I, which is transcriptionally activated by p53 following DNA damage. The anti-apoptotic protein, bcl-2, seemed to have no effect in inhibiting apoptosis following treatment with the two or three drug regimens in this in vitro leukemia model. The three-drug combination induced greater cellular apoptosis than the two-drug combination or Taxotere monotherapy. We conclude that the greater drug synergism observed in human leukemic cells, sensitive or resistant to ara-C, by Fludarabine + ara-C + Taxotere can be explained by the greater oligonucleosomal DNA fragmentation indicative of increased cellular apoptosis. The mechanism of this increased cytotoxic action is due to the upregulation of p53 and p21/WAF-1 with a down regulation of bcl-2. These studies are encouraging, and testing this three drug regimen in a clinical setting may result in improved antileukemic therapies.

The synergism of 6-mercaptopurine plus cytosine arabinoside followed by PEG-asparaginase in human leukemia cell lines (CCRF/CEM/0 and (CCRF/CEM/ara-C/7A) is due to increased cellular apoptosis.

BACKGROUND: The only effective drug against ALL that inhibits protein synthesis is Asparaginase (ASNase). The drug depletes asparagine (Asn) in serum and cells and since the leukemic T-cells (thymic origin cells) lack asparagine synthetase, the amino acid starvation leads to apoptosis. When PEG-ASNase is combined with antimetabolite drugs such as ara-C, or combinations of 6-MP followed by ara-C, it augments the cytotoxic effect synergistically against human T-leukemia cells. MATERIALS AND METHODS: Synergism studies with two- or three-drug combination regimens in the human leukemia cell lines, CEM/0 and CEM/ara-C/7A have been investigated along with its effect in inducing apoptosis. RESULTS: The IC50 (approximately Dm) values of ara-C were 0.032 microM and 0.11 microM, and that of PEG-ASNase were 0.002 IU/ml and 1.52 IU/ml against CEM/0 and CEM/ara-C/7A cells, respectively. Thus, CEM/ara-C/7A cell line that is partially resistant to ara-C exhibited 681-fold cross-resistant to PEG-ASNase as compared to CEM/0. The concurrent drug exposure of ara-C and PEG-ASNase for 48 hours resulted in IC50 values of 0.56 nM for ara-C and 0.56 mIU/ml for PEG-ASNase respectively, in CEM/0 cells which represents a 57.4-fold synergism compared to ara-C alone. In the CEM/ara-C/7A cell line, the co-incubation with these two drugs resulted in IC50 value of 0.015 microM for ara-C and 0.015 IU/ml for PEG-ASNase respectively, or a 7.25-fold synergism as compared to ara-C and 101.1-fold synergism in comparison with PEG-ASNase alone. Pre-clinical studies involving three-drug combination consisting of 6-MP, ara-C and PEG-ASNase in a sequence-specific manner showed a 15.6-fold synergism against CEM/0 cell line over the two-drug combination of 6-MP followed by ara-C or approximately 160-fold syneryism over ara-C alone. CONCLUSION: The two-drug combination of ara-C and PEG-ASNase or the three-drug combination of 6-MP, ara-C and PEG-ASNase in the ara-C sensitive and resistant cell line showed significant drug synergism and CEM/ara-C/7A cells exhibited collateral sensitivity to PEG-ASNase. The three-drug combination also induced dose-dependent apoptotic DNA fragmentation which was higher than the two-drug combination of 6-MP and ara-C. We also conclude that the sequence specific use of PEG-ASNase in combination with the nucleoside analog drugs may benefit leukemia patients in early relapse.

Pharmacokinetic and pharmacodynamic studies of fludarabine and cytosine arabinoside administered as loading boluses followed by continuous infusions after a phase I/II study in pediatric patients with relapsed leukemias. The Children's Cancer Group.

The sequential administration of fludarabine followed by cytosine arabinoside (ara-C) has demonstrated significant synergistic effects against the CEM human leukemic cell line. This in vitro synergism was investigated in a Phase I trial in pediatric patients with relapsed acute leukemia. The optimum concentrations of 9-beta-D-arabinofuranosyl 2-fluoroadenine and ara-C necessary to achieve significant drug synergism from in vitro studies were between 10 and 20 microM. Fludarabine was infused at a dose to attain a target plasma concentration of 10 microM for 48 h, followed by a continuous infusion of escalated ara-C doses to maintain plasma ara-C concentrations of 10, 12.5, 15, or 17.5 microM for 72 h. Thirteen patients with acute lymphocytic leukemia and 18 with acute myelocytic leukemia were entered into the study, 30 of whom were clinically evaluable for toxicity. Pharmacokinetic and pharmacodynamic studies were performed on specimens from 20 patients. The optimal 9-beta-D-arabinofuranosyl 2-fluoroadenine and ara-C concentrations in plasma were easily achieved after continuous infusion regimens of both drugs. Cellular ara-CTP is augmented 5-8-fold in leukemic cells from patients receiving fludarabine phosphate treatment followed by ara-C. The maximum tolerated plasma concentrations for this combination regimen was 10 microM fludarabine for 48 h followed by 72 h of 15 microM ara-C, which were achieved at dose level 3. A significant number of responses were also seen. Nine of 18 evaluable patients (50%) with acute myelocytic leukemia achieved complete or partial responses, and 3 of 9 evaluable patients with acute lymphocytic leukemia achieved complete or partial responses. Fludarabine and ara-C successfully eradicated bone marrow disease in 16 of 27 patients (59%), 23 patients of which had been treated previously with high-dose ara-C. These results verified the synergistic effect fludarabine exhibited in augmenting ara-CTP concentrations in patients' leukemic blasts, thus improving the clinical response in relapsed pediatric leukemias.

Phase I/II study of idarubicin given with continuous infusion fludarabine followed by continuous infusion cytarabine in children with acute leukemia: a report from the Children's Cancer Group.

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.

Acute lymphoblastic leukaemia: a guide to asparaginase and pegaspargase therapy.

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 Erwinia chrysanthemi. The half-lives (t((1/2))) of these forms are approximately 1.2 and 0.6 days, respectively. In order to increase the biological t((1/2)), pegaspargase was synthesised by the covalent attachment of monomethoxypolyethylene glycol (PEG) to native E. coli L-asparaginase: it has a t((1/2)) of approximately 5.7 days. The duration of asparagine depletion, the substrate amino acid of the drug, is directly related to asparaginase t((1/2)). 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 t((1/2)) 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.

Treatment of human B-cell precursor leukemia in SCID mice using a combination of the investigational biotherapeutic agent B43-PAP with cytosine arabinoside.

Combined immunochemotherapy regimens using the investigational biotherapeutic agent B43(anti-CD19)-poke-weed antiviral protein (PAP) immunotoxin may offer an effective treatment for refractory B-cell precursor leukemias. The purpose of the present study was to explore and identify effective combinations of B43-PAP with standard chemotherapeutic drugs, including the anthracyclin doxorubicin, the epipodophyllotoxin etoposide, the nitrosurea carmustine, and the antimetabolite cytosine arabinoside. Here, we report that the B43-PAP plus cytosine arabinoside combination has potent antileukemic activity against human B-cell precursor leukemia in SCID mice and leads to 100% long-term event-free survival from an otherwise invariably fatal leukemia. Surprisingly, none of the other treatment protocols tested, including combinations of B43-PAP with carmustine, doxorubicin, or etoposide, proved more effective than B43-PAP alone.

Pharmacokinetic studies of 13-cis-retinoic acid in pediatric patients with neuroblastoma following bone marrow transplantation.

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 levels > 10 microM, but only 13% (12/96) with peak serum levels < 10 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.

NONMEM population pharmacokinetic studies of cytosine arabinoside after high-dose and after loading bolus followed by continuous infusion of the drug in pediatric patients with leukemias.

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.