Integrating cell-based and clinical genome-wide studies to identify genetic variants contributing to treatment failure in neuroblastoma patients.

High-risk neuroblastoma is an aggressive malignancy, with high rates of treatment failure. We evaluated genetic variants associated with in vitro sensitivity to two derivatives of cyclophosphamide for association with clinical response in a separate replication cohort of neuroblastoma patients (n = 2,709). To determine sensitivity, lymphoblastoid cell lines (LCLs) were exposed to increasing concentrations of 4-hydroperoxycyclophosphamide (4HC; n = 422) and phosphoramide mustard (PM; n = 428). Genome-wide association studies were performed to identify single-nucleotide polymorphisms (SNPs) associated with sensitivity to 4HC and PM. SNPs consistently associated with LCL sensitivity were analyzed for associations with event-free survival (EFS) in patients. Two linked SNPs, rs9908694 and rs1453560, were found to be associated with (i) sensitivity to PM in LCLs across populations and (ii) EFS in all patients (P = 0.01) and within the high-risk subset (P = 0.05). Our study highlights the value of cell-based models to identify candidate variants that may predict response to treatment in patients with cancer.

Intraneoplastic polymer-based delivery of cyclophosphamide for intratumoral bioconversion by a replicating oncolytic viral vector.

rRp450 is an oncolytic herpesvirus that expresses the CYP2B1 cDNA, responsible for bioconverting cyclophosphamide (CPA) into the active metabolites 4-hydroxyCPA/aldophosphamide (AP). However, formal proof of prodrug activation is lacking. We report that activation of CPA in cells infected with rRp450 generates a time-dependent increase of diffusible 4-hydroxyCPA/AP. For in vivo applications, a CPA-impregnated polymer was implanted into human tumor xenografts inoculated with rRp450. The area under the curve for 4-hydroxyCPA/AP was 806 microg/g of tumor tissue/h when CPA was administered via intraneoplastic polymer and 3 microg/g of tumor tissue/h when CPA was administered i.p. Therefore, (a) a lytic virus expressing a "suicide" gene can activate a prodrug; and (b) within rRp450-infected tumor, more prolonged and higher concentrations of activated metabolites are generated by intraneoplastic compared with systemic administration of prodrug.

Effect of O6-benzylguanine on nitrogen mustard-induced toxicity, apoptosis, and mutagenicity in Chinese hamster ovary cells.

O6-Benzylguanine (BG) inactivates O6-alkylguanine-DNA alkyltransferase (AGT), resulting in an increase in the sensitivity of cells to the toxic effects of O6-alkylating agents. BG significantly enhances the cytotoxicity and decreases the mutagenicity of nitrogen mustards [i.e., phosphoramide mustard (PM), melphalan, and chlorambucil], a group of alkylating agents not known to produce O6-adducts in DNA. The enhancement is observed in cells irrespective of AGT activity. Exposure of Chinese hamster ovary cells to 100 microM BG results in enhancement in the cytotoxicity of PM (300 microM), chlorambucil (40 microM), and melphalan (10 microM) by 9-, 7-, and 18-fold, respectively. In contrast, mutation frequency after treatment with 300 microM PM is decreased from 259 mutants/10(6) cells to 22 mutants/10(6) cells when cells are pretreated with BG. The enhancement of toxicity of these bis-alkylating agents appears to involve cross-link formation, because neither cytotoxicity nor mutagenicity of a monoalkylating PM analogue is significantly altered when combined with BG. Enhanced cytotoxicity and decreased mutagenicity is concomitant with a dramatic increase in the number of cells undergoing apoptosis when BG is combined with PM, melphalan, or chlorambucil at 72-94 h after treatment. Cell cycle analysis demonstrates that BG alone or combined with nitrogen mustards arrests cells in G1 phase of the cell cycle. At 16 h after treatment, 11 and 57% of cells treated with PM alone or with BG plus PM are in G1 phase, respectively. Our data suggest that treatment with BG causes G1 arrest and drives noncycling cells treated with nitrogen mustards into apoptosis, thus protecting against mutagenic DNA damage introduced by nitrogen mustards.

Effect of O6-benzylguanine on alkylating agent-induced toxicity and mutagenicity. In Chinese hamster ovary cells expressing wild-type and mutant O6-alkylguanine-DNA alkyltransferases.

The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) has been shown to protect cells from the toxic and mutagenic effect of alkylating agents by removing lesions from the O6 position of guanine. O6-Benzylguanine (BG) is a potent inactivator of AGT, resulting in an increase in the sensitivity of cells to the toxic effects of chemotherapeutic alkylating agents. Chinese hamster ovary (CHO) cells and CHO cells transfected with wild-type AGT (CHOWTAGT) and a mutant AGT [P138 M/V139I/P140K (CHOMIK)] known to be resistant to BG were treated with BG and various alkylating agents. BG treatment alone dramatically decreased AGT activity in CHOWTAGT cells but resulted in no depletion in AGT activity in CHOMIK cells. In the absence of AGT, these cells are highly sensitive to the toxic and mutagenic effects of temozolomide and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and no further sensitization occurs in the presence of BG. In contrast, CHOWTAGT cells are resistant to temozolomide and BCNU, and treatment with BG resulted in a significantly higher cell killing and mutation frequency. CHOMIK cells were completely resistant to temozolomide or BCNU in the presence and absence of BG. Both cell killing and mutation frequency of 4-hydroperoxycyclophosphamide (4-HC) in CHO, CHOWTAGT, and CHOMIK cells were increased in the presence of BG. 4-HC generates two active metabolites, phosphoramide mustard (PM) and acrolein. BG had no effect on 4hydroperoxydidechlorocyclophosphamide (which generates acrolein and a nonalkylating form of PM) in CHO cells and CHOMIK cells, but enhancement of toxicity was observed with PM in both these cell lines. Therefore, we attribute the enhancement to the PM metabolite of 4-HC. Our results demonstrate that wild-type AGT plays an important role in protecting against the toxic and mutagenic effect of O6 alkylating agents and that a mutant AGT resistant to inactivation by BG effectively prevents BG-enhanced toxicity and mutagenicity induced by these agents. Expression of the AGT protein contributes to resistance of 4-HC. BG also enhances the toxicity of 4-HC and PM by a mechanism that may not involve the AGT repair protein.

Evidence for a role of chloroethylaziridine in the cytotoxicity of cyclophosphamide.

UNLABELLED: A number of investigators have observed that the use of 4-hydroperoxycyclophosphamide (4-HC) in multiwell plate cytotoxicity assays can be associated with toxicity to cells in wells that contain no drug. Previous reports have implicated diffusion of 4-HC decomposition products, and acrolein in particular, as the active species. PURPOSE: The purpose of this study was to elucidate the species responsible for the airborne cytotoxicity of 4-HC, and to devise ways to minimize such effects in chemosensitivity assays. METHODS: To this end, analogues of 4-HC were synthesized to identify the contributions of individual cyclophosphamide metabolites to cytotoxicity. The analogues were then tested for activity against three human breast tumor cell lines (including a line resistant to 4-HC), and one non-small-cell lung carcinoma line. Cytotoxicity was evaluated by assays that quantitate cellular metabolism and nucleic acid content. RESULTS: Didechloro-4-hydroperoxycyclophosphamide, a compound that generates acrolein and a nontoxic analogue of phosphoramide mustard, gave no cross-well toxicity. In contrast, a significant neighboring well effect was observed with phenylketophosphamide, a compound that generates phosphoramide mustard but not acrolein. Addition of authentic chloroethylaziridine reproduced the airborne toxicity patterns generated by 4-HC and phenylketophosphamide. Increasing the buffering capacity of the growth medium and sealing the microtiter plates prevented airborne cytotoxicity. CONCLUSION: Since it is unlikely that phosphoramide mustard is volatile, these findings implicate chloroethylaziridine rather than acrolein as the volatile metabolite of 4-HC that is responsible for airborne cytotoxicity. The fact that chloroethylaziridine is generated in amounts sufficient to volatilize, diffuse across wells and cause cytotoxicity indicates that it is an important component in the overall cytotoxicity of 4-HC in vitro. Furthermore, these findings suggest that chloroethylaziridine may also contribute to the toxicity of cyclophosphamide in vivo.

The chemistry of the metabolites of cyclophosphamide.

This is primarily an overview of the spontaneous (non-enzymatic) chemistry of the metabolites of cyclophosphamide, viz., cis- and trans-4-hydroxycyclophosphamide, aldophosphamide (and its hydrate), iminophosphamide, phosphoramide mustard, acrolein, and chloroethylaziridine. A brief description of detoxification products obtained through enzyme catalyzed reactions appears. Included as the historical basis for the development of cyclophosphamide is the chemistry of nitrogen mustards. Among the topics covered are: perturbations to metabolite distributions and half-lives effected by buffer, structure, pH and nucleophiles; effects of pH on mechanism; alkylation versus P-N bond hydrolysis; the influence of nucleophiles on alkylation product distributions; the influence of substituents on alkylation rates; and preactivated forms of cyclophosphamide as metabolite precursors (4-hydroperoxycyclophosphamide and mafosfamide). A review with 66 references.

Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity.

Because hematopoietic stem cells are rich in aldehyde dehydrogenase (ALDH) activity, we developed a fluorescent substrate for ALDH, termed BODIPY aminoacetaldehyde (BAAA), and tested its potential for isolating primitive human hematopoietic cells. A population of cells with low orthogonal light scattering and bright fluorescence intensity (SSC(lo)ALDH(br) cells) could be readily fractionated from human umbilical cord blood cells costained with BAAA and the multidrug-resistance inhibitor verapamil. The SSC(lo)ALDH(br) population was depleted of lineage-committed cells, 40-90% pure for CD34(+)CD38(lo/-) cells, and enriched 50- to 100-fold for primitive hematopoietic progenitors detected in short- and long-term culture analyses. Together, these observations indicate that fractionating human hematopoietic stem cells on the basis of ALDH activity using BAAA is an effective method for isolating primitive human hematopoietic progenitors. This technique may be useful for isolating stem cells from other tissues as well.

Role of O6-alkylguanine-DNA alkyltransferase in protecting against cyclophosphamide-induced toxicity and mutagenicity.

Cyclophosphamide is used to treat a wide range of human malignancies. However, it is also a known carcinogen associated with induction of therapy-related leukemia and bladder cancer. The DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), protects cells from the toxic and mutagenic effects of O6-alkylating agents. We report here the contribution of AGT in protecting against the toxic and mutagenic effects of cyclophosphamide. CHO cells transduced with wild-type human AGT (CHO(AGT)) and pcDNA3 (CHOpcDNA3) were treated with activated cyclophosphamide derivatives, 4-hydroperoxycyclophosphamide (4-HC), 4-hydroperoxydidechlorocyclophosphamide (4-HDC), a progenitor of acrolein, and phosphoramide mustard (PM). The results show that CHO(AGT) is 7- or 20-fold less sensitive to the toxic effects of 30 microM 4-HC or 300 microM 4-HDC, respectively, than CHOpcDNA3 cells as measured by cell survival using a colony-forming assay. CHO(AGT) cells treated with 20 microM 4-HC or 200 microM 4-HDC produced 4- or 7-fold lower mutation frequency as measured at the HPRT locus than CHOpcDNA3 cells treated with the same dose of drugs. At 30 microM acrolein, the cell survival for CHO(AGT) was 30% compared with 18.7% for CHOpcDNA3. The mutation frequency of acrolein at the same dose was 57 mutants/10(6) cells in CHOpcDNA3 compared with no mutants in CHO(AGT). In contrast, CHO(AGT) and CHOpcDNA3 cells treated with PM had similar survival curves and exhibited no difference in mutation frequency. The present study demonstrates that AGT plays an important role in protecting against the toxic and mutagenic effect of cyclophosphamide and suggests that acrolein, not PM, is responsible for generating the toxic and mutagenic lesion(s) protected by the AGT protein.

Modulation of cyclophosphamide activity by O6-alkylguanine-DNA alkyltransferase.

PURPOSE: The human medulloblastoma cell line D283 Med (4-HCR), a line resistant to 4-hydroperoxycyclophosphamide (4-HC), displays enhanced repair of DNA interstrand crosslinks induced by phosphoramide mustard. D283 Med (4-HCR) cells are cross-resistant to 1,3-bis(2-chloroethyl)- -nitrosourea, but partial sensitivity is restored after elevated levels of O6-alkylguanine-DNA alkyltransferase (AGT) are depleted by O6-benzylguanine (O6-BG). Studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) after AGT is depleted by O6-BG. METHODS: Limiting dilution and xenograft studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide with or without O6-BG. RESULTS: The activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) was increased after AGT depletion by O6-BG preincubation. Similar studies with Chinese hamster ovary cells, with or without stable transfection with a plasmid expressing the human AGT protein, revealed that the AGT-expressing cells were significantly less sensitive to 4-HC and 4-hydroperoxydidechlorocyclophosphamide. Reaction of DNA with 4-HC, phosphoramide mustard, or acrolein revealed that only 4-HC and acrolein caused a decrease in AGT levels. CONCLUSIONS: We propose that a small but potentially significant part of the cellular toxicity of cyclophosphamide in these cells is due to acrolein, and that this toxicity is abrogated by removal of the acrolein adduct from DNA by AGT.

The partitioning of phosphoramide mustard and its aziridinium ions among alkylation and P-N bond hydrolysis reactions.

NMR (1H and 31P) and HPLC techniques were used to study the partitioning of phosphoramide mustard (PM) and its aziridinium ions among alkylation and P-N bond hydrolysis reactions as a function of the concentration and strength of added nucleophiles at 37 degrees C and pH 7.4. With water as the nucleophile, bisalkylation accounted for only 10-13% of the product distribution given by PM. The remainder of the products resulted from P-N bond hydrolysis reactions. With 50 mM thiosulfate or 55-110 mM glutathione (GSH), bisalkylation by a strong nucleophile increased to 55-76%. The rest of the PM was lost to either HOH alkylation or P-N bond hydrolysis reactions. Strong experimental and theoretical evidence was obtained to support the hypothesis that the P-N bond scission observed at neutral pH does not occur in the parent PM to produce nornitrogen mustard; rather it is an aziridinium ion derived from PM which undergoes P-N bond hydrolysis to give chloroethylaziridine. In every buffer studied (bis-Tris, lutidine, triethanolamine, and Tris), the decomposition of PM (with and without GSH) gave rise to 31P NMR signals which could not be attributed to products of HOH or GSH alkylation or P-N bond hydrolysis. The intensities of these unidentified signals were dependent on the concentration of buffer.

A structural basis for a phosphoramide mustard-induced DNA interstrand cross-link at 5'-d(GAC).

Phosphoramide mustard-induced DNA interstrand cross-links were studied both in vitro and by computer simulation. The local determinants for the formation of phosphoramide mustard-induced DNA interstrand cross-links were defined by using different pairs of synthetic oligonucleotide duplexes, each of which contained a single potentially cross-linkable site. Phosphoramide mustard was found to cross-link dG to dG at a 5'-d(GAC)-3'. The structural basis for the formation of this 1,3 cross-link was studied by molecular dynamics and quantum chemistry. Molecular dynamics indicated that the geometrical proximity of the binding sites also favored a 1,3 dG-to-dG linkage over a 1,2 dG-to-dG linkage in a 5'-d(GCC)-3' sequence. While the enthalpies of 1,2 and 1,3 mustard cross-linked DNA were found to be very close, a 1,3 structure was more flexible and may therefore be in a considerably higher entropic state.

Effect of stereochemistry on the oxidative metabolism of the cyclophosphamide metabolite aldophosphamide.

31P NMR and cell perfusion techniques were used to investigate the conversion of the individual enantiomers of aldophosphamide (AP) to carboxyphosphamide (CBP) as catalyzed by aldehyde dehydrogenase in human erythroleukemia K562 cells. R- and S-cyclophosphamides (CPs) were treated with ozone and hydrogen peroxide to yield Rp- and Sp-cis-4-hydroperoxycyclophosphamides (Rp- and Sp-cis-4-HO2-CP); reduction of each hydroperoxide gave the corresponding enantiomer of AP [along with its tautomer 4-hydroxycyclophosphamide (4-HO-CP)]. In separate experiments, K562 cells embedded in agarose gel threads were perfused at pH 7.4, 21 +/- 1 degrees, with solutions of 1.4 mM Rp- and Sp-4-HO-CP/AP, both with and without added mesna (an acrolein scavenger). A comparison of the 31P NMR spectral data derived from the experiments revealed little statistical difference (+/- 10-20% error limits) in the normalized intensities of the CBP peaks arising from the individual AP enantiomers [with added mesna, the ratio Rp-CBP:Sp-CBP was 1.00:1.24 +/- 0.13 (average deviation); without mesna, the same ratio was 1.00:1.35]. Using conventional methods for evaluating the in vitro drug toxicities, CP-resistant L1210 cells were treated in separate experiments with Rp- and Sp-cis-4-HO2-CP; there were no significant differences between the toxicities exhibited by the stereoisomers.

Comparison of the protonation of isophosphoramide mustard and phosphoramide mustard.

The alkylating agent isophosphoramide mustard (IPM) spontaneously forms a relatively stable aziridine derivative which can be directly observed using NMR spectroscopy. The protonations of IMP and its aziridine were probed using 1H, 31P, 15N, and 17O NMR spectroscopy. The positions of the 31P, 15N, and 17O resonances of IPM between pH 2 and 10 each exhibit a single monobasic titration curve with the same pKa of 4.31 +/- 0.02. On the basis of a comparison with other compounds and our earlier work with phosphoramide mustard, the NMR results for IPM indicate that protonation occurs at nitrogen and not oxygen. Over this same pH range, each of the 1H, 31P, and 15N resonances of IPM-aziridine also show a single monobasic titration with a pKa of 5.30 +/- 0.09. The magnitude of the change in chemical shifts suggests that the protonation of the IPM-aziridine occurs at the ring nitrogen. Theoretical gas-phase calculations of PM, IPM, and IPM-aziridine suggest O-protonation to be more likely; however, aqueous phase calculations predict the N-protonated forms to be most stable. Furthermore, for PM and IPM-aziridine, which contain nonequivalent nitrogens, the theoretical calculations and experimental data both agree as to which nitrogen undergoes protonation. These results suggest that the IMP-aziridine remains unprotonated under physiological conditions and may, in part, explain the lower alkylating activity of IPM as compared to PM.

Assessment of aldehyde dehydrogenase in viable cells.

Cytosolic aldehyde dehydrogenase (ALDH), an enzyme responsible for oxidizing intracellular aldehydes, has an important role in ethanol, vitamin A, and cyclophosphamide metabolism. High expression of this enzyme in primitive stem cells from multiple tissues, including bone marrow and intestine, appears to be an important mechanism by which these cells are resistant to cyclophosphamide. However, although hematopoietic stem cells (HSC) express high levels of cytosolic ALDH, isolating viable HSC by their ALDH expression has not been possible because ALDH is an intracellular protein. We found that a fluorescent aldehyde, dansyl aminoacetaldehyde (DAAA), could be used in flow cytometry experiments to isolate viable mouse and human cells based on their ALDH content. The level of dansyl fluorescence exhibited by cells after incubation with DAAA paralleled cytosolic ALDH levels determined by Western blotting and the sensitivity of the cells to cyclophosphamide. Moreover, DAAA appeared to be a more sensitive means of assessing cytosolic ALDH levels than Western blotting. Bone marrow progenitors treated with DAAA proliferated normally. Furthermore, marrow cells expressing high levels of dansyl fluorescence after incubation with DAAA were enriched for hematopoietic progenitors. The ability to isolate viable cells that express high levels of cytosolic ALDH could be an important component of methodology for identifying and purifying HSC and for studying cyclophosphamide-resistant tumor cell populations.

Quantitation of 4-hydroxycyclophosphamide/aldophosphamide in whole blood.

There is considerable interest in determining 4-hydroxycylcophosphamide/aldophosphamide (4-HO-CP/AP) blood levels in patients receiving the prodrug, cyclophosphamide (CP). Phosphoramide mustard (PM), the alkylating metabolite of CP, is relatively impermeable to cell membranes and it is generally believed that circulating intermediary metabolites, including aldophosphamide, the immediate precursor of PM, is transported by circulating blood to tumor tissue. Therefore, circulating 4-HO-CP/AP blood levels should more closely reflect the oncostatic and cytotoxic effects of CP than the parent drug. We have developed a gas chromatographic electron-impact mass spectrometric (GC-EIMS) method suitable for routine monitoring of 4-HO-CP/AP levels in whole blood over the range 0.085 microM (25 ng/ml) to 34 microM (10 micrograms/ml). The unstable metabolites were derivatized with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine-HCl to form a stable aldophosphamide oxime derivative (PBOX). [2H4]PBOX was used as an internal standard. For clinical samples, tubes were prepared prior to blood drawing, which contained the derivatizing reagent solution and the internal standard. These solutions were stable for up to 3 months when stored at room temperature. Following addition of blood to the reaction tubes, PBOX formation was rapid and the resulting derivative was stable under these conditions for up to 8 days at room temperature. Application of the method was demonstrated by quantitating 4-HO-CP/AP blood levels in patients receiving 4 g/m2 intravenous infusion of CP over a period of 90 min.

Oxime derivatives of the intermediary oncostatic metabolites of cyclophosphamide and ifosfamide: synthesis and deuterium labeling for applications to metabolite quantification.

There is ongoing interest in the selective, quantitative analysis of the cyclophosphamide metabolites 4-hydroxycyclophosphamide (2a) and aldophosphamide (3a) because these tautomers are generally believed to play a key role in oncostatic selectivity and metabolite transport. O-(2,3,4,5,6-Pentafluorobenzyl)hydroxylamine (C6F5CH2ONH2, 1 equiv) provided for the complete conversion (by 31P NMR, 60% reaction within 15 min at 20 degrees C) of 2a/3a (17 mM in H2O/CH3OH) to E/Z-aldophosphamide O-(2,3,4,5,6-pentafluorobenzyl)oxime [C6F5CH2ON = CHCH2CH2OP-(O)(NH2)N(CH2CH2Cl)2; E:Z = 54:46 (+/- 3% average deviation)]. Under these conditions, the oxime exhibited little (6%) decomposition over 3 weeks. Parallel studies showed that 4-hydroxyifosfamide/aldoifosfamide reacted completely to give the analogous aldoifosfamide oxime [C6F5CH2ON = CHCH2CH2OP(O)(NHCH2CH2Cl)2; E:Z = 52:48 (+/- 1% average deviation)] with 50% reaction within 15 min at 20 degrees C with no product decomposition over 3 weeks. In aqueous methanol and with 2 equiv C6F5CH2ONH2, clinically useful 4-hydroperoxycyclophosphamide (10 mM; tau 1/2 = 10 min, 37 degrees C) and its isomer 4-hydroperoxyifosfamide (10 mM; tau 1/2 = 25 min, 20 degrees C) underwent complete conversion to the corresponding aldehyde oximes. Each oxime was synthesized with deuterium in the chloroethyl moieties for use as internal standards in GC/MS applications.

Direct detection of the intracellular formation of carboxyphosphamides using nuclear magnetic resonance spectroscopy.

31P nuclear magnetic resonance (NMR) spectroscopy was used in conjunction with cell perfusion techniques to monitor the intracellular chemistry of the cyclophosphamide (CP, CAS 6055-19-2) metabolites 4-hydroxy-cyclophosphamide (4-HO-CP) and aldophosphamide (AP) in U937 human histiocytic (CP-sensitive) and K562 human erythroleukemia (CP-resistant) cells. Similar experiments were carried out using the ifosfamide (IF, CAS3778-73-2) metabolites 4-hydroxyifosfamide (4-HO-IF) and aldoifosfamide (AIF). The hydroxy and aldehydic metabolites were generated by the triphenylphosphine reduction of 4-hydroperoxycyclophosphamide (4-HO2-CP) or 4-hydroperoxyifosfamide (4-HO2-IF) or by a spontaneous elimination/addition reaction involving water and 4-thiocyclophosphamide analogs 4-(2-hydroxyethyl) thiocyclophosphamide (4-ESCP) or mafosfamide. Cell death resulting from 4-HO-CP/AP perfusions was mimicked by perfusion with acrolein or an acrolein producing but non-alkylating, dechloro-CP analog. Acrolein toxicity was minimized by the presence of 2-mercaptoethanol or mesna (sodium 2-mercaptoethanesulfonate) in perfusion solutions as well as by fractional dose drug perfusions (sequential 2.5-3.0 h perfusions separated by cell washes with drug-free medium). The intracellular half-life for phosphoramide mustard (PM) at an intracellular pH value of 7.1 +/- 0.1 and an ambient probe temperature of 23 +/- 1 degree C in U937 cells was 2.1 h [k = (5.4 +/- 0.3) x 10(-3) min-1] and in K562 cells was 3.1 h [k = (3.7 +/- 0.4) x 10(-3) min-1]. Similar half-lives (2-4 h) were determined for intracellular isophosphoramide mustard (IPM). Fractional dose perfusion of U937 or K562 cells with 1.5 mmol/l 4-HO-CP/AP (generated from 4-HO2-CP) and 0.3 mmol/l mesna allowed for the observation of intracellular carboxyphosphamide (CBP); CBP was formed in higher concentrations in the CP-resistant K562 cells. Similar results were obtained using 4-ESCP and mafosfamide as sources of 4-HO-CP/AP. Identification of CBP was based on chemical shift, chemical stability, and membrane permeability studies of synthetic CBP. Concentrations of carboxyifosfamide (CBIF) formed in K562 cells were also greater than that in U937 cells.

Protonation of phosphoramide mustard and other phosphoramides.

The chemistry of the bifunctional alkylating agent phosphoramide mustard and model phosphoramides was probed by multinuclear NMR spectroscopy as a function of pH. Between pH 1 and 11, both the 31P and 15N resonances for phosphoramide mustard displayed a single monobasic titration curve with a pKa of 4.9. The protonation below pH 4.9 correlates with the loss in reactivity of the mustard. The 17O NMR spectrum of 17O-enriched phosphoramide mustard shows little change with pH. The data on the mustard was compared to 15N and 31P NMR data on 15N-enriched phosphoramidic acid, phosphorodiamidic acid, and phosphoric triamide. Contrary to the conclusions of previous studies, our combined 31P, 15N, and 17O NMR results are more consistent with N-protonation of phosphoramide mustard rather than an O-protonation. Theoretical calculations on the phosphoramidic acid, phosphorodiamidic acid, and phosphoric triamide show O-protonation to be more stable in the gas phase. For the latter two compounds, the calculations suggest that N-protonation may be the most stable protonated form in the aqueous phase. These findings influence our understanding of the structure-activity relationships of phosphoramide mustards.

Cyclophosphamide resistance in medulloblastoma.

Mechanisms of tumor resistance to 4-hydroperoxycyclophosphamide (4-HC) were studied by using a panel of human medulloblastoma cell lines either passaged in the laboratory for resistance to 4-HC or established from tumors showing clinical resistance to cyclophosphamide. Multiple distinct mechanisms of resistance were demonstrated. Daoy (4-HCR), a line that was 6-fold more resistant than Daoy, contained elevated levels of aldehyde dehydrogenase (ALDH). Most of the difference in sensitivity between the Daoy (4-HCR) and Daoy cell lines was abolished when 4-HC was replaced with phenylketocyclophosphamide, a 4-HC analogue that cannot be detoxified by ALDH. Thus, elevated levels of ALDH appear to play a role in the resistance of Daoy (4-HCR). Several of the cell lines [D283 Med (4-HCR), D341 Med (4-HCR), Daoy (4-HCR), D458 Med] contained elevated levels of glutathione (GSH). No changes in glutathione-S-transferase activity or isozyme pattern were observed, but in two of these three lines, the elevation in GSH was accompanied by elevated levels of gamma-glutamyl transpeptidase. To confirm the role of elevated GSH content in 4-HC resistance, the sensitivity of the cell lines to 4-HC was repeated after depletion of GSH by treatment with L-buthionine-S,R-sulfoximine. In medulloblastoma cell lines without other mechanisms of resistance, a linear relationship was seen between GSH content and resistance to 4-HC. Moreover, cells with GSH content greater than 5 nmol/mg protein and no other overriding mechanism of resistance could be sensitized to 4-HC treatment with L-buthionine-S,R-sulfoximine. Finally, D283 Med (4-HCR) cells had mild elevations in both ALDH and GSH content, but were resistant to phenylketocyclophosphamide and were not significantly sensitized by L-buthionine-S,R-sulfoximine. This cell line appears to demonstrate a third mechanism of resistance to 4-HC. These results suggest that 4-HC resistance in medulloblastoma can be multifactorial.

Pharmacokinetics and pharmacodynamics of topotecan in patients with advanced cancer.

Topotecan, a semisynthetic water-soluble analog of camptothecin, is the first topoisomerase I-directed drug to enter clinical trial in the United States in over 20 yr. In this study, 30-min infusions of topotecan were administered daily for 5 days every 3 weeks at doses ranging from 0.5 to 2.5 mg/m2. Topotecan is reversibly hydrolyzed in a pH-dependent reaction in aqueous solutions to the ring-open hydroxy acid. The disposition of the closed ring lactone has been studied in 26 patients, and the disposition of both lactone and hydroxy acid has been studied in 12 patients. The clearance rate for topotecan lactone was 1220 ml/min/m2, with a range of 300-4760 ml/min/m2. The clearance rate for total topotecan (lactone and hydroxy acid) was 493 ml/min/m2, with a range of 163-815 ml/min/m2. A model for the disposition of lactone and hydroxy acid incorporating both reversible hydrolysis and elimination was developed. We have shown that topotecan is partially hydrolyzed prior to administration in parenteral solutions, and that clearance of the parent compound proceeds in vivo by conversion to hydroxy acid and elimination. Renal clearance accounted for 30 +/- 18% of drug elimination in patients. The relationship between topotecan dose and myelotoxicity is well fit by a sigmoidal Emax model, as is the relationship between total topotecan area under the concentration-time curve and myelotoxicity. The disposition of topotecan was also studied in mice. The clearance rate for total topotecan in mice was 330 ml/min/m2 after administration of topotecan lactone.(ABSTRACT TRUNCATED AT 250 WORDS)