Short-term exposure of human ovarian follicles to cyclophosphamide metabolites seems to promote follicular activation in vitro.

How chemotherapy affects dormant ovarian primordial follicles is unclear. The 'burnout' theory, studied only in mice, suggests cyclophosphamide enhances primordial follicle activation. Using 4-hydroperoxycyclophosphamide (4hc) and phosphoramide mustard (PM), this study assessed how the active cyclophosphamide metabolites 4-hydroxycyclophosphamide (4-OHC) and PM, affect human primordial follicles. Frozen-thawed human ovarian samples were sliced and cultured with basic culture medium (cultured controls) or with 4hc/PM (3 µmol/l/10 µmol/l) (treated samples) for 24-48 h. Follicular counts and classification, Ki67 and anti-Müllerian hormone (AMH) immunohistochemistry and an apoptosis assay were used for evaluation, and 17ß-oestradiol and AMH were measured in spent media samples. Generally, there was primordial follicle decrease and elevated developing follicle rates in treated samples compared with cultured (P = 0.04 to P < 0.0005) and uncultured controls (P < 0.05 to P < 0.0001). No traces of apoptosis were found. There were almost twicethe levels of AMH and 17ß-oestradiol in treated compared with untreated samples (AMH with 4hc 3 µmol/l; P = 0.04). All follicles stained positively for AMHincluded treated samples. Ki67 positive staining was noted in all samples. Cyclophosphamide metabolites seem to enhance human primordial follicle activation to developing follicles, in vitro. Study findings support the 'burnout' theory as the mechanism of chemotherapy-induced ovarian toxicity.

Labeled oxazaphosphorines for applications in mass spectrometry studies. 2. Synthesis of deuterium-labeled 2-dechloroethylcyclophosphamides and 2- and 3-dechloroethylifosfamides.

The prodrugs cyclophosphamide (CP) and ifosfamide (IF) each metabolize to an active alkylating agent through a cytochrome P450-mediated oxidation at the C-4 position. Competing with this activation pathway are enzymatic oxidations at the exocyclic α and α' carbons, which result in dechloroethylation of CP and IF. The incidence of oxidation at one position relative to another is believed to be at least one factor underlying the high degree of interpatient variability in both CP and IF pharmacokinetics. As standards for the mass spectrometry quantification of dechloroethylation, the following were synthesized: (1) [4,4,5,5-(2) H4 ]-2-dechloroethylcyclophosphamide (equivalent to [4,4,5,5-(2) H4 ]-3-dechloroethylifosfamide); (2) [α,α,4,4,5,5-(2) H6 ]-2-dechloroethylcyclophosphamide (equivalent to [α,α,4,4,5,5-(2) H6 ]-3-dechloroethylifosfamide); and (3) [α,α,4,4,5,5-(2) H6 ]-2-dechloroethylifosfamide. The common precursor to all of the target compounds was [2,2,3,3-(2) H4 ]-3-aminopropanol. A one-pot reaction of this compound with POCl3 and unlabeled or labeled 2-chloroethylamine hydrochloride gave the d4 and d6 labeled 2-dechloroethylcyclophosphamides. The construction of the 2-dechloroethylifosfamide from the aminopropanol required five discreet steps. Optimization of the synthetic pathways and stability studies are discussed.

Non-invasive monitoring of L-2-oxothiazolidine-4-carboxylate metabolism in the rat brain by in vivo 13C magnetic resonance spectroscopy.

The cysteine precursor L-2-oxothiazolidine-4-carboxylate (OTZ, procysteine) can raise cysteine concentration, and thus glutathione levels, in some tissues. OTZ has therefore been proposed as a prodrug for combating oxidative stress. We have synthesized stable isotope labeled OTZ (i.e. L-2-oxo-[5-(13)C]-thiazolidine-4-carboxylate, (13)C-OTZ) and tracked its uptake and metabolism in vivo in rat brain by (13)C magnetic resonance spectroscopy. Although uptake and clearance of (13)C-OTZ was detectable in rat brain following a bolus dose by in vivo spectroscopy, no incorporation of isotope label into brain glutathione was detectable. Continuous infusion of (13)C-OTZ over 20 h, however, resulted in (13)C-label incorporation into glutathione, taurine, hypotaurine and lactate at levels sufficient for detection by in vivo magnetic resonance spectroscopy. Examination of brain tissue extracts by mass spectrometry confirmed only low levels of isotope incorporation into glutathione in rats treated with a bolus dose and much higher levels after 20 h of continuous infusion. In contrast to some previous studies, bolus administration of OTZ did not alter brain glutathione levels. Even a continuous infusion of OTZ over 20 h failed to raise brain glutathione levels. These studies demonstrate the utility of in vivo magnetic resonance for non-invasive monitoring of antioxidant uptake and metabolism in intact brain. These types of experiments can be used to evaluate the efficacy of various interventions for maintenance of brain glutathione.

Drug focus: Pharmacogenetic studies related to cyclophosphamide-based therapy.

Cyclophosphamide is a cornerstone in the treatment of many pediatric and adult malignancies, as well as in the treatment of refractory autoimmune conditions. Genetic factors are thought to play a role in the interindividual variation in both response and toxicities associated with cyclophosphamide-based therapies. This drug focus reviews the most compelling studies conducted on the pharmacogenetics of cyclophosphamide-based therapies. Broader pharmacogenomic studies are needed and may reveal additional factors important in susceptibility to toxicity and/or response to therapy.

Pharmacodynamic and pharmacokinetic study of chronic low-dose metronomic cyclophosphamide therapy in mice.

Prolonged, frequently administered low-dose metronomic chemotherapy (LDM) is being explored (pre)clinically as a promising antiangiogenic antitumor strategy. Although appealing because of a favorable side effect profile and mostly oral dosing, LDM involves new challenges different from conventional maximum tolerated dose chemotherapy. These include possible altered pharmacokinetic characteristics due to long-term drug exposure potentially resulting in acquired resistance and increased risk of unfavorable drug interactions. We therefore compared the antitumor and antivascular effects of LDM cyclophosphamide (CPA) given to mice that had been pretreated with either LDM CPA or normal saline, obtained blood 4-hydroxy-CPA (activated CPA) concentrations using either gas chromatography/mass spectrometry or liquid chromatography/tandem mass spectrometry in mice treated with LDM CPA, and measured hepatic and intratumoral activity of enzymes involved in the biotransformation of CPA and many other drugs [i.e., cytochrome P450 3A4 (CYP3A4) and aldehyde dehydrogenase]. Exposure of mice to LDM CPA for >or=8 weeks did not compromise subsequent activity of LDM CPA therapy, and biologically active 4-hydroxy-CPA levels were maintained during long-term LDM CPA administration. Whereas the effects on CYP3A4 were complex, aldehyde dehydrogenase activity was not affected. In summary, our findings suggest that acquired resistance to LDM CPA is unlikely accounted for by altered CPA biotransformation. In the absence of reliable pharmacodynamic surrogate markers, pharmacokinetic parameters might become helpful to individualize/optimize LDM CPA therapy. LDM CPA-associated changes of CYP3A4 activity point to a potential risk of unfavorable drug interactions when compounds that are metabolized by CYP3A4 are coadministered with LDM CPA.

Role of MGMT in protecting against cyclophosphamide-induced toxicity in cells and animals.

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that protects cells from the biological consequences of alkylating agents by removing alkyl groups from the O(6)-position of guanine. Cyclophosphamide and ifosfamide are oxazaphosphorines used clinically to treat a wide variety of cancers; however, the role of MGMT in recognizing DNA damage induced by these agents is unclear. In vitro evidence suggests that MGMT may protect against the urotoxic oxazaphosphorine metabolite, acrolein. Here, we demonstrate that Chinese hamster ovary cells transfected with MGMT are protected against cytotoxicity following treatment with chloroacetaldehyde (CAA), a neuro- and nephrotoxic metabolite of cyclophosphamide and ifosfamide. The mechanism by which MGMT recognizes damage induced by acrolein and CAA is unknown. CHO cells expressing a mutant form of MGMT (MGMT(R128A)), known to have >1000-fold less repair activity towards alkylated DNA while maintaining full active site transferase activity towards low molecular weight substrates, exhibited equivalent CAA- and acrolein-induced cytotoxicity to that of CHO cells transfected with plasmid control. These results imply that direct reaction of acrolein or CAA with the active site cysteine residue of MGMT, i.e. scavenging, is unlikely a mechanism to explain MGMT protection from CAA and acrolein-induced toxicity. In vivo, no difference was detected between Mgmt-/- and Mgmt+/+ mice in the lethal effects of cyclophosphamide. While MGMT may be important at the cellular level, mice deficient in MGMT are not significantly more susceptible to cyclophosphamide, acrolein or CAA. Thus, our data does not support targeting MGMT to improve oxazaphosphorine therapy.

1,3- vs 1,5-intramolecular alkylation reactions in isophosphoramide and phosphoramide mustards.

It is well-established that at pH 7.4, intramolecular 1,3-N-alkylation reactions in isophosphoramide mustard (IPM) and phosphoramide mustard (PM) produce electrophilic alkylating agents with aziridinyl moieties. To investigate the role of 1,5-intramolecular cyclizations in the chemistry of IPM and PM, the five-membered ring phospholidine products of these reactions were independently synthesized and characterized by (31)P NMR. In 0.33 M BisTris, pH 7.4, 37 degrees C, the intramolecular O-alkylation product of IPM [2-(2-chloroethylamino)-2-tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (11)] had a chemical shift of delta 33.0 and a half-life of 3.3 h. The O-alkylation product of PM [2-amino-3-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (12)] displayed a chemical shift of delta 30.6 and a half-life of 26.9 h. For both IPM and PM, 1,5-N-alkylation provides the same product [1-(2-chloroethyl)-2-hydroxy-tetrahydro-2H-1,3,2-diazaphospholidine-2-oxide (13)]. Because of its instability, 13 was generated in situ and was not isolated; however, the chemical shift (delta 33.0) and reactivity (half-life 0.3 h at 25 degrees C) of the species attributed to 13 were consistent with the assigned structure. Resonances with (31)P NMR chemical shifts indicative of 11 or 12 did not appear in reaction solutions of IPM or PM. The compound assigned as 13 gave hydrolysis products that were not found in reaction solutions of IPM or PM. The collective data supported the conclusion that intramolecular 1,5-alkylations do not contribute to the chemistry of IPM or PM in aqueous solutions at pH 7.4, 37 degrees C. Conversely, 11 and 12 were found to be the major if not exclusive products formed in DMSO solutions of the respective cyclohexylammonium salts of IPM and PM. Both 11 and 12 were relatively noncytotoxic against a series of cell lines, but there were differences in mutagenicities. Chinese hamster ovary cells were exposed to 11 or 12 for one half-life of each compound; 11 was nonmutagenic up to 500 microM, while 12 (500 microM) was mutagenic with 246 mutant colonies/10(6) surviving cells.

Evaluation of an elastin-like polypeptide-doxorubicin conjugate for cancer therapy.

Thermally responsive elastin-like polypeptides (ELPs) were synthesized by recombinant DNA techniques and conjugated to doxorubicin through an acid-labile hydrazone bond to enable release of the drug in the acidic environment of lysosomes. The thermal properties, intracellular localization and cytotoxicity of the conjugate were investigated in this study. The conjugation procedure resulted in a mixed population of free ELP and ELP-doxorubicin (ELP-dox) conjugates that exhibit a broader transition than the parent ELP. A simple centrifugation procedure was developed to purify the ELP-dox conjugate from other reactants and resulted in a sharper thermal transition, similar to the parent ELP. The ELP was endocytosed by squamous cell carcinoma cells (FaDu) and trafficked into lysosomes, as observed by the colocalization of the ELP with a lysosome-specific dye through confocal fluorescence microscopy. Interestingly, both the ELP-dox conjugate and free drug exhibited near equivalent in vitro cytotoxicity, although their subcellular localization was significantly different. The free drug was largely concentrated in the nucleus, while the conjugate was dispersed throughout the cytoplasm with limited nuclear accumulation. These differences are significant because they suggest a different mechanism of cytotoxicity for the conjugate as compared with the free drug.

Selective enhancement of ifosfamide-induced toxicity in Chinese hamster ovary cells.

PURPOSE: O6-benzylguanine (BG) is a unique purine analog that has been shown to influence nitrogen mustard activity and increase cytotoxicity. Ifosfamide is a nitrogen mustard with growing clinical applications; effective modulation may lead to improved efficacy. We thus undertook a preliminary investigation of BG's effects on ifosfamide and ifosfamide derivatives in vitro. EXPERIMENTAL DESIGN: BG's effect on ifosfamide toxicity was studied in CHO cells transfected with O6-alkylguanine-DNA alkyltransferase (AGT) (CHOwtAGT) or control plasmid pcDNA3 (CHOpcDNA) using five ifosfamide derivatives and two control compounds: 4-hydroperoxyifosfamide (4HI), isophosphoramide mustard (IPM), phenylketoifosfamide (PKIF), 4-hydroperoxydidechloroifosfamide (4HDI), chloroacetaldehyde (CAA), didechloroisophosphoramide mustard (d-IPM), didechlorophenylketoifosfamide (d-PKIF). To further explore the mechanism of interaction, BG's effect on apoptosis (annexin V-FITC) and cell cycle distribution in cells exposed to ifosfamide was also analyzed. RESULTS: BG substantially enhanced cytotoxicity induced only by agents that produce IPM (4HI, IPM, PKIF) in both CHOwtAGT and CHOpcDNA cell lines. BG did not modulate 4HDI or CAA cytotoxicity. The addition of BG to IPM in CHO cells increased the percentage of apoptotic cells from 5.5% to 28.9% at 72 h after treatment. Cell cycle analysis showed that BG exposure was associated with G1 arrest. At 16 h following treatment with IPM, PKIF, or phosphoramide mustard (PM), BG increased the percentage of cells in G1 from 16-20% to 29-64%. CONCLUSIONS: BG's ability to increase 4HI-, IPM-, and PKIF-mediated cytotoxicity in cells devoid of AGT activity suggests a novel AGT-independent mode of action that is associated with increased apoptosis and may involve G1 arrest. BG selectively enhanced IPM toxicity without enhancement of acrolein and CAA toxicity. The data strongly support further investigation into combinations of BG and nitrogen mustards.

Exposure to a deuterated analogue of phenylbutyrate retards S-phase progression in HT-29 colon cancer cells.

Differentiation agents that induce neoplastic cells to regain a normal phenotype and/or cause growth arrest without significantly affecting normal cells represent an attractive option for cancer treatment. Analogues of short chain fatty acids, such as phenylbutyrate (PB), have been studied as clinically relevant agents. In an attempt to improve its pharmacokinetic profile, structural modifications of PB and other fatty acids have been studied. We hypothesize that strategic isotopic modification of PB would result in a longer half-life and thus translate into a more potent differentiation agent for clinical use. Using a colon cancer model, we demonstrated that 2,2,3,3-tetradeuterated PB (D4PB) significantly increased induction of apoptosis and inhibition of cell proliferation as compared with PB and butyrate. Difference in potency could not be explained by the effect of D4PB on the expression of specific regulatory proteins of the apoptotic cascade or from the inhibitory effect of D4PB on histone deacetylase activity. Interestingly, exposure of HT-29 colon cancer cells to D4PB resulted in a slowing of S transit, in contrast to butyrate and PB, which induced a G2/M cell cycle block. This difference in cell cycle effect may explain the differences seen in the potency of the phenotypic changes seen with treatment with D4PB. Further studies are needed to elucidate the mechanisms underlying effects of D4PB on the cell cycle.

Modified guanines representing O(6)-alkylation by the cyclophosphamide metabolites acrolein and chloroacetaldehyde: synthesis, stability, and ab initio studies.

Alkylation of DNA by acrolein and/or chloroacetaldehyde may result in the mutations that lead to the therapy-induced leukemia associated with cyclophosphamide (and ifosfamide) treatment. O(6)-(n-Propanalyl)guanine (O(6)-PAG) and O(6)-(ethanalyl)guanine (O(6)-EAG) were synthesized for use as authentic standards in investigations of DNA alkylation by acrolein and chloroacetaldehyde, respectively. Preparation of the O-methyl oximes of these aldehydes aided in confirming the structural assignments of O(6)-PAG and O(6)-EAG. HPLC was used to study the stability of O(6)-PAG under a variety of conditions. The decomposition of O(6)-PAG was attributed to an alpha,beta-elimination reaction resulting in the formation of guanine and acrolein. In 0.1 M phosphate-DMSO (9:1), O(6)-PAG (1-10 mM) had a half-life of approximately 1 h (pH 7.4, 37 degrees C). In 0.05 M Tris-DMSO (9:1), the apparent half-life of O(6)-PAG (1-10 mM) was approximately 16 h (pH 7.4, 37 degrees C). The increased lifetime under the latter conditions was attributed to a reversible reaction between Tris and the aldehydic functionality of O(6)-PAG to give a more stable oxazolidine. Under conditions similar to those that would be used for hydrolysis of DNA [0.1 M HCl-DMSO (98:2), pH 1.3, 70 degrees C, 30 min], there was an estimated 10-35% loss of O(6)-PAG. Under the same conditions, O(6)-EAG had apparent half-lives of 6.6 h (phosphate-DMSO) and 2.5 days (Tris-DMSO) and the estimated loss at pH 1.3 over 30 min (70 degrees C) was 15-20%. Ab initio quantum chemical calculations were used to understand the energy factors that underlie the occurrence of O- versus N-alkylations as well as possible, subsequent intramolecular cyclizations. Simulations of the free energies of reactions between acrolein and guanine indicated that N-alkylation was favored over O(6)()-alkylation and that cyclizations to tautomers were most favorable if they involved the N-1 or NH(2) positions.

Isophosphoramide Mustard and Its Mechanism of Bisalkylation.

To investigate the mechanism(s) of bisalkylation by isophosphoramide mustard (IPM), IPM-beta,beta,beta',beta'-d(4) was synthesized and the products of its reaction with thiosulfate (at pD 7.0) were analyzed by NMR. By both (1)H and (13)C NMR, the distribution of deuterium in the products was consistent with bisalkylation through sequential aziridinyl intermediates [(NCH(2)CD(2)S):(NCD(2)CH(2)S) = 53:47]. Under the given reaction conditions, label scrambling as a result of thiosulfate acting as a leaving group was ruled out through control experiments. The data gave a calculated kinetic isotope effect of 0.97 per deuterium. For the initial aziridine species formed from IPM, ab initio quantum chemical calculations gave a hybridization value of sp(2.4)(-)(2.5) for each of the C-H bonds of the reaction centers, and this correlated with the observed inverse isotope effect. Other structure and bond order data were also determined for this aziridine intermediate and related compounds.