Influence of the alkylating function of aldo-Ifosfamide on the anti-tumor activity.

The present work investigates the influence of different DNA damages caused by different isophosphoramide mustards on the 3-hydroxypropanal-assisted apoptotic antitumor activity of oxazaphosphorine cytostatics using I-aldophosphamide-perhydrothiazine (IAP) and mesyl-I-aldophosphamide-perhydrothiazine (SUM-IAP) for in-vitro and in-vivo experiments. IAP and SUM-IAP hydrolyze spontaneously to the corresponding I-aldophosphamide derivatives. They differ in the chemical structure of the alkylating moiety, whereas IAP has two chlorethyl groups in the SUM-IAP molecule, one chlorethyl group is substituted by a mesylethyl group. With both substances, cytotoxicity studies on P388 tumor cells in vitro and therapy experiments in mice bearing advanced growing P388 tumors were carried out. IAP was significantly more cytotoxic in-vitro than SUM-IAP, but the antitumor activity of SUM-IAP was by order of magnitude higher than the antitumor activity of IAP. The reason for these findings is discussed with respect to the enzymatic cleavage of the various I-aldophosphamide derivatives to the corresponding isophosphoramide mustards and 3-hydroxypropanal. Overall, the findings indicate that antitumor activity of ifosfamide and derivatives of ifosfamide can be improved considerably by altering the alkylating moiety of the molecule, but retaining the aldophosphamide structure.

Phase I trial of sorafenib in combination with ifosfamide in patients with advanced sarcoma: a Spanish group for research on sarcomas (GEIS) study.

BACKGROUND: This phase I trial assessed safety, pharmacokinetics (PK), dose limiting toxicity (DLT), maximum tolerated dose and recommended dose (RD) of the combination of sorafenib plus ifosfamide in patients with advanced sarcoma. METHODS: Twelve sarcoma patients (9 soft-tissue, 3 bone sarcoma) were treated with sorafenib plus ifosfamide (starting doses 200 mg bid and 6 g/m(2) respectively). A 3 + 3 dose escalation design with cohorts of 3-6 patients was used. A study to assess the in vitro efficacy of the combination was also conducted. RESULTS: Three DLTs were observed: fatigue grade 4 with sorafenib 400 mg bid plus ifosfamide 6 g/m(2) and encephalopathy and emesis grade 3 with sorafenib 400 mg bid plus ifosfamide 7.5 g/m(2). Other toxicities included diarrhea, hand-foot syndrome, mucositis, neutropenia, skin rash and thrombocytopenia. There were no relevant effects on PK of sorafenib but an increase in ifosfamide active metabolite 4-hydroxy-ifosfamide was observed. Eight patients achieved stable disease lasting more than 12 weeks. An additive effect was observed in vitro. CONCLUSIONS: RD was sorafenib 400 mg bid plus ifosfamide 6 g/m(2), allowing administration of active doses of both agents. Limited preliminary antitumor activity was also observed. A phase II study is currently ongoing.

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.

In vitro cytotoxicity testing of new generation oxazaphosphorines against human histiocytic lymphoma cells.

Oxazaphosphorines belong to a group of alkylating agents. Mafosfamide cyclohexylamine salt (D-17272), 4-hydro-peroxy-cyclophosphamide (D-18864) and glufosfamide (D-19575, beta-D-glucose-isophosphoramide mustard) are new generation oxazaphosphorines. The objective of the present study was to compare the cytotoxic action of these oxazaphosphorine compounds against human histiocytic lymphoma U937 cells. The chemical structures of the oxazaphosphorines were responsible for the different responses of U937 cells. The cytotoxic effects of D-17272, D-18864, and D-19575 on U937 cells depended on the agent tested, its dose, and the time intervals after the oxazaphosphorine application. Among the oxazaphosphorine agents, D-18864 appeared to be the most cytotoxic, and D-19575 was characterized by the lowest cytotoxicity. The in vitro cytotoxic activities of the oxazaphosphorines were strongly associated with their cell death inducing potential.

[An overview of glycoconjugates for cancer targeting therapy and diagnosis].

Because of the changed metabolic behaviors of cancer cells, tumor cells uptake a corresponding larger amount of glucose in physiological condition when compared with normal cells. And they were prone to metabolize glucose for generating energy in anaerobic glycolysis ways in order to grow quickly. Anaerobic glycolysis consumes more glucose than aerobic way when the same amount of energy is obtained, which also results in large demand of glucose in tumor cells. This review briefly describes therapy methods related to characteristic mentioned above, and summarizes the research progress of drugs, diagnostic reagents and carriers conjugated with glucose, glucose derivatives or other kinds of sugars for cancer targeting. Furthermore, typically relative research reports from 2012 till now were listed and analyzed.

Anticancer activity of stabilized palifosfamide in vivo: schedule effects, oral bioavailability, and enhanced activity with docetaxel and doxorubicin.

Palifosfamide, the DNA-alkylating metabolite of ifosfamide (IFOS), has been synthesized as a stabilized tris or lysine salt and found to have preclinical and clinical antitumor activity. Stabilized palifosfamide overcomes limitations of IFOS because of patient-to-patient variability in response resulting from variable prodrug activation, resistance and toxicities of metabolic byproducts, acrolein and chloroacetaldehyde. Palifosfamide represents an effective alternative to IFOS and other DNA-alkylating prodrugs. The antitumor activities of stabilized palifosfamide were investigated in vivo. Dose response, route and schedule of administration, and interaction with docetaxel or doxorubicin were investigated in NCr-nu/nu mice bearing established orthotopic mammary MX-1 tumor xenografts. Oral activity was investigated in P388-1 leukemia in CD2F1 mice. Oral and intraperitoneal bioavailabilities were compared in Sprague-Dawley rats. Stabilized palifosfamide administered by optimized regimens suppressed MX-1 tumor growth (P<0.05) by greater than 80% with 17% complete antitumor responses and up to three-fold increase in time to three tumor doublings over controls. Median survival in the P388-1 (P<0.001) model was increased by 9 days over controls. Oral bioavailability in rats was 48-73% of parenteral administration, and antitumor activity in mice was equivalent by both routes. Treatment with palifosfamide-tris combined with docetaxel or doxorubicin at optimal regimens resulted in complete tumor regression in 62-75% of mice. These studies support investigation of stabilized palifosfamide in human cancers by parenteral or oral administration as a single agent and in combination with other approved drugs. The potential for clinical translation of the cooperative interaction of palifosfamide-tris with doxorubicin by intravenous administration is supported by results from a recent randomized Phase-II study in unresectable or metastatic soft-tissue sarcoma.

Glufosfamide as a new oxazaphosphorine anticancer agent.

Glufosfamide (ß-D-glucose-isophosphoramide mustard, D-19575) belongs to the oxazaphosphorine class. Glufosfamide is a novel glucose conjugate of ifosfamide in which isophosphoramide mustard, the alkylating metabolite of ifosfamide, is glycosidically linked to the ß-D-glucose molecule. Glufosfamide represents an attractive new agent for cancer therapy. Its mode of action on normal and pathological cells is still under experimental and clinical investigations. An assessment of the anticancer potential of glufosfamide is of key importance in therapy. The researchers reviewed the current knowledge available on glufosfamide tested in the preclinical studies/clinical trials, based on a collection of the original papers and conference abstracts published and relevant articles searched in the SCOPUS and MEDLINE database and websites.

An in vitro cytotoxicity of glufosfamide in HepG2 cells relative to its nonconjugated counterpart.

BACKGROUND: Glufosfamide (ß-D-glucosylisophosphoramide mustard, GLU) is an alkylating cytotoxic agent in which ifosforamide mustard (IPM) is glycosidically linked to the ß-D-glucose molecule. GLU exerted its cytotoxic effect as a targeted chemotherapy. Although, its cytotoxic efficacy in a number of cell lines, there were no experimental or clinical data available on the oncolytic effect of oxazaphosphorine drugs in hepatocellular carcinoma. Therefore, the main objective of the current study is to assess the cytotoxic potential of GLU for the first time in the hepatocellular carcinoma HepG2 cell line model. METHODS: Cytotoxicity was assayed by the MTT method, and half-maximal inhibitory concentration (IC50) was calculated. Flow cytometric analysis of apoptosis frequencies was measured by using Annexin V/PI double stain, an immunocytochemical assay of caspase-9, visualization of caspase-3, and Bcl2 gene expression were undertaken as apoptotic markers. Mitochondrial membrane potential was measured using the potentiometric dye; JC-1, as a clue for early apoptosis as well as ATP production, was measured by the luciferase-chemiluminescence assay. RESULTS: Glufosfamide induced cytotoxicity in HepG2 cells in a concentration- and time-dependent manner. The IC50 values for glufosfamide were significantly lower compared to ifosfamide. The frequency of apoptosis was much higher for glufosfamide than that of ifosfamide. The contents of caspase-9 and caspase-3 were elevated following exposure to GLU more than IFO. The anti-apoptotic Bcl2 gene expression, the mitochondrial membrane potential, and the cellular ATP levels were significantly decreased than in case of ifosfamide. CONCLUSIONS: The current study reported for the first time cytotoxicity activity of glufosfamide in HepG2 cells in vitro. The obtained results confirmed the higher oncolytic activity of glufosfamide than its aglycone ifosfamide. The generated data warrants further elucidations by in vivo study.

Induction of DNA breakage in U937 cells by oxazaphosphorines.

Oxazaphosphorines are a class of DNA alkylating agents. The aim of the present study was to compare the possible influence of three new generation oxazaphosphorines, D-17272 (mafosfamide cyclohexylamine salt), D-18864 (4-hydro-peroxy-cyclophosphamide), and D-19575 (glufosfamide, beta-D-glucose-isophosphoramide mustard) on DNA damage induction in the human histiocytic lymphoma U937 cells. The flow cytometry APO-BRDU assay, based on the TUNEL method, was used for the in situ detection of DNA strand breaks. After exposure of U937 cells to the oxazaphosphorines, the patterns of temporary changes in the frequency of TUNEL positive U937 cells, expressing DNA breakage, were determined. The effects of the oxazaphosphorines on U937 cells were dependent on the agent tested and its dose, and the time intervals after the drug application. The different potential of D-17272, D-18864 and D-19575 to induce DNA strand breakage in the human histiocytic lymphoma U937 cells was shown.

New ifosfamide analogs designed for lower associated neurotoxicity and nephrotoxicity with modified alkylating kinetics leading to enhanced in vitro anticancer activity.

Ifosfamide is a well known prodrug for cancer treatment with cytochrome P450 metabolism. It is associated with both antitumor activity and toxicities. Isophosphoramide mustard is the bisalkylating active metabolite, and acrolein is a urotoxic side product. Because acrolein toxicity is limited by coadministration of sodium mercaptoethanesulfonate, the incidence of urotoxicity has been lowered. Current evidence suggests that chloroacetaldehyde, a side-chain oxidation metabolite, is responsible for neurotoxicity and nephrotoxicity. The aim of our research is to prevent chloroacetaldehyde formation using new enantioselectively synthesized ifosfamide analogs, i.e., C7,C9-dimethyl-ifosfamide. We hypothesize that reduced toxicogenic catabolism may induce less toxicity without changing anticancer activity. Metabolite determinations of the dimethyl-ifosfamide analogs were performed using liquid chromatography and tandem mass spectrometry after in vitro biotransformation by drug-induced rat liver microsomes and human microsomes expressing the main CYP3A4 and minor CYP2B6 enzymes. Both human and rat microsomes incubations produced the same N-deschloroalkylated and 4-hydroxylated metabolites. A coculture assay of 9L rat glioblastoma cells and rat microsomes was performed to evaluate their cytotoxicity. Finally, a mechanistic study using (31)P NMR kinetics allowed estimating the alkylating activity of the modified mustards. The results showed that C7,C9-dimethyl-ifosfamide exhibited increased activities, although they were still metabolized through the same N-deschloroalkylation pathway. Analogs were 4 to 6 times more cytotoxic than ifosfamide on 9L cells, and the generated dimethylated mustards were 28 times faster alkylating agents than ifosfamide mustards. Among these new ifosfamide analogs, the 7S,9R-enantiomer will be assessed for further in vivo investigations for its anticancer activity and its toxicological profile.

HPV 16-associated tumours: IL-12 can repair the absence of cytotoxic and proliferative responses of tumour infiltrating cells after chemotherapy.

We have examined the effect of IL-12-producing cellular vaccines on the cytotoxicity and proliferative potential of CD45+ tumour-infiltrating cells (TIL) in mice carrying syngeneic TC-1 and TC-1/A9 HPV 16-associated tumours after chemotherapy with CBM-4A ifosfamide derivative. The chemotherapy resulted in the decrease of the CD4+ and CD8+ TIL, increase of the Gr-1+/CD11b+ TIL, no changes in the infiltration with CD4+/CD25+ Treg TIL, and decrease of the cytolytic and proliferative potential of the CD45+ TIL. Subsequent immunotherapy with the IL-12-producing, genetically modified TC-1 (TC-1-IL-12) cells increased tumour infiltration with CD8+ and CD4+ cells, decreased the Gr-1+/CD11b+ cells, and increased the cytolytic and proliferative potential of the CD45+ TIL. Taken together, these findings suggest that peritumoral administration of the IL-12-producing cellular vaccine can restore the cytolytic potential and inhibit immunosuppressive TIL-dependent mechanisms in the individuals bearing HPV 16-associated tumours, and explain our previously described tumour-inhibitory effects of the vaccine in mice with minimal residual disease after the tumour chemotherapy.

Detection and quantification of (R) and (S)-dechloroethylifosfamide metabolites in plasma from children by enantioselective LC/MS/MS.

Ifosfamide (IF), a potent chemotherapeutic agent for solid tumors, is known to cause high rates of nephrotoxicity in children with cancer, which is most likely due to the renal production of the metabolite chloroacetaldehyde. Using plasma samples obtained from pediatric oncology patients, we developed a simple nonderivatizing enantioselective liquid chromatography mass spectrometry method to detect the (R) and (S)-2- and 3-dechloroethylifosfamide metabolites. The (R) and (S)-enantiomers of the 2- and 3-DCEIF (N-3-dechlroethylifosfamide) were detectable in all 22 patients' samples with levels ranging from 9.9 to 238.7 ng/ml for (R)-2-DCEIF, 15.8 to 663.0 ng/ml for (S)-2-DCEIF, 20.8 to 852.8 ng/l for (R)-3-DCEIF and 28.0 to 862.0 ng/ml for (S)-3-DCEIF. In addition, the lower limit of quantification for this method is 1 ng/ml. Future studies should concentrate on (R) or (S) production of the 2-DCEIF and 3-DCEIF and subsequently chloroacetaldehyde formation with the aim of considering the administration of only the (R)-IF as its metabolism results in a lower production of chloroacetaldehyde.

A Phase 1 dose-escalation trial of glufosfamide in combination with gemcitabine in solid tumors including pancreatic adenocarcinoma.

PURPOSE: To evaluate safety and pharmacokinetics and to establish the maximum tolerated dose of glufosfamide when administered in combination with gemcitabine in advanced solid tumors. METHODS: This Phase 1 dose-escalation study evaluated the combination of glufosfamide + gemcitabine in patients with advanced solid tumors. Cohorts of three to six patients were treated with glufosfamide doses from 1,500 to 4,500 mg/m(2) i.v. over 4 h on Day 1 and gemcitabine 1,000 mg/m(2) i.v. over 30 min on Days 1, 8 and 15 of every 28-day cycle. Detailed PK sampling was performed on days 1 and 8 of the first two cycles. RESULTS: Nineteen patients were enrolled. Two patients had dose-limiting toxicity: Grade 3 fatigue at 2,500 mg/m(2) and Grade 4 thrombocytopenia at 4,500 mg/m(2). Five patients completed six cycles and one patient remained on study for ten cycles. Two patients discontinued for adverse events. Grade 3/4 neutropenia and thrombocytopenia occurred in seven patients and five patients, respectively. The CrCL fell below 60 mL/min in two patients. There was one unconfirmed partial response and 10 of 19 (52.6%) patients had stable disease or better at 8 weeks and three patients had continuing stable disease at 24 weeks. Pharmacokinetic analyses suggest no interaction between glufosfamide and gemcitabine. CONCLUSION: Phase I data indicate that full dose glufosfamide (4,500 mg/m(2)) can be given safely in combination with gemcitabine. A Phase II study in patients with pancreatic adenocarcinoma is ongoing.

Enantioselective liquid chromatography-mass spectrometry assay for the determination of ifosfamide and identification of the N-dechloroethylated metabolites of ifosfamide in human plasma.

A sensitive and specific liquid chromatography-mass spectrometry (LC-MS) method has been developed and validated for the enantioselective determination of ifosfamide [(R)-IF and (S)-IF] in human plasma and for the detection of the N-dechloroethylated metabolites of IF, 2-N-dechloroethylifosfamide [(R)-2-DCl-IF and (S)-2-DCl-IF] and 3-N-dechloroethylifosfamide [(R)-3-DCl-IF and (S)-3-DCl-IF]. IF, 2-DCl-IF and 3-DCl-IF were extracted from plasma using solid-phase extraction and resolved by liquid chromatography on a column containing a Chirabiotic T chiral stationary phase. The enantioselective separations were achieved using a mobile phase composed of 2-propanol:methanol (60:40, v/v) and a flow rate of 0.5 ml/min. The observed enantioselectivities (alpha) for IF, 2-DCl-IF and 3-DCl-IF were 1.20, 1.17 and 1.20, respectively. The calibration curve was linear in the concentration range of 37.50-4800 ng/ml for each ifosfamide enantiomer (r(2)>0.997). The lower limit of detection (LLOD) was 5.00 ng/ml. The inter- and intra-day precision ranged from 3.63 to 15.8% relative standard deviation (R.S.D.) and 10.1 to 14.3% R.S.D., respectively, and the accuracy ranged from 89.2 to 101.5% of the nominal values. The method was applied to the analysis of plasma samples obtained from a cancer patient who received 3.75 g/m(2)/day dose of (R,S)-ifosfamide as a 96-h continuous infusion.

Poly-isoprenylated ifosfamide analogs: Preactivated antitumor agents as free formulation or nanoassemblies.

Oxazaphosphorines including cyclophosphamide, trofosfamide and ifosfamide (IFO) belong to the alkylating agent class and are indicated in the treatment of numerous cancers. However, IFO is subject to limiting side-effects in high-dose protocols. To circumvent IFO drawbacks in clinical practices, preactivated IFO analogs were designed to by-pass the toxic metabolic pathway. Among these IFO analogs, some of them showed the ability to self-assemble due to the use of a poly-isoprenyloxy chain as preactivating moiety. We present here, the in vitro activity of the nanoassembly formulations of preactivated IFO derivatives with a C-4 geranyloxy, farnesyloxy and squalenoxy substituent on a large panel of tumor cell lines. The chemical and colloidal stabilities of the geranyloxy-IFO (G-IFO), farnesyloxy-IFO (F-IFO) and squalenoxy-IFO (SQ-IFO) NAs were further evaluated in comparison to their free formulation. Finally, pharmacokinetic parameters and maximal tolerated dose of the most potent preactivated IFO analog (G-IFO) were determined and compared to IFO, paving the way to in vivo studies.

Comparison of In Vitro Antileukemic Activity of 4-Hydroperoxyifosfamide and 4-Hydroperoxycyclophosphamide.

BACKGROUND/AIM: The oxazaphosphorines, ifosfamide and cyclophosphamide, represent a class of alkylating agents. The aim of the present in vitro study was to compare antileukemic activity of 4-hydroperoxyifosfamide (4-OOH-IF) and 4-hydroperoxycyclophosphamide (4-OOH-CP). MATERIALS AND METHODS: The experiments were performed on MOLT-4 and ML-1 cells. The research was conducted using flow cytometry fluorescein diacetate/propidium iodide (PI), fluorescein-conjugated annexin V/PI, CaspGLOW Red Active Caspase-8 and -9, CellEvent™ Caspase-3/7 Green assays, and tetramethylrhodamine ethyl ester test. RESULTS: 4-OOH-IF and 4-OOH-CP distinctly reduced cell viability and triggered apoptosis and necrosis, causing changes in intracellular esterase activity, plasma membrane structure and integrity, caspase activation, and mitochondrial membrane potential. The oxazaphosphorines were responsible for the different antileukemic activities. 4-Hydroperoxyifosfamide appeared to be less cytotoxic against the leukemia cells than 4-hydroperoxycyclophosphamide. MOLT-4 cells were more sensitive to the action of the oxazaphosphorines than ML-1 cells. CONCLUSION: The findings provide a new insight on the mechanisms of cytotoxic action of 4-OOH-IF and 4-OOH-CP on the human acute lymphoblastic and myeloblastic leukemia cells.

Chemotherapy, IL-12 gene therapy and combined adjuvant therapy of HPV 16-associated MHC class I-proficient and -deficient tumours.

Moderately immunogenic HPV 16-associated murine tumour cell line mimicking human HPV 16-associated neoplasms TC-1 (MHC class I(+)) and its variants, TC-1/P3C10 and TC-1/A9, with a marked down-regulation of MHC I molecules, were used to examine the effect of local interleukin 12 (IL-12) gene therapy for the treatment of early tumour transplants and minimal residual tumour disease obtained after cytoreductive chemotherapy (CMRTD). Experiments were designed to examine whether down-regulation of MHC class I molecules plays a role during chemotherapy and gene therapy of early tumour transplants. It was found that peritumoral administration of IL-12-producing tumour cell vaccines (single dose, day 8 after tumour cell administration) inhibited the growth of both TC-1 (MHC class I positive) tumours and their MHC class I-deficient variants. To investigate the antitumour effects in a clinically relevant setting, IL-12 gene therapy was utilised for the treatment of minimal residual tumour disease after cytoreductive chemotherapy. Intra-peritoneal treatment of tumour-bearing mice with ifosfamide derivative, CBM-4A, produced a significant tumour-inhibitory effect. This treatment was followed by peritumoral s.c. administration of genetically modified TC-1 (MHC class I positive) or MK16/I/IIIABC (MHC class I negative) vaccines producing IL-12 (single dose, day 7 after chemotherapy) or with recombinant interleukin 12 (rIL-12) in two cycles of 5 daily doses (days 8-19) after chemotherapy. This combined therapy significantly inhibited the growth of TC-1 and TC-1/A9 (MHC class I-) tumours. When the combined therapy of TC-1 (MHC class I positive) tumours was followed by peritumoral administration of bone marrow dendritic cell (BMDC) vaccines, the IL-12-mediated inhibitory effect was significantly boosted. In the next set of experiments, the impacts of chemotherapy and IL-12 adjuvant therapy on MHC class I surface expression were assessed. Chemotherapy and gene therapy of tumours led to the up-regulation of MHC I expression on MHC class I-deficient tumours (TC-1/A9 and TC-1/P3C10) and to down-regulation on MHC I-proficient tumours (TC-1). These findings indicate that the MHC I phenotype is not stable during tumour progression and treatment. Collectively, these results illustrate the efficacy of IL-12 gene therapy in combination with chemotherapy on HPV-associated tumours regardless of the level of MHC class I expression on the tumour cells.

Stability of glufosfamide in phosphate buffers and in biological samples.

Glufosfamide is a new, potential chemotherapeutic agent currently under investigation. Stability of glufosfamide was investigated in sodium phosphate buffers with different pH and temperature and in biological samples. Glufosfamide and isophosphamide mustard were quantified simultaneously using a liquid chromatography-ion trap mass spectrometric method; precision and accuracy were within 15% for each analyte. Glufosfamide was stable in neutral buffers, but decomposed to form isophosphoramide mustard under acidic and basic conditions, which was pH- and temperature-dependent. The stability of glufosfamide varied in different biological samples. Results indicated that glufosfamide was unstable in some biological samples, such as the small intestine, smooth muscles, pancreas and urine, especially in the small intestine homogenate, with a half-life of 1.1 h. But the pH (<8) and beta-glucosidase of the tissue homogenate was found to have negligible contribution to the degradation of glufosfamide. The enzymatic inhibition experiment with the specific inhibitor, saccharo-1,4-lactone, demonstrated that it was glucuronidase that resulted in the degradation of glufosfamide in small intestine homogenate. Methanol was recommended to be used to homogenize the tissue in an ice water bath, and the container for urine collection should also be maintained in an ice water bath, and all the biological samples collected should be preserved in frozen condition until analysis.

[Identification of glufosfamide metabolites in rats].

AIM: To elucidate the metabolic pathway of glufosfamide in rats. METHODS: In this study, a liquid chromatography-tandem mass spectrometric method was developed and applied to characterize the metabolites of glufosfamide in rat urine, after an i.v. administration of 50 mg x kg(-1). The analysis was performed under two ionization modes in two different chromatographic systems, separately. To make sure that the compounds detected in rat urine were metabolites or degradation products, the stability of glufosfamide, isophosphoramide mustard (M1), and the degradation products of M1 in urine were investigated. RESULTS: In positive ionization mode, besides glufosfamide, two metabolites, isophosphoramide mustard and monoaziridinyl derivative of isophosphoramide mustard, were detected. In negative ionization mode, only glufosfamide itself was detected, while derivatives of isophosphoramide mustard have no response in such condition. CONCLUSION: Glufosfamide was mainly unchanged excreted in urine, and two metabolites were detected as isophosphoramide mustard and monoaziridinyl derivative of isophosphoramide mustard.

Quantification by gas chromatography of N,N'-di-(2-chloroethyl)-phosphorodiamidic acid in the plasma of patients receiving isophosphamide.

A sensitive method, based on gas chromatography using a phosphorus-specific flame photometric detector, has been developed for quantifying N,N'-di-(2-chloroethyl)phosphorodiamidic acid (isophosphoramide mustard), the putative active metabolite of isophosphamide, in human plasma. Phosphoramide mustard was used as internal standard, and the two compounds were converted into separable trimethyl derivatives by reaction with methyliodide in the presence of silver oxide. The chemistry of the derivatization process has been elucidated using gas chromatography-electron impact mass spectrometry and selected ion monitoring. Levels of isophosphamide and of isophosphoramide mustard were measured in the plasma of patients receiving isophosphamide (2 g/sq m). Peak plasma levels of isophosphoramide mustard of 18.6 to 30.3 nmol/ml occurred at 2 to 4 hr, and levels were still appreciable (6.3 to 11.3 nmol/ml) at 24 hr.