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Objective: The objective of this study was to evaluate the frequency of genetic lesions in pharmacists and nurses who prepare and/or handle antineoplastic agents and to evaluate whether there are traces of contaminants in the urine of these professionals., Methods: A total of 59 professionals participated in the study, of which 10 were non-exposed professionals (controls), 25 were pharmacists, and 24 were nurses., Key Findings: There was a significant increase in genetic damage in lymphocytes and cells of the oral mucosa in both pharmacists and nurses. The levels of cyclophosphamide and ifosfamide were also increased in the urine samples from those individuals., Conclusions: These results demonstrate the growing need for genetic biomonitoring and biomonitoring of trace antineoplastic agents in the urine of health professionals who prepare and/or handle antineoplastic agents., (© 2019 Royal Pharmaceutical Society.)

New multi-walled carbon nanotubes supported on Ti 3 C 2 -MXene and chitosan (chit) composite film-based electrochemical sensor for ifosfamide (IFO), acetaminophen (ACOP), domperidone (DOM), and sumatriptan (SUM) have been developed. Ti 3 C 2 -MXene was synthesized by a fluoride method. Structural and chemical characterizations suggested the successful preparation of Ti 3 C 2 -MXene with clearly seen layered morphology, defined 0 0 2 diffraction peak at 7.5° and complete absence of 1 0 4 plane at 39°. The electrochemical performance of the sensor was investigated by cyclic voltammetry and adsorptive stripping differential pulse voltammetry. The Ti 3 C 2 /MWCNT/Chit modified glassy carbon electrode exhibits enhanced electrocatalytic activities toward the oxidation of target analytes. Excellent conductivity, large surface area, and high catalytic properties of the Ti 3 C 2 -MXene showed synergistic effects with MWCNTs and helped in achieving low detection limits of targets with high selectivity and reproducibility. The assay allows determination of IFO, ACOP, DOM, and SUM in the concentration ranges 0.0011-1.0, 0.0042-7.1, 0.0046-7.3, and 0.0033-61 μM with low detection limits of 0.00031, 0.00028, 0.00034, and 0.00042 μM, respectively. The sensor was successfully applied for voltammetric screening of target analytes in urine and blood serum samples with recoveries > 95.21%. Schematic illustration of the synthesis of self-assembled MXene/MWCNT/chitosan nanocomposite is given and its application to the voltammetric determination of ifosfamide, acetaminophen, domperidone, and sumatriptan described. Graphical abstract.

Grapefruit juice (GFJ) and naringin when consumed previously or together with medications may alter their bioavailavility and consequently the clinical effect. Ifosfamide (IF) is an antitumoral agent prescribed against various types of cancer. Nevertheless, there is no information regarding its interaction with the ingestion of GFJ or naringin. The aims of the present report were validating a method for the quantitation of IF in the plasma of mouse, and determine if mice pretreated with GFJ or naringin may modify the IF pharmacokinetics. Our HPLC results to quantify IF showed adequate intra and inter-day precision (RSD < 15%) and accuracy (RE < 15%) indicating reliability. Also, the administration of GFJ or naringin increased C max of IF 22.9% and 17.8%, respectively, and decreased T max of IF 19.2 and 53.8%, respectively. The concentration of IF was higher when GFJ (71.35 ± 3.5 µg/mL) was administered with respect to that obtained in the combination naringin with IF (64.12 ± µg/mL); however, the time required to reach such concentration was significantly lower when naringin was administered (p < 0.5). We concluded that pre-administering GFJ and naringin to mice increased the T max and decreased the C max of IF.

The simultaneous measurement of the concentration of anticancer drugs with a fast, sensitive and accurate method in biological samples is a challenge for better monitoring of drug therapy and better determine the pharmacokinetics. An electrochemical sensor was developed for the simultaneous determination of anticancer drugs, Ifosfamide (IFO) and Etoposide (ETO) based on pencil graphite electrode modified with Au/Pd@rGO nanocomposite decorated with poly (L-Cysteine). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were utilized to study the properties of the modified electrode. The electrochemical behavior of IFO and ETO on the Au/Pd@rGO@p(L-Cys) modified electrode was investigated by cyclic voltammetry and differential pulse voltammetry (DPV) techniques and the obtained results confirmed its efficiency for the individual and simultaneous sensing of IFO and ETO. After optimization of electrochemical parameters, the fabricated sensor presented excellent performance in simultaneous determination of IFO and ETO with a wide linear range from 0.10 to 90.0 μM and 0.01 to 40.0 μM and low detection limits (3 S b /m) of 9.210 nM and 0.718 nM, respectively. In addition, this study proved that the constructed sensor could be useful to simultaneous analysis of IFO and ETO in biological samples and pharmaceutical compounds., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ifosfamide blood concentrations are necessary to monitor its therapeutic response, avoiding any adverse effect. We developed and validated an analytical method by UPLC-MS/MS to quantify ifosfamide in dried blood spots (DBS). Blood samples were collected on Whatman 903® filter paper cards. Five 3 mm disks were punched out from each dried blood spot. Acetonitrile and ethyl acetate were used for drug extraction. Chromatographic separation was carried out in an Acquity UPLC equipment with a BEH-C18 column, 2.1 x 100 mm, 1.7 μm (Waters®). The mobile phase consisted in 5 mM ammonium formate and methanol:acetonitrile (40:48:12 v/v/v) at 0.2 mL/min. LC-MS/MS detection was done by ESI+ and multiple reaction mode monitoring, ionic transitions were m/z1+ 260.99 > 91.63 for ifosfamide and 261.00 > 139.90 for cyclophosphamide (internal standard). This method was linear within a 100-10000 ng/mL range and it was accurate, precise and selective. Ifosfamide samples in DBS were stable for up to 52 days at -80°C. The procedure was tested in 14 patients, ages 1 month to 17 years (9 males and 5 females), with embryonic tumours treated with ifosfamide, alone or combined, at a public tertiary referral hospital. Ifosfamide blood levels ranged from 11.1 to 39.7 μmol/L at 12 hours after the last infusion, while 24-hour levels ranged from 0.7-19.7 μmol/L. The median at 12 hours was 19.5 μmol/L (Q25 14.4-Q75 29.0) and 3.8 μmol/L (Q25 1.5-Q75 9.9) at 24 hours, p<0.001. This method is feasible to determine ifosfamide plasma levels in paediatric patients.

The antitumor drug, ifosfamide (IFO), requires activation by cytochrome P450 (CYP) to form the active metabolite, 4-hydroxyisfosfamide (4-OHIFO), leading to toxic by-products at high dose. In order to overcome these drawbacks, preactivated ifosfamide derivatives (RXIFO) were designed to release 4-OHIFO without CYP involvement. A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the simultaneous quantification of 4-OHIFO, IFO and four derivatives RXIFO in mouse plasma using multiple reaction monitoring. Because of its instability in plasma, 4-OHIFO was immediately converted to the semi-carbazone derivative, 4-OHIFO-SCZ. For the six analytes, the calibration curves were linear from 20 to 5000ng/mL in 50μL plasma and the lower limit of quantitation was determined at 20ng/mL with accuracies within ±10% of nominal and precisions less than 12%. Their recoveries ranged from 62 to 96% by using liquid-liquid extraction. With an improved assay sensitivity compared to analogues, the derivative 4-OHIFO-SCZ was stable in plasma at 4°C for 24h and at -20°C for three months. For all compounds, the assay was validated with accuracies within ±13% and precisions less than 15%. This method was applied to a comparative pharmacokinetic study of 4-OHIFO from IFO and three derivatives RXIFO in mice. This active metabolite was produced by some of the novel conjugates with good pharmacokinetic properties., (Copyright © 2015 Elsevier B.V. All rights reserved.)

This study investigated the effects of umbelliferone (UF) on alcoholic fatty liver and its underlying mechanism. Rats were fed a Lieber-DeCarli liquid diet with 36% of calories as alcohol with or without UF (0.05 g/L) for 8 weeks. Pair-fed rats received an isocaloric carbohydrate liquid diet. UF significantly reduced the severity of alcohol-induced body weight loss, hepatic lipid accumulation and droplet formation, and dyslipidemia. UF decreased plasma AST, ALT, and γGTP activity. UF significantly reduced hepatic cytochrome P450 2E1 activities and increased alcohol dehydrogenase and aldehyde dehydrogenase 2 activities compared to the alcohol control group, which resulted in a lower plasma acetaldehyde level in the rats that received UF. Chronic alcohol exposure inhibited hepatic AMPK activation compared to the pair-fed rats, which was reversed by UF supplementation. UF also significantly suppressed the lipogenic gene expression (SREBP-1c, SREBP-2, FAS, CIDEA, and PPARγ) and elevated the fatty acid oxidation gene expression (PPARα, Acsl1, CPT, Acox, and Acaa1a) compared to the alcohol control group, which could lead to inhibition of FAS activity and stimulation of CPT and fatty acid β-oxidation activities in the liver of chronic alcohol-fed rats. These results indicated that UF attenuated alcoholic steatosis through down-regulation of SREBP-1c-mediated lipogenesis and up-regulation of PPARα-mediated fatty acid oxidation. Therefore, UF may provide a promising natural therapeutic strategy against alcoholic fatty liver., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Ifosfamide (IFO), which is used in the treatment of pediatric solid tumors, causes high rates of nephrotoxicity. N-acetylcysteine (NAC), an antidote for acetaminophen overdose, has been shown to prevent IFO-induced renal cell death and nephrotoxicity in both LLCPK-1 cells and a rat model. To facilitate the use of NAC in preventing IFO-induced nephrotoxicity in children, the authors compared the systemic exposure to NAC in children treated for acetaminophen overdose to the systemic exposure of the therapeutically effective rat model. The mean systemic exposure in the rat model was 18.72 mM·h (range, 9.92-30.02 mM·h), compared to the mean systemic exposure found in treated children (14.48 mM·h; range, 6.22-32.96 mM·h). They also report 2 pediatric cases in which NAC-attenuated acute renal failure associated with IFO when given concurrently with their chemotherapy treatment. Systemic exposure to NAC measured in 1 of these cases was comparable to that in the children treated for acetaminophen overdose. These results corroborate NAC's potential to protect against IFO-induced nephrotoxicity in children when used in its clinically approved dose schedule and supports a clinical trial in children.

Among antitumor oxazaphosphorine drugs, the prodrug ifosfamide (IFO) and its analogs require metabolic activation by specific liver cytochrome P450 (CYP) enzymes to become therapeutically active. New 7,9-dimethyl-ifosfamide analogs have shown greater cytotoxic activity than IFO, whereas side-chain oxidation still occurred leading to monochloroacetone after N-dechloropropylation. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the simultaneous quantitation of the prodrug 7S,9S-dimethyl-ifosfamide (diMeIFO) and its two inactive metabolites, N(2)- and N(3)-deschloropropyl-dimethylifosfamide (N(2)-DCP-diMeIFO and N(3)-DCP-diMeIFO) in mouse plasma. After protein precipitation with methanol, the analytes were separated by isocratic reversed-phase chromatography with (methanol/ammonium formate pH 5.5, 60:40, v/v) and detected by tandem mass spectrometry using multiple reaction monitoring of transitions ions m/z 289→168 for diMeIFO, m/z 213→168 for N(2)-DCP-diMeIFO, m/z 213→92 for N(3)-DCP-diMeIFO and m/z 261→154 for IFO (internal standard). The calibration curves were linear over the concentration range of 20-10,000ng/mL for the three analytes. Mean extraction recoveries from mouse plasma were 99, 96, 99 and 100% for diMeIFO, N(2)-DCP-diMeIFO, N(3)-DCP-diMeIFO and IFO, respectively. The lower limit of quantitation for diMeIFO and its metabolites was 20 ng/mL in 50 μL plasma. The method was accurate with calculated bias from -5.8 to 4.0% for diMeIFO, from -1.1 to 10.6% for N(2)-DCP-diMeIFO and from -6.9 to 9.8% for N(3)-DCP-diMeIFO, and precise with coefficients of variation lower than 6.8%, 7.8% and 14.3%, respectively. The assay was successfully applied to a preliminary pharmacokinetic study of diMeIFO and of its metabolites in mice., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

Dexamethasone induces the hepatic cytochrome P450 3A and, therefore, is predicted to change the pharmacokinetics, activities, and side effects of drugs metabolized by cytochrome P450 3A. The aim of this study was to determine whether the pharmacokinetics of the cytochrome P450 3A-dependent oxazaphosphorine cytostatic drug ifosfamide is influenced by short-term antiemetic use of dexamethasone in patients. The peak concentration and area under the curve (AUC) were determined for the parent compound and the metabolites 4-hydroxyifosfamide and chloracetaldehyde in eight patients who received two cycles of ICE chemotherapy (ifosfamide 5 g/m(2) day 1, carboplatin 300 mg/m(2) day 1, etoposide 100 mg/m(2) days 1-3). One cycle included concomitant administration of dexamethasone (40 mg over 30 min, 16 h and 1 h before chemotherapy), whereas the other did not. The half-lives of ifosfamide, 4-hydroxyifosfamide, and chloracetaldehyde were shorter with concomitant administration of dexamethasone, but the differences were not statistically significant. In addition, there were no significant differences in the ifosfamide and active 4-hydroxyifosfamide peak concentrations and AUCs when dexamethasone was included. After dexamethasone administration, the chloracetaldehyde peak concentration was slightly increased by 1.5-fold and the AUC by 1.3-fold; however, these increases were not statistically significant. In conclusion, dexamethasone comedication in ICE chemotherapy did not change the ifosfamide pharmacokinetics. Thus, dexamethasone can be used safely as an antiemetic drug in ifosfamide chemotherapy.

In man, neurotoxicity associated to ifosfamide treatment can be reversed by intravenous thiamine administration. Trying to explain this clinical finding, we decided to study possible changes in thiamine availability and activation in patients exposed to ifosfamide. Free thiamine and its phosphate esters levels were measured in plasma, erythrocytes and urine by an ion-pair HPLC method with pre-column derivatization, which allowed separation of the fluorescent compounds in less than 10 min. The method was validated by linearity, sensitivity and reproducibility studies, whose values met the demands for bioanalytical assays. This method was applied to assess thiamine status in cancer patients exposed to ifosfamide therapy for advanced disease.

The combination of irinotecan (daily for 5 days for 2 consecutive weeks) and ifosfamide (daily on days 1 through 3) was investigated in children with osteosarcoma. Irinotecan pharmacokinetic investigations were performed before ifosfamide (day 1), after 3 days of ifosfamide (day 3), and 9 days after the end of ifosfamide (day 12). On day 3, the concentrations of irinotecan's active metabolite, SN-38, were below the limit of quantitation in two patients and were decreased in a third patient. The SN-38 area under the concentration-time curve remained below the day 1 value in two patients on day 12. The reduced area under the curve to the active metabolite SN-38 during ifosfamide therapy predicts a compromised efficacy of irinotecan in this combination.

Trofosfamide (TRO), like cyclophosphamide (CYCLO) and ifosfamide (IFO), is a prodrug oxazaphosphorine derivative that requires hepatic biotransformation to form the cytotoxically active 4-hydroxy derivative (4-hydroxy-TRO). Individual 4-hydroxyoxazaphosphorines and 4-hydroxy-TRO itself have not been demonstrated in humans up to now. For investigation of the principal pharmacokinetics of TRO and its metabolites, six tumour patients (49-65 years of age, Karnofsky index >70%) with normal liver and renal function were given a single oral dose of 600 mg/m(2) TRO. Plasma was sampled using a bedside technique. Individual 4-hydroxyoxazaphosphorines and TRO together with further metabolites were determined by a specially developed HPLC-UV method and a HPLC-MS method, respectively. With a short apparent half-life (1.2 h) and high apparent clearance (Cl/F 4.0 l/min), TRO was very quickly eliminated from plasma and highly converted to its metabolites, mainly 4-hydroxy-TRO and IFO. In relation to the AUC values of TRO (1.0) the following molar quotients were calculated: 1.59 (4-hydroxy-TRO), 0.40 (4-hydroxy-IFO), 6.90 (IFO) and 0.74 (CYCLO). C(max) values were in the range 10-13 micromol/l for TRO, 4-hydroxy-TRO and IFO and in the range 1.5-4.0 micromol/l for CYCLO, 2- and 3-dechloroethyl-IFO and 4-hydroxy-IFO. Kinetic data indicate that 4-hydroxy-IFO is formed by both hydroxylation of TRO and exocyclic N-dechloroethylation of 4-hydroxy-TRO. 4-hydroxy-CYCLO was not detected above the quantification limit of the method. Only mild haemodepressive side effects were observed after oral administration of 600 mg/m(2) TRO. In relation to known data for IFO, TRO is much more 4-hydroxylated than IFO. The high 4-hydroxy-TRO/TRO ratio found suggests that TRO is a promising tumourstatic agent.

This phase I study was designed to evaluate the activity toxicity and pharmacokinetics of docetaxel combined with ifosfamide in the treatment of hormone-refractory prostate cancer. Ten patients received a median of 4.6 treatment cycles. Docetaxel was administered at a dose of 40 mg/m2 in a 1-hour infusion followed by ifosfamide 3,000 mg/m2 in a 24-hour infusion every 3 weeks. The optimal sequence of chemotherapeutic agents was investigated by reversing the order of administration in the second cycle and by collecting a total of six pharmacokinetic blood samples per cycle from all patients during the first and second cycles. The sequence of administration did not influence the pharmacokinetics of docetaxel. Prostate-specific antigen (PSA) responses were observed in four out of nine patients, with a PSA response rate of 44.4% (complete response + partial response). The treatment was well tolerated. No grade IV toxicities were recorded and grade III leucopenia resulted in dose-reductions in 6 cycles (13.3%). The pharmacokinetic parameters of docetaxel were similar in both sequences. Our recommendation for further phase II studies is ifosfamide followed by low-dose docetaxel. Further phase II efficacy studies are warranted.

Background: The therapeutic efficacy of intratumoral instillation of genetically engineered, CYP2B1-expressing, microencapsulated cells in combination with ifosfamide had been previously demonstrated in xenografted human pancreatic ductal carcinomas [Gene Ther 1998;5:1070-1078]. Prior to a clinical study, the feasibility of an intra-arterial application of microencapsulated cells to the pancreas and its consequences to the organ had to be evaluated., Material and Methods: Microencapsulated, CYP2B1-producing cells were instilled both in vivo (transfemoral angiographical access) and in vitro (perfusion model) in the splenic lobe of the pig pancreas. In vivo, animals were monitored clinically for 7 days, then treated with ifosfamide and sacrificed. In vitro, ifosfamide was administered intra-arterially., Results: In all animals, 100 microcapsules could be instilled safely via the femoral route without clinical, biochemical or histological signs of pancreatitis. Histological examination revealed partial obstruction of small arteries by the capsules, without causing any parenchymal damage. In vitro, instillation reduced blood flow by half. Ifosfamide, also in combination with the capsules, did not add any damage to the pancreas., Conclusion: Intra-arterial instillation of microencapsulated cells to the pig pancreas is feasible and safe. Neither pancreatitis, foreign body reactions nor circulatory disturbances were observed. Clinical application of this genetically enhanced chemotherapeutic method seems possible., (Copyright 2003 S. Karger AG, Basel and IAP)

Objective: This study focuses on the pharmacokinetics of trofosfamide (TRO) and metabolites after oral administration of TRO., Methods: Twelve patients with solid tumors and non-Hodgkin lymphomas were treated with 450 mg TRO orally for 7 days. TRO and the stable metabolites ifosfamide (IFO), cyclophosphamide (CYC), 2- and 3-dechloroethylifosfamide (2-DCE, 3-DCE) were determined by GC and the sum of the 4-OH-metabolites was measured by HPLC., Results: A fast metabolism of TRO with a half-life of about 1 h was observed. IFO was the main stable metabolite, whereas CYC was only detected in minor quantities. The peak levels and the AUC of the 4-OH-metabolites were 9.5 and 4.3 times higher than observed after an equimolar IFO dose. Only 6% of the administered dose was recovered in urine within 24 hours as stable metabolites. TRO was under limit of detection., Conclusions: Our results confirm that dechloroethylation of TRO to IFO is a major metabolic pathway. Additionally, we found considerable 4-hydroxylation not shown previously. With respect to the low levels of IFO and CYC observed, the sum of 4-OH-metabolites cannot be explained by hydroxylation of these metabolites only. Hence, we assume a direct 4-hydroxylation of TRO occurring to a high extent. Bioavailability of TRO could not be calculated directly, because TRO is only available as an oral formulation. The bioavailability of oral IFO, however, is reported to be almost 100%. Therefore, after normalization of the dose, a bioavailability of 32% for IFO after oral TRO could be calculated. Thus, in contrast to previous reports, direct 4-hydroxylation of TRO seems to be the main metabolic pathway.

Objective: The population pharmacokinetics and pharmacodynamics of the cytostatic agent ifosfamide and its main metabolites 2- and 3-dechloroethylifosfamide and 4-hydroxyifosfamide were assessed in patients with soft tissue sarcoma., Methods: Twenty patients received 9 or 12 g/m2 ifosfamide administered as a 72-h continuous intravenous infusion. The population pharmacokinetic model was built in a sequential manner, starting with a covariate-free model and progressing to a covariate model with the aid of generalised additive modelling., Results: The addition of the covariates weight, body surface area, albumin, serum creatinine, serum urea, alkaline phosphatase and lactate dehydrogenase improved the prediction errors of the model. Typical pretreatment (mean +/- SEM) initial clearance of ifosfamide was 3.03 +/- 0.18 l/h with a volume of distribution of 44.0 +/- 1.8 l. Autoinduction, dependent on ifosfamide levels, was characterised by an induction half-life of 11.5 +/- 1.0 h with 50% maximum induction at 33.0 +/- 3.6 microM ifosfamide. Significant pharmacokinetic-pharmacodynamic relationships (P = 0.019) were observed between the exposure to 2- and 3-dechloroethylifosfamide and orientational disorder, a neurotoxic side-effect. No pharmacokinetic-pharmacodynamic relationships between exposure to 4-hydroxyifosfamide and haematological toxicities could be observed in this population.

Background: The autoinducible metabolic transformation of the anticancer agent ifosfamide involves activation through 4-hydroxyifosfamide to the ultimate cytotoxic ifosforamide mustard and deactivation to 2- and 3-dechloroethylifosfamide with concomitant release of the neurotoxic chloroacetaldehyde. Activation is mediated by cytochrome P450 (CYP) 3A4 and deactivation by CYP3A4 and CYP2B6. The aim of this study was to investigate modulation of the CYP-mediated metabolism of ifosfamide with ketoconazole, a potent inhibitor of CYP3A4, and rifampin (INN, rifampicin), an inducer of CYP3A4/CYP2B6., Methods: In a double-randomized, 2-way crossover study a total of 16 patients received ifosfamide 3 g/m(2) per 24 hours intravenously, either alone or in combination with 200 mg ketoconazole twice daily (1 day before treatment and 3 days of concomitant administration) or 300 mg rifampin twice daily (3 days before treatment and 3 days of concomitant administration). Plasma pharmacokinetics and urinary excretion of ifosfamide, 2- and 3-dechloroethylifosfamide, and 4-hydroxyifosfamide were assessed in both courses. Data analysis was performed with a population pharmacokinetic model with a description of autoinduction of ifosfamide., Results: Rifampin increased the clearance of ifosfamide at the start of therapy at 102%. The fraction of ifosfamide metabolized to the dechloroethylated metabolites was increased, whereas exposure to the metabolites was decreased as a result of increased elimination. The fraction metabolized and the exposure to 4-hydroxyifosfamide were not significantly influenced. Ketoconazole did not affect the fraction metabolized or the exposure to the dechloroethylated metabolites, whereas both parameters were reduced with 4-hydroxyifosfamide., Conclusions: Coadministration of ifosfamide with ketoconazole or rifampin did not produce changes in the pharmacokinetics of the parent or metabolites that may result in an increased benefit of ifosfamide therapy.

The distribution of ifosfamide (IF) and its metabolites 2-dechloroethylifosfamide (2DCE), 3-dechloroethylifosfamide (3DCE), 4-hydroxyifosfamide (4OHIF) and ifosforamide mustard (IFM) between plasma and erythrocytes was examined in vitro and in vivo. In vitro distribution was investigated by incubating blood with various concentrations of IF and its metabolites. In vivo distribution of IF, 2DCE, 3DCE and 4OHIF was determined in 7 patients receiving 9 g/m(2)/72 h intravenous continuous IF infusion. In vitro distribution equilibrium between erythrocytes and plasma was obtained quickly after drug addition. Mean (+/-sem) in vitro and in vivo erythrocyte (e)-plasma (p) partition coefficients (P(e/p)) were 0.75+/-0.01 and 0.81+/-0.03, 0.62+/-0.09 and 0.73+/-0.05, 0.76+/-0.10 and 0.93+/-0.05 and 1.38+/-0.04 and 0.98+/-0.09 for IF, 2DCE, 3DCE and 4OHIF, respectively. These ratios were independent of concentration and unaltered with time. The ratios of the area under the erythrocyte and plasma concentration--time curves (AUC(e/p)) were 0.96+/-0.03, 0.87+/-0.07, 0.98+/-0.06 and 1.34+/-0.39, respectively. A time- and concentration-dependent distribution--equilibrium phenomenon was observed with the relative hydrophilic IFM. It is concluded that IF and metabolites rapidly reach distribution equilibrium between erythrocytes and plasma; the process is slower for IFM. Drug distribution to the erythrocyte fraction ranged from about 38% for 2DCE to 58% for 4OHIF, and was stable over a wide range of clinically relevant concentrations. A strong parallelism in the erythrocyte and plasma concentration profiles was observed for all compounds. Thus, pharmacokinetic assessment using only plasma sampling yields direct and accurate insights into the whole blood kinetics of IF and metabolites and may be used for pharmacokinetic-pharmacodynamic studies., (Copyright 2001 John Wiley & Sons, Ltd.)

Aims: To determine the population pharmacokinetic (PK) parameters of doxorubicin (Dox), etoposide (Eto) and ifosfamide (Ifo) in small cell lung cancer (SCLC) patients, to assess the potential relationship between those parameters and to estimate the impact of individual morphological and biological covariates on patients' PK parameters., Methods: Twenty-four patients with either SCLC limited to the thorax or extensive SCLC entered the study. All but one received at least two 3 day courses of the standard AVI (Dox 50 mg m-2 day 1, Eto 120 mg m-2 day 1,2,3, Ifo 2000 mg m-2 day 1,2) regimen. Individual blood samples were collected during each course and data on 47 courses were available. Data were analysed with the NONMEM program. Dox, Eto and Ifo plasma concentrations were studied with multicompartment (3, 2 and 2, respectively) models. Inter-individual and interoccasion (course-to-course) variabilities were estimated. The influence of individual covariates (age, sex, stage of the disease, weight, height, body-surface area, serum creatinine, total protein, LDH, ASAT, ALAT, alkaline phosphatase, gamma-GT, bilirubin) on PK parameters was also assessed. Correlations between individual PK parameters of Dox, Eto and Ifo were explored by using Pearson's correlation coefficient., Results: Multiple data were available for each patient. Dox clearance (CL) and volume of distribution (Vd) were 32.0 l h-1 and 9.3 l (Inter-individual variability: 17.2% and 19.2%). Eto CL (l h-1) and Vd were, respectively, 3.34-0.0083* serum creatinine (micromol l-1) and 6.38 l (interindividual variability: 15.6% and 18.7%). Ifo CL and Vd at day 1 were 5.6 l h-1 and 26.0 l (interindividual variability: 10.1% and 17.2%, respectively). Estimation of course-to-course variability improved the precision of PK models in some cases. No correlation was observed between the respective PK parameters of each drug. Of individual covariates tested, only serum creatinine correlated with Eto CL (r = -0.37, P < 0.001). Self-induction of the metabolism of Ifo was apparent (mean CL increase from day 1 to day 2 : 42%) and individually correlated with the CL value at day 1 (r = -0.61, P < 0.001)., Conclusions: Assessment of potential relationships between individual systemic exposure of chemotherapy and therapeutic endpoints (tumour response, toxicity and survival) will be required to adjust drugs dosages based on individual PK parameters rather than questionable body-surface area. However, all three drugs in the AVI regimen should be monitored simultaneously.

The pharmacokinetics of the combination of docetaxel and ifosfamide were studied in a phase I study. Docetaxel was given to cancer patients as a 1-hour infusion followed by a 24-hour infusion of ifosfamide (schedule A). After the dose-limiting toxicity of the combination was reached, ifosfamide was administered as a 24-hour infusion followed after 24 hours by a 1-hour infusion of docetaxel (schedule B). Cycle duration was 21 days. Docetaxel was determined by high-performance liquid chromatography, and ifosfamide and its metabolites, by gas chromatography-mass spectrometry. Twenty-seven patients were treated according to schedule A, and 6 according to schedule B. Combining the two drugs did not change their respective plasma half-lives. The sequence of drug administration did not affect the clearance and the area under the curve (AUC) of docetaxel. There was a decrease in the AUC of ifosfamide in schedule A compared with schedule B, resulting from an increase in the clearance of ifosfamide. The pharmacokinetics of docetaxel are not influenced by combination with ifosfamide, regardless of the drug sequence, but ifosfamide pharmacokinetics are changed by docetaxel, depending on the sequence of administration. The increase of clearance in schedule A may be due to the pretreatment with corticosteroids.

Metabolic activation of ifosfamide (IFO) leads to the active 4-hydroxy-metabolite and to a substantial liberation of chloroacetaldehyde (CAA). CAA has been presumed responsible for side effects of IFO. We recently have shown cytotoxic effects of CAA against human tumor cells in vitro. The aim of this study was to demonstrate antitumor effects of CAA in vivo, and to compare its potency to 4-OH-IFO. Pharmacokinetics of IFO and metabolites were evaluated after infusion of 250 mg/kg IFO in mice. The area under the curve (AUC) for 4-hydroxyifosfamide (4-OH-IFO) and CAA were 138. 5 and 102.4 micromol. h/liter, respectively. To compare pharmacokinetics and antitumor effects, the mice received isolated infusion of 4-OH-IFO or CAA in equimolar doses to IFO. Administration of 4-OH-IFO yielded AUC values comparable with those obtained after administration of the parent drug. In contrast, infusion of isolated CAA via tail vein gave a low AUC value of 51.5 micromol. h/liter due to slow flow in the tail vein and rapid degradation. Administration of the parent drug gave highly cytotoxic intratumoral peak concentrations of 25 and 12 micromol/kg tumor weight for 4-OH-IFO and CAA in MX1 xenotransplanted nude mice. Both IFO and isolated 4-OH-IFO led to complete remissions. Administration of isolated CAA (75 mg/kg) delayed tumor growth significantly. The equitoxic dose of isolated 4-OH-IFO was 40 mg/kg. On a molar basis CAA was seven times less potent as 4-OH-IFO. However, on the basis of achieved AUC values, CAA seems to exhibit a similar antitumor activity to 4-OH-IFO.

The stereoselective pharmacokinetics of ifosfamide (IF) were investigated in male and female Sprague-Dawley rats. Following intravenous administration of IF deuterium-labeled pseudoracemates into rats at 40 mg/kg, IF enantiomers and their metabolites, 4-hydroxyIF (HOIF), N2-dechloroethylIF (N2D), N3-dechloroethylIF (N3D), and isophosphoramide mustard (IPM) were quantitated in plasma and urine using gas chromatographic-mass spectrometry techniques with appropriately deuterium-labeled analogs as the internal standards. In addition, the intrinsic clearances of IF isomers in rat liver microsomes were estimated by the in vitro metabolism study. Following drug administration in male rats, (R)-IF exhibited a lower area under the curve value and a shorter half-life of 34.2 minutes than (S)-IF, which gave a half-life of 41.8 minutes. In female rats, the half-lives of (R)- and (S)-IF were found to be 62.1 and 75.1 minutes, respectively, significantly longer than those in male rats. No change in volume of distribution or renal clearance for IF enantiomers in all rats was observed, and the protein binding value was low, with no enantioselectivity. Both in vitro and in vivo studies showed that metabolism of (R)-IF proceeded in favor of the 4-hydroxylation pathway, whereas (S)-IF preferentially underwent N2- and N3-dechloroethylation. The observed stereoselectivity and gender difference in pharmacokinetics of IF in the rat are mainly attributed to its stereoselective metabolism.

Purpose: This study was designed to determine individual variation in the metabolism of ifosfamide (IF) and any influence this may have on the degree of DNA damage produced in both peripheral blood lymphocytes (PBL) and in tumour tissue., Methods: The pharmacokinetics and metabolism of IF and also of doxorubicin (DOX) were determined in patients receiving IF/DOX neoadjuvant chemotherapy for the treatment of advanced breast cancer. The DNA-damaging effects of this regimen were measured using the comet assay in PBL and in breast tumour tissue obtained by fine needle aspirate. Parallel in vitro studies were carried out in order to establish if DNA damage caused by IF metabolites or DOX was predictive of cytotoxicity in breast cancer cell lines., Results: The median AUC, half-life and clearance of IF were found to be 291 microM.min, 5.2 h and 66 ml/min per m2, respectively. A high degree of interpatient variability (up to sevenfold) was observed in the metabolism of both IF and DOX and also in their metabolites. Treatment-related changes in the amount of DNA damage were observed in both PBL and tumour cells. That in PBL peaked 48 h after the end of IF infusion (median 17% damaged cells at 48 h compared to 4% damaged before treatment). DNA damage in tumour cells was not elevated above low pretreatment values (median 1.5% damaged cells) until 3 weeks after IF and DOX treatment (median 30% damaged cells), by which time damage in PBL showed almost complete resolution to basal levels. The DNA damage in PBL determined 24 h after the start of chemotherapy was found to be related to the AUC of 4-hydroxyifosfamide (4OHI; P = 0.05). The amount of damage in either tissue did not significantly correlate with clinical response or toxicity, but lower amounts of damage were observed in the tumour cells 3 weeks after treatment in those patients that subsequently relapsed, compared to those that remained disease free. DNA damage (more than 20% damaged cells) was observed after exposure to active IF metabolites at concentrations equal to or greater than the IC50 in MCF-7 and MDA-MB231 cell lines. At concentrations of 4OHI similar to those determined in vivo, an equivalent level of DNA damage was observed in PBL and in cell lines and was associated with significant growth inhibition. DNA damage induced by DOX was not predictive of cytotoxicity., Conclusion: Systemic DNA damage appeared to be related to levels of the active metabolite, consistent with the results of in vitro investigations of DNA damage. Further studies are warranted to substantiate this observation and to explore the relationship between metabolism, DNA damage and antitumour activity.

Background: The aim of this study was to assess the pharmacology, toxicity and activity of high-dose ifosfamide mesna +/- GM-CSF administered by a five-day continuous infusion at a total ifosfamide dose of 12-18 g/m2 in adult patients with advanced sarcomas., Patients and Methods: Between January 1991 and October 1992 32 patients with advanced or metastatic sarcoma were entered the study. Twenty-seven patients were pretreated including twenty-three with prior ifosfamide at less than 8 g/m2 total dose/cycle. In 25 patients (27 cycles) extensive pharmacokinetic analyses were performed., Results: The area under the plasma concentration-time curve (AUC) for ifosfamide increased linearly with dose while the AUC's of the metabolites measured in plasma by thin-layer chromatography did not increase with dose, particularly that of the active metabolite isophosphoramide mustard. Furthermore the AUC of the inactive carboxymetabolite did not increase with dose. Interpatient variability of pharmacokinetic parameters was high. Dose-limiting toxicity was myelosuppression at 18 g/m2 total dose with grade 4 neutropenia in five of six patients and grade 4 thrombocytopenia in four of six patients. Therefore the maximum tolerated dose was considered to be 18 g/m2 total dose. There was one CR and eleven PR in twenty-nine evaluable patients (overall response rate 41%)., Conclusion: Both the activation and inactivation pathways of ifosfamide are non-linear and saturable at high-doses although the pharmacokinetics of the parent drug itself are dose linear. Ifosfamide doses greater than 14-16 g/m2 per cycle appear to result in a relative decrease of the active metabolite isophosphoramide mustard. These data suggest a dose-dependent saturation or even inhibition of ifosfamide metabolism by increasing high dose ifosfamide and suggest the need for further metabolic studies.

Chloroacetaldehyde (CA) is a nephrotoxic and neurotoxic metabolite of the anticancer drug ifosfamide (IFA) and is a dose-limiting factor in IFA-based chemotherapy. Plasma levels of CA in IFA-treated cancer patients are often difficult to determine due to the lack of a sufficiently sensitive and specific analytical method. We have developed a simple and sensitive HPLC method with fluorescence detection to measure CA formation catalyzed by liver cytochrome P450 enzymes, either in vivo in IFA-injected rats or in vitro in liver microsomal incubations. This method is based on the formation of the highly fluorescent adduct 1-N(6)-ethenoadenosine from the reaction of CA with adenosine (10 mM) at pH 4.5 upon heating at 80 degrees C for 2 h. The derivatization mixture is directly injected onto a C18 HPLC column and is monitored with a fluorescence detector. Calibration curves are linear (r > 0.999) over a wide range of CA concentrations (5-400 pmol). The limit of detection of CA in plasma using this method is <0.1 microM and only 50 microl of plasma is required for the assay. By coupling this method with a recently described HPLC-fluorescent method to determine acrolein, a cytochrome P450 metabolite of IFA formed during the activation of the drug by 4-hydroxylation, the two major, alternative P450-catalyzed pathways of IFA metabolism can be monitored from the same plasma samples or liver microsomal incubations and the partitioning of drug between these two pathways thereby quantitated. This assay may prove to be useful for studies of IFA metabolism aimed at identifying factors that contribute to individual differences in CA formation and in developing approaches to minimize CA formation while maximizing IFA cytotoxicity., (Copyright 1999 Academic Press.)

After oral or parenteral administration of chemotherapeutic agents, these drugs are transported to the tissues by the blood in different fractions: plasma water, plasma proteins or cells. In this review, the role of the red blood cell in storage, transport and metabolism of different anti-cancer drugs is described.

The Authors have developed a novel and easily applicable HPLC method for ifosfamide (IF) determination. This method involves on-line sample processing and its solid-phase extraction by means of an automatic preparator integrated with the chromatographic system. The calibration graph of the method is linear in the concentration range 6-200 microg/ml; minimum detectable concentration is 6 microg/ml. This highly accurate and easily reproducible method was used by the Authors in the treatment of osteosarcoma with slow infusion of ifosfamide.

A method using reversed-phase high-performance liquid chromatography (RP-HPLC) is described for the measurement of the stabilized activated metabolite of ifosfamide, 4-hydroxyifosfamide (4-OHIF), in human plasma and erythrocytes. Immediately after sample collection and plasma-erythrocyte separation at 4 degrees C, 4-OHIF was stabilized by derivatization with semicarbazide (SCZ). The sample pretreatment involved liquid-liquid extraction with ethyl acetate. RP-HPLC was executed with a C8 column and acetonitrile-0.025 M potassium dihydrogenphosphate buffer (pH 7.40)-triethylamine (13.5:86:0.5, v/v) as mobile phase. The analyte was determined with UV detection at 230 nm. Complete validation, optimisation and stability studies were performed and the method proved to be specific, sensitive and with a stable analyte in the range of clinically relevant concentrations (0.1-10 microg/ml) after conventional dosing. The lower limit of quantitation was 100 ng/ml using 1.00 ml of sample. Accuracy was between 94.1 and 107.0%. Within-day and between-day precisions were less than 6.2% and 7.2%, respectively. 4-OHIF-SCZ was found to be stable in the biological matrix at -20 degrees C for at least 1 month. A pharmacokinetic study conducted in a patient receiving 9 g/m2 over 3 days by means of a continuous infusion, demonstrated the applicability of this method.

Plasma and urinary levels of ifosfamide (IF) enantiomers and their metabolites 2-dechloroethylifosfamide, 3-dechloroethylifosfamide, 4-hydroxyifosfamide, and isophosphoramide mustard were determined for control and phenobarbital-treated male Sprague-Dawley rats by using pseudoracemates and GC/MS and stable-isotope dilution analytical methods. For the control rats, the mean AUC for (S)-IF in plasma was greater than that for (R)-IF (R/S AUC ratio, 0.78) and the mean half-life of 41.8 min for (S)-IF was slightly longer than that of 34.3 min for (R)-IF. Phenobarbital pretreatment significantly decreased the AUC values for (R)-IF and (S)-IF, to 21 and 30% of the control values, respectively, and shortened plasma half-lives for both enantiomers [half-life for (R)-IF, 19.8 min; half-life for (S)-IF, 19.4 min]. The urinary excretion values for (R)-IF and (S)-IF were decreased to 41 and 30% of the control values, respectively. The overall amounts of the metabolites in urine were concomitantly increased. Additionally, there were significant reversals in both the R/S AUC ratio and the urinary excretion of 3-dechloroethylifosfamide. Moreover, the enantioselectivity for the generation of 4-hydroxyifosfamide and isophosphoramide mustard disappeared after phenobarbital treatment. These results strongly suggested that the 4-hydroxylation and dechloroethylation of IF enantiomers were mediated by different P450 isozymes or the same isozyme with different stereochemical selectivities.

In a randomized cross-over trial, 11 patients received ifosfamide (IFOS) in 21-day cycles, which alternated between 3 g m(-2) x (2 or 3) days given as a 1-h bolus doses, or the same total dose as a continuous infusion. Patients who received four or more cycles also alternated between two cycles on dexamethasone 4 mg 8 hourly for 3 days starting 8 h before IFOS, and two cycles off dexamethasone. A total of 34 patient cycles were studied and serum and urinary levels of IFOS, 2 dechloroethylifosfamide (2DC), 3 dechloroethylifosfamide (3DC), carboxyifosfamide (CX) and isophosphoramide mustard (IPM) were measured by thin-layer chromatography. No significant differences could be detected in the areas under the curve (AUCs) of serum concentration, nor in the proportion of IFOS or its metabolites found in the urine. There was no significant effect of dexamethasone on IFOS metabolism. These results indicate that there is no identifiable pharmacokinetic basis for insistence on either bolus or infusional methods of IFOS administration.

Purpose: Oxazaphosphorines are metabolised by a variety of pathways, one of which leads to activation and the formation of alkylating compounds. However, the transport forms conveying activated oxazaphosphorines to the tumour cell have not been fully characterised. There is increasing recognition of the importance of the erythrocyte as a carrier of compounds in the circulation, and we have recently described higher concentrations of 4-hydroxycyclophosphamide within the erythrocyte compartment compared to plasma. We have now determined the concentrations of ifosfamide and seven of its metabolites in the plasma and erythrocytes of patients receiving a six-hour intravenous infusion of ifosfamide., Patients and Methods: Red cells from five patients, receiving a total of eight cycles of ifosfamide, were separated from plasma using the MESED instrument, and analysis of red cells and plasma performed using Gas Chromatography-Mass Spectrometry (GC/MS)., Results: The concentration of all compounds in the erythrocyte compartment was higher than or equal to those in plasma, and isophosphoramide mustard and carboxyifosfamide showed a particular affinity for the erythrocyte. The red cell fraction can contain as much as 77% of the total blood concentration of isophosphoramide mustard., Conclusions: Erythrocyte associated isophosphoramide mustard is an important transport form of activated ifosfamide. Red cells may have a role in the delivery of activated oxazaphosphorines to tissues.

The enantiomers of the cytostatic drug ifosfamide and the two metabolites 2- and 3-dechloroethylifosfamide were isolated from plasma and urine by liquid-liquid extraction with ethyl acetate, resolved on a Chirasil-L-val gas chromatographic column and detected by a nitrogen-phosphorus-selective flame ionisation detector. Resolution of the racemic compounds for identification purposes was also accomplished with high-performance liquid chromatography on a chiral column. The validated gas chromatographic method was suitable to determine the total concentrations and the enantiomeric composition of ifosfamide and its dechloroethylated metabolites in plasma and urine samples from treated patients. Some metabolic preferences in the metabolism of ifosfamide were found.

Purpose: We designed a phase I dose-escalation study of vinorelbine on a novel (daily-times-three) schedule with ifosfamide with granulocyte colony-stimulating factor (G-CSF) support to define the dose-limiting toxicity (DLT) and maximum-tolerated dose (MTD) of vinorelbine in this combination., Patients and Methods: Cohorts of patients with stage IIIB or IV non-small-cell lung cancer (NSCLC) and no prior chemotherapy received vinorelbine starting at 15 mg/m2 on days 1, 2, and 3, and ifosfamide starting at 2.0 g/m2 on days 1, 2, and 3 with G-CSF support for all patients. Cycles were repeated every 21 days. Plasma vinorelbine concentrations were also analyzed., Results: Forty-two patients were treated. The median age was 58 years (range, 34 to 75) and 41 had a performance status of 0 or 1. The DLT was neutropenia and sepsis at a maximum-administered vinorelbine dose of 35 mg/m2 for 3 days. The recommended phase II dose was vinorelbine 30 mg/m2 with ifosfamide 1.6 g/m2 both given on 3 consecutive days. The overall response rate was 40% (17 of 42; all partial responders). The median survival duration was 50 weeks, with a 1-year survival rate of 48%. Pharmacokinetic analysis showed that vinorelbine in this combination and on this schedule is cleared 1.5 to two times faster than in single-agent once-weekly studies., Conclusion: Myelosuppression is the DLT of this regimen with no major subjective toxicities. With tolerable toxicity and an encouraging 1-year survival rate of 48%, further investigation of this new vinorelbine schedule is warranted in this and other combination regimens.

A high-performance liquid chromatographic method has been developed for the determination of 4-hydroxyifosfamide, a metabolite of ifosfamide, in plasma of cancer patients. The analyte is derivatized to 7-hydroxyquinoline, which can be detached fluorimetrically. The calibration graph is linear in the concentration range 0.05-25 microM, the limit of detection being 40 nM. Any inference from acrolein, another metabolite of ifosfamide, was ruled out. 4-Hydroxyifosfamide is very unstable in plasma and a stabilization procedure by adding citric acid has been developed. Thus treated, the samples were stable for 4 days. Analysis of a patient's plasma samples revealed that the 4-hydroxifosfamide concentration did not exceed 10 microM.

Purpose: Ifosfamide is becoming an important clinical anticancer drug. Meaningful pharmacology studies require quantification of its activated and active metabolites, 4-hydroxyifosfamide (HOIfos) and isophosphoramide mustard (IPM), respectively., Methods: Current methodology for quantifying the unstable HOIfos in biological fluids consists of trapping acrolein as it is produced during the decomposition of this metabolite. However, unlike cyclophosphamide, ifosfamide is extensively metabolized to two dechloroethylated metabolites, which are susceptible to 4-hydroxylation and similarly are capable of yielding acrolein upon decomposition. Because the current method has the potential to yield higher than actual values for HOIfos, it was compared with an HOIfos-specific method that traps the first stage degradation product of HOIfos, aldoifosfamide, as its semicarbazone, and depends on the use of radiolabeled drug for quantification. Six experiments in mice were conducted with blood collection 15 or 30 min after drug treatment followed by determination of HOIfos in plasma by the two methods., Results: Comparison of plasma levels of HOIfos determined by the two methods indicated only minor differences between the two., Conclusion: These results suggest the possibility that the nonspecific method may be acceptable as a first estimation of levels of this metabolite in biological fluids until the development of a specific method that does not require radiolabeled drug, such as high-performance liquid chromatography or gas chromatography/mass spectrometry, has been developed.

The cytotoxic drug ifosfamide is subject to an extensive metabolism. This study reports the results of a pharmacokinetic study of the parent drug and the two dechloroethylated metabolites in 22 patients on a 10-day continuous infusion of ifosfamide. Ifosfamide causes a substantial induction of the enzymes responsible for its metabolism, resulting in a two-fold increase of the clearance. The maximal IF concentration is reached after 24 h, after which the concentration decreases to a steady-state. The dechloro-ethylated compounds can be detected in the plasma about 8 h after the start of the infusion with plasma half-lives longer than for IF. Urinary excretion studies revealed that at least a quarter of the IF dose is excreted as inactive compounds.

A recently developed GC-MS analytical method for the quantitative determination of oxazaphosphorines and their metabolites in blood plasma, using stable trifluoroacetyl derivatives and electron capture negative chemical ionization detection, was applied to measure the partitioning of the antitumor drug ifosfamide and its metabolites between plasma and red blood cells for four cancer patients. The separation of a constant volume of red blood cells was performed using a special instrument, MESED, through centrifugation of blood samples. The measured compounds were ifosfamide, 2- and 3-dechloroethylifosfamide, 4-ketoifosfamide, carboxyifosfamide, ifosfamide mustard, 2-chloroethylamine and 1,3-oxazolidin-2-one. Concentration-time profiles for the metabolites in the two blood fractions and partitioning factors between erythrocytes and plasma were obtained. For ifosfamide itself, and metabolites with an intact ring system, a partitioning factor between 1 and 2 was observed for the concentration ratio between red blood cells and plasma in the patients studied. However, for the compounds with an open structure, carboxyifosfamide and ifosfamide mustard, partitioning factors higher than 3 were obtained. The active antitumor metabolite ifosfamide mustard showed a strong preference for the red blood cells in the measured patient samples. This means that erythrocytes may play an important role in the transport and the subsequent release of the active alkylating agent to the tumor cells.

A sensitive and specific method for the simultaneous quantitation of ifosfamide (IF), 4-hydroxylifosfamide (4-OHIF), N2-dechloroethylifosfamide (N2D), N3-dechloroethylifosfamide (N3D) and iphosphoramide mustard (IPM) has been developed using gas chromatography-mass spectrometry (GC-MS) with an ion-trap mass spectrometer. Deuterium labeled analogues for each of these analytes were synthesized as the internal standards. The labile 4-OHIF in plasma was first converted to the more stable cyanohydrin adducts before dichloromethane extraction. IPM was extracted by C18 reversed-phase resin. All analytes were converted to their silyl derivatives before GC-MS analysis. The sensitivity limits ranged from 0.1 to 0.5 microgram/ml when 100 microliters of plasma was used. This method was validated with within-run coefficients of variation less than 5% (n = 8) and between-run coefficients of variation less than 12% (n = 6). The method was applied to the determination of plasma levels of IF and metabolites in the rat.

Ifosfamide is a chiral pro-drug which is administered clinically in its racemic form. Serum concentrations of rac-ifosfamide and its enantiomers were measured in 12 patients with lung carcinoma following a mean (+/- s.d.) intravenous dose of 4.2 (0.83) g infused over 1 h. The mean (+/- s.d.) volumes of distribution (VSS) of rac, (R)- and (S)-ifosfamide were 0.61 (0.17), 0.60 (0.16) and 0.61 (0.19) l kg-1, respectively. The mean (+/- s.d.) half-lives and clearances were 6.57 (1.69), 7.12 (1.92) and 5.98 (1.52) h and 0.065 (0.013), 0.060 (0.013) and 0.072 (0.014) l h-1 kg-1 for rac, (R)- and (S)-ifosfamide, respectively. The half-life of (S)-ifosfamide was significantly (P < 0.001) shorter than that of (R)-ifosfamide and it had a significantly higher clearance (P < 0.001). There was no significant difference in the volumes of distribution of the enantiomers. The clinical significance of the faster elimination of (S)-ifosfamide is not known.

An autologous bone marrow transplant regimen of ifosfamide, carboplatin, and etoposide (ICE) has been developed as treatment for certain malignancies. At maximum tolerated doses renal insufficiency precludes dose escalation. The objective was to examine whether measurement of plasma drug levels early during treatment would provide warning of renal failure. Nine patients received a 96-h continuous infusion of ifosfamide 16,000 mg/m2, carboplatin 1600 mg/m2, and etoposide 1200 mg/m2. Pharmacokinetics, including drug levels and plasma concentration-time curves, of ifosfamide, ultrafiltrable platinum (uPt) and etoposide were analyzed and correlated with renal function. One of the nine patients developed anuric renal failure requiring hemodialysis. By 17 h from the start of infusion, this patient showed substantially higher drug levels of ifosfamide (200 vs mean 217 microM) and uPt (19 vs mean 10 microM) than those patients with preserved renal function. The 95% confidence intervals suggested that a 16-22 h ifosfamide level > 153 microM and an uPt level > microM predict the development of significant renal dysfunction. Although drug levels were substantially higher at 56 h, the serum creatinine did not yet reflect kidney injury. This study suggests that high plasma ifosfamide and uPt levels, analyzed early in the course of a 96-h infusion of high-dose ICE, provide warning of severe and potentially fatal renal injury. Since ICE has substantial activity in a number of malignancies, but significant renal morbidity, real-time pharmacokinetic-guided dosing may reduce treatment-related toxicity.

A method to measure racemic, R- and S-ifosfamide concentrations from the serum of patients receiving ifosfamide chemotherapy has been developed. The racemic ifosfamide concentrations are quantified on a separate system and then the ratio of the enantiomers is determined using an achiral-chiral coupled system. Racemic ifosfamide is separated on the achiral system using a C1 spherisorb stationary phase and the eluent containing analyte is selectively transferred to the chiral system for separation of the two enantiomers by an alpha 1 glycoprotein column. On both systems the mobile phase is 1% acetonitrite in 0.015 M phosphate buffer (pH 4) at a flow-rate of 1 ml/min. The retention times of S- and R-ifosfamide were 11.6 and 13.0 mins, respectively, with a resolution factor of 1.53. Serum concentrations at least three to four half-lives post-infusion were detected by this method. In ten patients, following a mean +/- S.D. 1-h infusion of 3.9 +/- 0.32 g racemic ifosfamide, the mean +/- S.D. clearances of R- and S-ifosfamide were 0.061 +/- 0.013 and 0.072 +/- 0.014 1 h-1 kg-1.

A rapid, sensitive, enantioselective gas chromatographic method has been developed for the quantitation of the enantiomers of ifosfamide (IFF) and its 2- and 3-dechloroethylated metabolites (2-DCE-IFF and 3-DCE-IFF) in human and animal plasma and human urine. IFF and the two dechloroethylated metabolites were extracted into chloroform, enantioselectively resolved by gas chromatography on a chiral stationary phase based upon heptakis(2,6-di-O-methyl- 3-O-pentyl)-beta-cyclodextrin and quantitated using mass-selective detection with selected-ion monitoring. The limits of quantitation for the enantiomers of IFF, 2-DCE-IFF and 3-DCE-IFF in plasma were 250 and 500 ng/ml respectively. In urine, the limits of quantitation for the enantiomers of IFF, 2-DCE-IFF and 3-DCE-IFF were 500 ng/ml. The method can detect concentrations as low as 250 ng/ml of each enantiomer of 2- and 3-DCE-IFF in plasma and urine. The intra- and inter-day coefficients of variation for this method were with one exception less than 8%. The assay was validated for enantioselective pharmacokinetic studies in humans and rats and is the first reported enantioselective assay for the measurement of the enantiomers of 2- and 3-DCE-IFF in plasma.

The pharmacokinetics and metabolism of ifosfamide was investigated in a group of 16 pediatric patients (5 girls) aged 1-17 years. Each received a dose of 3 g/m2/day for up to 3 days by continuous infusion. Plasma and urine were collected, and concentrations of ifosfamide and its principal metabolites were determined by a quantitative high-performance thin layer chromatography method. During 3 days of continuous infusion, the plasma concentrations of parent drug decreased. This was accompanied by a continuous increase in dechloroethylated products in plasma but not in urine. Estimated pharmacokinetic parameters (clearance, volume of distribution, and half-life) were dependent on body size and age but not any other patient variable. Renal clearance was a relatively minor route of elimination for parent drug and corresponded to < 25% of glomerular filtration rate. Metabolite data from plasma and urine indicated a high degree of interindividual variation in metabolism. Comparison of metabolite recoveries in urine indicated a positive correlation between activation and inactivation routes of metabolism. Prior exposure to ifosfamide was associated with a higher recovery in urine of dechloroethylated metabolites. The severity of hematological toxicity was inversely correlated with glomerular filtration rate but not to parameters of ifosfamide metabolism. There was marked variation in levels of the carboxy metabolite, which could not be detected in the plasma of 5 subjects. However, evidence for a polymorphism in metabolism to this metabolite was weaker than that seen with the isomeric oxazaphosphorine cyclophosphamide. There appeared to be a higher clearance of ifosfamide in pediatric patients compared to adults. The significance of this, and of the variation in metabolism of ifosfamide, for clinical outcome remains to be established, but the increase in the dechloroethylation route of metabolism may be associated with an increased risk of toxicity.

The metabolic oxidation of one of the chloroethyl groups of the antitumour drug ifosfamide leads to the formation of the inactive metabolites 2- and 3-dechloroethylifosfamide together with the neurotoxic metabolite chloroacetaldehyde. A very sensitive capillary gas chromatographic method, requiring only 50 microliters of plasma or urine, has been developed to measure the amounts of the drug and the two inactive metabolites in a single run. Calibration curves were linear (r > 0.999) in the concentration ranges from 50 ng/ml to 100 micrograms/ml in plasma and from 100 ng/ml to 1 mg/ml in urine.

The cytostatic drug ifosfamide (IFF) and its main metabolites 2-dechloroethyl ifosfamide (2-D-IFF) and 3-dechloroethyl ifosfamide (3-D-IFF) were isolated from the plasma matrix with high recovery by solid phase extraction using Bakerbond C18-cartridges. A further cleaning of the extracts is not necessary. An effective separation of IFF and metabolites from interfering compounds present in urine samples prior gc was performed with Extrelut-1 and dichloromethane/isopropanol (95:5, v/v). The described assays may be used for pharmacokinetic studies as well as drug monitoring in the clinical laboratory.

A method has been devised for the determination of the anticancer drug ifosfamide and its principal metabolites in urine, plasma and cerebrospinal fluid (CSF). The urine and CSF samples are absorbed onto Amberlite XAD-2 eluting the compounds of interest with methanol. Plasma is deproteinated using cold acetonitrile and centrifuged to yield a clear supernatant. The eluate and supernatant are analyzed by thin-layer chromatography, with spot visualization using 4-(4-nitrobenzyl)pyridine. The plates are photographed for subsequent densitometeric analysis. The intra-assay coefficient of variation for each compound in both urine and plasma was less than 10% and the lower limit of detection was 1 microgram/ml. The method provides a means of determining the full spectrum of metabolic products of ifosfamide in patients and will allow detailed investigation of variability in metabolism and pharmacokinetics of this drug.

The pharmacokinetics of ifosfamide were studied in 20 patients with soft tissue and bone sarcomas. Drug was administered as a 30-60 min i.v. infusion at 1.2 or 2.0 mg/m2/day for five consecutive days. Some patients also received 1.5 g/m2 of N-acetylcysteine (NAC) administered 3 times per day during the course of therapy. NAC had no effect on ifosfamide pharmacokinetics. There were significant differences in plasma half-life, area under the concentration-time curve and plasma clearance on day 1 versus day 5 of ifosfamide administration. Myelosuppression and granulocytopenia correlated better with day 1 versus day 5 ifosfamide pharmacokinetics suggesting that the alteration of ifosfamide pharmacology with multiple dosing has a significant effect on drug activity.