Trofosfamide metabolism in different species--ifosfamide is the predominant metabolite.

Trofosfamide (TRO) belongs to the group of oxazaphosphorines and is a congener of cyclophosphamide (CYC) and ifosfamide (IFO). The precondition for the cytotoxic effect of all oxazaphosphorines is their metabolic activation by "ring" oxidation at the hepatic mixed-function oxidase system. In addition, an inactivating metabolic pathway ("side chain" oxidation) is known for CYC and IFO. The metabolic pattern of the substances gains special interest in the discussion of a growing incidence of side effects. Therefore, the in vitro biotransformation of TRO was studied. Liver microsomes were prepared from different species, including the rat, rabbit, and mouse as well as from one human sample. Microsomal proteins were incubated for various periods and concentrations of TRO and its metabolites were analyzed by reversed-phase high-performance liquid chromatography (HPLC). In vitro metabolism resulted in the formation of activated metabolites by hydroxylation at position 4. In addition, side-chain oxidation resulted in the formation of IFO and CYC. IFO was the predominating metabolite of this pathway, with a 5- to 6-fold excess being noted as compared with CYC in rats and mice. The rabbit species showed similar CYC and IFO formation; in the single human sample, only IFO could be detected. In rats, the Michaelis constant (Km) for biotransformation to IFO was 398 microM, with the maximal volume (Vmax) being 70.8 nmol 120 min-1 mg protein-1, the corresponding values for biotransformation to CYC were 348 microM and 13.30 nmol 120 min-1 mg protein-1. On the basis of its structural similarity and the current knowledge of oxazaphosphorine metabolism, CYC was expected to be the main metabolite of TRO. The predominance of IFO was unexpected, but the observed metabolic profile promises numerous interesting aspects for the clinical use of TRO.

Urinary excretion of the enantiomers of ifosfamide and its inactive metabolites in children.

The precondition for the antineoplastic effect of ifosfamide (ifo) is the oxidation of the oxazaphosphorine ring system, which contains a chiral centre at the phosphorous atom. This "ring oxidation" leads to the formation of alkylating mustard via several steps. A second metabolic pathway produces the cytostatically inactive metabolites 2- and 3-dechloroethyl-ifosfamide (2-d- and 3-d-ifo). The urinary excretion of the optical isomers of unmetabolised ifo and of 2- and 3-d-ifo, which represents the amount of ifo that has not been activated, was investigated by capillary gas chromatography for 18 treatment cycles in 14 children on various therapeutic schedules. The total cumulative excretion in 12 completely sampled cycles ranged from 27% to 50% of the ifo dose. Between 14% and 34% of the dose could be detected as ifo; 9% to 29%, as 3-d-ifo; and 2% to 8%, as 2-d-ifo. At 24 h after the end of therapy, excretion was nearly complete. Without exception, slightly more R-ifo (53%-61%) than S-ifo was excreted. S-2-d-ifo (50%-73%) was the main 2-d-metabolite. S-3-d-ifo (deriving from R-ifo) predominated in 6 of 14 children and R-3-d-ifo, in 8. Enantiomer-specific excretion increased after the end of infusion (up to 73% for R-ifo and 27% for S-ifo). We demonstrated stereospecific metabolism of ifo in children, with two different patterns of side-chain oxidation being observed. There was no evidence of important stereospecific ring oxidation in most children. A benefit should not be expected from the therapeutic application of pure enantiomers.

Pharmacokinetics of trofosfamide and its dechloroethylated metabolites.

To contribute to effective and safe outpatient treatment, we investigated the metabolism of trofosfamide (Trofo) after oral administration. We analyzed Trofo metabolism in 15 patients aged from 3 to 73 years who were treated with 150 or 250 mg/m2 Trofo in combination with etoposide. Serum samples were collected with 13 patients after oral administration, and Trofo and its dechloroethylated metabolites were quantified by gas chromatography. Urine samples were collected from five patients and analyzed by same method. Ifosfamide (Ifo) was the main metabolite in serum and urine (AUCTrofo:AUCIfo 1:13), whereas cyclophosphamide (Cyclo) was formed in smaller amounts (AUC(Ifo):AUC(Cyclo) 18:1). Ifo and Cyclo were further oxidized in the chloroethyl side chains to form 2- and 3-dechlorethylifosfamide in varying quantities. The urinary excretion of Trofo and its dechloroethylated metabolites amounted to about 10% of the total dose. Our results confirm former in vitro observations about the metabolism of Trofo. The main side-chain metabolites Ifo and Cyclo can be further activated by oxidation and formation of their respective phosphoramide mustards. Hence, Trofo is an interesting agent for oral chemotherapy.

Investigation of the major human hepatic cytochrome P450 involved in 4-hydroxylation and N-dechloroethylation of trofosfamide.

Trofosfamide and its congeners ifosfamide and cyclophosphamide are cell-cycle-nonspecific alkylating agents that undergo bioactivation catalyzed by liver cytochrome P450 (CYP) enzymes. Two NADPH-dependent metabolic routes for the anticancer drug trofosfamide, i.e., 4-hydroxylation and N-dechloroethylation, were studied in human liver microsomes and in seven recombinant human CYP isoforms (i.e., CYP1A1, 1A2, 2A6, 2B6, 2D6, 2E1, and 3A4-OR) to identify the CYP enzymes involved. Recombinant human CYP3A4 and CYP2B6 exhibited catalytic activity with respect to both pathways of trofosfamide. Enzyme kinetic analyses revealed the dominant role of human CYP3A4 in 4-hydroxylation and N-dechloroethylation of trofosfamide. This was confirmed by the observation that only the CYP3A4 contents of five samples of human liver microsomes correlated with both pathways of trofosfamide. Furthermore, ketoconazole, a selective inhibitor of CYP3A4, substantially inhibited microsomal trofosfamide 4-hydroxylation and N-dechloroethylation (50% inhibitory concentration < 1 microM for both reactions). The present study indicates that human liver microsomal CYP3A4 preferentially catalyzes the two NADPH- dependent metabolic routes of trofosfamide, which emphasizes the necessity for awareness of potential interactions with any coadministered drugs that are CYP3A4 substrates.

Enantioseparations in capillary electromigration techniques: recent developments and future trends.

This review summarizes the current status of enantioseparations using capillary electromigration techniques and gives the authors insights on the selected fundamental aspects and future trends in this field. The most recent developments in the field of chiral separations using capillary electrophoresis (CE) and capillary electrochromatography (CEC) are summarized. The status of chiral electromigration techniques is evaluated tacking into account the most recent developments in related techniques such as chiral HPLC, GC and SFC.

Enantiomer separation of drugs by capillary electromigration techniques.

The review summarizes the most recent developments in the field of enantioseparation of chiral drugs using capillary electromigration techniques. The basic principles of enantioseparations in CE are discussed. Recent developments in sample introduction, separation and detection in capillary electrophoresis and capillary electrochromatography are summarized. The applications are arbitrarily divided into the following three groups: (a) racemates and artificial mixtures of enantiomers, (b) drug forms and (c) chiral drugs and their metabolites in biological fluids. Among the various techniques involved the relatively new developments such as CEC in aqueous and nonaqueous buffers, on-line CE-MS coupling, etc. are emphasized.

Investigation of the stereoselective metabolism of the chiral H1-antihistaminic drug terfenadine by high-performance liquid chromatography.

The enantiomers of the racemic H1-antihistaminic drug terfenadine (1) have been resolved by fractional crystallization of the diastereomeric salts with optically active 2-chlorotartranilic acid. The enantiomeric excess of both terfenadine enantiomers was determined using an achiral and a chiral HPLC system after formation of diastereomers with S-(+)-naphthylethylisocyanate. To investigate the metabolism of terfenadine after oral administration, an achiral HPLC system, equipped with a conventional reversed-phase column, was used to quantify the main metabolite MDL 16.455 (2) in human serum and urine. The determination of the enantiomeric composition of 2 was achieved using an Ultron ES-OVM column as chiral stationary phase. Metabolite 2, extracted from human blood plasma, was found to be enriched in the R-enantiomer, but was excreted in urine as racemate. The results of a study including six volunteers are presented.

Separation of ergot alkaloids and their epimers and determination in sclerotia by capillary electrophoresis.

Capillary electrophoresis has been shown to be a very useful analysis technique for secondary metabolites of plants. In the present study a method is described for the qualitative and quantitative determination of ergot alkaloids and their epimers. The extraction from the biological matrix yields recoveries of 50-97%, depending on the individual alkaloid. Using a mixture of 20 mM beta-cyclodextrin (CD), 8 mM gamma-CD, 2 M urea and 0.3% (w/w) poly(vinyl alcohol) to phosphate buffer at pH 2.5 the simultaneous separation of all analytes was achieved. A 37 cm (30 cm) fused-silica capillary, at a voltage of 25 kV and a temperature of 20 degrees C, was used for the analysis. Overall analysis time for the separation was 12 min. The limit of detection of the alkaloids using UV detection at 214 nm can be improved 30-fold to about 9.10(-8) M when laser-induced fluorescence detection is applied.

Enantioseparations in non-aqueous capillary electrochromatography using polysaccharide type chiral stationary phases.

Enantioseparations of chiral compounds with different structures were studied in non-aqueous capillary electrochromatography (NAQ CEC). Three different polysaccharide derivatives, cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD), amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD) and cellulose tris(4-methylbenzoate) (Chiralcel OJ) were used as chiral stationary phases (CSPs). Methanolic or ethanolic ammonium acetate solutions served as a mobile phase. The effect of the type of the CSP, the loading of the chiral selector on wide-pore aminopropyl derivatized silica gel and operational parameters such as apparent pH, applied voltage, etc. on the EOF and chromatographic characteristics (alpha, N, Rs) were studied. NAQ CEC represents a valuable alternative and an extension to chiral separations by HPLC with common-size columns as well as to capillary LC and CEC in aqueous buffers.

Simultaneous separation and enantioseparation of thalidomide and its hydroxylated metabolites using high-performance liquid chromatography in common-size columns, capillary liquid chromatography and nonaqueous capillary electrochromatography.

The separation of thalidomide (TD) and its hydroxylated metabolites including their simultaneous enantioseparation was studied using three different polysaccharide-type chiral stationary phases (CSPs) in combination with polar organic mobile phases. Three different techniques, high-performance liquid chromatography in common-size columns, capillary LC and nonaqueous capillary electrochromatography were compared in terms of separation. As this study illustrates, polar organic mobile phases represent a valuable extension for less polar and polar aqueous-organic mobile phases in combination with polysaccharide CSPs. Chiralpak AD consisting of 25% of amylose-tris(3,5-dimethylphenylcarbamate) coated on wide-pore aminopropylsilanized silica gel exhibited higher resolving ability compared to the similar cellulose derivative (Chiralcel OD) as well as to cellulose-tris(4-methylbenzoate) (Chiralcel OJ) CSPs for this particular set of chiral analytes. Baseline separation and simultaneous enantioseparation of all three compounds could be achieved under optimized separation conditions.

Enantioseparation of 3,4-dihydroxyphenylalanine and 2-hydrazino-2-methyl-3-(3,4-dihydroxyphenyl)propanoic acid by capillary electrophoresis using cyclodextrins.

The enantiomeric separations of 3,4-dihydroxyphenylalanine (dopa) and 2-hydrazino-2-methyl-3-(3,4-dihydroxyphenyl)propanoic acid (carbidopa) by capillary electrophoresis were studied using several native, neutral and anionic cyclodextrins as chiral additives and uncoated fused-silica capillaries. The effect of the type and concentration of the cyclodextrin added to 20 mM phosphate buffer (pH 2.5) on enantioseparation and migration times was studied. A high resolution value of 15.63 was obtained for dopa enantiomers with a buffer containing 20 mM single isomer, heptakis(2,3-diacetyl-6-sulfato)-beta-cyclodextrin. The enantiomers of carbidopa were separated using 20 mM carboxymethyl-beta-cyclodextrin as a chiral resolving agent. Both methods allowed the determination of 0.1% of the D-enantiomer (second migrating) in the presence of the L-enantiomer (first migrating) of dopa and carbidopa with a good precision. These methods also gave good results in terms of precision for both peak area, migration time, linearity and accuracy.

Separation and identification of etodolac and its urinary phase I metabolites using capillary electrochromatography and on-line capillary electrochromatography-electrospray ionisation mass spectrometry coupling.

Capillary high-performance liquid chromatography (capillary HPLC), pressure-assisted capillary electrochromatography (pCEC) and capillary electrochromatography (CEC) were performed in the same capillary packed with 5 microm octadecylsilica (C18) as stationary phase. These three separation modes were compared from the viewpoint of peak efficiency and separation selectivity in order to critically evaluate the advantages which CEC may offer compared to capillary HPLC for the solution of practical biomedical problems. The separation of the non-steroidal anti-inflammatory drug etodolac (ET, 1) and its phase I metabolites, 6-hydroxy etodolac (6-OH-ET, 2), 7-hydroxy etodolac (7-OH-ET, 3) and 8-(1'-hydroxyethyl) etodolac (8-OH-ET, 4) was selected as an example. Baseline separation of all compounds was achieved in different modes and conditions. The effect of pure electrophoretic separation mechanism on the overall separation selectivity observed in CEC has been shown. A high electroosmotic flow (EOF) was observed in C18 packed capillary even at pH 2.5 in various buffers. Furthermore, these separations were coupled on-line with electrospray ionisation mass spectrometry (ESI-MS) and the parent drug and its metabolites were identified in urine. For the coupling of CEC with ESI-MS a laboratory-made electrophoretic device was used in order to overcome some technical disadvantages of commercial instrumentation.

Determination of paclitaxel in biological fluids by micellar electrokinetic chromatography.

A method has been developed for the determination of paclitaxel (Taxol) in plasma and urine using capillary electrophoresis with sodium dodecyl sulfate as additive in the run buffer. The samples are extracted and preconcentrated with tert.-butyl methyl ether. Taxotere has been used as the internal standard. The limit of detection of paclitaxel is 20 ng/ml. In comparison to high-performance liquid chromatography, the capillary electrophoresis method is simple and needs less organic solvents.

Mechanistic study of opposite migration order of dimethindene enantiomers in capillary electrophoresis in the presence of native beta-cyclodextrin and heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin.

The possible mechanisms of the opposite affinity pattern of the enantiomers of dimethindene [(R,S)-N,N-dimethyl-3[1(2-pyridyl)ethyl]indene-2-ethylamine] (DIM) towards native beta-cyclodextrin (beta-CD) and heptakis(2,3,6-tri-O-methyl-)-beta-CD (TM-beta-CD) were studied using capillary electrophoresis (CE), NMR spectrometry, electrospray ionization mass spectrometry (ESI-MS) and X-ray crystallography. NMR spectrometry allowed to estimate the stoichiometry of the complex and to determine the binding constants. As found using ESI-MS, together with more abundant 1:1 complex, a complex with 1:2 stoichiometry may also be present in a rather small amount in a solution of DIM and beta-CD. One-dimensional ROESY experiments indicated that the geometry of the complexes of DIM with native beta-CD depends on the ratio of the components in the solution. In the 1:1 solution of DIM and beta-CD the complex may be formed by inclusion of the indene moiety of DIM into the cavity of beta-CD on the primary side and into the cavity of TM-beta-CD into the secondary side. The most likely structural reason for lower affinity of the enantiomers of DIM towards the cavity of TM-beta-CD compared to native beta-CD could be elucidated. The indene moiety does not enter the cavity of TM-beta-CD as deeply as the cavity of beta-CD. This may be the most likely explanation of significantly higher affinity constants of DIM enantiomers towards the latter CD compared to the former one. The marked difference between the structure of the complexes may also be responsible for the opposite affinity pattern of the DIM enantiomers towards beta-CD and TM-beta-CD.

Separation of brompheniramine enantiomers by capillary electrophoresis and study of chiral recognition mechanisms of cyclodextrins using NMR-spectroscopy, UV spectrometry, electrospray ionization mass spectrometry and X-ray crystallography.

Opposite migration order was observed for the enantiomers of brompheniramine [N-[3-(4-bromphenyl)-3-(2-pyridyl)propyl]-N,N-dimethylamine] (BrPh) in capillary electrophoresis (CE) when native beta-cyclodextrin (beta-CD) and heptakis(2,3,6-tri-O-methyl)-beta-CD (TM-beta-CD) were used as chiral selectors. NMR spectrometry was applied in order to obtain information about the stoichiometry, binding constants and structure of the selector-selectand complexes in solution. The data were further confirmed by UV spectrometry and electrospray ionization mass spectrometry. The structure of the complexes in the solid state was determined using X-ray crystallography performed on the co-crystals precipitated from the 1:1 aqueous solution of selector and selectand. This multiple approach allowed an elucidation of the most likely structural reason for a different affinity (binding strength) of BrPh enantiomers towards beta-CD and TM-beta-CD. However, the question about a force responsible for the opposite affinity pattern of BrPh enantiomers towards these CDs could not be answered definitely.

Capillary electrophoresis with laser-induced fluorescence in clinical drug development routine application and future aspects.

The clinical bioanalytical setting is characterized by sample volumes of < 1 ml biological fluid (e.g. plasma, urine), a range of 3-4 decades of concentrations to be quantified and a limit of quantitation in the microg/l-ng/l range for sets of 100-5000 individual samples. Setup of capillary electrophoresis (CE) for routine application in this analytical field was successful for analytes accessible to fluorescence detection by using laser-induced fluorescence (LIF) detection. Empowerment of CE-LIF for routine serial analysis of thousands of samples includes improvement in autosampler techniques, thorough procedures for capillary treatment and particularly more advanced detection technology. Introduction of multi-capillary systems with charge-coupled device cameras and frequency doubled Ar-ion laser (lambda = 257 nm) offers this technique the chance of superiority over classical analytical assays - especially in the field of (new) low volume samples e.g. capillary blood or microdialysate encouraging clinicians to search for meaningful non-invasive samples.

Enantiomers of benzothiadiazine diuretics by direct chromatographic resolution of the racemic drugs.

A number of racemic benzothiadiazine diuretics and two carbonyl analogue drugs were resolved into their optical isomers by liquid chromatography on chiral polyacrylamides 1. Enantiomeric resolution, which was, in some cases, almost complete, depended considerably on the substitution of the heterocyclic moiety of the drug molecules. Synthesis of the new adsorbent lb is described. The enantiomers of the benzothiadiazines penflutizide (2a) and bendroflumethiazide (2b) in high optical purity, as well as enriched (+)-buthiazide (2j) were obtained by repeated chromatography on a semipreparative scale. Chiroptical data, optical purity employing the chromatographic method, and first-order racemization kinetics as a function of pH in aqueous solutions were determined.

Interactions of antiarrhythmics with artificial phospholipid membranes.

Binding of antiarrhythmics to phospholipids has been quantified by measuring the drug-induced fluorescence increase in 8-anilino-1-napthalenesulfonate (ANS)-treated phosphatidylcholine membranes. The ability of test compounds to increase fluorescence intensity to 50% varies between 1.3 and 88 X 10(-6) M, exhibiting a potency ratio of 1.8 log units. The antiarrythmics tested in this study exhibited a wide spectrum of lipophilicity ranging between sigma f = 6.66 for the most lipophilic compound asocainol, to sigma f = 1.21 for the most hydrophilic compound procainamide. The pKa values ranged from 10.80 for quinacainol to 6.26 for ethmozine. Consequently, the test compounds are 99.9 to 6.8% protonated at physiological pH. Binding of antiarrythmics to phosphatidylcholine membranes appears to be determined mainly by their lipophilicity (r = 0.91), irrespective of the pKa, as demonstrated for ethmozine and lidocaine which showed an excellent fit to the regression line. Binding of a variety of antiarrhythmics (n = 8) to phosphatidylcholine appeared to correlate significantly with the mean daily dosage for these drugs (r = 0.97).

Analysis of charged cyclomalto-oligosaccharides (cyclodextrin) derivatives by ion-spray, matrix-assisted laser-desorption/ionization time-of-flight and fast-atom bombardment mass spectrometry, and by capillary electrophoresis.

The use of electrospray-ionization mass spectrometry (ESIMS), matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDITOFMS), and fast-atom bombardment mass spectrometry (FABMS) for the rapid determination of molecular weight, degree of substitution (ds), and purity is demonstrated for charged derivatives of cyclomalto-heptaose (beta CD) and -octaose (gamma CD). The access to anionic sulfoalkyl ethers (alkyl: ethyl, n-propyl, and n-butyl) and to beta CD-2-hydroxy-3-trimethylammoniumpropyl ether chlorides (HTAP-beta CD) leads only to mixtures of products, the compositions of which can be determined directly from ESI and MALDITOF mass spectra. All charged derivatives consist of a mixture of unreacted and higher substituted compounds. The substitution patterns obtained by MS are in good agreement with the results of experiments on the separation of beta CD-sulfoalkyl ethers by capillary electrophoresis (CE).

Excretion kinetics of ifosfamide side-chain metabolites in children on continuous and short-term infusion.

Ifosfamide (IFO) requires metabolic activation by hydroxylation of the ring system to exert cytotoxic activity. A second metabolic pathway produces the cytostatically inactive metabolites 2-dechloroethyl-ifosfamide (2-D-IFO) and 3-dechloroethyl-ifosfamide (3-D-IFO) under release of chloroacetaldehyde. This side-chain metabolism has been suggested to be involved in CNS- and renal toxicity. The total urinary excretion of ifosfamide and its metabolites was investigated during 23 cycles in 22 children at doses ranging from 400 mg/m2 to 3 g/m2. The kinetics of the excretion were compared following short-term and continuous ifosfamide infusion at a dosage of 3 g/m2. IFO and side-chain metabolites were analyzed by gas chromatography, the active metabolites by indirect determination of acrolein (ACR) and IFO mustard (IFO-M) with the NBP test. 59+/-15% of the applied dose could be recovered in the urine, 23+/-9% as unmetabolized IFO. The main metabolite was 3-D-IFO (14+/-4%) followed by isophosphoramide mustard (IFO-M) (13+/-4%) and 2-D-IFO (8+/-3%). Neither the total amount recovered nor the excretion kinetics of ifosfamide and side-chain metabolites showed obvious schedule dependency. The excretion kinetics of side-chain metabolites as well as unmetabolized IFO were nearly superimposable on short-term and continuous infusion. Even after 1-hour infusion there was a lag of 3 - 6 hours until dechloroethylation became relevant. Therefore, differences in toxicity and efficacy cannot be explained by an influence of the application time on the metabolic profile of ifosfamide.