The application of microbore UPLC/oa-TOF-MS and 1H NMR spectroscopy to the metabonomic analysis of rat urine following the intravenous administration of pravastatin.

The metabonomic effects of hepatotoxic doses of pravastatin on the urinary metabolic profiles of female rats have been investigated using ultra performance liquid chromatography (UPLC)-oa-TOF-MS and, independently, by (1)H NMR spectroscopy. UPLC was performed using a 1 mm microbore column packed with 1.7 microm particles. Examination of the data obtained from the individual animals, aided by statistical interpretation of the data, made it possible to identify potential markers for toxicological effects, with both NMR and UPLC-MS analysis highlighting distinct changes in the urinary metabolite profiles. These markers, which included elevated taurine and creatine, as well as bile acids, were consistent with hepatotoxicity in some animals, and this hypothesis was supported by histopathological and clinical chemistry findings. The analytical data from both techniques could be used to define a metabolic "trajectory" as toxicity developed and to provide an explanation for the lack of hepatotoxicity for one of the animals. The two analytical approaches (UPLC-MS and NMR) were found to be complementary whilst the use of a 1mm i.d. x 100 mm column reduced the amount of sample required for analysis to 2 microL, compared with 10 microL for a 2.1mm i.d. x 100 mm column. The 1mm i.d. column also provided increased signal-to-noise without loss of chromatographic efficiency.

Identification of phenacetin metabolites in human urine after administration of phenacetin-C2H3: measurement of futile metabolic deacetylation via HPLC/MS-SPE-NMR and HPLC-ToF MS.

The metabolism of acetyl-labelled phenacetin-C2H3 was investigated in man following a single (150 mg) oral dose. Urine samples were collected at predose, 0-2 h and >2-4 h post-dose, and samples from each time-point were then analysed directly using 1H-nuclear magnetic resonance (NMR) spectroscopy. The phenacetin metabolites acetaminophen (paracetamol) glucuronide, sulphate and the N-acetyl-L-cysteinyl conjugate were identified by this method, and all showed clear evidence of the loss of the original 2H3-acetyl label and its replacement with 1H3 (futile deacetylation). The observed percentage futile deacetylation by 1H-NMR spectroscopy was measured as approximately 20% in each metabolite (about 2% of the recovered dose). After sample preparation by solid-phase extraction on a C18 solid-phase extraction (SPE) cartridge, further profiling was performed using high-performance liquid chromatography/mass spectrometry-solid-phase extraction-nuclear magnetic resonance (HPLC/MS-SPE-NMR) confirming futile deacetylation had taken place as indicated by NMR spectroscopy on both the isolated acetaminophen glucuronide and L-cysteinyl-metabolites. Additional analysis by high-performance liquid chromatography-time-of-flight mass spectrometry (HPLC-ToF MS) identified further phenacetin metabolites, and from these data the mean percentage of futile deacetylation was measured as 31% +/- 2% for the acetylated phenacetin metabolites. A number of non-acetylated metabolites were also detected in the sample via HPLC-ToF MS. The results showed that phenacetin underwent a transient formation via a number of toxic intermediates to a much greater extent than had been observed in similar studies on acetaminophen. These results may contribute to the understanding of the analgesic nephropathy reported following chronic phenacetin consumption.

Formation of unusual glutamate conjugates of 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423) and its analogs: the role of gamma-glutamyltranspeptidase in the biotransformation of benzylamines.

The role of gamma-glutamyltranspeptidase (GGT) in transferring glutamate from endogenous glutathione (GSH) to the benzylamine moiety of a compound, such as 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423), is described. Studies were performed with structurally related analogs of DPC 423 to demonstrate that this type of reaction was common to compounds possessing a benzylamine group. Synthesizing appropriate standards and confirming by liquid chromatography (LC)/mass spectroscopy and LC/NMR made unambiguous assignments of the structures of glutamate conjugates of DPC 423. The use of stable isotope-labeled GSH for metabolism studies has not been described before. In the present study, we report the novel use of deuterated GSH in conjunction with mass spectral analysis to demonstrate the glutamate transfer to the benzylamines in the presence of GGT. To further demonstrate that the alpha protons on the benzylamines and glutamate (as part of glutathione) were unaffected during the transpeptidation, these protons were replaced with deuterium. Acivicin (AT-125), a potent and selective inhibitor of GGT, was used to abolish the formation of the glutamate conjugates of DPC 423 in vitro and in vivo. This provided further evidence of the role of GGT in forming the glutamate conjugates of benzylamines. This study demonstrated conclusively that GGT was responsible for mediating the transfer of glutamic acid from GSH to the benzylamine moiety of a series of structurally related compounds.

Metabonomic investigations into hydrazine toxicity in the rat.

The systemic biochemical effects of oral hydrazine administration (dosed at 75, 90, and 120 mg/kg) have been investigated in male Han Wistar rats using metabonomic analysis of (1)H NMR spectra of urine and plasma, conventional clinical chemistry, and liver histopathology. Plasma samples were collected both pre- and 24 h postdose, while urine was collected predose and daily over a 7 day postdose period. (1)H NMR spectra of the biofluids were analyzed visually and via pattern recognition using principal component analysis. The latter showed that there was a dose-dependent biochemical effect of hydrazine treatment on the levels of a range of low molecular weight compounds in urine and plasma, which was correlated with the severity of the hydrazine induced liver lesions. In plasma, increases in the levels of free glycine, alanine, isoleucine, valine, lysine, arginine, tyrosine, citrulline, 3-D-hydroxybutyrate, creatine, histidine, and threonine were observed. Urinary excretion of hippurate, citrate, succinate, 2-oxoglutarate, trimethylamine-N-oxide, fumarate and creatinine were decreased following hydrazine dosing, whereas taurine, creatine, threonine, N-methylnicotinic acid, tyrosine, beta-alanine, citrulline, Nalpha-acetylcitrulline and argininosuccinate excretion was increased. Moreover, the most notable effect was the appearance in urine and plasma of 2-aminoadipate, which has previously been shown to lead to neurological effects in rats. High urinary levels of 2-aminoadipate may explain the hitherto poorly understood neurological effects of hydrazine. Metabonomic analysis of high-resolution (1)H NMR spectra of biofluids has provided a means of monitoring the progression of toxicity and recovery, while also allowing the identification of novel biomarkers of development and regression of the lesion.

Directly coupled HPLC-NMR and HPLC-MS approaches for the rapid characterisation of drug metabolites in urine: application to the human metabolism of naproxen.

High resolution nuclear magnetic resonance (NMR) spectroscopy is a very powerful tool for the structural identification of xenobiotic metabolites in complex biological matrices such as plasma, urine and bile. However, these fluids are dominated by thousands of signals resulting from endogenous metabolites and it is advantageous when investigating drug metabolites in such matrices to simplify the spectra by including a separation step in the experiment by directly-coupling HPLC and NMR. Naproxen (6-methoxy-alpha-methyl-2-naphthyl acetic acid) is administered as the S-enantiomer and is metabolised in vivo to form its demethylated metabolite which is subsequently conjugated with beta-D-glucuronic acid as well as with sulfate. Naproxen is also metabolised by phase II metabolism directly to form a glycine conjugate as well as a glucuronic acid conjugate at the carboxyl group. In the present investigation, the metabolism of naproxen was investigated in urine samples with a very simple sample preparation using a combination of directly-coupled HPLC-1H NMR spectroscopy and HPLC-mass spectrometry (MS). A buffer system was developed which allows the same chromatographic method to be used for the HPLC-NMR as well as the HPLC-MS analysis. The combination of these methods is complementary in information content since the NMR spectra provide evidence to distinguish isomers such as the type of glucuronides formed, and the HPLC-MS data allow identification of molecules containing NMR-silent fragments such as occur in the sulfate ester.

Application of directly coupled LC-NMR-MS to the structural elucidation of metabolites of the HIV-1 reverse-transcriptase inhibitor BW935U83.

The human in vivo metabolism of the HIV-1 reverse transcriptase inhibitor 5-chloro-1-(2',3'-dideoxy-3'-fluoro-erythro-pentofuranosyl)uracil (BW935U83) was studied using 19F NMR spectroscopy, directly coupled LC-NMR and LC-NMR-MS. The number and relative proportions of the drug metabolites were obtained from 19F NMR spectra of whole human urine. The novel use of the continuous-flow 19F detected LC-NMR experiment yielded chromatographic retention times and 19F chemical shifts for the parent drug, the glucuronide conjugate of the parent and an early eluting polar metabolite. The parent drug and its glucuronide conjugate were easily characterised by directly coupled 1H LC-NMR spectroscopy and two-dimensional TOCSY experiments. The identification of the second metabolite was achieved using 19F NMR and directly coupled 1H LC-NMR-MS which furnished the molecular weight, and through the use of MS-MS techniques, information on the fragment ions. This species was identified as 3-fluoro-ribolactone.

Characterization of novel glutathione adducts of a non-nucleoside reverse transcriptase inhibitor, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3, 4-dihydro-2(1H)-quinazolinone (DPC 961), in rats. Possible formation of an oxirene metabolic intermediate from a disubstituted alkyne.

The postulated formation of oxirene-derived metabolites from rats treated with a disubstituted alkyne, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3, 4-dihydro-2(1H)-quinazolinone (DPC 961), is described. The reactivity of this postulated oxirene intermediate led to the formation of novel glutathione adducts whose structures were confirmed by LC/MS and by two-dimensional NMR experiments. These metabolites were either excreted in rat bile or degraded to mercapturic acid conjugates and eliminated in urine. To demonstrate the oxidation of the triple bond, an analogue of DPC 961 was synthesized, whereby the two carbons of the alkyne moiety were replaced with (13)C stable isotope labels. Rats were orally administered [(13)C]DPC 961 and glutathione adducts isolated from bile. The presence of an oxygen atom on one of the (13)C labels of the alkyne was demonstrated unequivocally by NMR experiments. Administration of (14)C-labeled DPC 961 showed that biliary elimination was the major route of excretion with the 8-OH glucuronide conjugate (M1) accounting for greater than 90% of the eliminated radioactivity. On the basis of radiochemical profiling, the glutathione-derived metabolites were minor in comparison to the glucuronide conjugate. Studies with cDNA-expressed rat enzymes, polyclonal antibodies, and chemical inhibitors pointed to the involvement of P450 3A1 and P450 1A2 in the formation of the postulated oxirene intermediate. The proposed mechanism shown in Scheme 1 begins with P450-catalyzed formation of an oxirene, rearrangement to a reactive cyclobutenyl ketone, and a 1,4-Michael addition with endogenous glutathione to produce two isomeric adducts, GS-1 and GS-2. The glutathione adducts were subsequently catabolized via the mercapturic acid pathway to cysteinylglycine, cysteine, and N-acetylcysteine adducts. The transient existence of the alpha,beta-unsaturated cyclobutenyl ketone was demonstrated by incubating the glutathione adduct in the presence of N-acetylcysteine and monitoring the formation of N-acetylcysteine adducts by LC/MS. Epimerization of GS-1 to GS-2 was also observed when N-acetylcysteine was omitted from the incubation.

Disposition of glutathione conjugates in rats by a novel glutamic acid pathway: characterization of unique peptide conjugates by liquid chromatography/mass spectrometry and liquid chromatography/NMR.

With the advent of liquid chromatography/mass spectrometry and liquid chromatography/NMR, it has become easier to characterize metabolites that were once difficult to isolate and identify. These techniques have enabled us to uncover the existence of an alternate pathway for the disposition of glutathione adducts of several structurally diverse compounds. Studies were carried out using acetaminophen as a model compound to investigate the role of the glutamic acid pathway in disposition of the glutathione adducts. Although the mercapturic acid pathway was the major route of degradation of the glutathione adducts, it was found that the conjugation of the glutathione, cysteinylglycine, and cysteine adducts of acetaminophen with the gamma-carboxylic acid of the glutamic acid was both interesting and novel. The coupling of the glutathione adduct and the products from the mercapturic acid pathway with the glutamic acid led to unusual peptide conjugates. The natures of these adducts were confirmed unequivocally by comparisons with synthetic standards. This pathway (addition of glutamic acids) led to larger peptides, in contrast to the mercapturic acid pathway, in which the glutathione adducts are broken down to smaller molecules. The enzyme responsible for the addition of glutamic acid to the different elements of the mercapturic acid pathway is currently unknown. It is postulated that the gamma-carboxylic acid is activated (perhaps by ATP) before enzymatic addition to the alpha-amino group of cysteine or glutamate takes place. The discovery of these peptide conjugates of acetaminophen represents a novel disposition of glutathione adducts of compounds. The formation of such conjugates may represent yet another pathway by which drugs could produce covalent binding via their reactive intermediates.

Mass spectrometric and NMR characterization of metabolites of roxifiban, a potent and selective antagonist of the platelet glycoprotein IIb/IIIa receptor.

1. The methyl ester prodrug roxifiban is an orally active, potent and selective antagonist of the platelet glycoprotein GPIIb/IIIa receptor and is being developed for the prevention and treatment of arterial thrombosis. 2. Roxifiban was rapidly hydrolyzed to the zwitterion XV459 in vivo and by liver slices from the rat, mouse and human and by intestinal cores from dog. XV459 was metabolized to only a small extent in vitro and in vivo. 3. Studies with rat and dog given radiolabelled roxifiban showed limited oral absorption with the majority of the radiolabel being excreted in faeces. After i.v. doses of 14C-roxifiban, most of the radioactivity was recovered in the urine of rat whereas the dog excreted significant amounts of radioactivity in bile and urine. 4. XV459 could be metabolized extrahepatically by dog gut flora to produce an isoxazoline ring-opened metabolite. In vitro hepatic metabolism of XV459 was mainly by hydroxylation at the prochiral and chiral centres of the isoxazoline ring. These hydroxylated metabolites were not detected in the urine and plasma of human volunteers administered roxifiban. 5. Initial LC/MS identification of metabolites was achieved by dosing the rat with an equimolar mixture of d0:d4 roxifiban and detecting isotopic clusters of pseudomolecular ions. Unequivocal characterization of these metabolites was achieved by LC/MS, LC/NMR and high-field NMR techniques using synthetic standards of the metabolites. 6. The synthesis of one hydroxylated metabolite enabled the assignment of the correct stereochemistry of the substituted hydroxyl group on the isoxazoline ring.

Accelerated toxicity screening using NMR and pattern recognition-based methods.

(1)H-NMR spectroscopy has proved to be a powerful and efficient means of monitoring the interaction of pharmacological agents with cells and tissues. The application of this technique to biofluid analysis, gives rise to a comprehensive metabolic profile of the low molecular weight components of biofluids, that reflect concentrations and fluxes of endogenous metabolites involved in key intermediary cellular pathways, thereby giving an indication of an organism's physiological or pathophysiological status. Recent developments in spectrometer technology have resulted in increased sensitivity and dispersion. Together with the increased capacity for sample throughput (~ 300 samples/day), arising from the latest advances in flow probe technology and in robotic transfer systems, (1)H-NMR spectroscopic techniques have become viable in terms of toxicological screening. However, the complexity of high-field biofluid spectra in conjunction with the increased capacity for sample handling, leading to a rapid growth in the size of toxicological spectral databases, has placed greater emphasis on the need to develop improved automated procedures for data processing and interpretation. By harnessing chemometric tools to the analysis of complex spectral data, the toxicological consequences of xenobiotic exposure can be evaluated efficiently on-line. Automation of spectral processing procedures and the construction of mathematically-based 'expert systems' for the prediction of drug-induced toxicity founded on 1H-NMR spectral profiles, have now been achieved. In this article, we review the recent developments in NMR and pattern recognition analysis and consider their application in toxicological screening.

Protein and nonprotein cysteinyl thiol modification by N-acetyl-p-benzoquinone imine via a novel ipso adduct.

N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen (APAP), can arylate and oxidize protein and nonprotein thiols in the pathogenesis of APAP-induced hepatotoxicity. We report the first direct evidence for the formation of a labile ipso adduct between glutathione (GSH) and NAPQI using a combination of techniques including liquid chromatography/tandem mass spectrometry and liquid chromatography/NMR spectroscopy. Decomposition kinetics of the GSH-NAPQI ipso adduct and product ratios suggested that the ipso adduct was readily reversible back to NAPQI under neutral and basic conditions. The significance of the ipso adduct is that it may migrate from its site of formation to other cell compartments where it can either oxidize protein thiols or covalently modify them. Ipso adduct formation with protein thiols was demonstrated with a cysteine protease, papain, whose catalytic activity relies on the presence of an active site cysteinyl thiol. The formation and reactions of cysteinyl thiol ipso adducts of NAPQI provides significant new insights into possible reactions of quinone imines with cellular peptides and proteins.

Directly-coupled HPLC-NMR spectroscopic studies of metabolism and futile deacetylation of phenacetin in the rat.

The metabolism and futile deacetylation of phenacetin has been investigated in the rat via 1H NMR spectroscopic analysis of urine. Animals were dosed with either phenacetin or phenacetin-C2H3 and urine samples were collected for -24-0 (pre-dosing), 0-8. 8-24, and 24-48 h post-dosing. Drug metabolites of the two compounds were concentrated from the urine using solid-phase extraction prior to the use of directly-coupled HPLC-1H NMR spectroscopy for separation and identification. Following dosing of phenacetin, the metabolites identified were paracetamol glucuronide, paracetamol and N-hydroxyparacetamol, whilst paracetamol and N-hydroxyparacetamol sulphate were identified following dosing of phenacetin-C2H3. Quantitatively the percentage futile deacetylation of phenacetin-C2H3 metabolites was found to be 32% in both paracetamol and N-hydroxyparacetamol sulphate. This study further indicated the importance of futile deacetylation in simple analgesics and the value of directly-coupled HPLC-NMR spectroscopy for the study of this process.

NMR and HPLC-NMR spectroscopic studies of futile deacetylation in paracetamol metabolites in rat and man.

HPLC-NMR spectroscopy has been used to investigate the level of deacetylation followed by reacetylation (futile deacetylation) of metabolites of paracetamol detected in human and rat urine. This has been achieved through the synthesis and administration of paracetamol isotopically labeled at the acetyl group with C2H3, 13CH3 and 13CO-13CH3. Using paracetamol-C2H3 it had been shown that in the rat the sulphate metabolite present in the urine shows 10-13% futile deacetylation depending on the dose, whereas for paracetamol-13CO-13CH3 the corresponding value was about 8%. After solid phase extraction, it was also possible to determine the level of futile deacetylation in the glucuronide metabolite using directly-coupled HPLC-NMR. This approach was facilitated by the use of acetonitrile-d3 as an HPLC eluent and the HPLC-NMR analyses showed that the level of futile deacetylation in the sulphate and glucuronide metabolites were equal at about 9%. The glucuronide of paracetamol-C2H3 was the predominant metabolite in man and following separation using HPLC-NMR, the level of futile deacetylation was shown to be 1% for the glucuronide and 2% for the sulphate, these values being equal within experimental error. This work demonstrates the utility of NMR and HPLC-NMR spectroscopy for isotope exchange studies.

Combined HPLC, NMR spectroscopy, and ion-trap mass spectrometry with application to the detection and characterization of xenobiotic and endogenous metabolites in human urine.

The direct coupling of HPLC with NMR spectroscopy has been extended by splitting the HPLC eluent after conventional UV detection and sending part to a NMR spectrometer and part to an ion-trap mass spectrometer in a "triplehyphenated" HPLC-NMR-MS system. Combined UV, 1H NMR, and positive-ion electrospray MS detection was achieved in the continuous-flow mode using whole human urine from a subject dosed with acetaminophen. By means of HPLC-NMR-MS, the structural information available from the complementary spectroscopic techniques provided rapid confirmation of the identity of the acetaminophen glucuronide and sulfate metabolites, together with a number of endogenous metabolites. In particular, the HPLC-NMR-MS approach allowed the unequivocal identification of phenylacetylglutamine in human urine, an endogenous metabolite not previously observed in 1H NMR spectra of urine because of extensive overlap with resonances from other metabolites. The analytical advantages and complementarity of NMR and MS techniques in direct hyphenation with HPLC are discussed. The new technique of HPLC-NMR-MS will provide the scope for more comprehensive and fully automated analysis of biofluids and other complex mixtures than was previously available from single hyphenation of these instruments.

Hplc-nmr identification of the human urinary metabolites of (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine, a nucleoside analogue active against human immunodeficiency virus (HIV).

1. Human urine samples from a clinical trial of the anti-HIV compound (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-cyto sin e (BW524W91) have been analysed using 19F-nmr and 1H-hplc-nmr spectroscopy. 2. The identities and relative levels of the xenobiotic species in the urine have been determined by 470-MHz 19F-nmr spectroscopy and by directly coupled 600-MHz 1H-hplc-nmr in the stop-flow mode with confirmation of the metabolite identities being made by comparison with nmr spectra of synthetic standard compounds. 3. The principal urinary xenobiotic was the unchanged drug, but the glucuronide ether conjugate at the 5' position of BW524W91, one of the two diastereomeric sulphoxides and the deaminated metabolite were also characterized. 4. The detection limit of directly coupled hplc-600-MHz 1H-nmr spectroscopy was evaluated by measuring two-dimensional nmr spectra of the glucuronide conjugate of BW524W91 and shown to be approximately 1 microgram material for 1H-1H-TOCSY and 20 micrograms metabolite for 1H-13C-HMQC spectra for overnight (16 h) acquisition.

Characterization of in vitro metabolites from human liver microsomes using directly coupled hplc-nmr: application to a phenoxathiin monoamine oxidase-A inhibitor.

1. The metabolism of 1-ethylphenoxathiin-10,10-dioxide (BW1370U87), an experimental compound designed as an inhibitor of monoamine oxidase-A for use as a possible anti-depression agent, has been studied in a human liver microsome preparation. 2. The identities of the metabolites have been determined using directly coupled hplc-1H nmr at 600 MHz in the stop-flow mode in this first study of in vitro xenobiotic metabolism using hplc-nmr. 3. The xenobiotic substances that were identified comprised the parent compound BW1370U87 (with -CH2CH3 at C1) together with sidechain-oxidized metabolites with C1 substituents of -CHOH.CH3, -CH2.CH2OH, -CHOH.CH2OH and -CH2.COOH, plus the unsubstituted phenoxathiin-10,10-dioxide.

Identification of an N-acetyl keto derivative of fumonisin B1 in corn cultures of Fusarium proliferatum.

A method is presented for the separation and identification of a new N-acetyl keto derivative of fumonisin B1 (FB1) produced in solid corn culture. Cultures of Fusarium proliferatum (M-1597) were purified using preparative hplc, and the new fumonisin was detected by negative-ion esms. Structures were confirmed by 1H- and 13C-nmr spectroscopy. The new fumonisin differs from FB1 in that the tricarballylic acid functionality at the C-15 position of the eicosane backbone is replaced by a ketone and the amino group is acetylated. Direct analysis of the culture material by negative-ion electrospray lc/ms confirmed that the new fumonisin is produced naturally by the fungus.

Balance/excretion of 3H- and 14C-tyloxapol in the male rabbit after intratracheal administration.

1. Tyloxapol, trace-labelled (50-100 microCi/animal) with 3H or 14C, was administered intratracheally in a surfactant formulation (EXOSURF Neonatal) to the male rabbit in a total tyloxapol dose of 5 mg/kg. Urine, faeces, expired air, and blood were collected for up to 10 days following tyloxapol administration. 2. Over 5 days, 3H-tyloxapol-related radioactivity in the urine (13.4%) and faeces (27.4%) accounted for a major fraction of the labelled dose. However, urine also contained an additional 13% of the dose as tritiated water. Expired air accounted for only 4.2% of the dose. At the end of the study, an additional 35.6% of the radioactive dose was found in tissues and the carcass, mainly in the lung (27.4%) and to a lesser extent in the liver (2.8%) and kidney (0.4%). Levels of radioactivity in other tissues, including whole blood, were low. 3. Over a separate 10-day study, faecal (30.4%) and renal (9.7%) elimination of 14C-tyloxapol accounted for 40% of the radioactive dose, with expired air accounting for much less (2.7%). At the end of the study, additional radioactivity was recovered from the lung (43.9%) and to a lesser extent from the liver (3.8%) and kidney (0.3%). The half-life for the elimination of total radioactivity from the lung was estimated to be 10-12 days. 4. These data indicate that, following intratracheal administration, tyloxapol and metabolites were retained by the lung, released slowly into the systemic circulation, and eliminated through faecal and renal excretion.

Pharmacokinetics and disposition in the rat of a DNA intercalator 502U83.

The pharmacokinetics, metabolism, and qualitative tissue distribution of 502U83, a compound with antineoplastic activity, were examined in the rat. After an oral dose, average maximum plasma concentrations of 5.3 micrograms/ml were achieved at 0.28 hr, indicating rapid absorption of the compound; the bioavailability was estimated to be 62%. After intravenous administration the half-life was 1.73 hr. Autoradiographs of rats dosed intravenously with [14C]502U83 showed the presence of significant levels of radioactivity in the bone marrow, salivary gland, thymus, and lung; highest levels were in the gastrointestinal tract. There was no evidence of penetration of radioactivity into the brain. After an intravenous administration of [14C]502U83, a mean of 94.1% of the dose was recovered in 72 hr, with 46.6% in the urine and 47.5% in the feces. HPLC analysis of the radiocarbon in urine and feces revealed the presence of at least six common radioactive peaks, each representing approximately 2 to 12% of the dose. Biotransformation of 502U83 by the rat mainly involves oxidation of the hydroxyethyl group, and one or both of the hydroxymethyl groups leading to three major metabolites, common to urine and feces. Parent drug was the major component in both urine and feces, respectively, accounting for 18% and 10% of the dose in 48 hr. The glucuronic acid conjugate of the parent drug was a minor metabolite (< 2% of the dose). There was no evidence of metabolism on the anthracene ring.