Measuring antibiotic levels and their relationship with the microbiome in chronic rhinosinusitis.

BACKGROUND: The evidence supporting the efficacy of antibiotic therapy in the treatment of chronic rhinosinusitis is not compelling. A limited number of studies show that the changes in the nasal microbiome in patients following drug therapy are unpredictable and variable. The evidence for the impact of oral antibiotics on the gut microbiota is stronger, possibly as a result of differences in drug distribution to various sites around the body. There are few studies on sinus mucosal and mucus levels of oral antibiotics used in the treatment of chronic rhinosinusitis. The distribution dependent effects of antibiotics on the sinonasal microbiome is unclear. CONCLUSION: This review highlights that relative drug concentrations and their efficacy on microbiota at different sites is an important subject for future studies investigating chronic rhinosinusitis.

Comparative bioactivation of the novel anti-tuberculosis agent PA-824 in Mycobacteria and a subcellular fraction of human liver.

BACKGROUND AND PURPOSE: PA-824 is a 2-nitroimidazooxazine prodrug currently in Phase II clinical trial for tuberculosis therapy. It is bioactivated by a deazaflavin (F(420) )-dependent nitroreductase (Ddn) isolated from Mycobacterium tuberculosis to form a des-nitro metabolite. This releases toxic reactive nitrogen species which may be responsible for its anti-mycobacterial activity. There are no published reports of mammalian enzymes bioactivating this prodrug. We have investigated the metabolism of PA-824 following incubation with a subcellular fraction of human liver, in comparison with purified Ddn, M. tuberculosis and Mycobacterium smegmatis. EXPERIMENTAL APPROACH: PA-824 (250 µM) was incubated with the 9000 × g supernatant (S9) of human liver homogenates, purified Ddn, M. tuberculosis and M. smegmatis for metabolite identification by liquid chromatography mass spectrometry analysis. KEY RESULTS: PA-824 was metabolized to seven products by Ddn and M. tuberculosis, with the major metabolite being the des-nitro product. Six of these products, but not the des-nitro metabolite, were also detected in M. smegmatis. In contrast, only four of these metabolites were observed in human liver S9; M3, a reduction product previously proposed as an intermediate in the Ddn-catalyzed des-nitrification and radiolytic reduction of PA-824; two unidentified metabolites, M1 and M4, which were products of M3; and a haem-catalyzed product of imidazole ring hydration (M2). CONCLUSIONS AND IMPLICATIONS: PA-824 was metabolized by des-nitrification in Ddn and M. tuberculosis, but this does not occur in human liver S9 and M. smegmatis. Thus, PA-824 was selectively bioactivated in M. tuberculosis and there was no evidence for 'cross-activation' by human enzymes.

Pharmacokinetics of 'party pill' drug N-benzylpiperazine (BZP) in healthy human participants.

There have been many reports of benzylpiperazine (BZP) and trifluoromethylphenylpiperazine (TFMPP) being used as recreational drugs which have been widely marketed in the form of 'party pills' since the late 1990's. However, there is no information currently available describing the pharmacokinetics of these drugs in humans. Human plasma concentrations of BZP were measured in blood and urine samples taken from healthy adults (n=7) over 24h following a 200mg oral dose of BZP. Plasma concentrations of BZP were found to peak at 262 ng/mL (C(max)) and 75min (T(max)). Plasma concentrations of the major metabolites of BZP, 4-OH BZP and 3-OH BZP, were found to peak at 7 ng/mL (at 60 min) and 13 ng/mL (at 75 min) respectively. The elimination half-life (t(1/2)) for BZP was found to be 5.5h. Clearance (Cl/F) was found to be 99L/h. The results of this study indicate that BZP may be detectable in plasma for up to 30 h following an oral dose. Additionally, several urinary metabolites can be detected.

Bioavailability and pharmacokinetics of a praziquantel bolus in kingfish Seriola lalandi.

Oral praziquantel (PZQ) preparations have recently been investigated for the treatment of monogeneans that infect the skin and gills of kingfish Seriola lalandi cultured in sea-cages. To evaluate an oral PZQ dosing strategy, the pharmacokinetics of a dissolved and in feed oral PZQ preparation (40 mg kg(-1) body weight) were compared with an intravenous bolus in kingfish plasma and skin using HPLC. Compared with intravenous administration, PZQ bioavailability (area under curve, AUC0-24h) was slightly improved when the drug was administered with food in both kingfish plasma (56.8% in feed vs. 50.8% in solution) and skin (55.5% in feed vs. 50.3% in solution). After oral dosing, maximum drug concentrations in skin were approximately one-third of those achieved in plasma and higher when the drug was administered in solution (5.26 microg ml(-1)) than in feed (3.96 microg ml(-1)); additionally, the time to achieve maximum PZQ concentration was similar in plasma and skin, although markedly reduced when the drug was administered in solution (1 h) than in feed (6 h). However, clearance of the drug was delayed in skin; administered as an oral formulation, PZQ concentrations in the systemic circulation fell below the limit of quantification after 24 h, but remained quantifiable (0.3 microg g(-1)) in skin at this time. These initial studies indicate that a daily treatment interval will lead to the exposure of parasites to highly variable anthelmintic concentrations, which may be sub-optimal for the treatment of monogeneans in this finfish species.

Can in vitro drug metabolism studies with human tissue replace in vivo animal studies?

Animals provide a physiologically relevant system for evaluation of drug metabolism, but marked inter-species differences limit extrapolation to humans. Liver microsomes are used extensively as an in vitro human drug metabolising system, and with appropriate selection of parameters, such as substrate and enzyme concentrations, may predict both routes and rate of metabolism. However, variable enzyme expression between donors and overlapping substrate specificity influence reproducibility, hence recombinant human CYP enzymes expressed in human, yeast or insect cells have been developed. For complex metabolic profiles involving sequential or competing pathways, isolated hepatocytes and liver slices are of value. Altered enzyme activity and restricted availability constrain their use. Cryopreservation or culture increase availability, but changes in enzyme activity remain a constraint. To date, human in vitro systems do not predict all aspects of drug metabolism, thus a combination of in vivo animal and in vitro human studies will be required for the foreseeable future.

Species differences in the metabolism of the antitumour agent 5,6-dimethylxanthenone-4-acetic acid in vitro: implications for prediction of metabolic interactions in vivo.

1. Mouse studies have indicated that the antitumour effects of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) are dramatically potentiated in combination with other drugs, and it has been proposed that optimization of the therapeutic potential of DMXAA would exploit combination therapy. The aim was to identify the most appropriate animal model for further investigations of the pharmacokinetics of possible DMXAA-drug combinations and their extrapolation to patients. 2. Qualitatively, the metabolic profile for DMXAA in liver microsomes was similar in mouse, rat, rabbit and humans, with glucuronidation and 6-methylhydroxylation the two major metabolic pathways. In all species, the intrinsic clearance by glucuronidation was at least 2-fold that due to hydroxylation. There was significant variability in the in vitro kinetic parameters (Km, Vmax), with the mouse being the least efficient DMXAA metabolizer compared with the other species. 3. Mouse, rat and rabbit renal microsomes exhibited DMXAA glucuronidation activity, but only the rabbit showed 6-methylhydroxylation. For the total in vitro CL(int) (Vmax/Km) by glucuronidation and 6-methylhydroxylation, the ratio of kidney:liver was 0.67, 0.03 and 0.34 in the mouse, rat and rabbit respectively. However, taking into account the liver and kidney weight difference, it is apparent that the in vivo renal metabolism would not be a major contributor to the overall elimination of DMXAA. 4. The inhibitory profile for liver DMXAA glucuronidation was similar across species, but there was remarkable interspecies variability in the inhibition of liver DMXAA 6methylhydroxylation. 5. Extrapolation of in vitro intrinsic clearance to in vivo gave a significant underestimation of plasma clearance for all species. However, there was a significant allometric relationship for plasma clearance and volume of distribution, but not for maximum tolerated dose across species. 6. The results indicate that animal models may have a limited role in the extrapolation to patients of drug interactions with agents such as DMXAA that have immunomodulating activity that may vary widely between species.

Effects of anticancer drugs on the metabolism of the anticancer drug 5,6-dimethylxanthenone-4-acetic (DMXAA) by human liver microsomes.

AIMS: To investigate the effects of various anticancer drugs on the major metabolic pathways (glucuronidation and 6-methylhydroxylation) of DMXAA in human liver microsomes. METHODS: The effects of various anticancer drugs at 100 and 500 microM on the formation of DMXAA acyl glucuronide (DMXAA-G) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA) in human liver microsomes were determined by high performance liquid chromatography (h.p.l.c.). For those anticancer drugs showing significant inhibition of DMXAA metabolism, the inhibition constants (Ki) were determined. The resulting in vitro data were extrapolated to predict in vivo changes in DMXAA pharmacokinetics. RESULTS: Vinblastine, vincristine and amsacrine at 500 microM significantly (P < 0.05) inhibited DMXAA glucuronidation (Ki = 319, 350 and 230 microM, respectively), but not 6-methylhydroxylation in human liver microsomes. Daunorubicin and N-[2-(dimethylamino)-ethyl]acridine-4-carboxamide (DACA) at 100 and 500 microM showed significant (P < 0.05) inhibition of DMXAA 6-methylhydroxylation (Ki = 131 and 0.59 microM, respectively), but not glucuronidation. Other drugs such as 5-fluoroucacil, paclitaxel, tirapazamine and methotrexate exhibited little or negligible inhibition of the metabolism of DMXAA. Pre-incubation of microsomes with the anticancer drugs (100 and 500 microM) did not enhance their inhibitory effects on DMXAA metabolism. Prediction of DMXAA-drug interactions in vivo based on these in vitro data indicated that all the anticancer drugs investigated except DACA appear unlikely to alter the pharmacokinetics of DMXAA, whereas DACA may increase the plasma AUC of DMXAA by 6%. CONCLUSIONS: These results indicate that alteration of the pharmacokinetics of DMXAA appears unlikely when used in combination with other common anticancer drugs. However, this does not rule out the possibility of pharmacokinetic interactions with other drugs used concurrently with this combination of anticancer drugs.

Identification and reactivity of the major metabolite (beta-1-glucuronide) of the anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in humans.

1. The novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is extensively metabolized by glucuronidation and 6-methylhydroxylation, resulting in DMXAA acyl glucuronide (DMXAA-G) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA). 2. The major human urinary metabolite of DMXAA was isolated and purified by a solid-phase extraction (SPE) method. The isolated metabolite was hydrolysed to free DMXAA by strong base, and by beta-glucuronidase. Liquid chromatography-mass spectrometry (LC-MS) and spectral data indicated the presence of a molecular ion [M + 1]+ at m/z 459, which was consistent with the molecular weight of protonated DMXAA-G. 3. The glucuronide was unstable in buffer at physiological pH, plasma and blood with species variability in half-life. Hydrolysis and intramolecular migration were major degradation pathways. 4. In vitro and in vivo formation of DMXAA-protein adducts was observed. The formation of DMXAA-protein adducts in cancer patients receiving DMXAA was significantly correlated with plasma DMXAA-G concentration and maximum plasma DMXAA concentration. 5. At least five metabolites of DMXAA were observed in patient urine, with up to 60% of the total dose excreted as DMXAA-G, 5.5% as 6-OH-MXAA and 4.5% as the glucuronide of 6-OH-MXAA. 6. These data suggest that the major metabolite in patients' urine is DMXAA beta-1-glucuronide, which may undergo hydrolysis, molecular rearrangement and covalent binding to plasma protein. The reactive properties of DMXAA-G may have important implications for the pharmacokinetics, pharmacodynamics and toxicity of DMXAA.

A difference between the rat and mouse in the pharmacokinetic interaction of 5,6-dimethylxanthenone-4-acetic acid with thalidomide.

PURPOSE: Coadministration of thalidomide, cyproheptadine or diclofenac has been shown to increase the area under the plasma concentration-time curve (AUC) of the novel antitumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in mice. The aim of this study was to further investigate these pharmacokinetic DMXAA-drug interactions in the rat model. METHODS: The effects of coadministration of L-thalidomide, cyproheptadine or diclofenac on the pharmacokinetics of DMXAA were investigated in male Wistar Kyoto rats. The effects of L-thalidomide, cyproheptadine and diclofenac on microsomal metabolism and plasma protein binding of DMXAA were also investigated. RESULTS: No significant alteration in the plasma concentration profile for DMXAA was observed following L-thalidomide pretreatment in rats. In contrast, when combined with diclofenac or cyproheptadine, the plasma AUC of DMXAA was significantly (P<0.05) increased by 48% and 88% and the T1/2 by 36% and 107%, respectively, compared to controls. Both diclofenac and cyproheptadine at 500 microM caused a significant inhibition of DMXAA metabolism in rat liver microsomes. In contrast, L-thalidomide had no or little inhibitory effect on DMXAA metabolism in rat liver microsomes except for causing a 32% decrease in 6methylhydroxylation at 500 microM. None of the drugs had a significant effect on the plasma protein binding of DMXAA in the rat. CONCLUSION: These studies showed that coadministration of L-thalidomide did not alter the plasma DMXAA AUC in rats, in contrast to previous studies in mice, whereas diclofenac and cyproheptadine significantly reduced the plasma clearance of DMXAA in rats in a similar manner to their effect in mice. The cause of the species difference in the pharmacokinetic response to thalidomide by DMXAA is unknown, and indicates difficulties in predicting the outcome of such a combination in patients.

Determination of the covalent adducts of the novel anti-cancer agent 5,6-dimethylxanthenone-4-acetic acid in biological samples by high-performance liquid chromatography.

The reversed-phase HPLC methods were developed to determinate the covalently bound protein adducts of the novel anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) via its glucuronides after releasing aglycone by alkaline hydrolysis in human plasma and human serum albumin (HSA). An aliquot of 75 microl of the mixture was injected onto a Spherex C18 column (150x4.6 mm; 5 microm) at a flow-rate of 2.5 ml/min. The mobile phase comprising of acetonitrile:10 mM ammonium acetate buffer (24:76, v/v, pH 5.8) was used in an isocratic condition, and DMXAA was detected by fluorescence. The method was validated with respect to recovery, selectivity, linearity, precision, and accuracy. Calibration curves for DMXAA were constructed in the concentration range of 0.5-40 microM in washed blank human plasma or HSA prior to alkaline hydrolysis. The difference between the theoretical and calculated concentration and the relative standard deviation were less than 10% at all quality control (QC) concentrations. The limit of detection for the covalent adduct in human plasma or HSA is 0.20 microM. The methods presented good accuracy, precision and sensitivity for use in the preclinical and clinical studies.

Determination of unbound concentration of the novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid in human plasma by ultrafiltration followed by high-performance liquid chromatography with fluorimetric detection.

The novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a highly protein bound drug with narrow therapeutic window. We report a simple HPLC method with fluorimetric detection for the determination of free DMXAA concentration in human plasma. Sample preparation involves the ultrafiltration of plasma by a Centrisart device for 30 min at 2000 g and extraction with acetonitrile: methanol mixture. The method was validated with respect to recovery, selectivity, linearity, precision, and accuracy. Calibration curves for DMXAA were constructed at the concentration range of 0.5-40 microM in blank plasma and phosphate buffer. The difference between the theoretical and calculated concentration and the relative standard deviation were less than 10% at all quality control (QC) concentrations. The HPLC method has been used for the analysis of preclinical studies.

In vitro and in vivo kinetic interactions of the antitumour agent 5,6-dimethylxanthenone-4-acetic acid with thalidomide and diclofenac.

BACKGROUND: Previous studies have demonstrated that coadministration of L-thalidomide with the novel antitumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) results in an increased area under the plasma concentration-time curve (AUC) of DMXAA, suggesting an explanation for the observed increase in the antitumour activity. The aims of this study were to investigate the effects of L-thalidomide on the in vitro metabolism of DMXAA in mouse and human liver microsomes using diclofenac as positive control, to examine the effects of L-thalidomide and diclofenac on the plasma protein binding of DMXAA in vitro, and to investigate whether the in vivo interactions can be predicted from in vitro data, particularly in humans. METHODS: Mouse and human liver microsomes were used to investigate the effects of L-thalidomide and diclofenac on DMXAA metabolism. The resulting in vitro data were extrapolated to predict in vivo changes in DMXAA, which were then compared with the results of in vivo mouse pharmacokinetic interaction studies. The protein binding of DMXAA in mouse and human plasma was determined using ultrafiltration followed by HPLC. RESULTS: Diclofenac at 100 microM caused significant inhibition of glucuronidation (> 70%) and 6-methylhydroxylation (> 54%) of DMXAA in mouse and human liver microsomes. In vivo diclofenac (100 mg/kg i.p.) resulted in a 24% and 31% increase in the plasma DMXAA AUC, and a threefold increase in T1/2 (P < 0.05) in male and female mice, respectively. In contrast, L-thalidomide at 100 microM had no inhibitory effect on DMXAA metabolism in vitro in either species, except for a decrease of about 25% in 6-methylhydroxylation in mice. L-Thalidomide at 500 microM resulted in further significant decreases in 6-methylhydroxylation in mice (30-60%) and human (30%) microsomes. Coadministration of L-thalidomide in male mice resulted in a 23% increase in DMXAA AUC and a twofold increase in T1/2 (P < 0.05). Neither L-thalidomide nor diclofenac at 50 or 500 microM had any significant effect on the in vitro plasma protein binding of DMXAA (500 microM) in mouse or human plasma. Based on our in vitro inhibition studies, we predicted a 20% increase in DMXAA AUC in mice with concomitant diclofenac, but little or no effect (< 5%) with L-thalidomide. CONCLUSION: Both L-thalidomide and diclofenac increased the plasma DMXAA AUC in mice. In the case of diclofenac, this appeared to be due to direct competitive inhibition of DMXAA metabolism, but this mechanism does not appear to be appropriate for L-thalidomide. From the in vitro human inhibition studies, it appears unlikely that concurrent diclofenac will cause an increase in the plasma AUC of DMXAA in patients. However, the effect of L-thalidomide on DMXAA could not be readily predicted from the in vitro data. Our study demonstrated that a predictive model based on direct inhibition of metabolism is appropriate for diclofenac-DMXAA interactions, but is inappropriate for the prediction of L-thalidomide-DMXAA interactions in mice and humans in vivo.

Reversible binding of the novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid to plasma proteins and its distribution into blood cells in various species.

The plasma protein binding and distribution in blood cells of the novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) has been investigated in-vitro using filtration and an HPLC method to measure DMXAA. DMXAA (500 microM) was extensively bound in plasma from all species with an unbound fraction (fu) of 4.61+/-1.10 (mouse), 2.59+/-0.32 (rat), 2.02+/-0.48 (rabbit) and 2.07+/-0.23% (human). The binding was concentration dependent with DMXAA concentrations > or = 1,000 microM markedly increasing the fu in the plasma from all species. The estimated number of binding sites in plasma were 2.4+/-0.2 (mouse), 1.7+/-0.2 (rat), 0.8+/-0.1 (rabbit) and 2.1+/- 0.2 (human). The major binding protein in human plasma was albumin, with negligible binding to gamma-globulin and alpha1-acid glycoprotein. There was a significant linear relationship between the bound:free DMXAA concentration ratio (Cb/Cu) and albumin concentration in human serum albumin solution (r = 0.955; P < 0.05) and in healthy human plasma (r = 0.998; P< 0.05), but not in plasma from cancer patients (n = 5), nor across species. In cancer patients (n = 5) DMXAA had a significantly higher (P < 0.05) fu (4.60+/- 0.42%) compared with healthy human plasma (2.07+/-0.23%). In human plasma, the fu of DMXAA (500 microM) was significantly reduced by 500 microM diazepam (P < 0.05), but not by warfarin, phenylbutazone, salicylic acid, ibuprofen or clofibric acid at that concentration. DMXAA significantly reduced the binding of dansylsarcosine (a Site-II binder) to HSA, but significantly increased the binding of dansylamide (a Site-I binder). Within species, the blood:plasma concentration ratio (CBL/CP) of DMXAA was relatively constant (mouse, 0.581+/-0.005; rat, 0.667+/-0.025; rabbit, 0.637+/-0.019; human, 0.673+/-0.103) over the range 50-1000 microM, but increased significantly at DMXAA concentrations > 1000 microM in all species except the rabbit. These results indicate that significant alterations in DMXAA plasma binding and distribution into blood cells occur with increasing concentrations of DMXAA in all species, and also that significant interspecies differences exist. It would be more appropriate to compare plasma unbound concentrations when assessing DMXAA exposure in cancer patients or when extrapolating across species.

Contrasting effects of acute and chronic dietary exposure to 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) on xenobiotic metabolising enzymes in the male Fischer 344 rat: implications for chemoprevention studies.

BACKGROUND: 2-Amino-3-methyl-imidazo[4,5-f]quinoline (IQ) is a mutagen produced in cooked food. It is commonly present in the human diet and often used as a (pro)carcinogen for chemoprevention studies. Many foodstuffs act as chemopreventers by altering xenobiotic metabolising enzyme expression in favour of detoxication over bioactivation pathways. However, IQ itself can also affect enzyme expression, which may be a confounding factor in chemoprevention studies. AIM OF STUDY: Chronic low dose IQ exposure is intuitively closest to the human dietary situation. The aim was to investigate the effects of chronic dietary exposure to IQ on the expression of enzymes involved in the bioactivation and detoxification of xenobiotics and to compare this with acute exposure, often used in chemoprevention studies. METHODS: Male Fischer rats received IQ (300 ppm) in the diet (AIN-76) for 52 weeks or were given IQ (20 mg.kg-1) orally for 3 days. Animals were killed, livers removed and subcellular fractions prepared. A range of enzymes was selected to allow investigation of several cellular mechanisms. Enzyme expression and activity were determined by Western blotting and the use of selective probe substrates as appropriate. RESULTS: Chronic exposure to IQ led to an increase in phase II detoxifying enzymes. Both the activity and expression of glutathione S-transferase (GST-A1/2) were increased, as were NADPH: Quinone oxidoreductase (NQO), UDP-glucuronosyl transferase (UGT) and beta-glucuronidase activities. There were no statistically significant changes in the potential for bioactivation by three cytochrome P450s. In contrast, acute IQ exposure significantly increased the expression and activity of some cytochrome P450 (CYP1A1 and CYP1A2), UGT and beta-glucuronidase, but significantly decreased glutathione S-transferase expression and activity. There was a non-significant decrease in NQO but no change in CYP3A2 and CYP2E1 activities. CONCLUSIONS: The changes after acute exposure suggest an interaction through the Ah receptor and xenobiotic response element, modified by the glucocorticoid response element. In contrast, the pattern of effects after chronic exposure suggests activation of the antioxidant response element (ARE). Although the acute model is more practically convenient for short-term chemoprevention screening, the data suggest that an entirely new mechanism is being invoked that completely masks effects of the ARE that occur during chronic exposure. There is a danger that chemopreventive strategies developed using acute models may be misleading, since the mechanism is unlikely to occur during human dietary exposures.

Identification of the human liver cytochrome P450 isoenzyme responsible for the 6-methylhydroxylation of the novel anticancer drug 5,6-dimethylxanthenone-4-acetic acid.

In vitro studies were conducted to identify the hepatic cytochrome P450 (CYP) isoenzyme involved in the 6-methylhydroxylation of 5, 6-dimethylxanthenone-4-acetic acid (DMXAA) by using a human liver library (n = 14). The metabolite 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA) was determined by HPLC with fluorescence detection. The metabolite formed in human liver microsomes and by cDNA-expressed CYP isoform was identified by liquid chromatography mass spectrometry as 6-OH-MXAA. In human liver microsomes (n = 14), 6-methylhydroxylation of DMXAA followed monophasic Michaelis-Menten kinetics, with a mean apparent K(m) of 21 +/- 5 microM and V(max) of 0.043 +/- 0.019 nmol/min/mg. An approximate 10-fold interindividual variation in the intrinsic clearance (V(max)/K(m)) of DMXAA 6-methylhydroxylation in human liver microsomes was observed. The involvement of CYP1A2 in DMXAA metabolism by human livers was demonstrated by the following: 1) the potent inhibition of DMXAA metabolism by furafylline (k(inact) = 0.23 +/- 0.04 min(-1), K'(app) = 15.6 +/- 6.7 microM) and alpha-naphthoflavone (K(i) = 0.036 microM), but not by cimetidine, ketoconazole, tolbutamide, quinidine, chlorzoxazone, diethyldithiocarbamate, troleandomycin, and sulfaphenazole; 2) when incubated with human lymphoblastoid cell microsomes containing cDNA-expressed CYP isoenzymes, DMXAA was metabolized only by CYP1A2, with an apparent K(m) of 6.2 +/- 1.5 microM and V(max) of 0.014 +/- 0.001 nmol/min/mg, but not by CYP2A6, CYP2B6, CYP2C9 (Arg(144)), CYP2C19, CYP2D6 (Val(374)), CYP2E1, and CYP3A4; 3) a significant correlation (r = 0.90; P <.001) between 6-methylhydroxylation of DMXAA and 7-ethoxyresorufin O-deethylation; and 4) a significant correlation (r = 0.75; P <.01) between the CYP1A protein level determined by Western blots and DMXAA 6-methylhydroxylation.

Antimutagenic effects of wheat bran diet through modification of xenobiotic metabolising enzymes.

Diets containing wheat bran (WB) protect against cancers of the colon or breast in rats, and may be beneficial in humans. In a previous study of rats treated with the carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), inclusion of 10% wheat bran in the diet led to an apparent reduction in IQ metabolites but not of intact IQ in plasma. In the present study, male Wistar rats were fed diets containing 0, 10 or 20% wheat bran, and effects on xenobiotic metabolising enzymes compared. Wheat bran-supplementation showed differential effects on phase I enzymes, significantly increasing the activity of hepatic cytochrome P450 isozyme CYP3A2, but slightly reducing the activity of CYP1A1/2. The activities of both hepatic phase II detoxification enzymes glutathione-S-transferase and glucuronosyl transferase were also reduced. Western blotting revealed similar effects on expression of the proteins. Interestingly, the expression of xenobiotic metabolising enzymes (XME) in the colon appeared to be modulated independently of hepatic XME. Although the wheat bran-supplemented diet still led to an increased expression of CYP3A, it now slightly increased CYP1A in the colon. However, 20% wheat bran significantly increased the expression of both glutathione transferase isozymes, GST A1 & A2, in the colon. Natures Gold (NG) is a commercial wheat bran derivative which is lower than wheat bran in dietary fibre, but enriched in vitamins, minerals and various phytochemicals. Dietary supplementation with 20% Natures Gold led to similar trends as seen in wheat bran-fed rats, but more potent effects in both hepatic and colonic enzymes. The significance of these changes for activation of carcinogens to mutagenic metabolites was investigated using the Salmonella/mammalian microsome mutagenicity test. The activation of IQ and benzo[a]pyrene, but not cyclophosphamide, to a mutagen by hepatic S9 from wheat bran-fed or Natures Gold-fed rats was significantly reduced compared with S9 from animals on a diet lacking wheat bran. We suggest that modulation of xenobiotic metabolising enzymes may be an important component of cancer protection by wheat bran, and this effect may relate to micronutrients or cancer-protective non-nutrient phytochemicals rather more than to dietary fibre.

A highly sensitive fluorescent microplate method for the determination of UDP-glucuronosyl transferase activity in tissues and placental cell lines.

The fluorescent compound 4-methylumbelliferone (4MU) can be used to detect uridine diphosphate glucuronosyl transferase activity by observing the fall in fluorescence as the compound is converted to 4-methylumbelliferone glucuronide. A microplate assay has been developed that has improved sensitivity and is faster and cheaper than the historical extraction method. Activity is detectable with approximately 10% of the protein required in the extraction method. Absence of extraction and cleanup procedures and the ability to observe reaction rate directly are also of great advantage to the researcher. Michaelis-Menten kinetic data from one healthy female human liver is presented. The extraction method yielded a mean V(max) of 19.9 nmol/min/mg of protein and a mean K(m) of 652.5 microM on 1 day [n = 6, coefficients of variation (CV) 15 and 24%, respectively]. For the microplate method on 1 day, the mean V(max) was 36.21 +/- 1.3 nmol/min/mg of protein (CV = 3.7%), significantly (P <.0001) higher than for the extraction method. The mean K(m), 175. 4 +/- 24.2 microM (CV = 14.5%), was significantly lower (P <.0001) than observed in the extraction method. The assay was performed in replicates of six over 6 days; average intra- and interassay coefficients of variation were 9 and 22% for V(max) and 8 and 35% for K(m), respectively, for the microplate method. The microplate method has also detected activity in the placental trophoblast-derived cell lines JEG-3, JAr, and BeWo (5.5, 4.1, and 2. 6 nmol/min/mg of protein, respectively, at 200 microM 4MU concentration), indicating that placental cells may be capable of glucuronidating 4MU.

Determinaton of two major metabolites of the novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid in hepatic microsomal incubations by high-performance liquid chromatography with fluorescence detection.

High-performance liquid chromatographic methods have been developed and validated for the glucuronidated and oxidative metabolites of the novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA), produced in human liver microsomal incubations. Calibration curves for DMXAA acyl glucuronide (DMXAA-Glu) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA) were constructed over the concentration ranges of 0.25 to 20 and 0.5 to 40 microM, respectively. Assay performance was determined by intra- and inter-day accuracy and precision of quality control (QC) samples. The difference between the theoretical and measured concentration, and the coefficient of variation, were less than 15% at low QC concentrations, and less than 10% at medium and high QC concentrations for both analytes. The methods presented good accuracy, precision and sensitivity for use in kinetic studies of the glucuronidated and oxidative metabolites of DMXAA in human liver microsomes.

Effect of disposition of mannich antimalarial agents on their pharmacology and toxicology.

The use of the antimalarial agent amodiaquine has been curtailed due to drug-induced idiosyncratic reactions. These have been attributed to the formation of a protein-reactive quinoneimine species via oxidation of the 4-aminophenol group. Therefore, the effects of chemical modifications on the disposition of amodiaquine in relation to its metabolism, distribution, and pharmacological activity have been investigated. The inclusion of a group at the C-5' position of amodiaquine reduced or eliminated bioactivation, as determined by glutathione conjugate formation in vivo. This can be seen in two series of C-5'-substituted compounds: the bis-Mannich antimalarial agents, including cycloquine and pyronaridine, and mono-Mannich antimalarial agents containing a 5'-chlorophenyl group (tebuquine and 5'-ClPAQ). Chemical substitution at the C-5' position also resulted in compounds which underwent slower elimination (<5% of the dose excreted into bile and urine, compared with 50% for amodiaquine) and increased levels of accumulation in tissue (10% of the dose in the liver at 48 h compared with 1% with amodiaquine). This may be due to an increase in either the lipophilicity or the basicity of the analogs and may reflect the lack of metabolic clearance for these compounds. The alteration in the disposition following the introduction of the C-5' substituent resulted in an increased duration of antimalarial activity in the mouse compared with that for amodiaquine. While this is desirable in the treatment of malaria, repeated administration for prophylaxis may induce toxicity through accumulation. Therefore, by simple chemical modification it is possible to block the bioactivation of amodiaquine while maintaining and in some cases extending the duration of antimalarial activity.

The effect of 2,2'-substitution on the metabolism and toxicity of dapsone in vitro and in vivo.

The effect of 2,2'-substitution with fluorine, methyl or trifluoromethyl groups on the toxicity, metabolism and pharmacological activity of dapsone has been investigated in vitro and in vivo. There was marked inter-species variation in the bioactivation (N-hydroxylation) of the compounds, as determined by methemoglobin formation. However, the inclusion of fluorine significantly (P<0.01) reduced methemoglobin formation compared with dapsone in all species studied. All three analogs resulted in significantly (P<0.001) less methemoglobinemia than dapsone when given either intraperitoneally or intravenously to the male Wistar rat. Rapid plasma clearance of the analogs through increased lipophilicity and enhanced N-glucuronidation may account for the low toxicity compared with dapsone. Although trifluoromethyl substitution resulted in a loss of activity against respiratory burst in human neutrophils in an in vitro model, all three analogs retained pharmacological activity against Plasmodium berghei malaria in an in vivo mouse model.