Sulphone Metabolites Research Articles

Comparative plasma disposition of fenbendazole, oxfendazole and albendazole in dogs

The plasma disposition of fenbendazole (FBZ), oxfendazole (OFZ) and albendazole (ABZ); and the enantiospecific disposition of OFZ, and ABZSO produced were investigated following an oral administration (50 mg/kg) in dogs. Blood samples were collected from 1 to 120 h post-administration. The plasma samples were analysed by high performance liquid chromatography (HPLC). The plasma concentration of FBZ, OFZ, ABZ and their metabolites were significantly different from each other and depended on the drug administered. The sulphone metabolite (FBZSO 2) of FBZ was not detected in any plasma samples and the parent molecule ABZ did not reach quantifiable concentrations following FBZ and ABZ administration, respectively. OFZ and its sulphone metabolite attained a significantly higher plasma concentration and remained much longer in plasma compared with FBZ and ABZ and their respective metabolites. The maximum plasma concentrations ( C max), area under the concentration time curve (AUC) and mean residence time (MRT) of parent OFZ were more than 30, 68 and 2 times those of FBZ, respectively. The same parameters for ABZSO were also significantly greater than those of FBZSO. The ratio for total AUCs of both the parent drug and the metabolites were 1:42:7 for following FBZ, OFZ and ABZ administration, respectively. The enantiomers were never in racemic proportions and (+) enantiomers of both OFZ and ABZSO were predominant in plasma. The AUC of (+) enantiomers of OFZ and ABZSO was, respectively more than three and seven times larger than that of (−) enantiomers of both molecules. It is concluded that the plasma concentration of OFZ was substantially greater compared with FBZ and ABZ. The data on the pharmacokinetic profile of OFZ presented here may contribute to evaluate its potential as an anthelmintic drug for parasite control in dogs.

Plasma Disposition and Faecal Excretion of Netobimin Metabolites and Enantiospecific Disposition of Albendazole Sulphoxide Produced in Ewes

Netobimin (NTB) was administered orally to ewes at 20 mg/kg bodyweight. Blood and faecal samples were collected from 1 to 120 h post-treatment and analysed by high-performance liquid chromatography (HPLC). Using a chiral phase-based HPLC, plasma disposition of albendazole sulphoxide (ABZSO) enantiomers produced was also determined. Neither NTB nor albendazole (ABZ) was present and only ABZSO and albendazole sulphone (ABZSO(2)) metabolites were detected in the plasma samples. Maximum plasma concentrations (C(max)) of ABZSO (4.1 +/- 0.7 microg/ml) and ABZSO(2) (1.1 +/- 0.4 microg/ml) were detected at (t(max)) 14.7 and 23.8 h, respectively following oral administration of netobimin. The area under the curve (AUC) of ABZSO (103.8 +/- 22.8 (microg h)/ml) was significantly higher than that ABZSO(2)(26.3 +/- 10.1 (microg h)/ml) (p < 0.01). (-)-ABZSO and (+)-ABZSO enantiomers were never in racemate proportions in plasma. The AUC of (+)-ABZSO (87.8 +/- 20.3 (microg h)/ml) was almost 6 times larger than that of (-)-ABZSO (15.5 +/- 5.1 (microg h)/ml) (p < 0.001). Netobimin was not detected, and ABZ was predominant and its AUC was significantly higher than that of ABZSO and ABZSO(2), following NTB administration in faecal samples (p > 0.01). Unlike in the plasma samples, the proportions of the enantiomers of ABZSO were close to racemic and the ratio of the faecal AUC of (-)-ABZSO (172.22 +/- 57.6 (microg h)/g) and (+)-ABZSO (187.19 +/- 63.4 (microg h)/g) was 0.92. It is concluded that NTB is completely converted to ABZ by the gastrointestinal flora and absorbed ABZ is completely metabolized to its sulphoxide and sulphone metabolites by first-pass effects. The specific behaviour of the two enantiomers probably reflects different enantioselectivity of the enzymatic systems of the liver that are responsible for sulphoxidation and sulphonation of ABZ.

Assessment of the main metabolism pathways for the flukicidal compound triclabendazole in sheep

Triclabendazole (TCBZ) is an halogenated benzimidazole (BZD) compound worldwide used to control immature and adult stages of the liver fluke Fasciola hepatica. The purpose of this investigation was to characterize in vitro the patterns of hepatic and ruminal biotransformation of TCBZ and its metabolites in sheep. TCBZ parent drug was metabolized into its sulphoxide (TCBZSO), sulphone (TCBZSO2) and hydroxy derivatives by sheep liver microsomes. The same microsomal fraction was also able to oxidize TCBZSO into TCBZSO2 and hydroxy-TCBZSO (HO-TCBZSO). TCBZ sulphoxidation was significantly (P < 0.001) inhibited after inactivation of the flavin-monooxygenase (FMO) system (77% inhibition) as well as in the presence of the FMO substrate methimazole (MTZ) (71% inhibition). TCBZ sulphoxidative metabolism was also reduced (24% inhibition, P < 0.05) by the cytochrome P450 inhibitor piperonyl butoxide (PB). The rate of TCBZSO conversion into TCBZSO2 was also significantly inhibited by PB (55% inhibition), MTZ (52% inhibition) and also following FMO inactivation (58% inhibition). The data reported here indicate that the FMO is the main enzymatic pathway involved in TCBZ sulphoxidation (ratio FMO/P450 = 3.83 +/- 1.63), although both enzymatic systems participate in a similar proportion in the sulphonation of TCBZSO to form the sulphone metabolite (ratio FMO/P450 = 1.31 +/- 0.23). Additionally, ketoconazole (KTZ) did not affect TCBZ sulphoxidation but decreased (66% inhibition, P < 0.05) the formation of TCBZSO2. Similarly, inhibition of TCBZSO2 production was observed after incubation of TCBZSO in the presence of KTZ and erythromycin (ETM). Conversely, thiabendazole (TBZ) and fenbendazole (FBZ) did not affect the oxidative metabolism of both incubated substrates. The sheep ruminal microflora was able to reduce the sulphoxide (TCBZSO) into the parent thioether (TCBZ). The ruminal sulphoreduction of the HO-TCBZSO derivative into HO-TCBZ was also demonstrated. The rate of sulphoreduction of HO-TCBZSO was significantly (P < 0.05) higher than that observed for TCBZSO. The metabolic approach tested here contributes to the identification of the different pathways involved in drug biotransformation in ruminant species. These findings on the pattern of hepatic and ruminal biotransformation of TCBZ and its main metabolites are a further contribution to the understanding of the pharmacological properties of widely used anthelmintics in ruminants. Comprehension of TCBZ metabolism is critical to optimize its flukicidal activity.

A phase I/II dose‐escalation study of exisulind and docetaxel in patients with hormone‐refractory prostate cancer

To determine the safety and efficacy, in a dose-escalation study, of exisulind (an oral sulphone metabolite of sulindac thought to induce apoptosis in malignant cells by inhibiting cGMP-phosphodiesterase) combined with docetaxel in men with hormone-refractory prostate cancer (HRPC), as pre-clinical studies suggested activity against prostate cancer and synergy with cytotoxic agents. Thirty-four patients with HRPC were treated with oral exisulind twice daily for 21-day cycles and intravenous docetaxel given for 1 h on the first day of each cycle. Three dose levels were assessed, combining exisulind 150 and 250 mg twice daily with docetaxel at 60 or 75 mg/m2. Toxicity was then evaluated using standard criteria. The recommended phase II dose was determined to be exisulind 250 mg and docetaxel 60 mg/m2, with escalation to 75 mg/m2 after cycle 1, as tolerated. The most common grade 3-4 toxicities among all patients were neutropenia (56%), infection (24%) and hyperglycaemia (18%). Twelve of 32 evaluable patients (38%, 95% confidence interval, CI, 23-55%) had a decline in PSA by at least half. Only four of 17 evaluable patients (95% CI, 1-47%) treated at the phase II dose level had such a decline in PSA. The median (95% CI) overall survival of all patients was 16 (12.9-19.7) months and median progression-free survival 4.7 (2.7-5.2) months. The combination of exisulind and docetaxel was tolerable in patients with HRPC. The PSA response rates do not suggest an improvement over historical data with single-agent docetaxel in this population.

Plasma disposition and faecal excretion of oxfendazole, fenbendazole and albendazole following oral administration to donkeys

Fenbendazole (FBZ), oxfendazole (fenbendazole sulphoxide, FBZSO), and albendazole (ABZ) were administered orally to donkeys at 10 mg/kg bodyweight. Blood and faecal samples were collected from 1 to 120 h post-treatment. The plasma and faecal samples were analysed by high performance liquid chromatography (HPLC). The parent molecule and its sulphoxide and sulphone (FBZSO 2) metabolites did not reach detectable concentrations in any plasma samples following FBZ administration. ABZ was also not detected in any plasma samples, but its sulphoxide and sulphone metabolites were detected, demonstrating that ABZ was completely metabolised by first-pass mechanisms in donkeys. Maximum plasma concentrations ( C max) of FBZSO (0.49 μg/mL) and FBZSO 2 (0.60 μg/mL) were detected at ( t max) 5.67 and 8.00 h, respectively, following administration of FBZSO. The area under the curve (AUC) of the sulphone metabolite (10.33 μg h/mL) was significantly higher than that of the parent drug FBZSO (5.17 μg h/mL). C max of albendazole sulphoxide (ABZSO) (0.08 g/mL) and albendazole sulphone (ABZSO 2) (0.04 μg/mL) were obtained at 5.71 and 8.00 h, respectively, following ABZ administration. The AUC of the sulphoxide metabolite (0.84 μg h/mL) of ABZ was significantly higher than that of the sulphone metabolite (0.50 μg h/mL). The highest dry-faecal concentrations of parent molecules were detected at 32, 34 and 30 h for FBZSO, FBZ and ABZ, respectively. The sulphide metabolite was significantly higher than the parent molecule after FBZSO administration. The parent molecule was predominant in the faecal samples following FBZ administration. After ABZ administration, the parent molecule was significantly metabolised, probably by gastrointestinal microflora, to its sulphoxide metabolite (ABZSO) that showed a similar excretion profile to the parent molecule in the faecal samples. The AUC of the parent FBZ was significantly higher than that of FBZSO and ABZ in faeces. It is concluded that the plasma concentration of FBZSO was significantly higher than that of FBZ and ABZ. Although ABZ is not licensed for use in Equidae, its metabolites presented a greater plasma kinetic profile than FBZ which is licensed for use in horses. A higher metabolic capacity, first-pass effects and lower absorption of benzimidazoles in donkeys decrease bioavailability and efficacy compared to ruminants.

Accumulation and tissue distribution of selected polychlorinated biphenyl congeners in chickens

During one to three consecutive periods of 2 weeks, broiler chickens ( n = 108) received test diets to which different amounts of PCBs (7 congeners) were added. The relationship between exposure time and accumulation of individual congeners in different chicken tissues, such as breast, thigh and abdominal fat tissue, was observed. In all tissues, the vast majority of the PCB accumulation occurred during the first 2 weeks of exposure. After that, PCB concentrations only increased in the abdominal fat tissue of the animals. The individual PCBs were distributed differently in the various tissues. While CBs 28, 118, 138, 153 and 180 accumulated in the chickens, CBs 52 and 101 were metabolized, but no methyl sulphone metabolites of these congeners could be detected. Our results provide information on the absorption, tissue distribution and biotransformation of the individual PCB congeners and confirm the structure–activity relationships for metabolism of PCBs in birds, which are different from those in fish or mammalian species.

Dose-dependent activity of albendazole against benzimidazole-resistant nematodes in sheep: relationship between pharmacokinetics and efficacy

The relationship between the pharmacokinetic behaviour and the anthelmintic efficacy of albendazole (ABZ) against benzimidazole (BZD)-resistant nematodes was studied in sheep. A micronized ABZ suspension was orally administered at two different dose levels to sheep naturally infected with BZD-resistant gastrointestinal (GI) nematodes. The experimental animals were allocated into the following groups ( n=8): (a) untreated control; (b) orally treated with ABZ at 3.8 mg/kg b.w.; and (c) orally treated with ABZ at 7.5 mg/kg b.w. Plasma samples were obtained serially over 72 h post-treatment from both treated groups and analysed by HPLC to measure the concentrations of ABZ and its sulphoxide (ABZSO) and sulphone (ABZSO 2) metabolites. Faecal egg counts were performed prior to treatment and at the necropsy day. All experimental animals were sacrificed 10 days after treatment to perform GI worm counts. While ABZ parent drug was not recovered in the bloodstream, ABZSO and ABZSO 2 were the molecules found in plasma. ABZSO was the metabolite measured at the highest concentrations in the bloodstream for up to 36 (treatment at 3.8 mg/kg) or 60 h (treatment at 7.5 mg/kg) post-administration. There was a proportional relationship between the administered ABZ dose and the measured plasma concentrations of both ABZ metabolites. Over a 100% increment on the plasma AUC values for the anthelmintically active ABZSO metabolite was observed at the 7.5 mg/kg compared to the 3.8 mg/kg treatment. The low efficacy patterns (<24%) observed against the GI nematodes investigated indicate a high level of resistance to ABZ given at 3.8 mg/kg an efficacious therapeutic dose rate recommended in some countries. However, the higher and prolonged plasma drug concentration measured after the 7.5 mg/kg treatment resulted in an improved efficacy pattern (estimated by both faecal egg and adult worm counts) against most of the GI nematodes studied compared to that obtained at the lower dose rate. A direct relationship between drug pharmacokinetic behaviour and anthelmintic efficacy against BZD-resistant nematodes in sheep was shown in the current work, although individual variation precluded the observation of statistically significant differences in worm counts.

Triclabendazole biotransformation and comparative diffusion of the parent drug and its oxidized metabolites into Fasciola hepatica

1. Triclabendazole (TCBZ) is an halogenated trematodicidal benzimidazole compound extensively used in veterinary medicine. It is active against immature and adult stages of the liver fluke Fasciola hepatica.2. Free and conjugated TCBZ metabolites have been identified in the bile of treated sheep.3. The experimental aims were to characterize the in vitro patterns of TCBZ biotransformation both in the animal host (sheep liver microsomes) and target parasite (F. hepatica microsomal preparation); and to compare the ex vivo diffusion of TCBZ parent drug and its oxidized metabolites (TCBZ sulphoxide [TCBZSO], TCBZ sulphone [TCBZSO2], and TCBZ-hydroxy derivatives) into F. hepatica. Additionally, the octanol–water partition coefficients for TCBZ and all its metabolites were estimated as an indicator of the relationship between drug lipophilicity and diffusion into the target parasite.4. Drug/metabolites concentrations were quantified by HPLC after sample clean up and a solvent-mediated chemical extraction.5. Sheep liver microsomes metabolized TCBZ into its sulphoxide and sulphone metabolites after 30 min of incubation. The rate of TCBZ sulphoxidation in the liver was significantly greater (p<0.01) than that observed for the sulphonation of TCBZSO.6. The trematode parasite oxidized TCBZ into its sulphoxide metabolite after 60 min of incubation at a metabolic rate of 0.09 nmol min−1 mg protein−1.7. TCBZ and all its oxidized metabolic products were recovered from F. hepatica as early as 15 min after their ex vivo incubation in a Kreb's Ringer Tris buffer. However, the diffusion of the hydroxy-derivatives into the fluke was lower than that observed for TCBZ, TCBZSO and TCBZSO2. There was a high correlation (r = 0.82) between drug lipophilicity (expressed as octanol–water partition coefficients) and drug availability measured within the parasite.8. Unlike the uptake pattern previously observed for albendazole, the parent TCBZ and its sulphoxide and sulphone metabolites showed a similar ability to penetrate into the trematode parasite.9. Understanding the relationship between TCBZ metabolism, the relative pharmacological potency of its metabolic products and their ability to reach the target parasite may be critical to optimize its flukicidal activity, particularly when TCBZ resistant flukes have been already isolated in the field.

Evaluation of Fast Disintegrating Lansoprazole Tablet in Human Subjects

Fast disintegrating lansoprazole tablet (LFDT) has been developed as a multiple unit formulation and evaluated using human subjects as compared to the conventional lansoprazole (LPZ) capsule containing enteric coated granules. Twelve healthy male volunteers, who were confirmed as extensive metabolizers (EMs) based on the plasma levels of LPZ sulphone metabolite, were enrolled into the study and genotype of CYP2C19 was confirmed. They kept 30 mg LFDT in their mouths for 2 min and the saliva was recovered without swallow. Eight subjects did not show LPZ in their serum after intake. Although LPZ was detected in 4 subjects' serum, their concentrations were less than 5 ng/mL. LPZ was thought to be not absorbed from the oral cavity. LFDT was orally administered to 12 healthy male EMs at two doses, 15 mg and 30 mg, and serum LPZ concentrations were measured. The mean C(max) and AUC(0-24) were 474.1+/-254.0 ng/mL and 1105.3+/-1101.4 ng.h/mL (15 mg) and 992.8+/-384.3 ng/mL and 2216.5+/-1270.1 ng.h/mL (30 mg). By comparing to that obtained after oral administration of the same doses of LPZ capsule, serum LPZ concentration vs. time curve was almost the same level, i.e., C(max) and AUC(0-24) did not have significant differences. From these results, LFDT has been shown to be equivalent to LPZ capsule and will show the same acid suppressing effects in the clinical situation.

Aspects of the pharmacokinetics of albendazole sulphoxide in sheep.

The plasma disposition kinetics of albendazole sulphoxide (ABZSO), ((+)ABZSO and (-)ABZSO) and its sulphone metabolite (ABZSO2) were investigated in adult sheep. Six Corriedale sheep received albendazole sulphoxide by intravenous injection at 5 mg/kg live weight. Jugular blood samples were taken serially for 72 h and the plasma was analysed by high-performance liquid chromatography (HPLC) for albendazole (ABZ), ABZ sulphoxide (ABZSO) and albendazole sulphone (ABZSO2). Albendazole was not detected in the plasma at any time after the treatment, ABZSO and ABZSO2 being the main metabolites detected between 10 min and 48 h after treatment. A biexponential plasma concentration versus time curve was observed for both ABZSO and ABZSO2 following the intravenous treatment. The plasma AUC values for ABZSO and ABZSO2 were 52.0 and 10.8 (microg x h)/ml, respectively. The ABZSO2 metabolite was measurable in plasma between 10 min and 48 h after administration of ABZSO, reaching a peak concentration of 0.38 microg/ml at 7.7 h after treatment. Using a chiral phase-based HPLC method, a biexponential plasma concentration versus time curve was observed for both ABZSO enantiomers. The total body clearance was higher for the (-) than for the (+) enantiomer, the values being 270.6 and 147.75 (ml/h)/kg, respectively. The elimination half-life of the (-) enantiomer was shorter than that of the (+) enantiomer, the values being 4.31 and 8.33 h, respectively. The enantiomeric ratio (+)ABZSO/( )ABZSO at t0 was close to unity. However, the ratio in the plasma increased with time.

Fenbendazole pharmacokinetics, metabolism, and potentiation in horses.

The present study was designed to describe the pharmacokinetics and fecal excretion of fenbendazole (FBZ) and fenbendazole sulphoxide (FBZSO) and their metabolites in horses, to investigate the effects which concurrent feeding has on the absorption and pharmacokinetics of FBZ, and to determine the effect of coadministration of the metabolic inhibitor piperonyl-butoxide on the in vivo pharmacokinetics and in vitro liver microsomal metabolism of sulfide and sulfoxide benzimidazoles. The effect of piperonyl-butoxide on the enantiomeric genesis of the sulfoxide moiety was also investigated. Following administration of FBZSO and FBZ, the fenbendazole sulphone metabolite predominated in plasma, and the C(max) and area under the plasma curve (AUC) values for each moiety were larger (P < 0.001) following FBZSO than FBZ. In feces the administered parent molecule predominated. The combined AUC for active benzimidazole moieties following oral administration of FBZ (10 mg/kg) in horses was almost 4 times as high in unfed horses (2.19 microg x h/ml) than in fed horses (0.59 microg x h/ml), and coadministration of piperonyl-butoxide significantly increased the AUC and C(max) of active moieties following intravenous administration of FBZSO and oral administration of FBZ. When FBZSO was administered i.v. as a racemate, the first enantiomer of oxfendazole (FBZSO-1) predominated in plasma, however, following coadministration with piperonyl-butoxide, the second enantiomer of oxfendazole (FBZSO-2) predominated for 10 h. Piperonyl-butoxide significantly reduced the oxidative metabolism of FBZSO and FBZ in equine liver microsomes and altered the ratio of enantiomers FBZSO-1/FBZSO-2 from >4:1 to 1:1. It is concluded that in horses efficacy of FBZSO and FBZ could be improved by administration to unfed animals and coadministration with piperonyl-butoxide.

Metabolite of 2,2′,4′,5-tetrabromobiphenyl, 3-methylsulphonyl-2,2′,4′,5-tetrabromobiphenyl, a potent inducer of CYP2B1/2 in rat

1. 3-Methylsulphonyl- and 4-methylsulphonyl-2,2',4',5-tetrabromobiphenyls (3-MeSO2- and 4-MeSO2-TetraBrBs) were detected in the liver, lung, kidney, adipose tissue and faeces of the 2,2',4',5-tetrabromobiphenyl (TetraBrB)-dosed rat. 2. The administration of 0.05-2.0 µmol kg−1 doses of 3-MeSO2-TetraBrB produced corrresponding increases in the hepatic concentration of the methyl sulphone metabolite, corresponding increases in the content of total cytochrome P450, and corresponding increases in the activities of 7-benzyloxy-, 7-ethoxy- and 7-pentoxyresorufin O-dealkylases. The inducing effects of the 3-MeSO2-TetraBrB (0.2 µmol kg−1), both on the content of total P450 and on the activities of the three alkoxyresorufin O-dealkylases, were higher than that of the parent TetraBrB (342 µmol kg−1). 3. The major phenobarbital (PB)-inducible forms of P450, CYP2B1, CYP2B2, CYP3A2 and CYP2C6, were substantially induced by 3-MeSO2-TetraBrB, but CYP1A1 and CYP1A2 were not. On the other hand, the activities of drug-metabolizing enzymes and the four PB-inducible forms of P450 were unchanged by 4-MeSO2-TetraBrB treatment. 4. The induction profiles of these enzymes and P450 forms in rat treated with 3-MeSO2-TetraBrB were similar to those treated with PB. 5. The inducing ability of 3-MeSO2-TetraBrB (0.5 µmol kg−1) both on the activities of the three alkoxyresorufin O-dealkylases and on the contents of four PB-inducible forms of P450 was roughly equal to that of PB (431 µmol kg−1 twice at a 24-h interval) or 3-MeSO2-2,2',4',5-tetrachlorobiphenyl (1 µmol kg−1). It is noteworthy that the effects of 3-MeSO2-TetraBrB on the drug-metabolizing enzymes CYP2B1 and CYP2B2 were several thousand-fold higher than those of parent TetraBrB, while the effect of its isomeric 4-MeSO2-TetraBrB were not. 6. The extent of hepatic accumulation of the 3-MeSO2 metabolite after the administration of TetraBrB (342 µmol kg−1) was almost the same as that after the administration of 3-MeSO2-TetraBrB (0.1-0.2 µmol kg−1). The relationship between the hepatic concentration of the 3-MeSO2 metabolite and the extent of enzyme induction after the administration of TetraBrB or 3-MeSO2-TetraBrB suggests that 3-MeSO2-TetraBrB plays an important role in the induction of microsomal drug-metabolizing enzymes by TetraBrB.

HPLC analysis of 4-chlorophenyl methyl sulphide and diphenyl sulphide and their corresponding sulphoxides and sulphones in rat liver microsomes.

Simple high performance liquid chromatography (HPLC) methods for the analysis of 4-chlorophenyl methyl sulphide (CPMS), diphenyl sulphide (DPS) and their corresponding sulphoxide and sulphone metabolites in rat liver microsomes are described. The assay methods are based on a reversed phase HPLC column (Spherisorb(R) 5 ODS, 15 x 0.46 cm) using a mixture of water and tetrahydrofuran (THF) as mobile phase at a flow rate of 0.5 ml/min and ultraviolet detection at 260 nm. The compounds were extracted into diethyl ether (2 x 5 ml) from rat liver microsomal incubation mixture (2 ml) and the recoveries were more than 80%. The calibration curves for determining the sulphoxide and sulphone of CPMS or DPS were linear (r > or =0.995) in the range of 0-50 microg/ml and the assays were reproducible with low inter- and intra-assay variation of less than 13.5%. The lower limit of quantitation (LOQ) was 0.1 microg/ml for CPMSO and 0.025 microg/ml for CPMSO(2), diphenyl sulphoxide (DPSO) and diphenyl sulphone (DPSO(2)). The HPLC methods were successfully applied to measure enzymically formed CPMSO, CPMSO(2), DPSO and DPSO(2) in rat liver microsomes and to characterise the Michaelis-Menten kinetics associated with the metabolism of CPMS and DPS and their corresponding sulphoxides. About 20% of the initial CPMS (0.5 mM) concentration in the incubation was converted to the sulphoxide although the sulphone was not detected under these optimum incubation conditions. Similarly, about 15-20% of DPS was converted to the sulphoxide while less than 0.1% of DPS was converted to DPSO(2). Eadie-Hofstee plot of CPMS sulphoxidation was biphasic. This suggests that the sulphoxidation of CPMS is a consequence of at least two enzyme systems, one characterized by low affinity and high capacity (K(m)=0.1 mM; V(max)=2.1 nmoles/mg protein/min) and the other by high affinity and low capacity (K(m)=0.05 mM; V(max)=1.5 nmoles/mg protein/min). On the other hand, the Eadie-Hofstee plot of DPS sulphoxidation was monophasic with an apparent V(max) and K(m) of 1.8 nmoles/mg protein/min and 0.036 mM, respectively.

Monitoring antioxidant effects using methaemoglobin formation in diabetic erythrocytes †

Deficiencies in the reducing power of diabetic erythrocytes can be studied using the sulphone metabolite, monoacetyl dapsone hydroxylamine (MADDS-NHOH) to generate methaemoglobin, which is a form of oxidative stress. The effects of three antioxidants, dyhydrolipoic acid (DHLA), lipoic acid and ascorbate were compared using this method in erythrocytes of type 1 diabetic patients. During 2-hour incubations, DHLA and lipoic acid significantly reduced MADDS-NHOH-mediated methaemoglobin formation to the same extent in diabetic and non-diabetic erythrocytes. In contrast, ascorbate markedly increased MADDS-NHOH-mediated methaemoglobin formation at all time points up to 2 hours for diabetic cells and 15—60 minutes in non-diabetic erythrocytes. Ascorbate abolished the deficiency in methaemoglobin sensitivity normally seen in diabetic erythrocytes compared with non-diabetic erythrocytes. During a 1-hour pre-incubation period, neither DHLA, lipoic acid nor ascorbate significantly altered total thiol concentrations. After a 2-hour incubation with MADDS-NHOH, thiol concentrations did not significantly change in the non-diabetic erythrocytes, but they fell significantly in the diabetic cells. Diabetic and non-diabetic erythrocytes pre-incubated with ascorbate, DHLA and lipoic acid showed no changes in thiol levels in the presence of MADDS-NHOH. In summary, ascorbate, DHLA and lipoic acid showed contrasting effects on methaemoglobin generation, although they each abolished the diabetic erythrocytic deficit in total thiol status caused by hydroxylamine-mediated methaemoglobin formation. This work provides evidence for the potential future use of antioxidant supplements in diabetic management.

The effects of a synthetic diet on the pharmacokinetics of ethyl methyl sulphide and its sulphoxide and sulphone metabolites in rats.

Ethyl methyl sulphide (EMS) is a simple dialkyl sulphide, which occurs naturally and forms part structures of more complex drug molecules. EMS is oxidized to the corresponding sulphoxide (EMSO) and sulphone (EMSO2) derivatives both in vitro and in vivo. Two distinct enzymatic pathways appear to be involved in this sulphoxidation process; the flavin-containing monooxygenase (FMO) is largely responsible for the S-oxidation of EMS to its sulphoxide while both cytochrome P450 and FMO are involved in the further oxidation of the sulphoxide to the sulphone. The pharmacokinetics of EMS and its sulphoxide and sulphone metabolites were examined in male wistar rats placed on normal rat chow and those placed on a synthetic diet. Blood levels of EMS were analysed by a sensitive headspace gas chromatographic assay. A separate gas chromatographic assay was developed to monitor the blood levels of EMSO and EMSO2. The pharmacokinetics of EMS in control rats were linear from 10 to 40 mg/kg dose range. The blood concentration-time profile of EMS declined monoexponentially. EMS was rapidly eliminated from rat blood with a terminal half-life of 0.14 h and was not dytectable 1 h after administration. Following intravenous administration of EMSO (5 mg/kg), the blood concentration-time profile of EMSO declined with a terminal half-life (t 1/2) of 1.46 h, about ten times longer than that of the parent sulphide. After administration of EMSO2 (15 mg/kg), the sulphone was metabolically stable and was eliminated very slowly from the blood. The in vivo disposition of EMS and EMSO were clearly altered in rats maintained on a synthetic diet following administration of EMS or EMSO. The pharmacokinetic data were consistent with a diminished drug oxidising capacity in rats placed on the synthetic diet and could serve as a useful probe for monitoring the regulation of FMO in animals.

PCB methyl sulphones in rat liver after exposure to PCB (Clophen A50): analysis and radiosynthesis of selected methylsulphonyl-PCBs

1. The hepatic metabolism of polychlorinated biphenyls (PCBs) and formation of PCB methyl sulphone metabolites (MeSO2-PCBs) was determined in the male Sprague-Dawley rat 1, 2, 4 or 8 weeks after dosage with Clophen A50 (a commercial PCB mixture). 2. The total concentration of the PCB congeners examined (ΣPCB) decreased during the experimental period, from 40 μg g−1 lipid (l.w.) after 1 week to 4 μg g−1 l.w. after 8 weeks. A 50% decrease of PCB in the liver were estimated to be 28, 13 and 11 days for 2,2′,4,4′,5,5′-hexachlorobiphenyl (CB153), 2,2′,3,3′,4,4′,5-heptaCB (CB170) and 2,2′3,4′,5,5′,6-heptaCB (CB187), respectively. 3. The total MeSO2-PCB (ΣMeSO2-PCB) concentration increased from 800 to 1020 ng g−1 l.w. during the first 2 weeks of treatment and thereafter a decrease to 120 ng g−1 l.w. after 8 weeks. The relative concentration of both 3′-MeSO2-2,2′,4,4′,5-pentaCB (3′-MeSO2-CB101) and 3′-MeSO2-2,2′,3,4,5′-pentaCB (3′-MeSO2-CB87) in rat liver showed significant increases during the 8 weeks. In contrast, the relative concentrations of 4-MeSO2-2,4′,5,5-tetraCB (4-MeSO2-CB64), 3-MeSO2-2,3′,4′,5-tetraCB (3-MeSO2- CB70) and 4-MeSO2-2,2′,3,4,5′,6′-hexaCB (4′-MeSO2-CB132) decreased significantly. 4. A route for the synthesis of radiolabelled MeSO2-PCBs was developed employing the `Pummerer reaction’ to convert methylthio-PCBs (MeS-PCBs) to the corresponding mercapto-PCBs (SH-PCB). The SH-PCBs were methylated with radiolabelled methyl iodide and the resulting sulphides oxidized to yield the corresponding MeSO2-PCBs. Using this approach, 4-[14C]-MeSO2-2′,4′,5,5′,6-hexaCB (3-[14C]-MeSO2-CB149), 4-[14C]-MeSO2-2,2′,4′,5,5′,6-hexaCB (4-[14C]-MeSO2-CB149), 3′-[14C]-MeSO2-CB101,4′-[14C]-MeSO2-2,2′,4,5,5′-pentaCB (4′-[14C]-MeSO2-CB101) and 4′-[3C]-MeSO2-CB101 were synthesized in quantitative radiochemical yields.

Pharmacokinetic studies with esomeprazole, the (S)-isomer of omeprazole.

This article reviews the pharmacokinetics of esomeprazole, the (S)-isomer of the proton pump inhibitor (PPI) omeprazole. Esomeprazole is the first single isomer PPI developed for the treatment of patients with acid-related diseases. In vitro experiments in human liver microsomes demonstrated that the formation of the hydroxy and 5-O-desmethyl metabolites of esomeprazole is via cytochrome P450 (CYP) 2C19, whereas that of the sulphone metabolite is via CYP3A4. The formation rate of the hydroxy metabolite from esomeprazole is lower than for (R)-omeprazole, but that of the 2 other metabolites is higher, demonstrating stereoselective metabolism. The sum of the intrinsic clearances of all 3 metabo- lites for esomeprazole was one-third of that for (R)-omeprazole, suggesting lower clearance of esomeprazole in vivo. In vivo investigations demonstrated that esomeprazole is chirally stable after administration. Esomeprazole is 97% bound to plasma proteins. In normal (extensive) metabolisers with regard to CYP2C19, esomeprazole is metabolised more slowly than omeprazole, resulting in a higher area under the concentration-time curve (AUC) after administration of the same dose. This is more pronounced after repeated administration rather than after a single dose. In poor metabolisers, the AUC is lower for esomeprazole than for omeprazole, contributing to less overall interindividual variability for esomeprazole than for omeprazole. In general, esomeprazole and omeprazole are subject to the same metabolic transformations. Almost complete recoveries were reported and the ratio between urinary and faecal excretion is about 4:1 for both compounds. The dose-dependent increase in AUC of esomeprazole with repeated administration results from a combination of decreased first-pass elimination and decreased systemic clearance. Patients with gastro-oesophageal reflux disease exhibit a pharmacokinetic pattern similar to that in healthy individuals, whereas elderly individuals exhibited a slightly lower metabolism rate. Patients with a severe deficit in their liver function had a lower rate of metabolism, as would be expected, whereas those with mild to moderate liver disease did not exhibit any alteration in the pharmacokinetics. The pharmacokinetics of esomeprazole in individuals with impaired renal function is unlikely to differ from that in healthy individuals. A slight sex difference in the pharmacokinetics of esomeprazole was demonstrated in that the AUC and peak plasma drug concentration were slightly, but not statistically significantly, higher in females than in males.

Simultaneous determination of triclabendazole and its sulphoxide and sulphone metabolites in bovine milk by high-performance liquid chromatography

A simple method has been developed for the simultaneous determination of triclabendazole and its metabolites (sulphoxide and sulphone) in bovine milk by reversed-phase high-performance liquid chromatography (HPLC). A milk sample was homogenized with sodium sulfate anhydrous and acetonitrile, and centrifuged. The supernatant was isolated, rinsed with n-hexane saturated with acetonitrile, and evaporated. The residue was dissolved with 0.1 M potassium dihydrogenphosphate, and 0.1 M sodium hydrogencarbonate, and then cleaned up on a Bond Elut C 18 cartridge. The three compounds were separated on a Capcell Pak C 18 UG 120 (5 μm, 150×4.6 mm I.D.) column and determined by UV detection at 295 nm. The mobile phase was acetonitrile–0.05 M ammonium acetate (50:50), and the flow-rate was 0.8 ml/min at 40°C. The mean recoveries ( n=4) were 89.1–95.0% with a relative standard deviation of 1.1–2.6%. The detection limits were 0.004–0.006 μg/g in milk. The proposed method was used to monitor raw milk samples for the market, and applied to the analysis of milk samples from 10 cows which had been administered with triclabendazole to control the liver fluke. The confirmation of the triclabendazole and its metabolites in the above milk sample has been achieved by electrospray LC–MS for the first time.

PCBs, DDTs and methyl sulphone metabolites in various tissues of harbour porpoises from Swedish waters

Aryl methyl sulphones of polychlorinated biphenyls (PCBs) and 3- p,p′-DDE (MeSO 2-PCBs and 3-MeSO 2- p,p′-DDE), PCBs and ΣDDTs were analysed in five different tissues (blubber, nuchal fat, liver, muscle, brain) of adult male harbour porpoises from the west coast of Sweden. Two different methods for MeSO 2-PCBs and 3-MeSO 2- p,p′-DDE determination were used, gas chromatography–mass spectrometry and gas chromatography–atomic emission detection. Highest concentrations of ΣMeSO 2-PCBs were found in liver (0.15–0.49 μg/g lipid wt.), which corresponded to 2.0–2.8% of the ΣPCBs concentrations. Blubber and nuchal fat showed ΣMeSO 2-PCB concentrations that were three to five times lower than those in liver. Concentrations of 3-MeSO 2- p,p′-DDE in liver, muscle and brain corresponded to 0.26–4.6% of the p,p′-DDE concentrations, while in blubber and nuchal fat, 3-MeSO 2- p,p′-DDE constituted 0.033–0.21% of p,p′-DDE. The different tissues analysed showed similar levels (lipid wt.) of ΣPCBs and ΣDDTs, except for brain that had almost 10 times lower levels compared to the other tissues. Using the ΣPCBs/ΣMeSO 2-PCBs ratio to estimate MeSO 2-PCB formation and secondary metabolism capacity, the harbour porpoise showed a relatively low capacity of MeSO 2-PCB formation compared to other small toothed whales and seals. Blubber sampled from five different anatomical locations showed that concentrations of contaminants may be unevenly distributed in blubber in certain animals. This should be taken into account when choosing sampling sites on the porpoise.

Synthesis and Activity of Fluorinated Derivatives of Sulindac Sulphide and Sulindac Sulphone

The synthesis of fluorinated derivatives of the sulphide and sulphone metabolites of sulindac, a non-steroidal anti-inflammatory agent with chemopreventative activity, is reported. The key step in the synthesis is a Pummerer-rearrangement of the parent sulindac sulphoxide using (diethylamino)sulphur trifluoride as an activating agent and as a source of the fluoride nucleophile. The reaction leads to the formation of the 4-fluoromethylthio and 4-fluoromethylsulphonyl derivatives of sulindac (4a and 4b, respectively). Sulindac sulphide is 2.5 times more potent as a COX-1 inhibitor compared with its fluorinated counterpart 4a. The sulphones were inactive as COX-1 inhibitors, and none of the compounds inhibited COX-2 concentrations up to 0.1 mM. Cytotoxicity assays showed that 4a and 4b were as cytotoxic as sulindac sulphide and sulindac sulphone on 3719 colorectal carcinoma cell lines. Compound 4b was the most potent compound on RCA cells with an IC50 of 95 μM (sulindac sulphide 140μM, sulindac sulphone 175 μM). Fluorinated sulindac derivatives warrant further investigation since we have shown that cytotoxic activity can be retained or even increased independently from COX-inhibitory properties. This could help minimize the undesired side-effects associated with chronic sulindac administration.