Effects of liquorice on pharmacokinetics of aconitine in rats

Abstract

1. Aconite alkaloids are the main bioactive ingredients existing in Aconitum, for instance aconitine (AC), which exhibit potent analgesic, antirheumatic and other pharmacological effects. In this study, effects of long-term treatment with liquorice on pharmacokinetics of AC in rats were investigated.

2. Pharmacokinetics of AC after oral administration of AC at 1.5?mg/kg either with pre-treatment of liquorice water extracts at 0.433 or 1.299?g/kg (crude drug), respectively, for one week or not were studied. Additionally, LS-180 cells and human primary hepatocytes were utilized to explore the potential effects of bioactive ingredients of liquorice on P-glycoprotein (P-gp) and Cytochromes P450 (CYPs), respectively.

3. The results revealed that exposure of AC after pre-treatment with liquorice was altered remarkably. Area under the concentration-time curve (AUC) decreased from 161?±?37.8 to 58.8?±?8.97 and 44.7?±?8.20?ng/mL*h, respectively. Similarly, C_max decreased from 26.2?±?5.19 to 11.8?±?1.15 and 6.86?±?0.600?ng/mL, respectively. In addition, expressions of CYPs of human primary hepatocytes were enhanced to various contents after induction. Moreover, accumulation of AC and hypaconitine (HA), not mesaconitine (MA) inside of LS-180 cells were reduced after pre-treatment by comparison with control.

4. In conclusion, the exposure of AC in vivo declined after pre-treatment with liquorice extract, which may be highly associated with upregulated expression and/or function of CYPs and P-gp.

     

The multiple-dosing pharmacokinetics of artemether,artesunate, and their metabolite dihydroartemisinin in rats

1. The present study was designed to investigate the multiple-dosing pharmacokinetics of antimalarial drugs artemether (ARM), artesunate (ARS), and their metabolite dihydroartemisinin (DHA) in rats.

2. Rats were randomized into four groups. Two groups of rats received oral doses of ARM or ARS once daily for five consecutive days. And another two groups of rats were given a single oral dose of ARM or ARS. Plasma samples were analysed for artemisinin drugs and their active metabolite DHA, using a validated liquid chromatography/tandem mass spectrometric (LC/MS/MS) method.

3. ARM and ARS could be biotransformed to metabolite DHA almost immediately after oral administration to rats. The area under the plasma concentration–time curve (AUC_0–t) of ARM after 5-day oral doses significantly decreased from 50.3 to 23.4 ng×h/mL (P?<?0.05), and oral clearance (CL/F) of ARM increased from 10.5 to 27.2?L/min/kg (P?<?0.05). The AUC_0–t of its metabolite DHA of ARM significantly decreased from 42.1 to 16.4 ng×h/mL (P?<?0.05), and its CL/F increased from 11.7 to 33.4?L/min/kg (P?<?0.05). The 5-day oral doses of ARS did not result in significant changes (P?>?0.05) in pharmacokinetic parameters of ARS, whereas its metabolite DHA exhibited lower AUC (P?=?0.05), compared with that obtained after a single oral administration.

4. The results showed ARM and its metabolite DHA exhibited time-dependent pharmacokinetic characteristics with decreased plasma drug level after five consecutive days of oral administration to rats, whereas ARS and its metabolite DHA did not show similar characteristics.

     

Pharmacokinetics of antofloxacin hydrochloride,a novel fluoroquinolone,after single-dose intravenous administration in healthy Chinese male volunteers

1. The purpose of the study was to evaluate the pharmacokinetic characteristics of a single, intravenous dose of antofloxacin hydrochloride in healthy Chinese male volunteers.

2. Twelve subjects were randomly assigned to groups that received a single, intravenous dose of 200, 300, or 400?mg antofloxacin hydrochloride in a three-way crossover design study. The serum and urine concentrations of antofloxacin were then assayed with high-performance liquid chromatography (HPLC). Major pharmacokinetic parameters and urine excretion were obtained up to 96?h after administration.

3. All three dosages were well tolerated. No clinically adverse reactions or abnormal laboratory results were detected.

4. After single-dose intravenous administration, antofloxacin hydrochloride exhibited linear pharmacokinetic characteristics with increasing dosages. The C_max for groups treated with 200, 300, or 400?mg dosages were 2.05?±?0.38, 3.01?±?0.60, and 3.80?±?0.78?mg l^?1, respectively; the areas under the curve from zero to infinity (AUC_0–∞) were 25.14?±?2.95, 37.63?±?5.42, and 53.87?±?9.48?mg l^?1·h, respectively. The t_1/2β was around 20?h; and the urinary excretion was measured as being from 58% to 60% within 96?h.

5. Based on these results, 300?mg of antofloxacin hydrochloride administered once daily is the dose suggested for further investigation in multiple-dose administration studies.

     

Effects of verapamil on the pharmacokinetics of puerarin in rats

Abstract

1. The oral bioavailability of puerarin is poor which hindered its clinical performance.

2. This study investigates the effects of verapamil on the pharmacokinetics of puerarin in rats.

3. The pharmacokinetics of orally administered puerarin (50?mg/kg) with or without verapamil pretreatment (10?mg/kg/day for 7?days) were investigated. The plasma concentration of puerarin was determined using LC-MS/MS method, and the pharmacokinetics profiles were calculated and compared. Caco-2 cell transwell model was also used to investigate the effects of verapamil on the transport pf puerarin.

4. The results showed that when the rats were pretreated with verapamil, the maximum concentration (C_max) of puerarin increased from 683.7?±?51.2 to 933.5?±?75.8?ng/mL (p?<?0.05), and the area under the concentration-time curve from zero to infinity (AUC_0-inf) also increased from 3687.3?±?444.6 to 5006.1?±?658.6?μg·h/L (p?<?0.05). The Caco-2 cell transwell experiments indicated that verapamil could decrease the efflux ratio of puerarin from 1.90 to 1.19 through inhibiting the activity of P-gp.

5. In conclusion, these results indicated that verapamil could affect the pharmacokinetics of puerarin, possibly by increasing the systemic exposure of puerarin by inhibiting the activity of P-gp.

     

Pharmacokinetics of antofloxacin hydrochloride in healthy male subjects after multiple intravenous dose administration

1. The purpose of the study was to evaluate pharmacokinetic characteristics of antofloxacin hydrochloride, a new fluoroquinolone antibiotic, during a multiple, intravenous dosing regimen.

2. Twelve healthy, Chinese male volunteer subjects were each given 300?mg of antofloxacin by intravenous infusion once daily for 7 days. Blood and urine samples were taken at designated time points for analysis of antofloxacin concentration by high-performance liquid chromatography (HPLC). Safety and tolerability were assessed by evaluation of subject complaints, vital signs, electrocardiograms, electroencephalograms, clinical chemistry parameters, haematology and urinalysis and prothrombin time.

3. The serum steady concentration of antofloxacin was obtained in 96?h after the administration of a daily intravenous dose of 300?mg of the drug. In the present study, the following pharmacokinetic parameters after 7 days of treatment with antofloxacin were determined to be: C_max 3.81?±?0.66?mg/L, C_min 0.85?±?0.19?mg/L, AUC_0–24 60.51?±?8.30?mg/L·h, C_av 2.52?±?0.35?mg/L, PTF 87.45?±?3.37%, t_1/2β 20.34?±?1.88?h. The C_max and AUC_0–24 after 7-day treatment were both higher than after the first dose (by 43% and 110%, respectively). The cumulative urinary elimination of antofloxacin within 96?h after the last dose was about 56%.

4. During the study, there were neither subject complaints nor significant adverse clinical findings.

5. Antofloxacin, administered intravenously as a single, daily 300?mg dose for 7 days, demonstrated favourable pharmacokinetic characteristics and tolerability. The results of this study indicate that antofloxacin hydrochloride is suitable for further clinical study.

     

Measurements of caffeine and plasma metabolite/caffeine ratios as a test for hepatic drug-oxidizing capacity in goats

1. The aim of this investigation was to determine the pharmacokinetics and demethylation of caffeine (CF) and the metabolite/CF ratios that correlated best with CF clearance, which were used to evaluate hepatic drug-oxidizing capacity of CF after a single intravenous dose (5?mg/kg) in hair goats (n?=?9).

2. Pharmacokinetic parameters of CF and its metabolites, theobromine (TB), paraxanthine (PX) and theophylline (TP), were calculated. The plasma metabolic ratios TB/CF, PX/CF, TP/CF and TB+PX+TP/CF were determined at 6, 8 and 10?h after CF administration to evaluate their hepatic drug-oxidizing capacity.

3. The plasma concentration–time data of CF were fit to a two-compartment model in all animals. The clearance of CF was 0.08?±?0.02?L/h/kg, and the volume of distribution was 0.91?±?0.16?L/kg. The demethylation fractions of CF to TB, PX and TP were 0.24, 0.37 and 0.39, respectively.

4. Correlations between the metabolic ratios and CF clearance were quite high, except for the PX/CF ratio, particularly at 6?h (r?=?0.650–0.750, P?<?0.01, 0.05) and 10?h (r?=?0.650–0.767, P?<?0.01, 0.05).

5. Plasma metabolite/CF ratios, except for the PX/CF ratio, may be useful as an alternative to measurements of CF clearance for the determination of the hepatic drug-oxidizing capacity in goats.

     

Comparison of intrinsic metabolic clearance in fresh and cryopreserved human hepatocytes

1. We compared the intrinsic clearance (CL_int) of a number of substrates in suspensions of fresh and cryopreserved human hepatocytes from seven donors.

2. CL_int values for a cocktail incubation of phenacetin, diclofenac, diazepam, bufuralol, midazolam, and hydroxycoumarin were 4.9?±?3.4, 18?±?7.2, 5.1?±?4.9, 6.3?±?3.3, 9.8?±?5.8 and 22?±?14?μl min^?1/10⁶ cells, respectively, and they correlated well with corresponding CL_int values using cryopreserved hepatocytes from 25 different donors.

3. CL_int values of each cocktail substrate and 20 AstraZeneca new chemical entities were compared in fresh and cryopreserved hepatocytes from the same three donors. There was a statistically significant correlation between CL_int in fresh and cryopreserved hepatocytes for each of the three livers (p?int values was 1.03.

4. In conclusion, the results add further support to the use of cryopreserved human hepatocytes as a screening model for the intrinsic clearance of new chemical entities.

     

Rapid determination of isocorydine in rat plasma and tissues using liquid chromatography – tandem mass spectrometry and its applications to pharmacokinetics and tissue distribution

1. A rapid and sensitive method for the determination of isocorydine in rat plasma and tissues was developed using liquid chromatography–tandem mass spectrometry (LC–MS/MS).

2. The biological samples were processed by extracting with diethyl ether–dichloromethane (3:2, v/v) and tetrahydropulmatine was used as the internal standard (IS). Detection of the analytes was achieved using positive ion mode electrospray ionization in the multiple reaction monitoring mode. The MS/MS ion transitions monitored were m/z 342.0→279.0 and 356.0→191.9 for isocorydine and IS, respectively.

3. The maximum plasma concentration (C_max 2496.8?±?374.4 µg/L) was achieved at 0.278?±?0.113?h (T_max) and the half-life (t_1/2) of isocorydine was 0.906?±?0.222?h after a 20?mg/kg oral administration. As for a 2?mg/kg intravenous (i.v.) administration, the C_max and clearance (CL) were 1843.3?±?338.3 µg/L and 2.381?±?0.356?L/h/kg, respectively. Based on the AUC_0–∞ obtained from oral and i.v. administration, the absolute bioavailability (F) was estimated as 33.4%. Tissue distribution results indicated that isocorydine underwent a rapid and wide distribution into tissues and it could effectively cross the blood-brain barrier.

     

Comparison of formation of thiolactones and active metabolites of prasugrel and clopidogrel in rats and dogs

1. Prasugrel and clopidogrel are antiplatelet prodrugs that are converted to their respective active metabolites through thiolactone intermediates. Prasugrel is rapidly hydrolysed by esterases to its thiolactone intermediate, while clopidogrel is oxidized by cytochrome P450 (CYP) isoforms to its thiolactone. The conversion of both thiolactones to the active metabolites is CYP mediated. This study compared the efficiency, in vivo, of the formation of prasugrel and clopidogrel thiolactones and their active metabolites.

2. The areas under the plasma concentration versus time curve (AUC) of the thiolactone intermediates in the portal vein plasma after an oral dose of prasugrel (1 mg kg^?1) and clopidogrel (0.77 mg kg^?1) were 15.8 ± 15.9 ng h ml^?1 and 0.113 ± 0.226 ng h ml^?1, respectively, in rats, and 454 ± 104 ng h ml^?1 and 23.3 ± 4.3 ng h ml^?1, respectively, in dogs, indicating efficient hydrolysis of prasugrel and little metabolism of clopidogrel to their thiolactones in the intestine.

3. The relative bioavailability of the active metabolites of prasugrel and clopidogrel calculated by the ratio of active metabolite AUC (prodrug oral administration/active metabolite intravenous administration) were 25% and 7%, respectively, in rats, and 25% and 10%, respectively, in dogs.

4. Single intraduodenal administration of prasugrel showed complete conversion of prasugrel, resulting in high concentrations of the thiolactone and active metabolite of prasugrel in rat portal vein plasma, which demonstrates that these products are generated in the intestine during the absorption process.

5. In conclusion, the extent of in vivo formation of the thiolactone and the active metabolite of prasugrel was greater than for clopidogrel’s thiolactone and active metabolite.

     

Pharmacokinetics,disposition and lipid-modulating activity of 5-{2-[4-(3,4-difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid,a potent and subtype-selective peroxisome proliferator-activated receptor α agonist in preclinical species and human

1. 5-{2-[4-(3,4-Difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid (1) is a novel, potent, and selective agonist of the peroxisome proliferator-activated receptor alpha (PPAR-α).

2. In preclinical species, compound 1 demonstrated generally favourable pharmacokinetic properties. Systemic plasma clearance (CL_p) after intravenous administration was low in Sprague–Dawley rats (3.2?±?1.4?ml min^?1 kg^?1) and cynomolgus monkeys (6.1?±?1.6?ml min^?1 kg^?1) resulting in plasma half-lives of 7.1?±?0.7?h and 9.4?±?0.8?h, respectively. Moderate bioavailability in rats (64%) and monkeys (55%) was observed after oral dosing. In rats, oral pharmacokinetics were dose-dependent over the dose range examined (10 and 50?mg kg^?1).

3. In vitro metabolism studies on 1 in cryopreserved rat, monkey, and human hepatocytes revealed that 1 was metabolized via oxidation and phase II glucuronidation pathways. In rats, a percentage of the dose (approximately 19%) was eliminated via biliary excretion in the unchanged form.

4. Studies using recombinant human CYP isozymes established that the rate-limiting step in the oxidative metabolism of 1 to the major primary alcohol metabolite M1 was catalysed by CYP3A4.

5. Compound 1 was greater than 99% bound to plasma proteins in rat, monkey, mouse, and human.

6. No competitive inhibition of the five major cytochrome P450 enzymes, namely CYP1A2, P4502C9, P4502C19, P4502D6 and P4503A4 (IC₅₀’s?>?30 μM) was discerned with 1.

7. Because of insignificant turnover of 1 in human liver microsomes and hepatocytes, human clearance was predicted using rat single-species allometric scaling from in vivo data. The steady-state volume was also scaled from rat volume after normalization for protein-binding differences. As such, these estimates were used to predict an efficacious human dose required for 30% lowering of triglycerides.

8. In order to aid human dose projections, pharmacokinetic/pharmacodynamic relationships for triglyceride lowering by 1 were first established in mice, which allowed an insight into the efficacious concentrations required for maximal triglyceride lowering. Assuming that the pharmacology translated in a quantitative fashion from mouse to human, dose projections were made for humans using mouse pharmacodynamic parameters and the predicted human pharmacokinetic estimates.

9. First-in-human clinical studies on 1 following oral administration suggested that the human pharmacokinetics/dose predictions were in the range that yielded a favourable pharmacodynamic response.

     

Disposition of GDC-0879, a B-RAF kinase inhibitor in preclinical species

1. The pharmacokinetics and disposition of GDC-0879, a small molecule B-RAF kinase inhibitor, was characterized in mouse, rat, dog, and monkey.

2. In mouse and monkey, clearance (CL) of GDC-0879 was moderate (18.7–24.3 and 14.5?±?2.1?ml min^?1 kg^?1, respectively), low in dog (5.84?±?1.06?ml min^?1 kg^?1) and high in rat (86.9?±?14.2?ml min^?1 kg^?1). The volume of distribution across species ranged from 0.49 to 1.9?l kg^?1. Mean terminal half-life values ranged from 0.28?h in rats to 2.97?h in dogs. Absolute oral bioavailability ranged from 18% in dog to 65% in mouse.

3. Plasma protein binding of GDC-0879 in mouse, rat, dog, monkey, and humans ranged from 68.8% to 81.9%.

4. In dog, the major ketone metabolite (G-030748) of GDC-0879 appeared to be formation rate-limited.

5. Based on assessment in dogs, the absorption of GDC-0879 appeared to be sensitive to changes in gut pH, food and salt form (solubililty), with approximately three- to four-fold change in areas under the curve (AUCs) observed.

     

Pharmacokinetics of the prodrug thiamphenicol glycinate and its active parent compound thiamphenicol in beagle dogs following intravenous administration

1. This study investigated the pharmacokinetics of thiamphenicol glycinate (TG) and thiamphenicol (TAP) in beagles (n?=?6) after intravenous administration of 50?mg/kg TG hydrochloride. Plasma concentrations of TG and TAP were measured by a HPLC-UV method.

2. Two-compartment model was selected to describe the pharmacokinetic characteristics of TG and TAP in vivo. Main parameters were as follows: AUC_0–∞ of TAP and TG were 16,328?±?1682 µg·min/mL and 3943?±?546 µg·min/mL, respectively. The total plasma clearance (CL) of TG and TAP were 12.7?±?2.0?mL/min/kg and 2.5?±?0.3?mL/min/kg, respectively. Mean residence time (MRT) of TG and TAP were 27.5?±?3.5 and 207.2?±?20.2?min, respectively. The transformative rate constant (k_1M) from TG to TAP was 0.0477?±?0.0028?min^?1. The elimination rate constant (k_M10) from TAP was 0.0238?±?0.0044?min^?1. Coefficients of variation (CV) between observed values and predicted ones were 5.9% and 18.2%, respectively. The volume of distribution of the central compartment for TG (V_C) and TAP (V_CM) were 0.264?±?0.022?L/kg and 0.127?±?0.023?L/kg, respectively.

3. Pharmacokinetic parameters suggested that TG was presumably cleaved quickly by tissue esterase to release TAP for effectiveness in beagles after administration.

     

Modulation of transporter activity of OATP1B1 and OATP1B3 by the major active components of Radix Ophiopogonis

Abstract

1. Radix Ophiopogonis is often an integral part of many traditional Chinese formulas, such as Shenmai injection used to treat cardio-cerebrovascular diseases. This study aimed to investigate the influence of the four active components of Radix Ophiopogonis on the transport activity of OATP1B1 and OATP1B3.

2. The uptake of rosuvastatin in OATP1B1-HEK293T cells were stimulated by methylophiopogonanone A (MA) and ophiopogonin D′ (OPD′) with EC₅₀ calculated to be 11.33?±?2.78 and 4.62?±?0.64?μM, respectively. However, there were no remarkable influences on rosuvastatin uptake in the presence of methylophiopogonanone B (MB) or ophiopogonin D (OPD). The uptake of atorvastatin in OATP1B1-HEK293T cells can be increased by MA, MB, OPD and OPD′ with EC₅₀ calculated to be 6.00?±?1.60, 13.64?±?4.07, 10.41?±?1.28 and 3.68?±?0.85?μM, respectively.

3. The uptake of rosuvastatin in OATP1B3-HEK293T cells was scarcely influenced by MA, MB and OPD, but was considerably increased by OPD′ with an EC₅₀ of 14.95?±?1.62?μM. However, the uptake of telmisartan in OATP1B3-HEK293T cells was notably reduced by OPD′ with an IC₅₀ of 4.44?±?1.10?μM, and barely affected by MA, MB and OPD.

4. The four active components of Radix Ophiopogonis affect the transporting activitives of OATP1B1 and OATP1B3 in a substrate-dependent manner.

     

Metabolism,pharmacokinetics and excretion of the GABAA receptor partial agonist [14C]CP-409,092 in rats

1. The metabolism and excretion of a GABA_A partial agonist developed for the treatment of anxiety, CP-409,092; 4-oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxylic acid (4-methylaminomethyl-phenyl)-amide, were studied in rats following intravenous and oral administration of a single doses of [¹⁴C]CP-409,092.

2. The pharmacokinetics of CP-409,092 following single intravenous and oral doses of 4 and 15?mg kg^?1, respectively, were characterized by high clearance of 169?±?18?ml min^?1 kg^?1, a volume of distribution of 8.99?±?1.46 l kg^?1, and an oral bioavailability of 2.9% ± 3%.

3. Following oral administration of 100?mg kg^?1 [¹⁴C]CP-409,092, the total recovery was 89.1% ± 3.2% for male rats and 89.3% ± 0.58% for female rats. Approximately 87% of the radioactivity recovered in urine and faeces were excreted in the first 48?h. A substantial portion of the radioactivity was measured in the faeces as unchanged drug, suggesting poor absorption and/or biliary excretion. There were no significant gender-related quantitative/qualitative differences in the excretion of metabolites in urine or faeces.

4. The major metabolic pathways of CP-409,092 were hydroxylation(s) at the oxo-tetrahydro-indole moiety and oxidative deamination to form an aldehyde intermediate and subsequent oxidation to form the benzoic acid. The minor metabolic pathways included N-demethylation and subsequent N-acetylation and oxidation.

5. The present work demonstrates that oxidative deamination at the benzylic amine of CP-409,092 and subsequent oxidation to form the acid metabolite seem to play an important role in the metabolism of the drug, and they contribute to its oral clearance and low exposure.

     

Effect of genistein on the activities of cytochrome P450 3A and P-glycoprotein in Chinese healthy participants

1. To determine the effect of genistein on cytochrome P450 3A (CYP3A) and P-glycoprotein (P-gp) function using the probe substrates midazolam and talinolol, respectively. Eighteen healthy adult male participants were enrolled in a two-phase randomized crossover design. In each phase, the participants received placebo or genistein for 14 days. On the 15th day, midazolam and talinolol were administered and blood samples were obtained. Midazolam and talinolol pharmacokinetic parameter values were calculated and compared before and after genistein administration.

2. Co-administration of genistein decreased the area under the concentration–time curve from 0 to 36?h (AUC 0-36) (143.65?±?55.40?ng h/mL versus 126.10?±?40.14?ng h/mL, p?<?0.05), and the area under the concentration–time curve from zero to infinity (AUC 0-∞) (209.18?±?56.61?ng h/mL versus 180.59?±?43.03?ng h/mL, p?<?0.05), and also maximum concentration (Cmax) of midazolam (48.86?±?20.21?ng/mL versus 36.25?±?14.35?ng/mL p?<?0.05). Similarly, AUC 0-36 (2490.282?±?668.79?ng h/mL versus 2114.46?±?861.11?ng h/mL, p?<?0.05), AUC 0-∞ (2980.45?±?921.09?ng h/mL versus 2626.92?±?1003.78?ng h/mL, p?<?0.05) and Cmax of talinolol (326.58?±?197.67?ng/mL versus 293.42?±?127.19?ng/mL, p?<?0.05) were reduced by genistein co-administration. The oral clearance of midazolam (1.68?±?0.85 h-1 versus 3.98?±?0.59 h-1, p?<?0.05) and talinolol (3.34?±?1.24 h-1 versus 3.79?±?1.55 h-1, p<0.05) were increased by genistien significantly.

3. Administration of genistein can result in a modest induction of CYP3A and possibly P-gp activity in healthy volunteers.

     

Reactions of benzene oxide,a reactive metabolite of benzene,with model nucleophiles and DNA

1. Reactivity of benzene oxide (BO), a reactive metabolite of benzene, was studied in model reactions with biologically relevant S- and N-nucleophiles by LC-ESI-MS.

2. Reaction with N-acetylcysteine (NAC) in aqueous buffer solutions gave N-acetyl-S-(6-hydroxycyclohexa-2,4-dien-1-yl)cysteine (pre-phenylmercapturic acid, PPhMA), which was easily dehydrated in acidic solutions to phenylmercapturic acid (PhMA). The yield of PPhMA + PhMA increased exponentially with pH up to 11% in the pH range from 5.5 to 11.4.

3. Primary 6-hydroxycyclohexa-2,4-dien-1-yl (HC) adducts were detected also in reactions of purine nucleosides and nucleotides under physiological conditions. After a vigorous acidic hydrolysis, all HC adducts were converted to corresponding phenyl purines, which were identified as 7-phenylguanine (7-PhG), 3-phenyladenine (3-PhA) and N⁶-phenyladenine (6-PhA). The yield of 7-PhG amounted to 14?±?5 and 16?±?7?ppm for 2′-deoxyguanosine and 2′-deoxyguanosine-5′-monophosphate, respectively, that of 6-PhA was 500?±?70 and 455?±?75?ppm with 2′-deoxyadenosine and 2′-deoxyadenosine-5′-phosphate, respectively, with only traces of 3-PhA.

4. Reactions with the DNA followed by acidic hydrolysis yielded 26?±?11?ppm (mean ± SD; n?=?9) of 7-PhG as the sole adduct detected.

5. In contrast to the reactions with S-nucleophiles, the reactivity of BO with nucleophilic sites in the DNA is very low and can therefore hardly account for a significant DNA damage caused by benzene.

     

Bioavailability and pharmacokinetics of S-propargyl-L-cysteine,a novel cardioprotective agent,after single and multiple doses in Beagle dogs

1. As a novel hydrogen sulfide-modulated agent, S-propargyl-L-cysteine (SPRC) is proven to be a potent cardioprotective candidate. Bioavailability and pharmacokinetics of SPRC (20?mg/kg) in beagle dogs after oral and intravenous administrations were investigated in this study. Plasma concentrations of SPRC were measured by a LC-MS/MS method.

2. Intravenous administration of SPRC (single dose) to beagle dogs gave a mean plasma half-life of 14.7?h, mean clearance of 0.4?ml min^?1 kg^?1 and mean apparent volume of distribution of 0.56?L/kg. Single oral administration was completely, fast absorbed (T_max= 0.33?±?0.20?h) with a mean absolute availability of 112% and mean plasma half-life of 16.5?h.

3. Multiple oral administration (once daily for 10 consecutive days) of SPRC to dogs resulted in steady state plasma drug concentration being reached after seven doses and didn’t cause obvious accumulation. No significant difference was found between the single and multiple pharmacokinetic parameters.

     

Transport and metabolism of the antitumour drug candidate 2′-benzoyloxycinnamaldehyde in Caco-2 cells

1. The transport and metabolism of the antitumour drug candidate 2′-benzoyloxycinnamaldehyde (BCA) was characterized in Caco-2 cells.

2. BCA disappeared rapidly from the donor side without being transported to the receiver side during its absorptive transport across Caco-2 cells. Its metabolites 2′-hydroxycinnamaldehyde (HCA) and o-coumaric acid (OCA) were formed in both the donor and the receiver sides.

3. HCA, in a separate study, also disappeared rapidly from the donor side, mostly being converted to its oxidative metabolite OCA during its absorptive transport across Caco-2 cells.

4. OCA was transported rapidly in the absorptive direction across Caco-2 cells with a P_app of 25.4?±?1.0?×?10^?6?cm s^?1 (mean?±?standard deviation (SD), n?=?3). OCA was fully recovered from both the donor and the receiver side throughout the time-course of this study.

5. Formation of HCA from BCA was inhibited almost completely by bis(p-nitrophenyl)phosphate (BNPP), a selective inhibitor of carboxylesterases (CES), and phenylmethylsulfonyl fluoride (PMSF), a broad specificity inhibitor of esterases in Caco-2 cells, suggesting that this hydrolytic biotransformation was likely mediated predominantly by CES. Conversion of HCA to OCA was inhibited significantly by isovanillin, a selective inhibitor of aldehyde oxidase (AO). Inhibitors for xanthine oxidase (XO) and aldehyde dehydrogenase (ALDH), which are known to be involved in the oxidation of aldehydes to carboxylic acids, did not have a significant effect on the biotransformation of HCA to OCA in Caco-2 cells.

6. In summary, the present work demonstrates that BCA is hydrolysed rapidly to HCA, followed by subsequent oxidation to OCA, in Caco-2 cells. The results provide a mechanistic understanding of the poor absorption and low bioavailability of BCA after oral administration.

     

Inhibitory effects of commonly used herbal extracts on UGT1A1 enzyme activity

1. Commonly used herbal supplements were screened for their potential to inhibit UGT1A1 activity using human liver microsomes. Extracts screened included ginseng, echinacea, black cohosh, milk thistle, garlic, valerian, saw palmetto, and green tea epigallocatechin gallate (EGCG). Estradiol-3-O-glucuronide (E-3-G) formation was used as the index of UGT1A1 activity.

2. All herbal extracts except garlic showed inhibition of UGT1A1 activity at one or more of the three concentrations tested. A volume per dose index (VDI) was calculated to estimate the volume in which the daily dose should be diluted to obtain an IC₅₀-equivalent concentration. EGCG, echinacea, saw palmetto, and milk thistle had VDI values >2.0?L per dose unit, suggesting a higher potential for interaction.

3. Inhibition curves were constructed for EGCG, echinacea, saw palmetto, and milk thistle. IC₅₀ values were (mean ± SE) 7.8?±?0.9, 211.7?±?43.5, 55.2?±?9.2, and 30.4?±?6.9 µg/ml for EGCG, echinacea, saw palmetto, and milk thistle extracts, respectively.

4. Based on our findings, inhibition of UGT1A1 by milk thistle and EGCG and to a lesser extent by echinacea and saw palmetto is plausible, particularly in the intestine where higher extract concentrations are anticipated. Further clinical studies are warranted.

     

Pharmacokinetics of fexofenadine following LPS administration to rats

1. The function and expression of drug transporters, including P-glycoprotein (P-gp) and organic-anion transporting polypeptides (Oatps), have been investigated but it is not well established how variables such as disease processes affect them. Fexofenadine is a substrate of these transporters and it was previously shown that its clearance is reduced in the rat isolated perfused liver following treatment with E.coli lipopolysaccharide (LPS). However, whether this translates to altered fexofenadine pharmacokinetics in vivo is yet to be established.

2. E.coli LPS at 5?mg/kg or sterile saline (control) was injected intraperitoneally in rats. Oral or intravenous (IV) fexofenadine (10?mg/kg) was administered 24?h later and plasma and urine samples collected for pharmacokinetic analysis.

3. LPS treatment did not significantly change the pharmacokinetics of IV fexofenadine, although there was a good correlation between weight loss and clearance suggesting reduced clearance in more severely affected animals. However, AUC_0–∞ of oral fexofenadine was a significantly higher in LPS-treated animals (13.9?±?9.76 min·µg/ml) compared to controls (5.53?±?1.12 min·µg/ml).

4. In conclusion, LPS treatment increased the bioavailability of fexofenadine but did not affect other pharmacokinetic parameters. This is consistent with a reduction in hepatic Oatp and/or P-gp for a high extraction ratio drug such as fexofenadine.