In vitro-in vivo correlation for intrinsic clearance for CP-409,092 and sumatriptan: a case study to predict the in vivo clearance for compounds metabolized by monoamine oxidase

1. Oxidative deamination of the GABA_A partial agonist CP-409,092 and sumatriptan represents a major metabolic pathway and seems to play an important role for the clearance of these two compounds.

2. Similar to sumatriptan, human mitochondrial incubations with deprenyl and clorgyline, probe inhibitors of monoamine oxidase B and monoamine oxidase A (MAO-B and MAO-A), respectively, showed that CP-409,092 was metabolized to a large extent by the enzyme MAO-A.

3. The metabolism of CP-409,092 and sumatriptan was therefore studied in human liver mitochondria and in vitro intrinsic clearance (CL_int) values were determined and compared to the corresponding in vivo oral clearance (CL_PO) values. The overall objective was to determine whether an in vitro-in vivo correlation (IVIVC) could be described for compounds cleared by MAO-A.

4. The intrinsic clearance, CL_int, of CP-409,092 was approximately 4-fold greater than that of sumatriptan (CL_int, values were calculated as 0.008 and 0.002?ml/mg/min for CP-409,092 and sumatriptan, respectively). A similar correlation was observed from the in vivo metabolic data where the unbound oral clearance, CL(u)_PO, values in humans were calculated as 724 and 178?ml/min/kg for CP-409,092 and sumatriptan, respectively.

5. The present work demonstrates that it is possible to predict in vivo metabolic clearance from in vitro metabolic data for drugs metabolized by the enzyme monoamine oxidase.

     

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.

     

Organophosphorus pesticide degradation product in vitro metabolic stability and time-course uptake and elimination in rats following oral and intravenous dosing

1. Levels of urinary dialkylphosphates (DAPs) are currently used as a biomarker of human exposure to organophosphorus insecticides (OPs). It is known that OPs degrade on food commodities to DAPs at levels that approach or exceed those of the parent OP. However, little has been reported on the extent of DAP absorption, distribution, metabolism and excretion.

2. The metabolic stability of O,O-dimethylphosphate (DMP) was assessed using pooled human and rat hepatic microsomes. Time-course samples were collected over 2?h and analyzed by LC-MS/MS. It was found that DMP was not metabolized by rat or pooled human hepatic microsomes.

3. Male Sprague–Dawley rats were administered DMP at 20?mg kg^?1 via oral gavage and i.v. injection. Time-course plasma and urine samples were collected and analyzed by LC-MS/MS. DMP oral bioavailability was found to be 107?±?39% and the amount of orally administered dose recovered in the urine was 30?±?9.9% by 48?h.

4. The in vitro metabolic stability, high bioavailability and extent of DMP urinary excretion following oral exposure in a rat model suggests that measurement of DMP as a biomarker of OP exposure may lead to overestimation of human exposure.

     

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.

     

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.

     

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.

     

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.

     

Testosterone hydroxylation in bovine liver: enzyme kinetic and inhibition study

1. In order to sort out the involvement of cytochrome P450 (CYP) 3A and possibly CYP2B in testosterone hydroxylation in cattle, enzyme kinetic and inhibition studies were performed.

2. Most relevant kinetic constants (Km and Vmax) for 6β-, 16β- and 2β-testosterone hydroxylase (OHT) activities were determined and accounted for 93.4?±?13.8, 36.4?±?6.1 and 110.8?±?15.2?μM, respectively, for Km and 0.558?±?0.03, 0.280?±?0.013, and 0.338?±?0.017?nmol min–1 mg–1 protein, respectively, for Vmax. Eadie–Hofstee plot analysis pointed out how these enzymatic activities in cattle follow a monophasic kinetic pattern.

3. Preliminary inhibition studies conducted with the CYP3A inhibitor ketoconazole and the CYP2B inhibitors orphenadrine and 9-ethynylphenanthrene seemed to suggest the major involvement of CYP3A in testosterone hydroxylation in cattle.

4. Immuno-inhibition studies with an anti-peptide antibody against bovine CYP3A4 confirmed the predominant role of CYP3A in testosterone hydroxylation in bovine liver, proving the usefulness of anti-peptide antibodies in defining the contribution of specific P450 isoforms in drug metabolism in veterinary species.

     

Pharmacokinetics and first-pass effects of ϵ-acetamidocaproic acid after administration of zinc acexamate in rats

1. Zinc acexamate (ZAC) is ionized to zinc and ?-acetamidocaproic acid (AACA). Thus, the pharmacokinetics and tissue distribution of zinc and AACA after intravenous (50?mg kg^?1) and oral (100?mg kg^?1) administration of ZAC were evaluated in rats. Also the pharmacokinetics of AACA after intravenous (10, 20, 30, and 50?mg kg^?1) and oral (20, 50, and 100?mg kg^?1) administration of ZAC and the first-pass extractions of AACA at a ZAC dose of 20?mg kg^?1 were evaluated in rats.

2. After oral administration of ZAC (20?mg kg^?1), approximately 0.408% of the oral dose was not absorbed, the F value was approximately 47.1%, and the hepatic and gastrointestinal (GI) first-pass extractions of AACA were approximately 8.50% and 46.4% of the oral dose, respectively. The incomplete F value of AACA was mainly due to the considerable GI first-pass extraction in rats.

3. Affinity of rat tissues to zinc and AACA was low—the tissue-to-plasma (T/P) ratios were less than unity. The equilibrium plasma-to-blood cells partition ratios of AACA were independent of initial blood ZAC concentrations of 1, 5, and 10?µg ml^?1—the mean values were 0.481, 0.490, and 0.499, respectively. The bound fractions of zinc and AACA to rat plasma were 96.6% and 39.0%, respectively.

     

Absorption,distribution, metabolism and excretion (ADME) study with 2,2′,4,4′,5,6′-hexabromodiphenyl ether (BDE-154) in male Sprague–Dawley rats

1. A metabolism study of orally administered 2,2′,4,4′,5,6′-hexabromodiphenyl ether (BDE-154; 11.3 μmoles kg^?1) was conducted in conventional and bile duct-cannulated male Sprague–Dawley rats.

2. In conventional rats, approximately 31% of the radiolabelled dose was retained at 72 h, and lipophilic tissues were the preferred sites for disposition.

3. Urinary excretion of BDE-154 was very low (1.0%), and parent compound was detected.

4. Cumulative biliary excretion was 1.3%, and glutathione conjugates were suggested.

5. Over 62% of the dose in conventional male rats was excreted in faeces, and was composed of parent compound (7.3%), free metabolites (13.1%), and covalently bound residues (41.4%). Faecal metabolites characterized by gas chromatography/mass spectrometry included multiple isomers of monohydroxylated hexa-/penta-/tetrabromodiphenyl ethers, and di-hydroxylated hexa/pentabromodiphenyl ethers.

6. The adipose tissue ¹⁴C was extractable BDE-154, but 40% of liver ¹⁴C was bound to macromolecules.

7. The study demonstrated the importance of performing individual polybrominated diphenyl ether (PBDE) metabolism studies to understand fully PBDE pharmacokinetics.

     

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.

     

Absorption,distribution, metabolism and excretion of peginesatide,a novel erythropoiesis-stimulating agent,in rats

1. The pharmacokinetics (PK) (absorption, distribution, metabolism, excretion) of peginesatide, a synthetic, PEGylated, investigational, peptide-based erythropoiesis-stimulating agent (ESA), was evaluated in rats. The PK profile was evaluated at 0.1–5 mg·kg^?1 IV using unlabeled or [¹⁴C]-labeled peginesatide. Mass balance, tissue distribution and metabolism were evaluated following IV administration of 5 mg·kg^?1 [¹⁴C]-peginesatide, with tissue distribution also evaluated by quantitative whole-body autoradiography (QWBA) following an IV dose of 17 mg·kg^?1 [¹⁴C]-peginesatide.

2. Plasma clearance was slow and elimination was biphasic with unchanged peginesatide representing >90% of the total radioactivity of the total radioactive exposure. Slow uptake of the radiolabeled compound from the vascular compartment into the tissues was observed.

3. Biodistribution to bone marrow and extramedullary hematopoietic sites, and to highly vascularized lymphatic and excretory tissues occurred.

4. A predominant degradation event to occur in vivo was the loss of one PEG chain from the branched PEG moiety to generate mono-PEG.

5. Renal excretion was the primary mechanism (41%) of elimination, with parent molecule (67%) the major moiety excreted.

6. In conclusion, elimination of [¹⁴C]-peginesatide-derived radioactivity was extended, retention preferentially occurred at sites of erythropoiesis (bone marrow), and urinary excretion was the primary elimination route.

     

Mechanism-based inhibition of human cytochrome P4503A4 by domperidone

1. Domperidone was evaluated in direct and time-dependent cytochrome P450 (CYP) 3A inhibition assays in human liver microsomes with midazolam and testosterone as probe substrates.

2. Domperidone was found to be a modest mechanism-based inhibitor of human and rat CYP3A. For human CYP3A, the inactivation constant (K_I) is 12 μM, and the maximum inactivation rate (k_inact) is 0.037?min^?1.

3. A rat interaction study was conducted between midazolam and either a single dose or five daily doses of domperidone. Although a single oral dose of 10?mg kg^?1 domperidone did not affect the pharmacokinetics of 10?mg kg^?1 oral midazolam, five daily oral doses of domperidone almost doubled the area under the plasma concentration versus time curve (AUC) of midazolam, and increased the maximum plasma concentration (C_max) of midazolam by 72%.

4. Based on the simulation and rat in vitro–in vivo extrapolation, it is predicted that co-administration of domperidone in humans could modestly increase (approximately 50%) the exposure of drugs that are primarily cleared by CYP3A.

     

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.

     

Preclinical stereoselective disposition and toxicokinetics of two novel MET inhibitors

1. The R- and S-enantiomer of N-(4-(3-(1-ethyl-3,3-difluoropiperidin-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide are novel MET kinase inhibitors that have been investigated as potential anticancer agents. The effect of the chirality of these compounds on preclinical in vivo pharmacokinetics and toxicity was studied.

2. The plasma clearance for the S-enantiomer was low in mice and monkeys (23.7 and 7.8?mL min^?1 kg^?1, respectively) and high in rats (79.2?mL min^?1 kg^?1). The R/S enantiomer clearance ratio was 1.5 except in rats (0.49). After oral single-dose administration at 5?mg kg^?1 the R/S enantiomer ratio of AUC_inf was 0.95, 1.9 and 0.41 in mice, rats and monkeys, respectively.

3. In an oral single-dose dose-ranging study at 200 and 500?mg kg^?1 and multi-dose toxicity study in mice plasma AUC exposure was approximately 2- to 3-fold higher for the R-enantiomer compared to the S-enantiomer. Greater toxicity of the S-enantiomer was observed which appeared to be due to high plasma C_min values and tissue concentrations approximately 24?h after the final dose.

4. Both enantiomers showed low to moderate permeability in MDCKI cells with no significant efflux, no preferential distribution into red blood cells and similar plasma protein binding in vitro.

5. Overall, the differences between the enantiomers with respect to low dose pharmacokinetics and in vitro properties were relatively modest. However, toxicity results warrant further development of the R-enantiomer over the S-enantiomer.

     

Effect of ursodeoxycholic acid on the pharmacokinetics of midazolam and CYP3A in the liver and intestine of rats

1. The elimination half-life of midazolam administered intravenously (5 mg kg^?1) or orally (15 mg kg^?1) was significantly decreased by 70% and 73%, respectively, 24 h after a single oral administration of ursodeoxycholic acid (UDCA, 300 mg kg^?1) in rats. In the liver there was a significant enhancement of the hydroxylation of midazolam in the microsomes and expression of cytochrome P450 (CYP) 3A1 messenger RNA (mRNA) and CYP3A2 mRNA.

2. The C_max and area under the curve (AUC)_0–∞ of midazolam were significantly (1.8–2.3 fold) increased by the single oral treatment with UDCA (100 and 300 mg kg^?1). Thus, the oral bioavailability, estimated from the AUC_0–∞, of midazolam administered intravenously and orally was significantly (1.8- and 2.3-fold, respectively) increased by the treatment with UDCA.

3. Repeated administration of UDCA (300 mg kg^?1 day^?1) for 7 days did not alter the pharmacokinetics of midazolam administered intravenously or orally, and the expression of mRNA for CYP3As in the rat liver.

4. The study has shown that a single administration of UDCA in rats induces significant hepatic CYP3A activity and increases significantly the oral bioavailability of midazolam. Such effects on the pharmacokinetics of midazolam were little observed on the repeated administration of UDCA.

     

Pharmacokinetics of lipoyl vildagliptin,a novel dipeptidyl peptidase IV inhibitor after oral administration in rats

1. The pharmacokinetics of lipoyl vildagliptin, a novel dipeptidyl peptidase IV (DPP IV) inhibitor, was studied in rats after oral administration for developing it as an antidiabetic agent.

2. A liquid chromatography-tandem mass spectroscopy (LC-MS/MS) method was developed to determine lipoyl vildagliptin in rat plasma. After an overnight fasting, rats were orally given lipoyl vildagliptin. Following a single oral dose of 25, 50, and 100?mg·kg^?1, T_max values were from 1.25 to 1.84?h, CL/F values were around 100?l h^?1 kg^?1. In the dose range, C_max values (63.9–296?μg·l^?1) and AUC_0–∞values (260–1214?μg·h·l^?1) were proportional to the doses.

3. In conclusion, this LC-MS/MS method for the determination of lipoyl vildagliptin in rat plasma was selective and sensitive. In rats, lipoyl vildagliptin displayed linear pharmacokinetics after a single oral dose in the range of 25–100?mg·kg^?1. Lipoyl vildagliptin might have very high CL/F values and V_d/F values, which indicated that the bioavailability of this drug might be low or lipoyl vildagliptin might distribute extensively or accumulate in tissues in view of its high liposolubility.

     

Pharmacokinetics of PSC 833 (valspodar) in its Cremophor EL formulation in rat

1. Valspodar is a P-glycoprotein inhibitor widely used in preclinical and clinical studies for overcoming multidrug resistance. Despite this, the pharmacokinetics of valspodar in rat, a commonly used animal model, have not been reported. Here, we report on the pharmacokinetics of valspodar in Sprague–Dawley rats following intravenous and oral administration of its Cremophor EL formulation, which has been used for humans in clinical trials.

2. After intravenous doses, valspodar displayed properties of slow clearance and a large volume of distribution. Its plasma unbound fraction was around 15% in the Cremophor EL formulation used in the study. After 10?mg kg^?1 orally it was rapidly absorbed with an average maximal plasma concentration of 1.48?mg l^?1 within approximately 2?h. The mean bioavailability of valspodar was 42.8%.

3. In rat, valspodar showed properties of low hepatic extraction and wide distribution, similar to that of its structural analogue cyclosporine A.

     

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.

     

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.