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The unbound concentrations of 14 commercial drugs, including five non-efflux/uptake transporter substrates-Class I, five efflux transporter substrates-class II and four influx transporter substrates-Class III, were simultaneously measured in rat liver, muscle, and blood via microanalysis. K puu,liver and K puu,muscle were calculated to evaluate the membrane transport activity and cell metabolism on the unbound drug concentrations in the skeletal muscle and liver. For Class I compounds, represented by antipyrine, unbound concentrations among liver, muscle and blood are symmetrically distributed when compound hepatic clearance is low. And when compound hepatic clearance is high, unbound concentrations among liver, muscle and blood are asymmetrically distributed, such as Propranolol. For Class II and III compounds, overall, the unbound concentrations among liver, muscle, and blood are asymmetrically distributed due to a combination of hepatic metabolism and efflux and/or influx transporter activity., (© 2021 Pharmaron Beijing Co Ltd. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

In the present study, a simple and rapid method for metamizole metabolite 4-methylamino antipyrine (MAA) determination in human plasma was developed, validated and successfully applied to a clinical trial. Chromatographic separation was achieved in HILIC mode on a YMC-Pack SIL column (100 × 2.0 mm; S-5 μm, 30 nm), with a mobile phase consisting of acetonitrile, water and formic acid. Protein precipitation of a small plasma volume using acetonitrile was selected for sample preparation. The multiple reaction monitoring transitions in the positive ionization mode were m/z 218.2 → 56.2 for MAA and m/z 221.2 → 56.2 for MAA-d3 (IS, internal standard). Concentration levels of MAA calibration standards were in the range of 0.100-20 μg/ml. Metamizole conversion into MAA in both water and organic media was investigated, and the level of the conversion in commercially available injection solutions was estimated., (© 2020 John Wiley & Sons, Ltd.)

Induction potentials of the pregnane X receptor (PXR) activator rifampin (RIF) on transporter genes [e.g., organic anion-transporting polypeptides (OATPs)] are still in its infancy or remain controversial in the field. The present investigations characterized changes in transporter gene expression by RIF in sandwich-cultured hepatocytes from multiple donors of human and cynomolgus monkey using real-time quantitative reverse transcription polymerase chain reaction method. Three-day treatment of RIF significantly induced CYP3A4 (∼60-fold induction), but not CYP1A2 and CYP2D6 genes. SLC51B was the most highly induced uptake transporter gene (>10-fold) in both human and monkey hepatocytes. A greater induction of CYP2C9 was observed in monkey hepatocytes than that in humans. ATP-binding cassette (ABC)B1 and ABCC2 were induced slightly above 2-fold in human and monkey hepatocytes and appeared to be dose-dependent. The induction of OATP and other transporter genes was generally less than 2-fold and considered not clinically relevant. SLCO2B1 was not detectable in monkey hepatocytes. To investigate in vivo OATP induction, RIF (18 mg/kg per day) was orally dosed to cynomolgus monkeys for 7 days. Pitavastatin and antipyrine were intravenously dosed before and after RIF treatment as exogenous probes of OATP and CYP activities, respectively. Plasma coproporphyrin-I (CP-I) and coproporphyrin-III (CP-III) were measured as OATP endogenous biomarkers. Although a significant increase of antipyrine clearance (CL) was observed after RIF treatment, the plasma exposures of pitavastatin, CP-I, and CP-III remained unchanged, suggesting that OATP function was not significantly altered. The results suggested that OATP transporters were not significantly induced by PXR ligand RIF. The data are consistent with current regulatory guidances that the in vitro characterization of transporter induction during drug development is not required. SIGNIFICANCE STATEMENT: Organic anion-transporting polypeptide (OATP) genes were not induced by rifampin in sandwich-cultured human and monkey hepatocytes OATP functions measured by OATP probe pitavastatin and endogenous marker coproporphyrins were not altered in monkeys in vivo by 7-day rifampin treatment. The data suggested that OATP transporters are unlikely induced by the pregnane X receptor ligand rifampin, which are consistent with current regulatory guidances that the in vitro characterization of OATP1B induction during drug development is not required., Competing Interests: The authors were all employees of Gilead Sciences during this research and declare no conflicts of interest., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Amyotrophic lateral sclerosis (ALS) affects persons of all races, and there continues to be a need for effective therapies to treat the disease., Objective: To compare the pharmacokinetics (PK) of edaravone between Japanese and Caucasian populations., Methods: Data from five PK studies among Japanese and Caucasian healthy volunteers were pooled and evaluated. In population PK (PPK) modelling, compartment models and other models with linear elimination were evaluated for appropriateness. Covariate effects by race, sex, weight, and age were investigated to explain variability in PK parameters. Simulations of the final PPK model were performed using a virtual population based on ALS clinical trials., Results: The analysis included 86 subjects. A three-compartment model with Michaelis-Menten plus linear elimination was selected as the best fit model. Race was statistically detected as a covariate for the second peripheral volume of distribution (V2), indicating a 26% increase for Caucasian subjects compared to Japanese subjects. However, based on simulation of PPK model for a virtual ALS population, the small difference of V2 was associated with a difference of C tau around 1 ng/mL after infusion, which was minimal compared to C max of approximately 1000 ng/ml., Conclusion: The PPK analyses demonstrated no clinically relevant difference in the PK profiles of edaravone by race, sex, weight, or age.

The objective of this study was to investigate the stability of edaravone in dog plasma by using an added antioxidant stabilizer, with an ultimate goal of developing and validating a sensitive, reliable and robust LC-MS-MS method for determination of edaravone in plasma samples. Edaravone was unstable in plasma, but it presented a good stability performance in the plasma with sodium metabisulfite (SMB), an effective antioxidant. The blood sample was collected in the heparinized eppendorf tube containing SMB and plasma sample was deproteinized using acetonitrile containing 20 ng/mL of phenacetin (Internal standard). The chromatographic separation was performed on a Zorbax Extend-C18 analytical column (2.1 mm × 150 mm I.D., particle size 3.5 µm, Agilent Technologies, USA). The mobile phase consisted of 0.1% formic acid in water (v/v) and methanol, and gradient elution was used. The analyte detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization by multiple reaction ion monitoring mode of the transitions at m/z [M + H]+ 175.1 → 77.1 for edaravone, and m/z [M + H]+ 180.2 → 110.0 for phenacetin. The linearity of this method was within the concentration range of 10-1000 ng/mL for edaravone in dog plasma. The lower limit of quantification was 10 ng/mL. The relative standard deviations of intra- and inter-precision were <10%. This method was successfully employed in the pharmacokinetics evaluation of edaravone in beagle dogs after intravenous administration., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

1-Aminobenzotriazole (ABT) is a well-known in vivo nonspecific inhibitor of cytochrome P450 (CYP) enzymes. An effective dosing regimen of ABT for a multiple-administration study is needed to conduct pharmacological studies for proof-of-concept, although it has been established for single-administration study, to characterize the pharmacokinetics of drug candidates. This study demonstrated a suitable dosing vehicle of ABT for continuous administration and increased exposure to antipyrine, which is a nonspecific probe of CYP, using ABT for a long period in mice. The dosing vehicle of ABT was 0.5% (w/v) hydroxypropyl methylcellulose and 0.5% (v/v) Tween 80 in N,N-dimethylacetamide/20% hydroxypropyl-β-cyclodextrin aqueous solution (2:8, v/v) based on the duration of apparent solubility. After implantation of an ALZET osmotic pump with ABT, the plasma concentrations of ABT were maintained at more than 4.1 μg/ml over 336 h. Compared with the vehicle group, the CLtot of antipyrine with ABT decreased to approximately one-fourth, and the BA of antipyrine with ABT increased up to 3-fold. In addition, the enhancement of exposure of antipyrine by ABT was maintained over the 336 h. The body weight, food consumption and hematological parameters of mice did not change with ABT administration for 16 days. These findings demonstrated that pretreatment of ABT can increase long-term exposure using continuous administration with the ALZET osmotic pump in mice with no overt toxicity. It is concluded that the in vivo use of 1-aminobenzotriazole can be applied to pharmacological studies for proof-of-concept, thus contributing to the selection of drug candidates at an early drug discovery stage. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

The effectiveness of controlled release 1-aminobenzotriazole (ABT) administration to inhibit cytochrome P450 (P450) enzymes has been evaluated in mice. To maximize the duration of P450 inhibition in vivo, ABT was administered via an osmotic pump. The degree of P450 inhibition was compared with that achieved with a single bolus dose of ABT. Two-hour prior subcutaneous treatment of mice with ABT (50 mg/kg) inhibited antipyrine clearance by 88%. A less pronounced inhibitory effect (29% reduction in clearance) was observed when ABT was administered 24-hours before antipyrine administration, indicating partial restoration of P450 activity during this longer pretreatment time. The duration of ABT in mice was very short (mean residence time = 1.7 hours) after subcutaneous bolus administration. When the inhibitor was delivered by an osmotic pump, maximum blood concentrations of the inhibitor were observed 24 hours after device implantation and were maintained at steady state for 6 days. Inhibition of P450 activity, as measured by antipyrine clearance, was confirmed at 24 hours and 120 hours after pump implantation, highlighting the utility of this method as a longer-term model for P450 inhibition in mice. The magnitude of P450 inhibition in ABT-treated mice was compared with that in hepatic P450 reductase null mice and both models were comparable. In vivo ABT administration by an osmotic pump offers an effective approach for longer-term P450 inhibition in mice and avoids the necessity for multiple dosing of the inhibitor., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

The sampling of study included 185 examined workers. Out of them 90 work at "Opitnii zavod Neftekhim" (67 females and 23 males) and 95--at "Kaustik" (64 females and 31 males) from various workshops of the given enterprises. To determine biochemical indicators samples of blood, saliva and urine were collected. The study was carried out in concordance with ethic principles of the Helsinki world medical association declaration, 2008 ed. with receiving written consent of patient to participate in study.

In this work, two high-performance liquid chromatography (HPLC) assays were developed and validated for the independent determination of edaravone and taurine using 3-methyl-1-p-tolyl-5-pyrazolone and L-glutamine as internal standards. In in vitro experiments, human plasma was separately spiked with a mixture of edaravone and taurine, edaravone or taurine alone. Plasma was precipitated with acetonitrile containing 0.1% formic acid. Ultrafiltration was employed to obtain the unbound ingredients of the two drugs. The factors that might influence the ultrafiltration effiency were elaborately optimized. Plasma supernatant and ultrafiltrate containing taurine were derivated with o-phthalaldehyde and ethanethiol in the presence of 40 mmol/L sodium borate buffer (pH 10.2) at room temperature within 1 min. Chromatographic separations were achieved on an InertSustain C18 column (250 × 4.6 mm, 5 µm). Isocratic 50 mmol/L ammonium acetate-acetonitrile and gradient 50 mmol/L sodium acetate (pH 5.3)-methanol were respectively selected as the mobile phase for the determination of edaravone and taurine. All of the validation data including linearity, extraction recovery, precision, accuracy and stability conformed to the requirements. Results showed that there were no significant alterations in the plasma protein binding rate of taurine and edaravone, implying that the proposed combination therapy was pharmacologically feasible., (Copyright © 2014 John Wiley & Sons, Ltd.)

Phenazone is a non-opioid analgesic used to treat acute mild to moderate pain, and is considered to be a safe drug. It is most often sold as a nonprescription/over-the-counter drug. Very few fatalities due to phenazone overdoses are reported in the literature. We present a case where a man in his early sixties was found dead in his home in the bottom of a staircase, the scene suggesting that death might have been caused by blunt force injury. However, in spite of the apparently dramatic scene, the gross findings at autopsy did not reveal lethal injuries. Whole blood from the femoral vein was collected during autopsy and screened for drugs of abuse and medicinal drugs. The only toxicological findings were a very high concentration of phenazone (280mg/L) and a high therapeutic concentration of caffeine (34mg/L). An UPLC-MS/MS method was used for quantification of the drugs., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

In pharmacokinetic evaluation of mice, using serial sampling methods rather than a terminal blood sampling method could reduce the number of animals needed and lead to more reliable data by excluding individual differences. In addition, using serial sampling methods can be valuable for evaluation of the drug-drug interaction (DDI) potential of drug candidates. In this study, we established an improved method for serially sampling the blood from one mouse by only one incision of the lateral tail vein, and investigated whether our method could be adapted to pharmacokinetic and DDI studies. After intravenous and oral administration of ibuprofen and fexofenadine (BCS class II and III), the plasma concentration and pharmacokinetic parameters were evaluated by our method and a terminal blood sampling method, with the result that both methods gave comparable results (ibuprofen: 63.8 ± 4.0% and 64.4%, fexofenadine: 6.5 ± 0.7% and 7.9%, respectively, in bioavailability). In addition, our method could be adapted to DDI study for cytochrome P450 and organic anion transporting polypeptide inhibition. These results demonstrate that our method can be useful for pharmacokinetic evaluation from the perspective of reliable data acquisition as well as easy handling and low stress to mice and improve the quality of pharmacokinetic and DDI studies., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Three liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were respectively developed and validated for the simultaneous or independent determination of taurine and edaravone in rat plasma using 3-methyl-1-p-tolyl-5-pyrazolone and sulfanilic acid as the internal standards (IS). Chromatographic separations were achieved on an Agilent Zorbax SB-Aq (100 × 2.1 mm, 3.5 µm) column. Gradient 0.03% formic acid-methanol, isocratic 0.1% formic acid-methanol (90:10) and 0.02% formic acid-methanol (40:60) were respectively selected as the mobile phase for the simultaneous determination of two analytes, taurine or edaravone alone. The MS acquisition was performed in multiple reaction monitoring mode with a positive and negative electrospray ionization source. The mass transitions monitored were m/z [M + H](+) 175.1 → 133.0 and [M + H](+) 189.2 → 147.0 for edaravone and its IS, m/z [M - H](-) 124.1 → 80.0 and [M - H](-) 172.0 → 80.0 for taurine and its IS, respectively. The validated methods were successfully applied to study the pharmacokinetic interaction of taurine and edaravone in rats after independent intravenous administration and co-administration with a single dose. Our collective results showed that there were no significant alterations on the main pharmacokinetic parameters (area under concentration-time curve, mean residence time, half-life and clearance) of taurine and edaravone, implying that the proposed combination therapy was pharmacologically feasible., (Copyright © 2014 John Wiley & Sons, Ltd.)

An LC-MS/MS method was developed and validated for the simultaneous quantification of edaravone and taurine in beagle plasma. The plasma sample was deproteinized using acetonitrile containing formic acid. Chromatographic separations were achieved on an Agilent Zorbax SB-Aq (100 × 2.1 mm, 3.5 μm) column, with a gradient of water (containing 0.03% formic acid) and methanol as the mobile phase at a flow rate of 0.3 mL/min. The analyte detection was carried out in multiple reaction monitoring mode and the optimized precursor-to-product transitions of m/z [M+H](+) 175.1 → 133.0 (edaravone), m/z [M+H](+) 189.1 → 147.0 (3-methyl-1-p-tolyl-5-pyrazolone, internal standard, IS), m/z [M-H](-) 124.1→80.0 (taurine), and m/z [M-H](-) 172.0 → 80.0 (sulfanilic acid, IS) were employed to quantify edaravone, taurine, and their corresponding ISs, respectively. The LOD and the lower LOQ were 0.01 and 0.05 μg/mL for edaravone and 0.66 and 2 μg/mL for taurine, respectively. The calibration curves of these two analytes demonstrated good linearity (r ＞ 0.99). All the validation data including the specificity, precision, recovery, and stability conformed to the acceptable requirements. This validated method has successfully been applied in the pharmacokinetic study of edaravone and taurine mixture in beagle dogs., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cyfluthrin effects on in vivo drug metabolizing enzymes were evaluated using the oxidative substrate antipyrine. Antipyrine pharmacokinetics in plasma and urinary excretion of its major metabolites with and without cyfluthrin oral treatment (20mg/kg/day for 6 days) were investigated in rats. Cyfluthrin increased the apparent intrinsic clearance and decreased the antipyrine half-life at β phase. Cyfluthrin also increased the clearance of the antipyrine metabolites, norantipyrine, 4-hydroxyantipyrine and 3-hydroxymethylantipyrine and the formation rate constants for each of the three metabolites measured in urine. These results suggest that cyfluthrin affects hepatic cytochrome P450 (CYP) system. In order to confirm, a second experiment was carried out. We evaluated the effects of repeated exposure to cyfluthrin on hepatic and renal CYP2E, CYP1A and CYP4A subfamilies and peroxisomal proliferation in rats following oral administration (10 and 20mg/kg/day for 6 days). At the highest dose, cyfluthrin increased renal and hepatic O-deethylation of ethoxyresorufin and O-demethylation of methoxyresorufin, metabolism mediated by the CYP1A subfamily. Liver and kidney were susceptible to cyfluthrin-dependent induction of 12- and 11-hydroxylation of lauric acid, suggesting CYP4A subfamily induction. Also cyfluthrin increased the β-oxidation of palmitoyl-coenzyme A and carnitine acetyltransferase activity, supporting cyfluthrin as a peroxisome proliferator. In conclusion, the demonstration that cyfluthrin induced hepatic CYP1A, CYP4A subfamilies and peroxisomal proliferation raises the possibility of cyfluthrin could produce changes in oxidative stress., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

A rapid, simple, and sensitive on-line solid-phase extraction HPLC-DAD method for simultaneous evaluation of the activity of five CYP450 isoforms (CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in vivo has been developed and validated. The five specific probe substrates include caffeine (1A2), metoprolol (2D6), dapsone (3A4), omeprazole (2C19) and chlorzoxazone (2E1). Automated pre-purification of plasma and enrichment of analytes were performed using a C18 on-line solid-phase extraction cartridge. After being eluted from the cartridge, the analytes and the internal standard antipyrine were separated on a C18 RP analytical column and analyzed by DAD. The method was validated to quantify the concentration ranges of 0.05-50.0 μg/ml for dapsone and omeprazole, 0.1-50.0 μg/ml for caffeine and 0.2-50.0 μg/ml for metoprolol and chlorzoxazone. The linearity (R(2)) for all analytes tested was exceeded 0.99. The intra-day precision ranged from 0.29 to 13% and the inter-day precision ranged from 5.0 to 15%, respectively. The intra-day and inter-day accuracy were between 86.7% and 113.6%. The extraction recoveries were in the range 82.8-109.9% for all the analytes and internal standard antipyrine. This method was successfully applied to evaluate the effects of TM208 on rat five CYP450 isoforms., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A sensitive and selective method for the simultaneous determination of non-steroidal anti-inflammatory drugs in bovine plasma was developed. Confirmatory analysis was carried out by liquid chromatography coupled with an electrospray ionisation tandem mass spectrometer (LC-ESI-MS/MS). Target compounds were acidified in plasma and extracted with acetonitrile. Sodium chloride was added to assist separation of the plasma and acetonitrile mixture. The acetonitrile extract is then subjected to liquid-liquid purification by the addition of hexane. Accuracy of the methods in plasma was between 93 and 102%. The precision of the method for the basic NSAIDs in plasma expressed as % RSD, for the within-laboratory reproducibility was less than 10%. Decision limit (CCα values) and detection capability (CCβ) values were established. The methods were validated according to Commission Decision 2002/657/EC., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objectives: The aim was to investigate the pharmacokinetic interaction between puerarin and edaravone, and the effect of borneol on the brain distribution kinetics of puerarin in rats., Methods: A reversed-phase high performance liquid chromatography method was developed and validated for the simultaneous determination of puerarin and edaravone in rat plasma. The detection method was successfully applied to compare the pharmacokinetic interaction and brain distribution kinetics of puerarin and edaravone using in-situ microdialysis sampling in rats after intravenous administration and co-administration with a single dose., Key Findings: The method gave good linearity and no endogenous material interfered with the two target compounds and internal standard peaks. The limit of detection of puerarin and edaravone was 0.03 and 0.05 microg/ml, respectively. The average recovery of the two compounds from rat plasma was >94%. The precision of the test was determined to be within 10%. The combination of puerarin and edaravone reduced drug elimination rates, gave a wider distribution, and the disposition of both drugs in rats was optimized. The distribution of puerarin in brain tissues was significantly increased and its elimination was noticeably slower with borneol pretreatment., Conclusions: The results provide important information for the improved combined use of puerarin and edaravone with borneol pretreatment in clinical practice.

We examined the in vitro dissolution-in vivo absorption correlation (IVIVC) for enteric-coated granules containing theophylline, antipyrine or acetaminophen as model drugs with high solubility and high permeability. More than 85% of each drug was released from granules coated with hypromellose acetate succinate (HPMCAS) (AS-LG grade, which dissolves at pH above 5.5) at a mean dissolution rate of more than 5 %/min after a lag time of less than 4 min in simulated intestinal fluid of pH 6.8. The lag time and the dissolution rate were significantly extended and reduced, respectively, when AS-LG was replaced with AS-HG (a grade of HPMCAS that dissolves at pH above 6.8). Enteric-coated granules were administered intraduodenally to anesthetized rats. Statistical significances of differences of in vitro lag time between AS-LG- and AS-HG-coated granules were consistent with those in vivo, for all drugs. Significant differences in dissolution rates between granules also corresponded to those in absorption rates calculated using a deconvolution method, and both parameters had comparable absolute values, except in the case of antipyrine-containing granules with relatively fast dissolution rates. Thus, a good IVIVC was generally obtained; however, the exception suggests the importance of developing a dissolution test that fully reflects the in vivo situation., ((c) 2009 Wiley-Liss, Inc. and the American Pharmacists Association)

Background: Dipyrone (metamizole) is a nonsteroidal anti-inflammatory drug used as an analgesic and antipyretic in both pediatric and adult patients. Dipyrone hydrolyses to 4-methylaminoantipyrine (MAA) in the stomach before absorption. There are several HPLC methods available for analysis of MAA from human plasma but no method has yet been developed on liquid chromatography-mass spectrometry (LC-MS) or LC tandem MS (LC-MS/MS), which are much more specific and sensitive techniques., Methodology: A high-performance LC-MS method for the quantification of 4-methylaminoantipyrine from human plasma has been developed, validated and applied to a pharmacokinetic study of 500 mg oral dose dipyrone. Following liquid-liquid extraction, the analyte was first separated on a reverse phase column using isocratic mobile phase and then analyzed by MS in selected ion monitoring mode using [M+H](+) ions, m/z 218.2 for 4-methylaminoantipyrine and 231.3 for 4-isopropylantipyrine (internal standard)., Results: The method exhibited a linear range from 0.2 to 10.0 µg/ml when only 100 µl human plasma sample was used. The lower limit of detection was 0.04 µg/ml (160 pg on column). The recovery was 80%. The accuracy and precision were obtained over the calibration curve range and were well within the limits specified under guidelines for bioanalytical method validation. The compound was stable under the experimental conditions., Conclusion: The method was demonstrated to be, simple, sensitive and rapid. It can be easily adopted in laboratories with access to LC-MS or MS/MS and applied to sample analysis in clinical settings where a large number of samples are generated.

The purpose of the study was to compare the disposition of pharmacologic markers for cytochrome P-450 (CYP) metabolism, glomerular filtration rate (GFR), and extracellular (ECFV) and total body fluid volumes (TBFV) of Greyhounds and Beagles. Six healthy Greyhound and six healthy Beagle dogs were studied. Antipyrine, a marker for CYP metabolism and TBFV, and inulin, a marker for the GFR and ECFV, were administered i.v. Samples were collected at predetermined times and plasma was analyzed by validated high-pressure liquid chromatography (HPLC) methods. There were no differences in the disposition or pharmacokinetic parameters for inulin between the dog breeds. However, the clearance of antipyrine (mean = 8.33 mL/min/kg) in Greyhounds was significantly slower than Beagles (13.42 mL/min/kg, P = 0.004). The volume of distribution of antipyrine was significantly larger in Greyhounds (0.789 L/kg) than in Beagles (0.644 L/kg, P = 0.01). The half-life of antipyrine was significantly longer in Greyhounds (1.09 h) compared with Beagles (0.55 h, P = 0.002). The in vitro plasma protein binding of antipyrine was significantly less in Greyhounds (28%) compared with Beagles (40.3%, P = 0.008). Greyhounds exhibited significantly slower CYP metabolism, higher TBFV, and lower in vitro protein binding of antipyrine compared with Beagles. No differences in GFR or ECFV were found.

The differential effects of endotoxin derived from Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli on hepatic cytochrome P450 (CYP)-dependent drug-metabolizing enzyme activity and on the expression of hepatic CYP3A2, CYP2C11, P-glycoprotein and multidrug resistance-associated protein 2 (Mrp2) was investigated in rats. Endotoxin from all three different pathogens significantly decreased the systemic clearance of antipyrine, reflecting reduced hepatic drug-metabolizing enzyme activity 24 h after intravenous injection (0.5 mg/kg). The degree of the decreased systemic clearance by P. aeruginosa endotoxin was smaller than that by both K. pneumoniae and E. coli endotoxin. Western blot analysis revealed that the down-regulation of CYP3A2 by K. pneumoniae and E. coli endotoxin was greater than that by P. aeruginosa endotoxin. However, the down-regulation of CYP2C11 by all three different endotoxin was almost the same. Both K. pneumoniae and P. aeruginosa endotoxin significantly down-regulated P-glycoprotein, but did not down-regulate Mrp2. E. coli endotoxin had no effect on the expression of either P-glycoprotein or Mrp2, probably due to the low dose used. The down-regulation of CYP3A2 by endotoxin was parallel to the decreased systemic clearance of antipyrine. These results suggest that endotoxin has a differential effect on the hepatic CYP-mediated drug-metabolizing enzyme activity, and on the protein levels of hepatic CYP3A2 and P-glycoprotein, probably due to bacterial source-differences in the production of some proinflammatory mediators. Endotoxin appears to regulate coordinately CYP3A2, CYP2C11 and P-glycoprotein, but not Mrp2.

Edaravone, a newly synthesized synthetic radical scavenger, has been identified and adopted as an anti-stroke agent. However, its mechanism and the effect of edaravone on lipoprotein oxidation are not fully understood. Therefore, whether edaravone could suppress oxidation of low-density lipoprotein (LDL) and be involved in the expression of endothelial nitric oxide synthase (eNOS) in relation to anti-atherogenesis by improving and conserving vascular circulation was investigated. We investigated the in vitro effects of edaravone on copper- and endothelial cell-mediated LDL oxidation, and the expression of eNOS in human umbilical vein endothelial cells (HUVEC) modulated by oxidized LDL. The in vivo effect of edaravone on antioxidative effect was also studied in male rats intravenously administered with edaravone. Edaravone apparently inhibited copper- and HUVEC-mediated LDL oxidation at the concentration equivalent to serum concentrations in clinical use. The intravenous administration of edaravone also enhanced serum radical-scavenging property in rats. We tested the effect of edaravone on protein and mRNA expression of eNOS in HUVEC. Edaravone enhanced eNOS expression in HUVEC, presumably because of increased stability of eNOS mRNA, and reversed eNOS expression reduced by oxidized LDL nearly to the control levels. The present study demonstrates for the first time that edaravone increases eNOS expression with the inhibition of LDL oxidation, and that edaravone can reverse oxidized LDL-mediated reduction in eNOS expression in endothelial cells. The preventive action of edaravone from ischemic disease consequence may be attributed to these eNOS up-regulation with decreased oxidation.

Single-dose pharmacokinetics of 1-aminobenzotriazole (ABT), a potent nonspecific inhibitor of cytochromes P450 (P450s), were characterized after oral administration to mice and guinea pigs at doses of 50, 100, and 150 mg/kg using serial sampling in both species. Only 30-microl blood samples were drawn from jugular vein-cannulated mice using Microvette capillary tubes containing lithium heparin. A comparison of the pharmacokinetics of antipyrine (AP) administered i.v. at 20 mg/kg to mice followed by serial and terminal sampling techniques yielded similar results. The ABT concentrations in plasma were sustained at high levels (5-100 microM) for at least 12 h in both species. Pretreatment of animals with ABT 2 h prior to AP administration decreased the plasma AP clearance by about 95% in mice at all ABT doses studied and 84, 95, and 95% in guinea pigs at a dose of 50, 100, and 150 mg/kg ABT, respectively. In vitro, the dissociation constants (KI) for ABT as the P450 mechanism-based inactivator were determined to be 45.6 and 193 microM, and the maximal inactivation rate constants (kinact) were determined to be 0.089 and 0.075 min(-1) for the mouse and guinea pig liver microsomes, respectively. The projected P450 inactivations at the plasma Cmax of ABT agreed with the inhibitions of P450-mediated AP clearance observed in vivo. For mechanistic studies in vivo overall, a 2-h prior oral pretreatment with ABT at 50 mg/kg in mice and 100 mg/kg in guinea pigs would provide significant systemic concentrations of the inhibitor over 24 h and inhibition of P450-dependent clearance of test compounds.

Purpose: The purpose of this study was to develop and validate a method for separately evaluating the roles of gastrointestinal absorption and hepatic extraction as barriers to oral bioavailability (BA). The method was validated using five reference compounds known to have different absorption and hepatic extraction properties. Dose-dependence was also investigated for one reference compound., Methods: Five reference compounds, amoxicillin, antipyrine, atenolol, propranolol, and testosterone, were administered as a cassette intravenouly (IV), via the hepatoportal vein (IPV), intraduodenally (ID), and intracolonically (IC) to male Sprague-Dawley rats. Blood samples were taken at nine time points, and the compounds were extracted from plasma using solid phase extraction. Plasma concentrations of each compound were determined using Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS). Pharmacokinetic parameters including bioavailability were calculated for each compound for each route of administration., Results: Testosterone BA was less than 10% by ID, IC, and IPV routes, due to high hepatic extraction, consistent with its high systemic clearance (63 ml x min(-1) x kg(-1)) and short terminal plasma half-life (23 min). The IPV BA of amoxicillin was 95%+/-6% indicating the absence of hepatic extraction in the rat, but with an ID BA of approximately 39% suggesting incomplete GI absorption to be the main barrier to bioavailability. Absorption was poor from the colon, demonstrating site-dependence consistent with literature reports of site-dependent absorption. Low oral BA of propranolol was due in part to first-pass hepatic extraction (IPV BA of 36%). The IPV BA of propranolol was dose-dependent, most likely due to saturation of the P450 enzymes. Atenolol was incompletely bioavailable due to incomplete intestinal absorption, with no contribution of hepatic first-pass metabolism. Antipyrine was highly bioavailable by all routes., Conclusions: This in vivo rat model is demonstrated to be useful for identifying and quantifying the causes of incomplete bioavailabilty. It separately evaluates intestinal absorption, hepatic extraction, and site-dependent absorption. Concentration-dependence of saturable processes can also be examined.

Objective: We tested whether fibrotic progression in chronic hepatitis C could be predicted by liver tests, antipyrine clearance, or platelet count., Methods: In 58 patients (6 untreated, 52 interferon-treated), a second liver biopsy was taken median 4.5 yr after first histologic diagnosis. We used receiver operating characteristic curves to determine whether changes in conventional liver tests, antipyrine clearance, or platelet count were predictive of altered hepatic fibrosis score., Results: Apart from a weak association with change in ALT, conventional liver tests (albumin, bilirubin, prothrombin time) failed to correlate with changes (Delta) in hepatic fibrosis, but there were significant correlations between deltaantipyrine clearance or deltaplatelet count and deltafibrosis score (p < 0.01). As indicated by areas under the receiver operating characteristic curves, the diagnostic accuracy of deltaantipyrine clearance for fibrotic progression was 68%; for Deltaplatelet count it was 80%. With defined cut-off values (-0.05 ml/min/kg for deltaantipyrine clearance; -41 x 10(9)/L for deltaplatelet count), the negative predictive values for fibrotic progression were 85% with antipyrine clearance and 89% with platelet count. Corresponding positive predictive values were 48% and 91%, respectively., Conclusions: Changes in antipyrine clearance and platelet count are more sensitive than conventional tests for indicating fibrotic change in chronic hepatitis C. Both could be used to reliably identify those who do not have fibrotic progression, and platelet count also has a high positive predictive value for disease progression.

This study was designed to assess the efficacy of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger that possesses anti-oxidant effects, on cardiac function and fine structure of the left ventricular myocardium in diabetes mellitus. Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of spontaneous development of type II diabetes (30 weeks; n = 15) were divided into two groups and treated with edaravone 30 mg/kg/d or vehicle for 2 weeks. OLETF rats showed hyperglycemia (352 +/- 71 mg/dl vs normal control; 128 +/- 52 mg/dl), increased thiobarbituric acid-reactive substances (TBARS; 6.9 +/- 2.5 nM/ml vs 2.8 +/- 0.6 nM/ml), and decreased superoxide dismutase activity (21.5 +/- 0.9 U/ml vs 25.8 +/- 0.7 U/ml). Increased left ventricular end-diastolic pressure (12 +/- 3 mm Hg vs 6 +/- 2 mm Hg) and hypertrophied cardiocytes (23.1 +/- 1.4 vs 17.6 +/- 1.0 microm) were also observed (P < 0.05, respectively). Edaravone could not improve plasma glucose level and hemodynamic parameters but significantly decreased TBARS values (3.8 +/- 0.5) and increased superoxide dismutase activity (24.5 +/- 0.8) (vs OLETF, P < 0.05, respectively). Moreover, edaravone effectively preserved cardiocyte diameter (18.2 +/- 0.9 microm) and the fine structure of mitochondria. Thus, edaravone exhibits modest cardiac protection in diabetes mellitus independent of blood sugar level.

The validation of a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the determination of the selective cyclooxygenase-2 inhibitor etoricoxib in human plasma with phenazone as internal standard is described. The plasma samples were extracted by solid-phase extraction using polymer-based cartridges. Chromatography was carried out on a short, narrow bore RP C(18) column (30x2 mm). Detection was achieved by a Sciex API 3000 triple quadrupole mass spectrometer equipped with a turbo ion spray source working in positive ion mode. The respective mass transitions used for quantification of etoricoxib and phenazone were m/z 359.2-->280.2 and m/z 189.0-->104.1. The analytical method was validated over the concentration range 0.2-200 ng/ml. The limit of quantification was 0.2 ng/ml. The method is applicable to pharmacokinetic studies in humans.

We investigated the effects of ursodeoxycholic acid (UDCA; 60 microg/day/100 g b.wt.) on the impairment induced by maternal obstructive cholestasis during pregnancy (OCP) in the rat placenta-maternal liver tandem excretory pathway. A blunted catheter was implanted in the common bile duct on day 14 of pregnancy, and the tip was cut on day 21. [(14)C]Glycocholate (GC) was then administered through the umbilical artery of "in situ" perfused placenta (placental transfer test) or through the maternal jugular vein (biliary secretion test), and GC bile output was measured. OCP impaired both GC placental transfer and maternal biliary secretion. UDCA moderately improved the latter but had a more marked beneficial effect on GC placental transfer. Histological examination revealed trophoblast atrophy and structural alterations, e.g., loss of apical membrane microvilli in OCP placentas. Gene expression level was investigated by real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis. OCP reduced both placental lactogen II (a trophoblast-specific gene) mRNA and the functional amount of epithelial tissue, determined by transplacental diffusion of antipyrin. Using a rapid filtration technique, impairment in the ATP-dependent GC transport across trophoblast apical plasma membranes obtained from OCP placentas was found. UDCA partially prevented all these changes. The expression level of organic anion transporters Oatp1, Oatp2, and Oatp4, and multidrug resistance-associated proteins Mrp1, Mrp2, and Mrp3 in whole placenta were not affected or were moderately affected by OCP but greatly enhanced by UDCA. In summary, UDCA partially prevents deleterious effects of OCP on the rat placenta-maternal liver tandem excretory pathway, mainly by preserving trophoblast structure and function.

Objective: We studied transplacental passage of lamotrigine (3,5-diamino-6-[2,3-dichlorophenyl]-1,2,4-triazine; LTG) using an ex vivo human placental perfusion method and in in vivo samples., Methods: Term placentas from healthy mothers without medications were perfused in a recirculating dual perfusion system. LTG (2.5 microg/ml, n=4; 10 microg/ml, n=4) and reference compound antipyrine (100 microg/ml) were added into the maternal circulation. The disappearance of drugs from the maternal circulation and appearance into the foetal circulation was followed every 15 min up to 2 h. Drug concentrations were analysed using high-performance liquid chromatography. In addition to human placental perfusions, we analysed LTG concentrations in maternal vein and cord blood samples after delivery from two epileptic mothers receiving LTG therapy during pregnancy., Results: LTG was detectable in the foetal circulation at 15 min in all of the perfusions, indicating rapid transfer. Maternal and foetal concentrations reached equilibrium at 60 min with both concentrations used. The feto-maternal ratio was 1.26+/-0.20 with 10 microg/ml LTG and 0.83+/-0.41 with 2.5 microg/ml LTG at the end of the perfusion. The transfer of LTG from the maternal to the foetal compartment at 120 min was 28.9+/-10.7% with 2.5 microg/ml LTG and 37.8+/-3.2% with 10 microg/ml LTG (p>0.05). In the serum samples from epileptic mothers, the cord blood maternal concentration ratio was 1.02 in one pair and 1.55 in the other., Conclusions: LTG crossed the placenta easily and rapidly, indicating that the maternal treatment leads to a considerable foetal exposure.

Introduction: Presently, no well-validated predictive tools are available for human placental transfer. We studied the transplacental passage of diazepam (DZP) in a recirculating dual human placental perfusion and compared the data with in vivo clinical data from the literature., Methods: Term placentas from healthy mothers without medication were used. The dual, recirculating perfusion technique was used. DZP (2 microg/ml, n = 4; 200 ng/ml, n = 3) and the reference compound antipyrine (100 microg/ml) were added into the maternal circulation simultaneously. The disappearance of drugs from the maternal circulation and appearance into the fetal circulation were followed every 15 min for 2 h., Results: DZP was detectable in the fetal circulation within 15 min in all of the perfusions indicating rapid transfer. DZP concentrations in the maternal circulation were higher than in the fetal circulation throughout the perfusion with both initial concentrations. At the end of the perfusion, the feto-maternal ratio was 0.48 +/- 0.11 (mean +/- S.D.) and the transfer from the maternal to the fetal compartment 18.4 +/- 3.6% with 2 microg/ml of DZP and 0.55 +/- 0.10 and 20.5 +/- 3.1% with 200 ng/ml of DZP, respectively. DZP concentrations in the perfused area of the placenta were in average 2 times higher than in the maternal perfusate and 3.6 times higher than in the fetal perfusate. Total recovery of DZP from samples, perfusion fluid, and perfused tissue was 37.6 +/- 21%., Discussion: Since animal studies in vivo do not accurately predict human placental transfer and it is problematic to study placental transfer of drugs in humans in vivo, the present human placental perfusion system could serve as one part of a test battery for fetotoxicity. However, although our earlier studies and those from the literature indicate a good correlation between in vivo and placental perfusion data, the present study shows this is not the case for all drugs.

1-Aminobenzotriazole (ABT) has been extensively used as a nonspecific inhibitor of cytochromes p450 (p450s) in animals for mechanistic studies, and antipyrine (AP) has been used as a probe for hepatic oxidative metabolic capacity determination in vivo. The method of use of ABT has been variable from lab to lab due largely to unknown pharmacokinetics of ABT itself and incomplete information on various p450s inhibited. The oral pharmacokinetic profiles of ABT were generated in rats, dogs, and monkeys in the dose range of 5 to 200 mg/kg. The results showed that after single oral doses of 50 mg/kg in rats, and 20 mg/kg in dogs and monkeys, the plasma concentrations were high and were sustained for over 24 h. In vitro, inhibition of various expressed p450s upon 30-min preincubation with ABT (0-500 micro M) showed that CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 were inhibited in a dose-dependent manner. The intravenous pharmacokinetics of AP also was affected in a dose-dependent manner in all species, treated 2 h earlier with ABT. Thus, the plasma clearance of AP was inhibited by 88% in rats pretreated with 50 mg/kg ABT and 96% in dogs and 83% in monkeys pretreated with 20 mg/kg ABT. Based on these data in rats, dogs, and monkeys, and the established safety profile of ABT in rats dosed up to 100 mg/kg, a pretreatment at 2 h with a single oral dose of ABT at 100 mg/kg in rats (providing 93% inhibition) and 20 mg/kg in dogs and monkeys effectively inhibited the clearance of the probe compound.

Studies of factors affecting the initial disposition of drugs with a rapid onset of effect following i.v. administration have used antipyrine as a surrogate for lipophilic drugs because it lacks cardiovascular effects. The present study tested the assumption that antipyrine is a useful surrogate for the flow-dependent tissue distribution of the lipophilic drug thiopental by comparing the recirculatory pharmacokinetic models of antipyrine and thiopental disposition after concomitant administration to five dogs anesthetized with 1.5% halothane. The pharmacokinetics of indocyanine green, a marker of the intravascular behavior of antipyrine and thiopental, and antipyrine in these dogs was nearly identical to that described previously in dogs anesthetized with 1.5% halothane but not given thiopental. The total volume of distribution of the highly lipophilic drug thiopental was more than 60% larger than that of antipyrine, 53 versus 33 liters, respectively. Nonetheless, the initial distribution kinetics of the two drugs, including the pulmonary tissue volume and the volume of the nondistributive pathway as well as the clearance to it, were nearly identical. As a result, the fraction of cardiac output involved in distribution of the two drugs to peripheral tissues was similarly identical, although the distribution of cardiac output between clearance to the rapidly equilibrating tissues and clearance to the slowly equilibrating tissues differed slightly. This study validates the assumption that antipyrine is a useful surrogate for lipophilic drugs in pharmacokinetic studies in which physiologic stability is desirable to meet the assumption of system stationarity.

Objectives: It has been suggested that regular physical activity might maintain and promote the antioxidant defense capacity against oxidative stress. Therefore, we assessed exercise-induced oxidative stress in relation to habitual physical activity level (PAL) in older adults., Design: The study included a 2-week observation period for the measurement of average daily metabolic rate (ADMR) and PAL. Exercise-induced oxidative stress was measured during a 45-minute cycling test at submaximal intensity., Setting: A university medical research center., Participants: Twenty-six subjects volunteered for the study (n = 26; mean age +/- standard deviation 60 +/- 1; body mass index 27 +/- 1 kg/m2)., Measurements: PAL was determined as ADMR combined with a measurement of basal metabolic rate (BMR): PAL = ADMR/BMR. ADMR was measured over 2 weeks with the doubly labeled water method, preceded by a BMR measurement with a ventilated hood. Antipyrine oxidation was used as marker for oxidative stress in vivo. Reaction of antipyrine with hydroxyl radicals results in the formation of para-hydroxyantipyrine (p-APOH) and ortho-hydroxyantipyrine (o-APOH), where o-APOH is not formed through alternative oxygenetic pathways., Results: PAL was inversely related to the exercise-induced increase in the ratio of o-APOH to native antipyrine (r = 0.49, P = .010). The relationship between PAL and exercise-induced increase in the ratio of p-APOH (r = 0.30, P = .140) or thiobarbituric acid reactive species (r = 0.31, P = .130) did not reach the level of significance., Conclusion: Physically active older adults have a reduced exercise-induced oxidative stress than older adults with a lower level of physical activity. It seems that regular physical activity improves the antioxidant defense capacity.

The effects of 4-hydroxyantipyrine (4-OH), a major metabolite of antipyrine and its sulfate, 4-hydroxyantipyrine O-sulfate (4-S), on the pharmacokinetics of citicoline and thiopental sodium were investigated in rats. The concomitant use of 4-OH increased significantly the tissue-to-plasma concentration ratio (Kp) of citicoline in the brain and liver and that of thiopental sodium in the brain, liver, and heart, while 4-S did not affect them. The permeability clearance of blood-brain barrier (BBB) (Kin) and the total distribution volume (Vdbr) of citicoline were not affected by either 4-OH or 4-S. However, those of thiopental sodium were significantly increased by not only 4-OH but also by 4-S. On the other hand, the plasma concentration of antipyrine was significantly decreased by the intravenous bolus coadministration of N-acetyl-p-aminophenyl O-sulfate (APAPS) at steady-state plasma concentration of antipyrine. A similar reduction was not observed with the intravenous coadministration of acetaminophen (APAP). The Kp value of antipyrine was significantly increased in the brain by the coadministration of APAPS, but was not affected by APAP. The increment in the drug distribution to the brain with the concomitant use of 4-OH (or APAPS) observed in this study is useful information for the application of drug combinations as biodistribution promoters.

Effects of 12 wk exercise training on oxidative stress were examined in elderly humans. We measured oxidative stress during a 45 min cycling test by using antipyrine hydroxylation products. Antipyrine breakdown is independent of blood flow to the liver, which is important during exercise. Furthermore, antipyrine reacts quickly with hydroxyl radicals to form para- and ortho-hydroxyantipyrine. Ortho-hydroxyantipyrine is not formed in man through the mono-oxygenase pathway of cytochrome P450. Twenty subjects (9 women; 60 +/- 3 y) participated in the training program. Thirteen subjects (5 women; 64 +/- 7 y) served as inactive controls. Subjects trained, twice a week for 1 h, at a fitness center. After 12 wk, maximal oxygen uptake (p < .005) and workload capacity (p < .001) were only significantly elevated in the training group. After 12 wk, both groups observed no change in the ratios of antipyrine hydroxylates, para- and ortho-hydroxyantipyrine, to native antipyrine. Furthermore, no differences were observed within or between groups in the exercise-induced increase in the plasma level of thiobarbituric acid reactive species. In conclusion, 12-wk training had no effect on exercise-induced oxidative stress in elderly humans as measured by free radical reaction products of antipyrine. Despite the fact that training in elderly humans improves functional capacity, it appears not to compromise antioxidant defense mechanisms.

Objective: The purpose of the present work was estimation of liver function using the phenazone test and commonly used biochemical tests in children with acute lymphoblastic leukemia (ALL) during anticancer treatment., Methods: Observations were carried out in the same 21 patients with ALL before the beginning of chemotherapy, after Protocol I and after Protocol M of the antileukemic treatment carried out according to the program BFM 86., Results: The applied chemotherapy inhibited phenazone elimination. Both phenazone half-life and metabolic clearance rate were significantly different in patients after treatment with anticancer drugs, especially with high-dose of methotrexate (MTX), from those in patients before the beginning of chemotherapy (p < 0.001). Moreover, after MTX administration transaminases activity and serum bilirubin concentration were significantly higher than before treatment (p < 0.05)., Conclusion: Our results showed that in children with acute lymphoblastic leukemia, anticancer chemotherapy decreased liver metabolic capacity. Particularly, high-dose methotrexate treatment altered the elimination of phenazone by inhibiting the activity of hepatic mixed function oxidase system. This change may lead to an increase in toxicity of active drugs which are metabolized by this enzyme system. In addition, altered activity of liver metabolic function can impair transformation of prodrugs to active forms. It should be considered in selection of individual drug dosages. The objective estimation of the type and degree of liver dysfunction can only be achieved by the combination of a quantitative phenazone dynamic test and static biochemical tests.

Recirculatory pharmacokinetic models for indocyanine green (ICG), inulin, and antipyrine facilitate description of intravascular mixing and tissue distribution following intravenous administration. These models characterized physiologic marker disposition in four awake dogs under control conditions and during phenylephrine, isoproterenol, and nitroprusside infusions. Systemic vascular resistance was more than doubled by phenylephrine and was decreased more than 50% by both isoproterenol and nitroprusside. Dye (ICG) dilution cardiac output (CO) was decreased nearly one-third by phenylephrine, was more than doubled by isoproterenol, and was largely unaffected by nitroprusside. Although phenylephrine reduced CO, the fraction of CO represented by nondistributive blood flow nearly doubled at the expense of blood flow to rapidly equilibrating tissues. The area under the blood antipyrine concentration versus time relationship for 3 min after administration (AUC(0-3 min)) during the phenylephrine infusion was nearly 75% larger than control due to both increased first-pass AUC and an increased fraction of CO represented by nondistributive blood flow. The large increase in CO produced by isoproterenol increased blood flow to rapidly equilibrating tissues and relatively decreased blood flow to slowly equilibrating tissues, because some appeared to equilibrate rapidly. Antipyrine AUC(0-3 min) during the isoproterenol infusion decreased more than 30%, due to decreased first-pass AUC. Nitroprusside changed antipyrine intercompartmental clearances in proportion to CO and, hence, had little effect on antipyrine AUC(0-3 min). These data provide further evidence that changes in antipyrine (a lipophilic drug surrogate) blood flow-dependent distribution after rapid i.v. administration are not proportional to changes in CO but depend on both CO and its distribution.

A novel and convenient in vitro method for predicting in vivo metabolic clearance in the liver (CL(H)) was developed. The CL(H) of a drug is usually predicted by using both the unbound fraction in serum and the intrinsic hepatic clearance of the unbound fraction, but this procedure is labor-intensive. We simplified the method by directly measuring intrinsic hepatic clearance using isolated rat hepatocytes suspended in rat serum and called this "the serum incubation method". Sixteen commercially available compounds reported to be mainly excreted by liver metabolism were evaluated using our method. The remaining ratio of the unchanged drug after incubation was measured to calculate the rate of metabolism, and then CL(H) was predicted based on the dispersion model. The predicted CL(H) values of the drugs estimated by the serum incubation method were in good agreement with their in vivo plasma clearance values. In addition, the intrinsic hepatic clearance values obtained by the serum incubation method were comparable with those obtained by conventional methods. Furthermore, oral bioavailability values were equal to or lower than hepatic availability values predicted from the serum incubation method. These results indicate that compounds showing poor oral bioavailability can be excluded before in vivo pharmacokinetic study by using this method. In conclusion, the serum incubation method is a convenient and useful tool at the early stage of drug discovery.

Here we present a method for measuring the permeability coefficient-surface area product (PS) values at the blood-brain barrier in mice, using the in situ brain perfusion technique originally developed for rats by Takasato et al. (Am J Physiol Heart Circ Physiol 247: H484-H493, 1984). Retrograde infusion into the right external carotid artery increased the carotid perfusion pressure in proportion to the perfusion rate. Intravascular volume and cerebral perfusion fluid flow at a perfusion rate of 1.0 ml/min in mice were similar to those in rats. In addition, the contribution of systemic blood to total flow in the hemisphere was small (only 3. 2%). These findings indicated that this perfusion rate is suitable for mice. The PS values of more than 20 different compounds were determined in mice by using the in situ brain perfusion technique, and comparisons were made with data from rats. There was a close relationship (1:1) between the PS values in mice and rats, indicating that brain capillary permeabilities are similar in mice and rats.

Autoradiographic measurement of local cerebral blood flow (CBF) with [14C]iodoantipyrine (IAP) is limited in mice by the difficulty in cannulating vessels and the blood loss for repeated blood sampling. The authors modified and validated the method to measure local CBF with [14C]IAP in mice by combining intraperitoneal tracer application with a single blood sampling from the heart at the end of the experiment. Experiments were carried out in male SV129 mice under halothane anesthesia. After intraperitoneal administration of 15 microCi [14C]IAP, arterial blood samples were collected repeatedly and anesthetized animals were immersed in liquid nitrogen. In addition, frozen blood from the heart was sampled to obtain the final blood [14C]radioactivity. Correlation analysis between the sampling time and [14C]radioactivity of the arterial blood revealed a highly significant linear relationship (P < 0.001, r = 0.978) and a lag time of the [14C]tracer in arterial blood of 3.3 +/- 0.6 seconds. [14C]radioactivity of the final arterial blood sample (444 +/- 264 nCi/mL) was almost equal to that of the heart blood (454 +/- 242 nCi/mL), and the absolute difference in each animal was 3.3 +/- 4.2% (mean +/- SD). The convolution integrals for the CBF calculation were determined either by integrating the radioactivity of individual arterial blood samples or by assuming a linear rise from [14C]tracer lag time after intraperitoneal [14C]IAP injection to the value measured in the blood sample from the frozen heart. Regional flow values calculated by the two methods differed by less than 11% (not significant). This method allows the quantitative measurement of local CBF in anesthetized mice without any vessel catheterization and will make mutant mice a more powerful tool to elucidate the molecular mechanisms of brain injuries by combining flow studies with molecular-biological methods.

Objective: To investigate the effects of fusidic acid therapy on the hepatic cytochrome P450 (CYP450) enzyme system., Methods: Thirty HIV-seropositive L-methadone-substituted i.v. drug abusers (stage CDC/WHO B2 - 3 with CD4+-counts ranging from 65 to 293/microl) were randomized into 3 groups (A - C). Ten patients were treated with fusidic acid 500 mg/day over a period of 14 (group A) or 28 days (group B), respectively. Patients in group C served as a control group and did not receive any medication apart from L-methadone. In order to investigate the hepatic monooxygenase system, pharmacokinetics were determined in all patients before initiation and 14 and 28 days after starting therapy with fusidic acid. The concentration of antipyrine and its 3 main metabolites (norantipyrine (NORA), 4-hydroxyantipyrine (OHA), 3-hydroxymethylantipyrine (HMA)) in plasma and urine were measured by high-performance liquid chromatography (HPLC)., Results: No effects on antipyrine pharmacokinetics and pharmacokinetics of antipyrine metabolites were found in group A after 14 days of fusidic acid intake and in the control group without therapy. However, in contrast an activation of the CYP450 enzyme system was observed in group B after 28 days of fusidic acid therapy with an increase of total antipyrine clearance (43.0 +/- 7.62 ml/min to 51.0 +/- 9.03 ml/min) as well as clearances to all metabolites (NORA 7.11 +/- 1.75 to 8.60 +/-2.10 ml/min, OHA 11.5 +/- 2.89 to 14.0 +/- 3.97 ml/min, HMA 4.05 +/- 0.99 to 4.94 +/- 1.27 ml/min). Antipyrine half-life was significantly reduced (12.3 +/- 2.8 h to 9.4 +/- 2.2 h) and some patients developed clinical signs of L-methadone underdosage., Conclusions: Our results suggest that fusidic acid has a time-dependent activating effect on the CYP450 enzyme system. Especially in treatment of patients who are frequently under multidrug regimens such as HIV-positive patients drug interactions should be taken into consideration.

To evaluate the potential activity of Schisandra chinensis in restoring hepatic drug metabolism in CCl4 damaged liver, antipyrine was employed as a probe for the possible effects of the herb on Phase I oxidative metabolism in rats. Schisandra lignan fraction (160 mg/kg) was given orally to male Sprague-Dawley rats (220-240 g) 30 min or 6 h before CCl4 intoxication (4 ml/kg, s.c.). Following a single oral dose of antipyrine (80 mg/kg) to the rats with damaged liver, the pharmacokinetics of antipyrine in whole blood were determined and levels of liver enzymes, e.g. SGPT, SGOT, and cytochrome P450 were measured. Pharmacokinetic parameters for antipyrine were estimated using noncompartmental analysis. Results indicated that CCl4 significantly increased the elimination half-life (t(1/2)) of antipyrine from 2.59 +/- 1.04 to 11.25 +/- 3.91 h (P < 0.001) and decreased its clearance (CL) from 65.94 to 10.84 ml/h as compared to control. Pretreatment with the Schisandra lignan fraction 30 min or 6 h before intoxication significantly (P < 0.001) improved antipyrine elimination by reducing its t(1/2) to 3.30 +/- 0.52 and 3.58 +/- 1.05 h, respectively. The corresponding improvements observed for CL, i.e. 49.06 +/- 21.75 ml/h (P < 0.01); 21.10 +/- 10.42 ml/h (P < 0.05), were also substantial. Moreover, normalization of SGPT, SGOT and P450 levels was observed with the two Schisandra pretreatment schedules. In conclusion, Schisandra lignans exhibited strong protective effect on Phase I oxidative metabolism in the liver damaged by CCl4. Furthermore, pretreatment of Schisandra 30 min before intoxication showed a more pronounced effect than that of the 6 h pretreatment. The current pharmacokinetic approach allowed the protective effects of Schisandra on oxidative drug metabolism in damaged liver to be systemically examined and will certainly help in the evaluation of hepato-protectants obtained from natural sources.

This manuscript describes a method to determine antipyrine and its phenolic derivatives in plasma by reversed-phase high-performance liquid chromatography-mass spectrometry (RP-HPLC-MS). The sample pretreatment consisted of a C18 solid-phase extraction (SPE), to remove the salts and proteins. The retention behavior of antipyrine and its phenolic derivatives in the SPE procedure was estimated by the k values determined on a C18 HPLC column at different pH values and with different buffer compositions. Recoveries of antipyrine and its phenolic products were 90% in water and 100% in plasma. Atmospheric pressure chemical ionization (APCI) was used to introduce the components into the mass spectrometer. The mass spectrometer was operated in the single ion monitoring mode (SIM mode) as well as in the selective reaction (SR) mode. The SR mode or tandem MS resulted in the best signal-to-noise ratio, with a detection limit for antipyrine of 6 pg in 20 microl. For the different phenolic antipyrines, different target ions were used and conditions were optimized for each.

The effect of gabapentin on antipyrine clearance was assessed in 12 healthy male volunteers, using a known enzyme inducer, phenytoin, as control. Subjects received gabapentin 400 mg or phenytoin 100 mg three times daily for 2 weeks. Antipyrine tests were performed before, during, and after treatment with gabapentin or phenytoin. In contrast to phenytoin, chronic administration of gabapentin did not affect antipyrine clearance. Gabapentin appears to have little potential for drug interactions.

Background: Hepatic drug metabolism was examined in patients with chronic pancreatitis and healthy controls by using a cocktail design with three different model compounds: antipyrine to express phase-I oxidation, oxazepam to express phase-II conjugation, and indocyanine green (ICG), a high-clearance compound., Methods: Eight patients with chronic pancreatitis and seven healthy controls participated. Patients were diagnosed by the presence of typical morphologic changes of the pancreas on imaging and had a moderately but significantly reduced exocrine function and no or only slight impairment of the glucose tolerance. No one had a history or clinical signs of liver disease. Clearance of the three model compounds was estimated after the administration of 1 g antipyrine and 15 mg oxazepam orally and a bolus of indocyanine green, 0.5 mg/kg body weight, intravenously., Results: The antipyrine clearance and ICG clearance were significantly decreased in the patients compared with the controls (mean, 27.2 ml/min; 95% confidence interval (CI), 19.4-35; versus 46.2 ml/min; 34.7-58.7, and 501 ml/min; 4014601, versus 771 mU/min; 677-865 (P < 0.05), respectively). The oxazepam clearance did not differ significantly between the two groups (181 ml/min (145-217) versus 178 ml/min (152-204)). The model drug clearance ratios between the patient and control clearances showed decreased values for antipyrine and ICG compared with the oxazepam data (0.59 and 0.65 versus 1.02, respectively). Patients and controls were characterized by a body weight of 58.2 kg (53.1-63.3) and 83.4 kg (72.7-94.1), respectively, and a body mass index (BMI) of 19.6 kg/m2 (17.9-21.3) versus 25.9 kg/m2 (23.4-28.4) (P < 0.05 for both)., Conclusions: Patients with chronic pancreatitis characterized by a moderately reduced exocrine function and absence of diabetes mellitus and overt liver disease had a decreased antipyrine oxidation and ICG clearance, whereas no difference was seen in oxazepam conjugation when compared with healthy volunteers. In chronic pancreatitis the hepatic phase-I oxidation is reduced compared with the phase-II conjugation, as shown by the model drug clearance ratios. The clearance of ICG was also affected, pointing at a reduced hepatic plasma flow, provided that the hepatic extraction fraction is normal for these patients.

The aim of this study was to determine the effect of sex on the metabolism of antipyrine by measuring the antipyrine plasma clearance as well as excretion of three major metabolites in urine in cattle of different ages. The experiment was carried out on 10 female and 10 male cattle of Black and White breed. The antipyrine test was carried out at 1, 2, 4, 6, 8, 12 and 18 months of age for each animal (single dose of 10 mg/kg antipyrine were given intravenously). The concentrations of antipyrine, 4-hydroxyantipyrine (4-OHA), 3-hydroxymethylantipyrine (HMA) and norantipyrine (NORA) were measured in plasma and urine by high performance liquid chromatography (HPLC). The apparent volume of distribution of antipyrine (aVd) decreased significantly between 1 and 18 months of age, but mean aVd values observed in males and females were not statistically different. The experimental period was characterised by a steady decrease (statistically significant) in antipyrine half-life (t1/2beta). These values did not differ significantly between males and females under 12 months. In 12 and 18 month-old animals the antipyrine half-life in the females was significantly shorter than in the males. The systemic clearance (Cls) of antipyrine increased significantly between 1 and 18 months of age. No significant differences were observed between systemic clearance of antipyrine in males and females under 12 months. In 12 and 18 month-old animals the Cls values were significantly higher in females than in males. Following intravenous administration, recovery of antipyrine and its three main metabolites increased significantly with age. These values did not differ significantly between males and females under 12 month of age. In 12 and 18 month-old females the excretion of 4-OHA and HMA in urine was significantly higher than in males at the same age. The excretion of NORA and unchanged antipyrine in males and females did not differ significantly. The partial clearances of antipyrine metabolites (Cl(m)) increased significantly between 1 and 18 months of age. No significant differences were observed between Cl(m) values in males and females under 12 months of age. In 12 and 18 month-old females the partial clearances of 4-OHA and HMA were significantly higher than in males. The clearance of NORA was significantly higher in 18 month-old females than in males. In conclusion, we report a sex-linked difference in plasma antipyrine clearance and urinary excretion of the main metabolites of antipyrine in cattle over 12 months of age, the females being the more active metabolizers.

The plasma pharmacokinetics of antipyrine, warfarin and paracetamol have been studied in the Australian brushtail possum (Trichosurus vulpecula). The plasma elimination half-lives (t1/2) were 1.2 h for antipyrine, 11.9 h for warfarin and 5.2-12.9 h for paracetamol. Our data indicate that the clearance of these three xenobiotics in the possum is similar to that reported in eutherian mammals. There was no dose-dependent increase in paracetamol plasma t1/2 over the dose range 100-1000 mg kg(-1), indicating a lack of capacity saturation. This observation may in part explain the unusual resistance of the possum to the hepatotoxic effect of high doses of paracetamol.

Objective: The aim of this work was to study the influence of neoplastic disease, especially acute myeloblastic leukemia (AML), and its chemotherapy on the activity of hepatic microsomal enzymes by using phenazone, as a marker of oxidative drug metabolizing activity., Methods: The observations were carried out in 21 patients with AML and in 53 healthy volunteers. The influence of disease on phenazone kinetics was studied before chemotherapy and the effect of anticancer drugs administration after the first cycle of chemotherapy., Results: The mean phenazone half-life time was significantly shorter in patients with AML (8.79 (3.01) h) than in control group (11.08 (3.61) h) (p < 0.012). Treatment with anticancer drugs, especially with epirubicine, inhibited phenazone elimination. The mean phenazone half-life time was significantly longer (18.08 (8.80) h) and the mean metabolic clearance rate was significantly smaller (33.92 (15.40) ml/min) after chemotherapy in comparison with the initial value, before treatment (10.22 (2.90) h), (p < 0.01) (50.33 (20.29) ml/min) (p < 0.008)., Conclusion: Our results lead to the conclusion that phenazone is an important index of hepatic metabolic capacity in patients with acute myeloblastic leukemia. The evaluation of its kinetics allowed to early recognition of the presence and the degree of drug oxidizing modification. Acceleration of phenazone elimination before treatment and its inhibition after chemotherapy, particulary epirubicine, may suggest that in patients with AML elimination of the other drugs metabolized by the pathway similar to phenazone also may be changed. It should be considered in individualization of their dosage regimen.

The acute phase response (APR) was induced by five separate intravenous (i.v.) injections of Escherichia coli lipopolysaccharide (LPS, 17 microg/kg each time) in rabbits, with intervals of 1 h. This model was used to study the effects of APR on the activities of hepatic microsomal cytochrome P450 (CYP)-dependent enzyme including drug metabolism. Five female rabbits were included in each of four groups, a control group and three LPS-treated groups (group I, II and III). The rabbits of the control, group I, II and III were killed at 1, 1, 3 and 7 days after saline (control only) or the LPS injection, respectively. The APR was confirmed by increases in rectal body temperature, plasma concentrations of interleukin-6 and C-reactive protein (CRP). Pharmacokinetics of antipyrine before death were examined in every group. Antipyrine was administered (5 mg/kg) at 24 h (control and group I), 3 days (group II) and 7 days (group III) after the first LPS injection. Total body clearance (Cl(tot)) of antipyrine tended to decrease in group I. All the livers were excised for measuring CYP-dependent activities. Total CYP content and several CYP-dependent activities (aminopyrine N-demethylation, aniline 4-hydroxylation and caffeine 3-demethylation) decreased in group I. The maximum velocity (Vmax) values of those enzymes, and the amount of CYP1A1/1A2 and CYP2E1 apoproteins appeared to decrease. Michaelis constant (Km) values of those enzymes were not affected by the APR. Rectal body temperature recovered to normal at 3 days after the first LPS injection in group II and III. The concentration of CRP, albumin, total CYP content and the plasma clearance of antipyrine returned to the control levels at 7 days after the first LPS injection. These results suggest that the metabolism of drugs, including CYP-dependent drug metabolizing activity, is suppressed markedly in incipient APR induction in rabbits, and the drug metabolizing capacity is returned to normal at 7 days after APR induction.

Purpose: The objectives of dermal application of drugs are not only systemic therapeutic, but also local ones. We would expect its intradermal kinetics to be dependent on its therapeutic purpose. To develop more efficient drugs for local or systemic therapeutics, it will be important to estimate quantitatively the intradermal disposition of drugs applied topically. We tried, therefore, to develop the compartment model to describe the intradermal disposition kinetics after topical application of drugs., Methods: In vivo percutaneous absorption study for antipyrine, a model compound, was performed using rats with tape-stripped skin, using the assumption that the stratum corneum permeability to drugs would be improved enough not to be a rate-limiting process., Results: To analyze the results obtained, a 4-compartment model, composed of donor cell, viable skin, muscle, and plasma compartments, was applied. Although the fitting lines obtained could describe the concentration-time profiles of antipyrine in each compartment very well, the concentration profiles in the contralateral tissues were extensively overestimated. Therefore, we developed a 6-compartment model which included the viable skin and muscle in the contralateral site, and analysed the concentration-time curve of each compartment. The fitting curves were in good agreement with the experimental data for all the compartments including the contralateral viable skin and muscle, and thus, this model was recognized to be adequate for the estimation of intradermal kinetics after topical application. Judging from the obtained values of clearance from viable skin to plasma and from viable skin to muscle, about 80% of antipyrine penetrated into viable skin, which suggested it was absorbed into circulating blood and 20% was transported to muscle under viable skin., Conclusions: Pharmacokinetic analysis using the 6-compartment model would be very useful for the estimation of local and systemic availability after topical application of drugs.