bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis, antiplatelet and vasorelaxing effects of monooxygenated flavones and flavonoxypropanolamines
Authors: Chung Mei-Ing , Han Feng-Wen , Lin Hsien-Cheng , Liou Shorong-Shii , Huang Pao-Ling , Ko Horng-Huey , Chang Ya-Ling , Kang Jaw-Jou , Teng Che-Ming , Lin Chun-Nan
Published: July 2003

A series of flavones and flavonoxypropanolamines were synthesized and tested in-vitro for their ability to inhibit aggregation of washed rabbit platelets and human platelet-rich plasma (PRP), and for vasoconstriction of rat thoracic aorta. The various substituted positions of the hydroxyl group in flavone ring B and the various oxypropanolamine side chains substituted at position C-2′ of flavone modified the antiplatelet effects. All the compounds tested in human PRP showed significant inhibition of secondary aggregation induced by adrenaline (epinephrine), suggesting that the antiplatelet effect of these compounds is mainly due to an inhibitory effect on thromboxane formation. Compounds 11 and 12 also had potent vasorelaxant effects in rat thoracic aorta. Phenylephrine- and high-K+-induced 45Ca2+ influx in aorta were both inhibited by the selected compound 11. This result indicates that the inhibitory effect of 11 on the contractile response caused by high-K+ medium and noradrenaline (norepinephrine) in rat thoracic aorta is mainly due to inhibition of Ca2+ influx through both voltage-dependent and receptor-operated Ca2+ channels.
The impact of P-glycoprotein efflux on enterocyte residence time and enterocyte-based metabolism of verapamil
Authors: Johnson Brendan M , Charman William N , Porter Christopher J H
Published: July 2003

P-glycoprotein (P-gp) can limit the intestinal permeability of a number of compounds and may therefore influence their exposure to metabolizing enzymes within the enterocyte (e.g. cytochrome P450 3A, CYP 3A). In this study, the intestinal metabolic profile of verapamil, the influence of P-gp anti-transport on the cellular residence time of verapamil, and the impact of this change in residence time on the extent of enterocyte-based metabolism have been investigated in-vitro, utilizing segments of rat jejunum and side-by-side diffusion chambers. Verapamil exhibited concentration-dependent P-gp efflux and CYP 3A metabolism. The P-gp efflux of verapamil (1 μm) increased the cellular residence time across the intestinal membrane (approximately 3-fold) in the mucosal to serosal (m to s) direction relative to serosal to mucosal (s to m), yielding significantly greater metabolism (approximately 2-fold), presumably as a result of the prolonged exposure to CYP 3A. Intestinal metabolism of verapamil generated not only norverapamil, but resulted also in the formation of an N-dealkylated product (D-617). Norverapamil and D-617 accumulated significantly in mucosal chambers, relative to serosal chambers, over the time course of the experiment. Based on these in-vitro data, it was apparent that P-gp efflux prolonged the cellular residence time of verapamil (m to s) and therefore increased the extent of intestinal metabolism, and also played a role in metabolite secretion from within the enterocyte.
Stereoselective pharmacokinetics and pharmacodynamics of disopyramide and its metabolite in rabbits
Authors: Horikawa Masato , Yasumuro Misao , Kanno Megumi , Hanada Kazuhiko , Hashiguchi Masayuki , Ogata Hiroyasu
Published: July 2003

The extent to which interactions between enantiomers of disopyramide and between disopyramide and its metabolite, mono-N-dealkylated disopyramide (MND), contribute to stereoselectivity of the anti-arrhythmic effect has been investigated in rabbits by measuring the prolongation of the QUc interval. The plasma unbound fraction of disopyramide enantiomers was constant at a concentration range of 1.44-28.9 μm. An intravenous infusion study of the disopyramide enantiomer or racemate suggested that the S-enantiomer had a pharmacological effect, determined by linear regression analysis, approximately 3.3-times more potent than that of the R-enantiomer. Furthermore, the effect caused by racemic disopyramide was the sum of that elicited by both enantiomers individually. No significant difference was observed between the slope of linear regression analysis of intravenous infusion and that of intravenous bolus injection. Single intravenous bolus injection of MND did not affect the QUc intervals. In conclusion, the S-enantiomer of disopyramide was approximately 3.3-times more potent pharmacologically than the R-enantiomer. The relationship between plasma concentration of the disopyramide enantiomers and pharmacological effect was the sum of each enantiomer individually.
Pharmacokinetics and interspecies scaling of a novel VEGF receptor inhibitor, SU5416
Authors: Sukbuntherng Juthamas , Cropp Gillian , Hannah Alison , Wagner Gregory S , Shawver Laura K , Antonian Lida
Published: July 2003

The pharmacokinetics and allometric relationships of SU5416, a novel small anti-angiogenesis agent, were studied. The pharmacokinetics of SU5416 were examined in mice, rats, dogs, and cancer patients. The in-vitro intrinsic clearance (CLint) was estimated from the in-vitro metabolism study in mouse, rat, dog, monkey and human liver microsomes. The parameters of interest were correlated across species as a function of bodyweight using an allometric approach. The steady-state volume of distribution (Vdss), plasma clearance (CLs), and CLint of SU5416 were well correlated across species. The exponent of the allometric relationship (b) of the corresponding parameters was 0.92, 0.80 and 0.66, respectively. The elimination half-life (t½) was consistent across species and independent of bodyweight. The prediction of CLs, Vdss, CLint, and t½ in humans using the data from mouse, rat, and dog, and monkey (for CLint) was reasonably good (within 4-fold of the observed values). However, an improved prediction (within 2-fold of the observed values) of the corresponding parameters in humans was obtained when extrapolation from only the rodent data was performed, suggesting that the rodent data are sufficient for the scale-up of SU5416 pharmacokinetic parameters in humans. Using allometry, it was possible to achieve reasonable predictions of the pharmacokinetic parameters of SU5416 in cancer patients with various solid tumours.
A validated method for quantitative determination of saponins in notoginseng (Panax notoginseng) using high-performance liquid chromatography with evaporative light-scattering detection
Authors: Li Wenkui , Fitzloff John F
Published: July 2003

A gradient high-performance liquid chromatographic (HPLC) method with evaporative light-scattering detection (ELSD) for the determination of major saponins in a Chinese traditional herbal medicine, Panax notoginseng, is described. Samples were analysed by means of a reverse-phase column (Waters Spherisorb ODS-2, C-18) using acetonitrile and water under gradient conditions as the mobile phase over 60 min. The ELSD used was set at an evaporating temperature of 35°C and pressurized air pressure of 3.4 bars. The detection limit (signal/noise > 3) of the saponins was 50 ng. The method was validated by inter- and intra-day assays and recovery tests.
Topical anti-inflammatory activity of Thymus willdenowii
Authors: Ismaili H , Tortora S , Sosa S , Fkih-Tetouani S , Ilidrissi A , Loggia R Della , Tubaro A , Aquino R
Published: July 2003

The topical anti-inflammatory activity of Thymus willdenowii Boiss (Labiatae) leaves, a herbal drug used in Moroccan folk medicine, has been studied using the croton oil ear test in mice. A bioassay-oriented fractionation procedure showed that the activity concentrates in the chloroform extract, which has a potency similar to that of indometacin, the non-steroidal anti-inflammatory drug used as reference (ID50 (dose giving 50% oedema inhibition) = 83 μg cm−2 and 93 μg cm−2, respectively). The main compounds responsible for the anti-inflammatory activity of T. willdenowii are ursolic acid and oleanolic acid. The flavonoids luteolin-3′-O-glucuronide and eriodictyol-7-O-glucoside were found for the first time in the genus Thymus.
Comparison of the growth-inhibitory effect of Hypericum perforatum L. extracts, differing in the concentration of phloroglucinols and flavonoids, on leukaemia cells
Authors: Hostanska Katarina , Bommer Silvia , Weber Michel , Krasniqi Blerim , Saller Reinhard
Published: July 2003

In this study we compared, simultaneously, the growth-inhibitory effect of Hypericum perforatum L. extracts, containing various amounts of hyperforin (A 3.25%; B 2.21%; C 0.21% w/w) and flavonoids (A and B 5.3%; C 10% w/w), but closely same amounts of naphthodiantrones (0.3%) on two leukaemic cell lines K562 and U937 in the WST-1 assay. The GI50 (concentration of extracts which caused 50% of cell growth inhibition) for H. perforatum extracts analysed and characterized by HPLC for their biologically active constituents was 248.3–621.3 μg mL−1 in K562 and 378.2–911.7 μg mL−1 in U937 cells. The corresponding values of the three main groups were 1.6–3.9 μm naphthodianthrones, 1.0–40.7 μm phloroglucinols and 30.5–68.5 μm flavonoids. The results of this study supported the hypothesis that, apart from hyperforin and flavonoids, other components of the extract could be involved in its growth-inhibitory effect that it exerts without light activation.
Effect of surfactant on fabrication and characterization of paclitaxel-loaded polybutylcyanoacrylate nanoparticulate delivery systems
Authors: Mitra Amitava , Lin Senshang
Published: July 2003

The feasibility of applying biodegradable polybutylcyanoacrylate (PBCA) nanoparticulate delivery systems (NDSs) for the controlled release of paclitaxel was investigated. Paclitaxel-loaded and unloaded PBCA-NDSs containing various surfactants (dextran 70, cholesterol, polyvinyl alcohol and lecithin) were prepared by anionic polymerization. The effects of surfactant (1% w/v), surfactant combination (1% w/v each), and surfactant concentration (0.05, 1.0 and 2.5% w/v) on PBCA-NDSs were evaluated and characterized by particle size, zeta potential, entrapment efficiency, and in-vitro paclitaxel release kinetics. The physicochemical characteristics of PBCA-NDSs incorporated with various surfactants were significantly improved compared with PBCA-NDS without any surfactant, by decreasing particle size at least 3-fold as well as by increasing the zeta potential up to 18-fold to minimize the agglomeration of nanoparticles. Moreover, PBCA-NDSs incorporated with various surfactants demonstrated higher entrapment efficiency of paclitaxel. Results from the in-vitro release kinetic studies indicated that a more controlled biphasic zero-order release pattern of paclitaxel was observed for PBCA-NDSs incorporated with various surfactants. Compared with dextran 70 and polyvinyl alcohol, the naturally occurring lipids, lecithin and cholesterol, indicated greater advantages in improving the physicochemical properties of PBCA-NDSs, in terms of smaller particle size, higher zeta potential and better drug entrapment efficiency, and better controlled release of paclitaxel, in terms of lower release rate and prolonged action from PBCA-NDSs.
Formulation and evaluation of metronidazole acid gel for vaginal contraception
Authors: El-Gizawy Sanaa A , Aglan Nagwa I
Published: July 2003

In this study, the efficacy of metronidazole as a local spermicidal agent was investigated. The drug was formulated in a concentration of 5% as an acid gel for vaginal application as a local contraception. The minimum spermicidal concentration of metronidazole was evaluated in-vitro and found to be 0.2% w/v. The formulated gel showed instant immobilization and death of all sperm within 30 s. A clinical study was conducted to determine the drug concentrations in vaginal secretions of healthy women and women with symptoms suggesting genital tract infection every 15 min for a 1-h period after application of 1 g of the gel in the vagina. Drug concentrations in the infected group were significantly lower (P<0.05) than those in the healthy group at all time intervals. The drug concentrations in vaginal secretions after 1 h of vaginal medication remained above the determined minimum spermicidal concentration (0.2%) in the two groups. Measurement of vaginal pH before and after medication revealed a significant drop (P<0.05) in pH to its normal value in both healthy and infected groups as a result of gel application. In conclusion, the designed gel has potential advantages of achieving a long retention time and effective drug concentrations in the vagina for at least 1 h after application, and of maintaining vaginal pH within its normal range.
Contribution of organic anion transporting polypeptide OATP-C to hepatic elimination of the opioid pentapeptide analogue [d-Ala2, d-Leu5]-enkephalin
Authors: Nozawa Takashi , Tamai Ikumi , Sai Yoshimichi , Nezu Jun-Ichi , Tsuji Akira
Published: July 2003

The objective of this study was to examine the transport activity of the human organic anion transporter OATP-C (SLC21A6) for oligopeptides that are eliminated rapidly from the systemic circulation. We focused on an opioid peptide analogue, [d-Ala2, d-Leu5]-enkephalin (DADLE), a linear pentapeptide modified to be stable. [3H]DADLE was taken up by rat isolated hepatocytes in a saturable manner and highly accumulated in the liver after intravenous administration to rats. The uptake of [3H]DADLE by the isolated hepatocytes was inhibited by several organic anions and pentapeptides, but not by tetra- or tripeptides. When OATP-C was expressed in Xenopus laevis oocytes, a significant increase in uptake of [3H]DADLE was observed. Moreover, the inhibitory effects of various compounds, including some peptides, on [3H]estrone-3-sulfate uptake by OATP-C were similar to those observed in [3H]DADLE uptake by rat isolated hepatocytes. In conclusion, it was demonstrated that OATP-C contributes to the rapid hepatic excretion of peptides and peptide-mimetic drugs.
Inhibitory effect of erythromycin on potassium currents in rat ventricular myocytes in comparison with disopyramide
Authors: Hanada Erika , Ohtani Hisakazu , Hirota Michiko , Uemura Noriko , Nakaya Haruaki , Kotaki Hajime , Sato Hitoshi , Yamada Yasuhiko , Iga Tatsuji
Published: July 2003

Disopyramide, a class la antiarrhythmic agent, has been reported to induce torsades de pointes (TdP) associated with excessive QT prolongation in electrocardiogram (ECG), especially when concomitantly administered with erythromycin, a macrolide antibiotic agent. In this study, we have evaluated the effects of erythromycin on action potential duration (APD) and potassium currents in rat ventricular myocytes in comparison with disopyramide. We have evaluated the relationship between in-vitro potassium current inhibition and in-vivo QT prolongation observed in a previous study. Action potentials and membrane potassium currents, including delayed rectifier current (IK) and transient outward current (Ito), were recorded using a whole-cell patch clamp method in enzymatically-dissociated ventricular cells. Erythromycin and disopyramide prolonged APD in a concentration-dependent manner. Disopyramide (10–100 μm) and erythromycin (100 μm) led to increases in the APD at 90% repolarization level. Disopyramide reduced IK (IC50 = 37.2 + 0.17 μm) and Ito (IC50 = 20.9 + 0.13 μm) while erythromycin reduced IK (IC50 = 60.1 + 0.29 μm) but not Ito. The observed prolongation of APD might be ascribed to the inhibition of potassium currents. Erythromycin produced the prolongation of APD and the inhibition of potassium currents with a lag time after addition of the drugs, which suggested that erythromycin might not reach potassium channels from outside the ventricular cells. The potency of disopyramide was almost equivalent under in-vitro and in-vivo conditions. However, potency of erythromycin in-vitro was far weaker than that in-vivo reported in a previous study, presumably due to a difference in the uptake of erythromycin into ventricular myocytes between in-vivo and in-vitro conditions. Therefore, when drug-induced risks of QT prolongation are to be evaluated, the difference of potencies between in-vitro and in-vivo should be taken into consideration.
Screening pharmaceutical preparations containing extracts of turmeric rhizome, artichoke leaf, devil's claw root and garlic or salmon oil for antioxidant capacity
Authors: Betancor-Fernández Alejandro , Pérez-Gálvez Antonio , Sies Helmut , Stahl Wilhelm
Published: July 2003

Pharmaceutical preparations derived from natural sources such as vegetables often contain compounds that contribute to the antioxidant defence system and apparently play a role in the protection against degenerative diseases. In the present study, commercial preparations containing extracts of turmeric, artichoke, devil's claw and garlic or salmon oil were investigated. The products were divided into fractions of different polarity, and their antioxidant activity was determined using the Trolox equivalent antioxidant capacity (TEAC) assay. This test is based on the efficacy of the test material to scavenge 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) derived radicals. Total phenols were determined in all fractions as well as specific carotenoids in the most lipophilic fraction to assess their contribution to the antioxidant activity. For comparison, the radical scavenging effect of selected constituents of the extracts such as curcumin, luteolin, kaempferol, chlorogenic acid, harpagoside, β-carotene and α-tocopherol was investigated and compared with that of Trolox. Curcumin, luteolin, kaempferol, chlorogenic acid and β-carotene showed an antioxidant activity superior to Trolox in the TEAC assay; harpagoside was barely active. All fractions of the turmeric extract preparation exhibited pronounced antioxidant activity, which was assigned to the presence of curcumin and other polyphenols. The antioxidant activity corresponding to the artichoke leaf extract was higher in the aqueous fractions than in the lipophilic fractions. Similarly, devil's claw extract was particularly rich in water-soluble antioxidants. Harpagoside, a major compound in devil's claw, did not contribute significantly to its antioxidant activity. The antioxidant capacity of the garlic preparation was poor in the TEAC assay. That of salmon oil was mainly attributed to vitamin E, which is added to the product for stabilization. In all test preparations, the antioxidant activity was significantly correlated with the content of total phenolic compounds.
Prediction of human pharmacokinetics from animal data and molecular structural parameters using multivariate regression analysis: volume of distribution at steady state
Authors: Wajima Toshihiro , Fukumura Kazuya , Yano Yoshitaka , Oguma Takayoshi
Published: July 2003

The aim of this study was to develop a regression equation for predicting volume of distribution at steady state (Vdss) in humans to enable application to various types of drugs using animal experimental data for rats and dogs and some molecular structural parameters. The Vdss data for rats, dogs and humans of 64 drugs were obtained from literature. The compounds have various structures, pharmacological activities and pharmacokinetic characteristics. In addition, the molecular weight, calculated partition coefficient (clogP), and the number of hydrogen bond acceptors were used as possible descriptors related to the Vdss in humans. Multivariate regression analyses, multiple linear regression analysis and the partial least squares (PLS) method were used to predict Vdss in humans. Interaction terms were also introduced into the regression analysis to evaluate the non-linear relationship. For the data set used in the present study, PLS with quadratic term descriptors gave the best predictive performance. The PLS model using Vdss data for only two animal species and using easily calculated structural parameters could generally predict Vdss in humans better than an allometric method. In addition, the PLS model with only animal data gave almost the same predictive performance as the PLS model with quadratic term descriptors. This model may be easier to use and be practical in a realistic situation, and could predict Vdss in humans better than the allometric method.
Regional transport and metabolism of ropivacaine and its CYP3A4 metabolite PPX in human intestine
Authors: Berggren Sofia , Lennernäs Pernilla , Ekelund Mats , Weström Björn , Hoogstraate Janet , Lennernäs Hans
Published: July 2003

The major aim of this study was to investigate the CYP3A4 metabolism and polarized transport of ropivacaine and its metabolite 2′,6′-pipecoloxylidide (PPX) in tissue specimens from the human small and large intestine. Ropivacaine has been shown to be effective in the treatment of ulcerative colitis in human colon. This study was conducted using a modified Ussing-chamber technique with specimens from jejunum, ileum and colon collected from 11 patients. The local kinetics of ropivacaine and PPX were assessed from their concentration–time profiles in mucosal and serosal compartments. The permeability (Papp) in the absorptive direction for both ropivacaine and PPX increased regionally in the order jejunum < ileum < colon. Ropivacaine was not found to be subjected to any carrier-mediated intestinal efflux. However, the CYP3A4 metabolite left the human enterocyte in a polarized manner and both the extent of CYP3A4 metabolism of ropivacaine and the extrusion of its metabolite to the mucosal chamber were more efficient in jejunum than in ileum. P-glycoprotein was probably not involved in the metabolite extrusion. No other metabolite than PPX was found. This in-vitro study with human intestinal tissues provides new mechanistic insights into regional transport and metabolism of drugs.
Interactions of a series of novel spiropyranocoumarin derivatives with reactive oxygen species
Authors: Panteleon Vassiliki , Marakos Panagiotis , Pouli Nicole , Mikros Emmanuel , Andreadou Ioanna
Published: July 2003

A series of new spiro-substituted pyranocoumarin derivatives have been synthesized starting from the commercially available 7-hydroxycoumarin and the conformation of the pyran ring was investigated. The antioxidant activity of the compounds was evaluated in-vitro, by means of three different tests: the interaction with the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), the competition with DMSO for hydroxyl radicals scavenging ability and the quenching of superoxide anions generated by the enzymic xanthine–xanthine oxidase system. In the DPPH test the spiroadamantane derivative 13 was the most active and possessed a 40% inhibition at a concentration of 400 μm. All compounds successfully compete with DMSO for hydroxyl radicals generated by the Fe3+/ascorbic acid system. Compound 13 inhibited the oxidation of DMSO (3.125mm) by 93% at 2 mm and by 71% at 0.25 mm. The corresponding second-order rate constants have been estimated and all compounds demonstrated higher rate constants compared with the reference compounds, 7-hydroxycoumarin and mannitol. Derivatives possessing extended conjugation showed the highest inhibitory activity for superoxide anions generated by the xanthine–xanthine oxidase system, although the results of this experiment possessed partial parallelism with the results observed in the other two tests. The overall obtained data indicate that the size of the different spiro- substituents influence the degree of free radical scavenging and demonstrate the importance of extended conjugation for the anti-oxidant activity. Due to its multiple mechanism of protective action, derivative 13 may serve as a lead for the development of analogues that could be useful for the treatment of pathophysiological processes dependent upon reactive oxygen species.
Synergistic Capsicum–tea mixtures with anticancer activity
Authors: Morré D James , Morré Dorothy M
Published: July 2003

We have demonstrated a synergy between a decaffeinated green tea concentrate and a vanilloid-containing Capsicum preparation obtained commercially. At a ratio of 25 parts green tea concentrate to 1 part Capsicum preparation, the resultant product exhibited efficacy in the killing of cancer cells in culture 100-times that of green tea on a weight basis. These studies were guided by assays of the putative catechin-vanilloid target protein tNOX, a cell surface growth-related enzymatic activity specific to cancer. The activity of the protein target was inhibited by the tea catechins and the Capsicum vanilloids. As with growth, the tea and Capsicum preparations evaluated were synergistic in their inhibition of the target enzymatic activity.
Efficient Biliary Excretion of Susalimod, Probably via the Bromosulphthalein Carrier, Studied in a Chronic Bile Fistula Model in Dogs
Authors: Påhlman Ingrid , Wilén Maria , Bowald Staffan
Published: July 2003

Susalimod is a structural analogue of sulphasalazine, known to be extensively excreted in the bile in various animal species and for inducing bile duct hyperplasia after long-term treatment of the dog with doses exceeding 25 mg kg−1. In this study local concentrations of susalimod in the bile duct were determined after oral administration in dogs.

A chronic bile fistula experimental model was designed to affect the bile duct as little as possible. The dogs received repeated oral doses of 25–150 mg kg−1 day−1 for 5 days; these doses had been used in previous toxicology studies. Extremely high biliary concentrations of unchanged susalimod (20 000–43 000 μm) were measured. Biliary excretion approached saturation at the higher doses, resulting in super-proportional increases in peripheral plasma concentrations as the dose was increased. The maximal bile/plasma concentration ratio was 4300. The high biliary clearance was indicative of almost complete first-pass elimination at doses below saturation of the elimination process. Interaction studies with the biliary excretion marker bromosulphthalein (BSP) demonstrated that susalimod and BSP probably share the same carrier transport system in biliary excretion. The elimination of BSP from plasma was prolonged 20 times and the biliary excretion rate was markedly reduced when susalimod was co-administered with BSP.

These results show that susalimod is highly enriched in the bile, in a saturable manner, after oral administration. The compound interacts with the biliary excretion of BSP, suggesting that it shares the same carrier-mediated transport system.
Dose-dependent Intestinal and Hepatic First-pass Metabolism of Midazolam, a Cytochrome P450 3A Substrate with Differently Modulated Enzyme Activity in Rats
Authors: Higashikawa Fumiko , Murakami Teruo , Kaneda Tatsuya , Kato Akiko , Takano Mikihisa
Published: July 2003

The dose-dependent first-pass metabolism of midazolam, a cytochrome P450 (CYP) 3A substrate, was separately estimated in the intestine and liver after administration into a jejunal loop of rats with differently modulated enzyme activity. Modulation of CYP3A enzyme activity of Sprague-Dawley rats was performed by pretreating the rats with inducers such as dexamethasone or by co-administering ketoconazole (an inhibitor) with midazolam.

Bioavailabilities of midazolam administered into the jejunal loop at a dose of 10 μmol were 12% in untreated (control) rats, and 2% in dexamethasone-pretreated rats. Coadministered ketoconazole (2 μmol) significantly increased the bioavailability to 53% and 7%, respectively, in these rats. The intestinal first-pass metabolism of midazolam administered into the jejunal loop at a dose of 50nmol in untreated and dexamethasone-pretreated rats, estimated by the mesenteric blood-collecting method in-situ, was 25% and 49% of absorbed amount, respectively. The intestinal first-pass metabolism of midazolam was reduced when ketoconazole (0.5 μmol) was co-administered or when the dose of midazolam was increased to 0.5 μmmol in these rats. Assuming that the contribution of intestinal first-pass metabolism could be negligible when midazolam was administered at a much higher dose of 10 μmol, the estimated hepatic first-pass metabolism of midazolam at a dose of 10 μmol in untreated rats, dexamethasone-pretreated rats, untreated rats given ketoconazole, and dexamethasone-pretreated rats given ketoconazole was, respectively, 86, 97, 46, and 92% of the amounts absorbed.

In conclusion, the dose-dependent intestinal first-pass metabolism and the hepatic first-pass metabolism of midazolam in rats with differently modulated CYP3A activities was quantitatively estimated by in-vivo and in-situ absorption studies.
Debromination of (α-Bromoiso-valeryl)urea Catalysed by Rat Blood
Authors: Kitamura Shigeyuki , Kuwasako Mie , Sugihara Kazumi , Tatsumi Kiyoshi , Ohta Shigeru
Published: July 2003

(α-Bromoiso-valeryl) urea, a sedative or hypnotic, is metabolized to (3-methylbutyryl)urea by reductive debromination. This study was designed to evaluate the role of blood in the debromination of (α-bromoiso-valeryl) urea.

Rat blood containing an electron donor had significant debrominating activity toward (α-bromoiso-valeryl)urea. This debromination proceeded by enzymatic and non-enzymatic processes which required both NADH (or NADPH) and flavin mononucleotide (FMN), under anaerobic conditions. The debrominating activity was sensitive to inhibition by carbon monoxide, and the pH optimum was 8.5. When FMN was replaced by flavin adenine dinucleotide (FAD) or riboflavin, similar results were obtained. The optimum concentration of flavins was 10−4m. The reductive debromination was also mediated by rat erythrocytes, but not by plasma. When the blood or erythrocytes were boiled, the debrominating activity was not abolished, but was enhanced, suggesting that the activity arises from the haemoglobin in erythrocytes, and haemoglobin had debrominating activity when supplemented with both a reduced pyridine nucleotide and a flavin. Furthermore, haematin had significant debrominating activity in the presence of these cofactors. The activity of haematin was also observed with the photochemically reduced form of FMN.

The results imply that the debromination proceeds in two steps—enzymatic or non-enzymatic reduction of a flavin such as FAD, FMN or riboflavin by NADPH or NADH, then non-enzymatic reductive debromination of (α-bromoiso-valeryl)urea to (3-methyl-butyryl)urea catalysed by the haem group of rat haemoglobin in the presence of the reduced flavin.
Reductive Debromination of (α-Bromoiso-valeryl)urea by Intestinal Bacteria
Authors: Kitamura Shigeyuki , Kuwasako Mie , Ohta Shigeru , Tatsumi Kiyoshi
Published: July 2003

The reductive debromination of the hypnotic (α-bromoiso-valeryl)urea to (3-methylbutyryl)urea by intestinal bacteria has been studied.

The caecal contents of rats, mice, hamsters, guinea-pigs and rabbits had significant debrominating activity toward (α-bromoiso-valeryl)urea. The cell-free extract of intestinal bacteria from the caecal contents of rats had debrominating activity in the presence of both flavin mononucleotide (FMN) and NADH (or NADPH) under anaerobic conditions. Seven pure strains of intestinal bacteria were also tested and the highest activity was observed with Clostridium sporogenes.The cell-free extract of Clostridium sporogeneshad debrominating activity in the presence of both FMN and NADH (or NADPH), and this activity was inhibited by sodium arsenite and potassium cyanide. The activity of the cellfree extract was also supported by the photochemically reduced form of FMN. The debromination in intestinal bacteria seems to proceed in two steps—reduction of flavins by bacterial flavin reductase(s) in the presence of NADPH or NADH, and then the reductive debromination of (α-bromoiso-valeryl)urea to (3-methylbutyryl)urea by bacterial dehalogenase(s) using the reduced flavins as an electron donor.

These results indicate that intestinal bacteria play a role in the reductive debromination of (α-bromoiso-valeryl)urea to (3-methylbutyryl)urea in animals. The debromination is inhibited by oxygen and dependent on flavins.
Interspecies Pharmacokinetic Comparisons and Allometric Scaling of Napsagatran, a Low Molecular Weight Thrombin Inhibitor
Authors: Lavé Thierry , Portmann Renée , Schenker Gabriela , Gianni Alvaro , Guenzi Alberto , Girometta Maria-Angela , Schmitt Monique
Published: July 2003

The objective of this work was to assess the pharmacokinetics of napsagatran, a low molecular weight thrombin inhibitor, after intravenous administration in a variety of laboratory animals, and prospectively to help design the first pharmacokinetic studies in man. Napsagatran is actively excreted into the bile and urine of various species and pronounced species-differences in its pharmacokinetics are observed. It is, therefore, an interesting compound to use in tests of the limitations of presently available inter-species scaling methods.

The present data suggest that allometric exponent values which are consistent with the values expected for physiological processes and small organic molecules are not necessarily associated with successful predictions in man when active transport processes are involved in the disposition of the compounds. For example, compared with the values observed in man, the clearance (CL), non-renal clearance (CLnr) and the volume of distribution at steady state (Vdss) were over-predicted by 3-, 7- and 2-fold, respectively, by use of allometry. Of the species tested, the cynomolgus monkey seemed to be the most useful for predicting kinetics in man when the approach based on concentration-time transformations was used. Thus, for half-life (t½), CL and Vdss, the observed mean values of 1.7 h, 459 mL min−1 and 24 L kg−1 in man were very close to the values predicted from the cynomolgus monkey (1.7h, 652 mL min−1 and 22 L kg−1, respectively).

The results show that there are large inter-species differences for kidney and liver excretion of napsagatran. This is probably because of the involvement of active transport processes, which compromised the kinetic extrapolation from animal to man, although a more thorough investigation of the transporters involved in the disposition of napsagatran is necessary to enable better understanding of the species differences observed.
Synthesis and Hypolipidaemic Evaluation of a Series of α-Asarone Analogues Related to Clofibrate in Mice
Authors: Labarrios Fernando , Garduño Leticia , Vidal Maria Del Rosario , Garcia Raul , Salazar Maria , Martinez Elizdath , Diaz Francisco , Chamorro German , Tamariz Joaquin
Published: July 2003

A series of α-asarone analogues related to clofibrate, containing an acetic acid group at C-2 of the aromatic ring, has been prepared as the acids or as the ethyl and methyl esters. The corresponding alcohols were also synthesized by reduction of the ethyl esters. The compounds were examined in hyperlipidaemic male mice to evaluate their ability to modify serum lipoprotein cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides after oral administration of 40 and 80 mg kg−1 for 6 days.

Except for methyl 2-methoxy-5-nitro-4-(2-propenyl)phenoxyacetate at either dose, these clofibrate-related phenoxyacetic acid derivatives were found to have significant hypocholesterolaemic activity. Levels of low-density lipoprotein cholesterol and triglycerides were significantly reduced and those of high-density lipoprotein cholesterol were elevated. 2-Methoxy-5-nitro-4-(2-propenyl)phenoxyacetic acid was active at both doses in all the tests. Clofibrate (150 mg kg−1) was more potent at reducing low-density lipoprotein cholesterol. No activity was detected for the alcohol derivatives.

These preliminary results suggest that this class of compound might have more promise as potential hypolipidaemic agents than other α-asarone derivatives. Further investigation and characterization should be performed to determine the mode of action of these agents on lipid metabolism.
Autocatalytic Degradation and Stability of Obidoxime
Authors: Rubnov Shai , Shats Igor , Levy Daniel , Amisar Shai , Schneider Hava
Published: July 2003

The degradation of obidoxime chloride (toxogonin), a reactivator of inhibited cholinesterase in organophosphorus poisoning, in concentrated (250 mg mL−1) acidic solutions was studied by HPLC at several temperatures to determine the degradation mechanism.

The degradation had an autocatalytic profile, which was found to result from the formation of formaldehyde during the degradation process. The activation energy of the hydrolysis was 26.2 kcal mol−1. The shelf-life (t90, the time by which 10% of the drug has degraded) at 25°C was calculated by several methods and found to be more than 37 years. Autocatalysis, a mechanism found only rarely in the degradation of pharmaceuticals, has not been reported in previous studies of obidoxime hydrolysis.
In-vivo Evaluation of Indium-111-Diethylenetriaminepentaacetic Acid-labelling for Determining the Sites and Rates of Protein Catabolism in Mice
Authors: Mukai Takahiro , Arano Yasushi , Nishida Koyo , Sasaki Hitoshi , Saji Hideo , Nakamura Junzo
Published: July 2003

Pharmacokinetic analyses of protein pharmaceuticals are of prime importance for their clinical application. Because many proteins have pharmacological activity at low concentrations, radiolabelling of proteins is widely used to identify the sites and determine the rates of protein catabolism in-vivo due to the high sensitivity of detection of radioactivity. Recently, a metallic radionuclide, 111In, has been used to trace the pharmacokinetics of proteins of interest after conjugation of the proteins with diethylenetriaminepentaacetic acid (DTPA). In this study, galactosyl-neoglycoalbumin (NGA) was reacted with the cyclic dianhydride of DTPA and labelled with 111In to estimate the validity of this radiolabelling procedure for pharmacokinetic analyses. For comparison, we also evaluated direct radioiodination, because directly-radioiodinated proteins are widely used to assess the pharmacokinetics of proteins of interest.

The hepatic radioactivity profile after intravenous injection of [131I]NGA or [111In]DTPA-NGA into mice was analysed pharmacokinetically, and the first-order rate constant representing the elimination of the respective radiometabolite from hepatic parenchymal cells was determined.

The results indicated that direct radioiodination is inappropriate for pursuing the pharmacokinetics of the proteins, because of rapid elimination of the radioactivity from the sites of protein catabolism. These findings also implied that the [111In]DTPA label could be used to identify the catabolic sites and determine the rates of catabolism of proteins with relatively short biological half-lives, although characterization of radiolabelled species at the sites of accumulation would be required for accurate determination of the catabolic sites of proteins.
Resistance to Glutathione Depletion in Diabetic and Non-diabetic Human Erythrocytes In-vitro
Authors: Coleman Michael D , Rustioni Charlotte V
Published: July 2003

We have investigated the resistance of erythrocytes from diabetics and non-diabetics to glutathione depletion caused by p-benzoquinone, 1-chloro-2,4-dinitrobenzene (CDNB), diethyl maleate and 4-aminophenol.

Incubation of erythrocytes with 4-aminophenol (2mm) caused a precipitous reduction (>80%) in cellular glutathione levels although there was no significant difference between 4-aminophenol-mediated glutathione depletion in the diabetic and non-diabetic cells. p-Benzoquinone and CDNB were both associated with a less severe initial reduction in glutathione levels (>50% at 30 min) although p-benzoquinone caused greater depletion (P < 0.001) at 4.5 h (21.1 ± 3.1%, non-diabetic; 20.0 ± 1.0%, diabetic) compared with CDNB (49.2± 2-2%, non-diabetic; 51.3 ± 1.1% diabetic). Although there was no significant difference between the two types of cell in terms of level of depletion, administration of diethyl maleate caused a significant reduction in glutathione levels at 30 min (P < 0.0005), 3.5h (P < 0.05) and 4.5h (P < 0.05) in erythrocytes from diabetic man compared with those from non-diabetic man. Co-administration of buthionine sulphoximine (20 mm) and 4-aminophenol (l mm) also led to a significant reduction in glutathione levels in diabetic cells at 30 min (P < 0.05), 3.5h (P < 0.02) and 4.5h (P < 0.007) compared with those in non-diabetic cells.

The observations that diabetic red cells' resistance to depletion was similar to that of non-diabetic cells for three of the four depletors, and that the combination of 4-aminophenol and buthionine sulphoximine-mediated inhibition of glutathione synthesis was required to illustrate differences suggests that diabetic complications might be a result of the long-term effect of small deficiencies in oxidative self-defence mechanisms such as glutathione.
Simultaneous Use of Sodium Deoxycholate and Dipotassium Glycyrrhizinate Enhances the Cellular Transport of Poorly Absorbed Compounds Across Caco-2 Cell Monolayers
Authors: Sakai Michinori , Imai Teruko , Ohtake Hiroshi , Azuma Hidekazu , Otagiri Masaki
Published: July 2003

The absorption-enhancing effect of a combination of sodium deoxycholate and dipotassium glycyrrhizinate in Caco-2 cell monolayers has been compared with that of the enhancers when used alone, and the mechanism of the enhancement was partially elucidated.

The effect of the combined compounds was evaluated by measurement of transepithelial electrical resistance (TEER) and the cellular permeability of the water-soluble model compounds sodium fluorescein (MW 376.3) and fluorescein isothiocyanate dextran (MW 4400). The TEER of the monolayers decreased with increasing concentrations of dipotassium glycyrrhizinate in combination with 0.02% (w/v) sodium deoxycholate for 20 min, and reached a minimum at 1% (w/v) dipotassium glycyrrhizinate. Although a combination of 0.02% (w/v) sodium deoxycholate and 1% (w/v) dipotassium glycyrrhizinate enhanced the cellular permeability of sodium fluorescein and fluoresceinisothiocyanate dextran, 0.02% (w/v) sodium deoxycholate and 1% (w/v) dipotassium glycyrrhizinate alone had no effect on either the TEER of the monolayers or the cellular transport of the water-soluble compounds. Sequential and separate exposure of the monolayers to each enhancer for 10 min had no effect on the TEER, but a marked decrease in TEER was observed when both compounds were used in combination. The enhancing effect of the combination of sodium deoxycholate and dipotassium glycyrrhizinate was inhibited by H7, a protein kinase C (PKC) inhibitor, suggesting that dipotassium glycyrrhizinate might enhance the activation of PKC via sodium deoxycholate. The combined use of these two enhancers had no toxic effects.

These results provide useful, basic information on the action of these absorption enhancers on drugs for which absorption is limited owing to polarity or molecular size, or both.
The Role of an α-Amino Group on H+-dependent Transepithelial Transport of Cephalosporins in Caco-2 Cells
Authors: Raeissi Shamsi D , Li Jibin , Hidalgo Ismael J
Published: July 2003

The role of an α-amino group on interaction with the intestinal and renal peptide carriers (PEPT 1 and PEPT 2, respectively) has been the subject of much investigation. Studies have differed in their conclusions about the role of an α-amino group on carrier-mediated absorption. Most studies have used brush-border membrane vesicles or perfused intestinal segments. These techniques enable the determination of membrane uptake and luminal disappearance, respectively, but not transepithelial transport. Transepithelial transport should be more predictive of absorption because it includes basolateral efflux, which could be the rate-limiting process in drug absorption. The objective of this study was to evaluate the influence of an α-amino group on PEPT 1-mediated transepithelial transport in Caco-2 cells.

The apical-to-basolateral permeability coefficients of cephalosporins with or without a free α-amino group were determined in the presence and absence of a pH gradient. Permeability coefficients obtained under these conditions were used to calculate a permeability ratio (i.e. Papp (pH 6.0)/Papp (pH 7.4)), which should indicate whether PEPT 1 is involved in transport. For cephalosporins with an α-amino group (cephalexin, cefaclor, cefadroxil, cephradine, cephaloglycin) the permeability ratios ranged between 1.77 and 2.77. In contrast, the permeability ratios for cephalosporins without an α-amino group were 1 (approx.; range = 0.74–1.26).

These data suggest that the presence of an α-amino group on cephalosporins increases their PEPT 1-mediated transepithelial transport in Caco-2 monolayers.
Determination of the Affinity of Drugs toward Serum Albumin by Measurement of the Quenching of the Intrinsic Tryptophan Fluorescence of the Protein
Authors: Epps Dennis E , Raub Thomas J , Caiolfa Valeria , Chiari Augusto , Zamai Moreno
Published: July 2003

Binding of new chemical entities to serum proteins is an issue confronting pharmaceutical companies during development of potential therapeutic agents. Most drugs bind to the most abundant plasma protein, human serum albumin (HSA), at two major binding sites. Excepting fluorescence spectroscopy, existing methods for assaying drug binding to serum albumin are insensitive to higher-affinity compounds and can be labour-intensive, time-consuming, and usually require compound-specific assays. This led us to examine alternative ways to measure drug-albumin interaction.

One method described here uses fluorescence quenching of the single tryptophan (Trp) residue in HSA excited at 295 nm to measure drug-binding affinity. Unfortunately, many compounds absorb, fluoresce, or both, in this UV wavelength region of the spectrum. Several types of binding phenomenon and spectral interference were identified by use of six structurally unrelated compounds and the equations necessary to make corrections mathematically were derived and applied to calculate binding constants accurately. The general cases were: direct quenching of Trp fluorescence by optically transparent ligands with low or high affinities; binding of optically transparent, non-fluorescent ligands to two specific sites where both sites or only one site result in Trp fluorescence quenching; and chromophores whose absorption either overlaps the Trp emission and quenches by energy transfer or absorbs light at the Trp fluorescence excitation wavelength producing absorptive screening as well as fluorescence quenching.

Unless identification of the site specificity of drug binding to serum albumin is desired, quenching of the Trp fluorescence of albumin by titration with ligand is a rapid and facile method for determining the binding affinities of drugs for serum albumin.
Protein Binding of Piroxicam Studied by Means of Affinity Chromatography and Circular Dichroism
Authors: Russeva Veska , Zhivkova Zvetanka , Prodanova Krassimira , Rakovska Rositza
Published: July 2003

The protein binding of piroxicam, a widely used non-steroidal anti-inflammatory drug has been investigated by high-performance liquid affinity chromatography, with phenylbutazone and diazepam used as markers for binding-site characterization, and by circular dichroism titration.

It was found that piroxicam binds to high-affinity phenylbutazone-binding sites and to high-affinity diazepam-binding sites. No binding to the low-affinity sites of either marker was established. High values of the primary (high-affinity) binding constants corresponding to both types of binding site were obtained by means of a mathematical method cited in the literature. The circular dichroic spectra of piroxicam were studied at a given albumin concentration and various drug concentrations. A new Cotton effect was observed and was ascribed to the binding of piroxicam to the protein molecule. The values of differential molar ellipticity (δθ)were treated by a new mathematical procedure for analysis of the data obtained. A high affinity constant was calculated for one class of binding site. Its value is in good agreement with the values obtained by affinity chromatography.

These results reveal that circular dichroism is an acceptable method for investigation of protein binding.