Changes in the excretion of endogenous glycine conjugate as a possible artifact in toxicological experiments

Changes in the urinary excretion of hippuric acid (HIA) and phenaceturic acid (PUA) as well as their metabolic precursors, i.e. benzoic (BA) and phenylacetic acid (PAA), in rats housed in glass metabolic cages for 4 days were monitored using gas-liquid chromatography. The amount of HIA excreted was 128±63 mol/kg for female and 79±43 mol/kg for male rats in the first 24 h and decreased to 11±7 mol/kg (p< 0.01) for female and 3.2±2.4 mol/kg (p< 0.001) for male rats on the 2nd day. These values remained nearly at the same level until the end of the experiment. The amount of PUA decreased from 48±12 mol/kg on the 1st day to 22±9 mol/kg (p< 0.05) on the 2nd day by male rats, whereas by the females the decrease from 30±9 mol/kg to 21±8 mol/kg was not significant. The decrease in the excretion of glycine conjugates was compensated by a parallel increase in the level of unconjugated BA and PAA.     

Mechanisms of the contractile effects of flosequinoxan

In guinea-pig papillary muscles the positive inotropic effect of flosequinoxan (BTS) starting at 100 mol/1 amounted to 287.6 ± 34.2% at 300 mol/l without any effects on time to peak tension (103.9 ± 2%) and relaxation time (107.1 ± 6.7% of predrug value, respectively). 10 mol/l carbachol attenuated the positive inotropic effect of 300 mol/l to 166.5 ± 11.6% (n = 10). The phosphorylation state of the inhibitory subunit of troponin (TnI) and phospholamban(PLB) in [³²P]-labeled guinea-pig ventricular myocytes was increased starting at 100 mol/l amounting to 142.5 ± 12.6% and 130.9 ± 2.2% at 300 mol/l, respectively (n = 5). Furthermore, BTS (300 mol/l) decreased phosphorylase phosphatase activity by 23.1%. It is concluded that the contractile effects of BTS are accompanied by enhanced phosphorylation of regulatory proteins which could in part be due to inhibition of phosphorylase phosphatase activity.     

In Vitro Cutaneous Disposition of a Topical Diclofenac Lotion in Human Skin: Effect of a Multi-Dose Regimen

Purpose. This study determines comparative bioavailability of diclofenac sodium lotion compared to an aqueous solution after topical application to viable human skin in vitro. In addition, the difference between a single dose and multiple doses (8 times) was also determined. Methods. An in vitro flow-through diffusion cell system was employed, using radiolabelled diclofenac sodium. Results. Multiple doses of lotion (2 l/cm² and 5 l/cm²) delivered a total of 40.1 ± 17.6 g and 85.6 ± 41.4 g diclofenac, respectively, at 48 h, compared to only 9.4 ± 2.9 g and 35.7 ± 19.0 g absorbed after topical application of diclofenac as an aqueous solution (P < 0.05). A single dose study showed no statistical difference between diclofenac delivered in lotion or an aqueous solution. Over 48 h the total absorption from lotion was 10.2 ± 6.7 g and 26.2 ± 17.6 g (2 l/cm² and 5 l/cm², respectively), compared to 8.3 ± 1.5 g and 12.5 ± 5.7 g from an aqueous solution. Both single doses of lotion and aqueous diclofenac showed decreased diclofenac absorption into the receptor fluid between 12 and 24 h. However, when applied multiple times, absorption from lotion was continually increasing up to 48 h. The total dose accountability ranged from 76.8 ± 8.2% to 110.6 ± 15.1% of the applied dose. Conclusions. Diclofenac lotion exhibited enhanced diclofenac percutaneous absorption rate through human skin (mass, flux and partition coefficient) when applied a multiple number of times and this enhanced absorption was maintained over 48 h. This suggests that a constituent of the lotion (DMSO) will enhance human skin absorption of diclofenac when used in a multi-dose regimen, but not after a single dose.     

Hepatic Disposition of Fexofenadine: Influence of the Transport Inhibitors Erythromycin and Dibromosulphothalein

Purpose. To examine the disposition of fexofenadine in the isolated perfused rat liver and the influence of erythromycin and dibromosulphthalein (DBSP) on the hepatic uptake and biliary excretion of fexofenadine. Methods. Livers from four groups of rats were perfused in a recirculatory manner with fexofenadine HCl added as a bolus (125, 250, 500, or 1000 g) to perfusate. Livers from another three groups of rats were perfused with 250 g of fexofenadine HCl. With one group as control, erythromycin (4.0 g/ml) or DBSP (136 g/ml) was added to the perfusate of the other groups. In all experiments, perfusate and bile were collected for 60 min; in addition, livers from the second experiment were retained for assay. Fexofenadine was determined in perfusate, bile, and homogenized liver by HPLC. Results. The area under the curve (AUC) of fexofenadine was linearly related to concentration. It was unchanged from control (12,800 ± 200 ng·h/ml) by erythromycin (14,400 ± 2000 ng·h/ml), but was increased 95% by DBSP (25,000 ± 2600 ng·h/ml, P <0.001). The ratios of the concentrations of fexofenadine in liver/perfusate were decreased significantly by DBSP; those for bile/liver were increased by erythromycin. Conclusions. Erythromycin reduced the canalicular transport of fexofenadine into bile, whereas DBSP reduced uptake across the sinusoidal membrane.     

Carrier Mechanisms Involved in the Transepithelial Transport of bis(POM)-PMEA and Its Metabolites Across Caco-2 Monolayers

Purpose. To investigate the role of carrier mechanisms in: [1] the polarized transport of the bis(pivaloyloxymethyl)- [bis(POM)-] ester prodrug of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine [PMEA] and [2] the directional secretion of its metabolites. Methods. Caco-2 monolayers were used to study the modulating effect of carriers on the transport of bis(POM)-PMEA and the efflux of intracellularly formed metabolites mono(POM)-PMEA and PMEA from the cells. The interaction of bis(POM)-PMEA and its metabolites with the efflux mechanisms present in Caco-2 monolayers was investigated by testing the effect of various concentrations of verapamil (30, 100, 300 M) or indomethacin (10-500 M) on transport and efflux. Results. Polarity in transport of bis(POM)-PMEA (50 M) across Caco-2 monolayers was noted: transport of total PMEA [=bis(POM)-PMEA, mono(POM)-PMEA and PMEA] was significantly higher in basolateral (BL) to apical (AP) direction (14.5 ± 0.4%) than transport in the opposite (AP to BL) direction (1.7 ± 0.2%). This difference was reduced in a concentration dependent way when verapamil (0–100 M) was included in both AP and BL incubation media. After loading the cells with bis(POM)-PMEA (100 M) for 1 hr, studies on efflux of PMEA and mono(POM)-PMEA from the Caco-2 monolayers over a 3 hr period, revealed that both metabolites were preferentially secreted towards the AP compartment. Efflux of PMEA towards AP and BL compartments amounted to 14.6 ± 1.1 % and 5.3 ± 0.4%, respectively, of the initial intracellular amount of total PMEA, while efflux of mono(POM)-PMEA towards AP and BL compartments was limited to 2.3 ± 0.1 % and 0.5 ± 0.1 %, respectively. When 10 M indomethacin was included in the AP incubation medium, efflux of PMEA was decreased to 7.8 ± 0.3% and 3.3 ± 0.3% towards the AP and BL compartments, respectively. The decrease in efflux by indomethacin was concentration-dependent up to 100 M. Transepithelial transport of total PMEA was also reduced in the presence of 30 M indomethacin, as reflected in smaller concentrations of PMEA and mono(POM)-PMEA in the acceptor compartment, irrespective of the transport direction. Conclusions. The data obtained in this study suggest that bis(POM)-PMEA is substrate for a P-glycoprotein-like carrier mechanism in Caco-2 monolayers, while its metabolites mono(POM)-PMEA and PMEA are transported by a non-P-glycoprotein efflux protein.     

Stereoselective analysis of the disposition of tosufloxacin enantiomers in man

Summary The pharmacokinetics of tosufloxacin enantiomers after oral administration of racemic tosufloxacin were examined in healthy volunteers. Only small differences were observed in time to peak concentration (2.6±0.3 [mean ± SEM] h for (+)-tosufloxacin vs 2.4±0.2 h for (–)-tosufloxacin), elimination half-life (3.61±0.24 h vs 3.49±0.23 h), and area under the curve (2.78±0.19 h·g/ml vs 2.87±0.19 h·g/ml); however, peak concentration (0.40±0.03 g/ml vs 0.44±0.03 g/ml), renal clearance (226±10 ml/min vs 202±10 ml/min), and urinary recovery (35.4±2.2% vs 32.4±1.9%) differed significantly between enantiomers.     

Pharmacokinetics of cefradine, sulfamethoxazole and trimethoprim and their metabolites in a patient with peritonitis undergoing continuous ambulatory peritoneal dialysis

Cefradine and co-trimoxazole pharmacokinetics were studied in a patient with peritonitis that complicated continuous ambulatory peritoneal dialysis (CAPD). Concentrations in the plasma reached after oral administration of 500 mg cefradine four times daily and 400/80 mg co-trimoxazole four times daily were for cefradine 100g/ml, for trimethoprim 15g/ml, and for sulfamethoxazole 100/ml, respectively. In the dialysate concentrations were reached of 35–70/ml cefradine, 2–5/ml trimethoprim and 8–17g/ml sulfamethoxazole. The values for sulfamethoxazole are regarded too low to be clinically effective. Half-lives protein binding values and CAPD clearances are presented. Low CAPD clearances were obtained during the night and high values during the day. The dosage yielded too high plasma trimethoprim concentrations, while sulfamethoxazole dialysate concentrations were too low. It seems questionable therefore whether co-trimoxazole can be used orally for the treatment of CAPD peritonitis.     

The Mechanism of Uptake of Biodegradable Microparticles in Caco-2 Cells Is Size Dependent

Purpose. To study the uptake of biodegradable microparticles in Caco-2 cells. Methods. Biodegradable microparticles of polylactic polyglycolic acid co-polymer (PLGA 50:50) of mean diameters 0.1 m, 1 m, and 10 m containing bovine serum albumin as a model protein and 6-coumarin as a fluorescent marker were formulated by a multiple emulsion technique. The Caco-2 cell monolayers were incubated with each diameter microparticles (100 g/ml) for two hours. The microparticle uptake in Caco-2 cells was studied by confocal microscopy and also by quantitating the 6-coumarin content of the microparticles taken up by the cells. The effects of microparticle concentration, and incubation time and temperature on microparticle cell uptake were also studied. Results. The study demonstrated that the Caco-2 cell microparticle uptake significantly depends upon the microparticle diameter. The 0.1 m diameter microparticles had 2.5 fold greater uptake on the weight basis than the 1 m and 6 fold greater than the 10 m diameter microparticles. Similarly in terms of number the uptake of 0.1 m diameter microparticles was 2.7 × 10³ fold greater than the 1 m and 6.7 × 10⁶ greater than the 10 m diameter microparticles. The efficiency of uptake of 0.1 m diameter microparticles at 100 g/ml concentration was 41% compared to 15% and 6% for the 1 m and the 10 m diameter microparticles, respectively. The Caco-2 cell microparticle (0.1 m) uptake increased with concentration in the range of 100 g/ml to 500 g/ml which then reached a plateau at higher concentration. The uptake of microparticles increased with incubation time, reaching a steady state at two hours. The uptake was greater at an incubation temperature of 37°C compared to at 4°C. Conclusions. The Caco-2 cell microparticle uptake was microparticle diameter, concentration, and incubation time and temperature dependent. The small diameter microparticles (0.1 m) had significantly greater uptake compared to larger diameter microparticles. The results thus suggest that the mechanism of uptake of microparticles in Caco-2 cell is particle diameter dependent. Caco-2 cells are used as an in vitro model for gastrointestinal uptake, and therefore the results obtained in these studies could be of significant importance in optimizing the microparticle-based oral drug delivery systems.     

Quantitative Evaluation of Capacity-Limited Hepatobiliary Transport Based on Hepatocellular Diffusion Model by MULTI(FEM)

The dose-dependency of hepatic uptake and hepatobiliary transport of a drug was evaluated by means of a nonlinear least square program incorporating the finite element method, MULTI(FEM). A perfusion experiment using isolated rat livers following a pulse input (i.e., under non-steady-state conditions) was performed at three dose levels of cefpiramide as a model drug. The hepatic extraction ratio (E_H) of cefpiramide decreased with an increase in dose, which demonstrates that the hepatic uptake is capacity-limited. The outflow time-profiles from the liver were represented by a two-compartment dispersion model with central Michaelis–Menten elimination, and the maximal elimination rate per central compartment volume (V_max) and the Michaelis constant (K_m) were estimated to be 1420 g/ml/min and 235 g/ml, respectively. The biliary mean transit time increased slightly with an increase in dose. The hepatocellular diffusion model under non-steady-state conditions considering nonlinear transport across the bile canalicular membrane was adopted to evaluate dose-dependency in the biliary excretion of cefpiramide. The maximal penetration velocity across the bile canalicular membrane per liver and the affinity constant of penetration across the bile canalicular membrane were estimated to be 40.1 g/min and 123 g, respectively. Considering that the volume of a rat liver (A_H.L) is approximately 10 ml, the Michaelis constant of penetration (k _m ^bmc ), which is an apparent parameter, was estimated to be approximately 12.3 g/ml. In conclusion, MULTI(FEM) is useful for evaluation of capacity-limited local disposition.     

Influence of N-allyl-normetazocine on acetylcholine release from brain slices: involvement of muscarinic receptors

Summary The effects of (±)N-allyl-normetazocine on the release of acetylcholine from different areas of guinea-pig and rat brain were investigated. 1. The drug did not modify the electrically (2 Hz) evoked tritium efflux from guinea-pig cerebral cortex, thalamus and caudate nucleus slices, preloaded with ³H-choline 0.1 mol/l and superfused with Krebs solution containing hemicholinium-3 10 mol/l. 2. (±)N-allyl-normetazocine 10 mol/l. enhanced the evoked ³H efflux from guinea-pig brain slices superfused with Krebs solution containing physostigmine 30 mol/l or oxotremorine 0.3 -1 gmol/l; the effect was naloxone-insensitive and was abolished by atropine 0.15 mol/l, but not by pirenzepine 1 mol/l. 3. (±)N-allyl-normetazocine 5 mol/l enhanced the electrically evoked release of endogenous acetylcholine as well, in a naloxone-insensitive way. 4. Both (±) and (+)N-allyl-normetazocine were without effect on ³H efflux from rat caudate nucleus slices electrically stimulated at 0.2 Hz frequency, after preloading with ³H-choline and during superfusion with hemicholinium-3. 5. The results are discussed in view of the antimuscarinic properties of the drug. Send offprint requests to A. Siniscalchi     

Characterization of P-glycoprotein Mediated Transport of K02, a Novel Vinylsulfone Peptidomimetic Cysteine Protease Inhibitor,Across MDR1-MDCK and Caco-2 Cell Monolayers

Purpose. Here we characterized the transport properties of morpholine-urea-phenylalanine- homophenylalanine-vinylsulfone-phenyl (K02), a newly developed peptidomimetic cysteine protease inhibitor, across monolayers of P-gp-expressed MDR1 transfected MDCK cells (MDR1-MDCK) and Caco-2 cells. Methods. MDR1-MDCK, MDCK and Caco-2 cells, grown to confluence on Transwell insert membranes, were used to investigate transcellular transport of [¹⁴C]-K02. Results. The basolateral to apical (B-A) flux of 10 M [^I4C]-K02 across MDR1-MDCK cells was markedly greater than its apical to basolateral (A-B) flux (ratio = 39). This specific B-A transport was temperature dependent and saturable, with an apparent Michaelis-Menten constant and maximum velocity of 69.1 ± 19.5 M and 148.9 ± 16.3 pmol/min/cm², respectively. This B-A flux was significantly inhibited by cyclosporine (IC₅₀ = 17.1 ± 0.7 M), vinblastine (IC₅₀ = 75.9 ± 13.0 M) and verapamil (IC₅₀ = 236 ± 63 M). In Caco-2 cell monolayers, the B-A flux was reduced about 50% compared to that in MDR1-MDCK and the A-B flux was increased about 8-fold. The apparent Michaelis-Menten constant and maximum velocity values for the B-A transport were 71.8 ± 45.9 M and 35.3 ± 9.0 pmol/min/ cm². This B-A flux was also significantly inhibited by P-gp substrates/ inhibitors. Western blots showed that the P-gp expression in MDR1-MDCK cells was about 10-fold that in Caco-2 cells. Conclusions. K02 is transported by P-gp in both MDR1-MDCK and Caco-2 cells, and the in vitro interactions between K02 and various P-gp substrates may provide strategies to overcome the bioavailability barrier by intestinal P-gp. __     

Investigation of the 5-hydroxytryptamine receptor mediating the “transient” short-circuit current response in guinea-pig ileal mucosa

5-Hydroxytryptamine (5-HT) stimulated an increase in short-circuit current (I_sc) in guinea-pig isolated ileal mucosa over a wide concentration range (0.1 nM-0.1 mM). The concentration-response relationship was biphasic, consisting of a high potency phase (0.1 nM–1 M) and a low potency phase (3–10 M). Stimulation of Isc observed at the high potency phase tended to be sustained while responses at the low potency phase (3–10 M) contained two components, an initial transient response followed by a maintained response. Both the high potency phase (maximum stimulation 30 A cm^–2) and the low potency phase (maximum stimulation 80 A cm ^–2) 5-HT response were antagonized by tetrodotoxin (TTX, 0.3 M) and atropine (1 M). However, another low potency (3 M-0.1 mM, maximum stimulation 30 A cm^–2) component of the 5-HT response was revealed in the presence of TTX or atropine.In the presence of methysergide (1 M), the concentration-response relationship of 5-HT was still biphasic and tropisetron (0.1 and 10 M) antagonized both phases of the 5-HT response. In the presence of methysergide, the high potency phase 5-HT response was mimicked by 5-methoxytryptamine (5-MeOT) and the selective 5-HT₄ agonist SC-53116 but not by BIMU 8. The potent 5-HT₄ antagonist GR 113808 antagonized the response to 5-MeOT in a surmountable manner with an affinity estimate of 9.6 ± 0.3 (n = 4). The 5-MeOT stimulated increase in I_sc was also antagonized in an unsurmountable manner by granisetron (1 M).In the presence of methysergide, desensitization of 5-HT₃ receptors with 2-methyl-5-hydroxytryptamine (10 M) abolished both phases of the 5-HT response. Under the same condition, desensitization of 5-HT₄ receptors with 5-MeOT (10 M) abolished only the high potency 5-HT response and dextrally shifted the low potency 5-HT response.These data show that neuronal and non-neuronal 5-HT receptors are involved in the regulation of secretion in ileal mucosa. We propose the presence of a neuronal 5-HT₄ receptor located upstream of the well characterized neuronal 5-HT₃ receptors to be responsible for the high potency 5-HT response. A schematic model is proposed to explain our findings and the relationship between this 5-HT₄ receptor and other 5-HT receptor subtypes regulating secretion that have been described in the literature.     

Phorbol 12,13-dibutyrate,an activator of protein kinase C,stimulates both contraction and Ca2+ fluxes in dog saphenous vein

Summary The vascular effects of phorbol 12,13-dibutyrate (PDBu) were studied in the dog saphenous vein. PDBu (1 M) caused contraction (0.58 ± 0.22 g/mg wet wt.) and Ca uptake (74.2 ± 41.2 mol/kg wet wt.) which were unaffected by 10 M phentolamine (N = 6). The PDBu-induced contraction was greatly (60–80%) inhibited in Ca²⁺-free solution. 15 Ca efflux measurements performed in Ca²⁺-free solution showed that PDBu did not cause Ca release from intracellular storage sites. The contractile response to PDBu (1 nM-1 M) was significantly correlated with the magnitude of Ca uptake; contraction and the rise in tissue Ca²⁺ also had a similar time course. Correlation between the two measures persisted when the responses to PDBu were augmented by co-administration with 20 mM KCl. However, no synergism occurred between the two agonists. Both the contraction and Ca uptake responses to PDBu were reduced by nifedipine and verapamil, each at 1 M. In the Triton X-100 skinned saphenous vein, where the voltage-dependent Ca channel is not functional, 10 M PDBu did not cause contractions in the presence of 0.1 M Ca²⁺. Thus, contraction of the intact saphenous vein by PDBu characteristically exhibits great Ca dependence and PDBu seems to activate the voltage-dependent Ca channel, presumably through stimulation of protein kinase C; the ensuing Ca entry is primarily responsible for contraction. However, the mechanism responsible for the PDBu-induced contractions that are resistant to Ca²⁺-free PSS or Ca entry blockers remains to be defined. It appears that the dog saphenous vein differs from dog femoral artery, rabbit aorta and pig carotid artery where PDBu contractions do not display dependence on external Ca²⁺. Send offprint requests to P. J. S. Chin at the above address     

Polymers with Thiol Groups: A New Generation of Mucoadhesive Polymers?

Purpose. To improve the mucoadhesive properties of polycarbophil by the introduction of sulfhydryl groups. Methods. Mediated by a carbodiimide, cysteine was covalently bound to polycarbophil (PCP) forming amide bonds between the primary amino group of the amino acid and the carboxylic acid moieties of the polymer. The amount of covalently attached cysteine and the formation of disulfide bonds within the modified polymer were determined by quantifying the share of thiol groups on the polymer conjugates with Ellman's reagent. The adhesive properties of polycarbophil-cysteine conjugates were evaluated in vitro on excised porcine intestinal mucosa by determining the total work of adhesion (TWA). Results. Depending on the weight-ratio of polycarbophil to cysteine at the coupling reaction, e.g., 16:1 and 2:1, 0.6 ± 0.7 mole and 5.3 ± 2.4 mole cysteine, respectively, were covalently bound per g polymer. The modified polymer displayed improved internal cohesive properties due to the formation of interchain disulfide bonds within the polymer in aqueous solutions at pH-values above 5. Adhesion studies revealed strongly improved adhesive properties. Whereas the TWA was determined to be 104 ± 21 J for the unmodified polymer, it was 191 ± 47 J for the polymer-cysteine conjugate 16:1 and 280 ± 67 J for the polymer-cysteine conjugate 2:1. Conclusions. Polymers with thiol groups might represent a new generation of mucoadhesive polymers displaying comparatively stronger adhesive properties.     

Pharmacokinetics and safety of ILX23-7553, a non-calcemic-vitamin D3 analogue, in a phase I study of patients with advanced malignancies

Purpose: Differentiation therapy is an alternative to chemotherapy with potentially less toxicity, improved quality of life, and survival. We conducted a phase I trial of ILX23-7553, a formulation of 1,25-dihydroxy-16-ene-23-yne-vitamin D₃, a 1,25-dihydroxyvitamin D₃ analog with preclinically demonstrated antitumor and differentiating effects and diminished hypercalcemic effects. Patients and methods: The protocol consisted of five daily oral treatments during 14-day cycles at 15 dose levels from 1.3 to 45.0g/m²/day. We treated 42 heavily pretreated patients who had a variety of malignancies with 162 treatment cycles, and obtained pharmacokinetics from three patients at the two highest dose levels. Results: There were no grade 3 or 4 toxicities. Grade 1–2 toxicities included diarrhea, nausea, fatigue, constipation, and one grade 1 hypercalcemia. Average day 6 calcium was 9.26 ± 0.55mg/dl in cycle 1 and 9.30 ± 0.67mg/dl in cycle 2. Pharmacokinetics at dose levels 14 (40g/m²/day) (1 patient) and 15 (45g/m²/day) (2 patients) demonstrated an average C _max of 30.4 ± 7.8pg/ml (0.07nM) and 104 ± 38.2pg/ml (0.25nM), and AUCs of 222.5 ± 225.2pg·h/ml and 855 ± 536pgh/ml, respectively. Eight patients (19%) had stable disease. While in vitro effects have been reported at these concentrations, they were at least 10-fold lower than ED₅₀s, and the study was terminated before an MTD was reached. Conclusion: The drug is safe and has potential benefits at serum concentrations where effects begin to be noted in vitro. Further study is needed with a reformulated higher unit dose compound to determine the safety and efficacy of higher serum concentrations.     

Disposition Kinetics of α-Tocopherol in Apolipoprotein B Knockout Mice

Purpose. We examined the effects of apolipoprotein B (apoB) on the disposition kinetics of -tocopherol by using apoB knockout mice. Methods. The concentrations of -tocopherol in plasma and tissues were measured by gas chromatography-mass spectrometry. Results. In apob (–/–) mice, the endogenous levels of -tocopherol in plasma and tissues (except liver) were significantly lower, and the liver concentration was significantly higher than those in wild-type mice. After single i.v. administration of -tocopherol (25 mg/kg), the area under the plasma concentration-time curve (AUC) and the distribution volume at steady state were significantly decreased, whereas the total clearance of -tocopherol was significantly increased in apob (–/–) vs. wild-type mice. -Tocopherol was highly distributed to the liver, compared with other tissues. After an oral administration of -tocopherol (100 mg/kg), the intestinal absorption of -tocopherol was very low in apoB knockout mice, as the value of AUC_0-32h for apob (–/–) mice (17.7 ± 8.3 g h/mL) was significantly less than that for apob (+/+) wild-type mice (96.5 ± 15.8 g h/mL, mean ± SD of five experiments, p < 0.01). The biliary excretion of -tocopherol was significantly greater in apob (+/–) mice than in apob (+/+) mice. Conclusions. These results show that apoB plays a role in hepatic secretion and intestinal absorption of -tocopherol.     

A method to estimate the potency of the μ-component of an opioid having mixedμ-, andκ- and/orδ-agonist activities

The SC administration of either typical-agonists such as morphine, pethidine, fentanyl and levorphanol or a mixed- and-agonist like [d-Ala²,d-Leu⁵]-enkephalin to 10-day-old rats produced loss of righting reflex. Additionally, the loss of righting reflex induced by these opioid agonists was antagonized by naloxone, an opioid antagonist having a preference for-receptors, but by neither nor-binaltorphimine nor naltrindole, a specific- or-antagonist, respectively, indicating that the loss of righting reflex was produced by the interaction of an opioid with-receptors. Moreover, the potency of each opioid agonist relative to that of morphine estimated by the present in vivo method was similar to that determined by the traditional in vitro isolated preparation. In contrast to-agonists, neither typical-agonists such as U-50, 488H, ketocyclazocine, pentazocine and butorphanol, nor a selective-agonist like [d-Pen²,d-Pen⁵]-enkephalin affected the righting reflex of 10-day-old rats, indicating that-agonists, but neither- nor-agonists, produced the naloxone-reversible loss of righting reflex in infant rats. By employing the present in vivo method to estimate the-agonist activity of an opioid with mixed agonist activities, it was indicated that the-agonist activity of ethylketocyclazocine, which had been employed as a representative-agonist, was essentially the same as that of morphine, a representative-agonist.     

Cytotoxicity of diethyldithiocarbamate in human versus rodent cell lines

Summary Diethyldithiocarbamate (DDTC) and other dithiocarbamates are currently receiving attention as potential adjuncts to traditional chemotherapy. In vitro studies with rodent cancer cell lines have consistently shown that DDTC concentrations of 0.1–1.0 g/ml are highly cytotoxic. Paradoxically, however, concentrations of 10–100 g/ml have been significantly less toxic.In the present study, such a biphasic pattern was reproduced when 3 rodent cell lines were exposed for 1 hour to 0.001 to 1000 g DDTC/ml. In contrast, in 7 human cell lines survival decreased steadily with increasing DDTC concentration (in the same dose range) without evidence of a biphasic pattern. These data might have implications for studies in which rodent cell lines are used to model the effects of dithiocarbamates in human tissues.     

Sulfinosine-induced cell growth inhibition and apoptosis in human lung carcinomas in vitro

In spite of tremendous effort for improvedtherapy, lung cancer remains the leadingcause of cancer-related deaths worldwide.In the present study, we used the novelpurine ribunocleoside sulfinosine andevaluated its antiproliferative andapoptotic outcome on the non-small celllung carcinoma cell line (NSCLC) and thesmall cell lung carcinoma cell line (SCLC).Using a BrdU incorporation-test sulfinosineinhibited cell growth in a dosedependent-manner. ID₅₀ values were4.65 ± 0.17 M in the case of NSCLCcells, and 3.59 ± 0.81 M in thecase of SCLC cells. MTT testing revealedthat IC₅₀ values were 6.24 ±0.77 M for NSCLC and 5.68 ±0.58 M for SCLC. Inhibitoryconcentrations (IC₅₀ and ID₅₀)for sulfinosine were nonsignificantly lowerin SCLC cells compared to NSCLC cells,indicating similar susceptibility of thecells. Flow-cytometric analysis, TUNELstaining, DNA laddering and cell deathELISA test were used to investigateapoptotic cell death. Our resultsdemonstrated that high concentrations ofsulfinosine can cause typical DNAladdering, a hallmark for apoptosis.Evidence of free nucleosomes and enzymaticlabeling of fragmented DNA confirmedapoptosis involvement in sulfinosinecytotoxicity. In addition, flow-cytometricanalysis showed that 25 M sulfinosinearrested cell cycle progression atthe G₂M phase and induction ofapoptosis in both cell lines. From theseresults, we concluded that sulfinosine mayact as an anticancer agent and furtherstudies may prove its efficacy in lungcancer cells. Thus the biological effectsof sulfinosine may be due to modulation ofcell growth, cell death, and cell cycleregulatory molecules.     

Investigations differentiating the mechanism of specific bradycardic agents from that of calcium channel blockers

Summary The effects of two specific bradycardic agents, falipamil (AQ-A 39) and the alinidine-congener STH 2148 2-[N-(cyclopropylmethyl)-N-(2,6-dibromophenyl)amino]2-imidazoline, on the spontaneous electrical discharge rate of intact guinea-pig sinus node preparations were investigated in comparison to that of the calcium channel blocker verapamil. Addition of falipamil (10 g/ml) to a maximally rate lowering concentration of STH 2148 (30 g/ml) exerted no further bradycardic effect. In contrast, verapamil (0.1 g/ml) added to either STH 2148 (30 gg/ml) or a maximally effective concentration of falipamil (30 gg/ml) resulted in a further, significant reduction of sinus rate. The results are compatible with the idea of a common mechanism of the two specific bradycardic agents, different from that of calcium channel blockers. Send offprint requests to W. Kobinger at the above address