Fluvoxamine pharmacokinetics in healthy elderly subjects and elderly patients with chronic heart failure.

AIMS: To investigate the effects of age and chronic heart failure (CHF) on the oral disposition kinetics of fluvoxamine. METHODS: A single fluvoxamine dose (50 mg) was administered orally to 10 healthy young adults, 10 healthy elderly subjects and 10 elderly patients with CHF. Fluvoxamine concentration in plasma was measured for up to 96 h. RESULTS: With the exception of apparent distribution volume, ageing modified all main pharmacokinetic parameters of fluvoxamine. Thus, peak concentration was about doubled {31 +/- 19 vs. 15 +/- 9 ng ml(-1); difference [95% confidence interval (CI)] 16 (3, 29), P < 0.05}, and area under the concentration-time curve was almost three times higher [885 +/- 560 vs. 304 +/- 84 ng h ml(-1); difference (95% CI) 581 (205, 957), P < 0.05]; half-life was prolonged by 63% [21.1 +/- 6.2 vs. 12.9 +/- 6.4 h; difference (95% CI) 8.2 (2.3, 14.1), P < 0.01], and oral clearance was halved (1.12 +/- 0.77 vs. 2.25 +/- 0.66 l h(-1) kg(-1); difference (95% CI) -1.13 (-1.80, -0.46), P < 0.001]. A significant inverse correlation was consistently observed between age and oral clearance (r=-0.67; P < 0.001). The coexistence of CHF had no significant effect on any pharmacokinetic parameters in elderly subjects. CONCLUSIONS: Ageing results in considerable impairment of fluvoxamine disposition, whereas CHF causes no significant modifications. Therefore, adjustment of initial dose and subsequent dose titrations may be required in elderly subjects, whereas no further dose reduction is necessary in elderly patients with CHF.

Comparison of the binding affinity of CGP-12177A at recombinant rat alpha(1D)-adrenoceptors expressed in BHK-21 cell membranes and alpha(1)-adrenoceptors present in rat cerebral cortex membranes.

Recent in vitro studies, performed in rat aorta, mesenteric and intrapulmonary arteries, and human pulmonary artery, demonstrated that the beta-adrenoceptor ligand CGP-12177A (4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one) is also provided with antagonist or partial agonist properties at alpha(1)-adrenoceptors. These observations were supported by estimates of CGP-12177A binding affinity at alpha(1)-adrenoceptors, which have been always performed in rat cerebral cortex membranes, as a surrogate of vascular tissue. Since alpha(1D)-adrenoceptors are predominant in both rat aorta and mesenteric artery, in the present study, we measured, for the first time, the binding affinity of CGP-12177A at recombinant rat alpha(1D)-adrenoceptors expressed in BHK-21 cell membranes. CGP-12177A binding affinity was also determined in rat cerebral cortex membranes, where various alpha(1)-adrenoceptor subtypes are present. By means of [(3)H]prazosin binding competition experiments, we found that CGP-12177A bound to alpha(1D)-adrenoceptor-expressing BHK-21 cell membranes, with a binding affinity (pK(i)=5.39+/-0.27) almost identical to that measured in cerebral membranes (pK(i)=5.44+/-0.07), indicating that it is a non-subtype selective alpha(1)-adrenoceptor ligand. Moreover, CGP-12177A binding affinity was very close to its functional affinity evaluated in rat aorta in terms of antagonist potency against phenylephrine-induced contraction (pK(B)=5.65+/-0.07). In conclusion, our results demonstrate that, in order to evaluate CGP-12177A binding affinity at aorta and mesenteric artery alpha(1)-adrenoceptors, estimates in rat cerebral membranes are as reliable as those in recombinant rat alpha(1D)-adrenoceptors, since both values are very close to CGP-12177A functional affinities in isolated vessels.

Carteolol, a non-conventional partial agonist of beta(1)-adrenoceptors, relaxes phenylephrine-constricted rat aorta through antagonism at alpha(1)-adrenoceptors.

This in vitro study was designed to investigate whether carteolol, a non-conventional partial agonists of beta(1)-adrenoceptors, relaxes phenylephrine-constricted rat aorta through activation of the low-affinity state of beta(1)-adrenoceptors or antagonist effect at alpha(1)-adrenoceptors. Carteolol-induced complete concentration-dependent relaxation of phenylephrine-contracted aorta (pD(2)=3.65+/-0.04), this effect not being modified by endothelium removal and not antagonised by NO-synthase inhibitor N(G)-nitro-l-arginine methyl ester (100 microM) or cyclo-oxygenase inhibitor indomethacin (10 microM). The effect of carteolol was unaffected by the non-selective beta-adrenoceptor antagonist propranolol (1 microM), or the beta(2)-adrenoceptor selective antagonist (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118,551, 1 microM). Increasing concentrations of carteolol produced a parallel rightward shift of the concentration-response curves for phenylephrine-induced contraction, exhibiting a pK(B) of 4.28+/-0.07. Carteolol affinity for alpha(1)-adrenoceptors was evaluated by means of competition experiments carried out in BHK-21 cell membranes expressing rat recombinant alpha(1D)-adrenoceptor, the alpha(1)-adrenoceptor subtype mainly present in rat aorta. Carteolol competed monophasically with [(3)H]prazosin, exhibiting a pK(i) value (3.39+/-0.31) similar to its pD(2) and not very far from its pK(B). In conclusion, this study indicates that carteolol relaxes phenylephrine-contracted aorta through its alpha(1)-adrenoceptor antagonist properties, excluding the possibility that the relaxant effect is due to the activation of beta-adrenoceptors, particularly of the low-affinity state of beta(1)-adrenoceptors, by the drug.

Pentoxifylline and its major oxidative metabolites exhibit different pharmacological properties.

Previous investigations indicate that some of the metabolites of the hemorheological agent pentoxifylline (PTX), namely 1-(5-hydroxyhexyl)-3,7-dimethylxanthine (M1), 1-(4-carboxybutyl)-3,7-dimethylxanthine (M4) and 1-(3-carboxypropyl)-3,7-dimethylxanthine (M5), concur to some of the biological effects of the drug. However, information on the bioactivity of the major circulating oxidative metabolites of PTX (M4 and M5) is scanty. Here, we compared the effects of M4 and M5 with that of PTX and its major reductive metabolite, M1, on TNF-alpha production and cytotoxicity, endothelial cell proliferation and on the ATPase activity related to some ATP-binding cassette (ABC) transporters. Unlike PTX and M1, M4 and M5 poorly inhibited lipopolysaccaride-stimulated tumor necrosis factor-alpha (TNF-alpha) release by RAW 264.7 murine macrophages, and did not affect at all cell proliferation and upregulation of TNF-alpha-induced vascular cell adhesion molecule-1 (VCAM-1) in H5V endothelioma cells. By contrast, M4 and M5 were more effective than PTX and M1 in protecting WC/1 murine fibrosarcoma cells from TNF-alpha cytotoxicity. Moreover, results from ATP hydrolase assays indicated that neither PTX nor its tested metabolites interacted significantly with the human multidrug resistance transporters p-glycoprotein/multidrug resistance 1 (MDR1), multidrug resistance-related protein 1 (MRP1), and breast cancer resistance protein (BCRP). Based on these results and literature data, M5, retaining some of the PTX effects but lacking in significant inhibition of TNF-alpha production, may be a promising candidate drug for certain pathologic conditions.

Formation and antitumor activity of PNU-159682, a major metabolite of nemorubicin in human liver microsomes.

PURPOSE: Nemorubicin (3'-deamino-3'-[2''(S)-methoxy-4''-morpholinyl]doxorubicin; MMDX) is an investigational drug currently in phase II/III clinical testing in hepatocellular carcinoma. A bioactivation product of MMDX, 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682), has been recently identified in an incubate of the drug with NADPH-supplemented rat liver microsomes. The aims of this study were to obtain information about MMDX biotransformation to PNU-159682 in humans, and to explore the antitumor activity of PNU-159682. EXPERIMENTAL DESIGN: Human liver microsomes (HLM) and microsomes from genetically engineered cell lines expressing individual human cytochrome P450s (CYP) were used to study MMDX biotransformation. We also examined the cytotoxicity and antitumor activity of PNU-159682 using a panel of in vitro-cultured human tumor cell lines and tumor-bearing mice, respectively. RESULTS: HLMs converted MMDX to a major metabolite, whose retention time in liquid chromatography and ion fragmentation in tandem mass spectrometry were identical to those of synthetic PNU-159682. In a bank of HLMs from 10 donors, rates of PNU-159682 formation correlated significantly with three distinct CYP3A-mediated activities. Troleandomycin and ketoconazole, both inhibitors of CYP3A, markedly reduced PNU-159682 formation by HLMs; the reaction was also concentration-dependently inhibited by a monoclonal antibody to CYP3A4/5. Of the 10 cDNA-expressed CYPs examined, only CYP3A4 formed PNU-159682. In addition, PNU-159682 was remarkably more cytotoxic than MMDX and doxorubicin in vitro, and was effective in the two in vivo tumor models tested, i.e., disseminated murine L1210 leukemia and MX-1 human mammary carcinoma xenografts. CONCLUSIONS: CYP3A4, the major CYP in human liver, converts MMDX to a more cytotoxic metabolite, PNU-159682, which retains antitumor activity in vivo.

Antioxidant defences in cybrids harboring mtDNA mutations associated with Leber's hereditary optic neuropathy.

Oxidative stress and imbalance between free radical generation and detoxification may play a pivotal role in the pathogenesis of Leber's hereditary optic neuropathy (LHON). Mitochondria, carrying the homoplasmic 11778/ND4, 3460/ND1 and 14484/ND6 mtDNA point mutations associated with LHON, were used to generate osteosarcoma-derived cybrids. Enhanced mitochondrial production of reactive oxygen species has recently been demonstrated in these cybrids [Beretta S, Mattavelli L, Sala G, Tremolizzo L, Schapira AHV, Martinuzzi A, Carelli V & Ferrarese C (2004) Brain 127, 2183-2192]. The aim of this study was to characterize the antioxidant defences of these LHON-affected cells. The activities of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutases (SOD) and catalase, and the amounts of glutathione (GSH) and oxidized glutathione (GSSG) were measured in cybrids cultured both in glucose-rich medium and galactose-rich medium. The latter is known to cause oxidative stress and to trigger apoptotic death in these cells. In spite of reduced SOD activities in all LHON cybrids, and of low GPx and GR activities in cells with the most severe 3460/ND1 and 11778/ND4 mutations, GSH and GSSG content were not significantly modified in LHON cybrids cultured in glucose medium. In contrast, in galactose, GSSG concentrations increased significantly in all cells, indicating severe oxidative stress, whereas GR and MnSOD activities further decreased in all LHON cybrids. These data suggest that, in cells carrying LHON mutations, there is a decrease in antioxidant defences, which is especially evident in cells with mutations associated with the most severe clinical phenotype. This is magnified by stressful conditions such as exposure to galactose.

Cytochrome P450 1A2 is a major determinant of lidocaine metabolism in vivo: effects of liver function.

OBJECTIVES: This study was designed (1) to evaluate the effect of a cytochrome P450 (CYP) 1A2 inhibitor, fluvoxamine, on the pharmacokinetics of intravenous lidocaine and its 2 pharmacologically active metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), to confirm recent in vitro results indicating that CYP1A2 is the main isoform responsible for lidocaine biotransformation and (2) to assess whether liver function has any influence on the fluvoxamine-lidocaine interaction. METHODS: The study was carried out in 10 healthy volunteers and 20 patients with cirrhosis, 10 with mild (Child grade A) and 10 with severe (Child grade C) liver dysfunction, according to a randomized, double-blind, 2-phase, crossover design. In one phase all participants received placebo for 6 days; in the other phase they received 50 mg fluvoxamine for 2 days and 100 mg fluvoxamine for the next 4 days. On day 6, a 1-mg/kg lidocaine dose was administered intravenously 2 hours after the last dose of fluvoxamine or placebo. Plasma concentrations of lidocaine, MEGX, GX, and fluvoxamine were measured up to 12 hours after lidocaine injection. RESULTS: The effects of fluvoxamine coadministration were dependent on liver function. Lidocaine clearance was decreased on average by 60% (from 12.1 mL/min.kg to 4.85 mL/min.kg, P <.001) in healthy subjects and by 44% (from 9.83 mL/min.kg to 5.06 mL/min.kg, P <.001) in patients with mild liver dysfunction, with proportional increases in terminal half-lives, whereas virtually no effect was produced in patients with severe liver dysfunction (4.21 mL/min.kg versus 3.65 mL/min.kg, P >.05). Analogous effects were observed on MEGX and GX formation kinetics, which were drastically impaired in healthy subjects and patients with mild liver cirrhosis but virtually unaffected in patients with severe cirrhosis. CONCLUSION: CYP1A2 is the enzyme principally responsible for the metabolic disposition of lidocaine in subjects with normal liver function. The extent of fluvoxamine-lidocaine interaction decreases as liver function worsens, most likely because of the concomitant decrease in the hepatic level of CYP1A2. These observations indicate that results obtained in healthy subjects cannot be extended a priori to patients with liver dysfunction, but the clinical consequences of inhibition of drug metabolism must also be assessed in such patients.

Oral administration of trans-resveratrol to guinea pigs increases cardiac DT-diaphorase and catalase activities, and protects isolated atria from menadione toxicity.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and wine. It has antioxidant and antiproliferative activities, and has been shown to induce NAD(P)H:quinone oxidoreductase, also known as DT-diaphorase, in cultured mouse hepatoma cells. DT-diaphorase is a detoxifying enzyme for quinone-containing substances, due to its ability to prevent their one-electron reduction and the consequent generation of reactive oxygen species (ROS). The aim of the present study was to investigate whether oral administration of trans-resveratrol to guinea pigs (60 mg/l in tap water for 16 days, ad libitum) increases cardiac DT-diaphorase and, consequently, reduces the response of isolated atria to 2-methyl-1,4-naphthoquinone (menadione), the positive inotropic effect of which is related to the amount of ROS generated by its cardiac metabolism. In the cardiac tissue of resveratrol-treated animals, DT-diaphorase activity was significantly higher than that measured in control animals, the V(max) of the enzyme reaction being 75.47 +/- 3.87 and 50.73 +/- 0.63 nmoles/mg protein/min, respectively (p < 0.05). Resveratrol administration also significantly increased the activity of cardiac catalase (32.20 +/- 2.39 vs. 25.14 +/- 3.85 units/mg protein in treated and control animals, respectively; p < 0.001). As a consequence, menadione metabolism by the cardiac homogenate obtained from resveratrol-treated animals generated a smaller amount of ROS and, in electrically driven left atria, menadione produced a significantly lower increase in the force of contraction than in atria isolated from control animals. These results indicate that oral administration of resveratrol exerts cardioprotection against ROS-mediated menadione toxicity.

Severe liver cirrhosis markedly reduces AhR-mediated induction of cytochrome P450 in rats by decreasing the transcription of target genes.

Although the induction of cytochrome P450 (CYP) has long been investigated in patients with cirrhosis, the question whether liver dysfunction impairs the response to CYP inducers still remains unresolved. Moreover, the mechanism underlying the possible effect of cirrhosis on induction has not been investigated. Since ethical constraints do not permit methodologically rigorous studies in humans, this question was addressed by investigating the effect of the prototypical inducer benzo[a]pyrene (BP) on CYP1A1 and CYP1A2 in cirrhotic rats stratified according to the severity of liver dysfunction. We simultaneously assessed mRNA level, protein expression and enzymatic activity of the CYP1A enzymes, as well as mRNA and protein expressions of the aryl hydrocarbon receptor (AhR), which mediates the BP effect. Basal mRNA and protein expressions of CYP1A1 were virtually absent in both healthy and cirrhotic rats. On the contrary, CYP1A2 mRNA, protein and enzyme activity were constitutively present in healthy rats and decreased significantly as liver function worsened. BP treatment markedly increased the concentrations of mRNA and immunodetectable protein, and the enzymatic activities of both CYP1A enzymes to similar levels in healthy and non-ascitic cirrhotic rats. Induced mRNA levels, protein expressions and enzymatic activities of both CYPs were much lower in ascitic rats and were proportionally reduced. Both constitutive and induced protein expressions of AhR were significantly lower in ascitic than in healthy rats. These results indicate that the inducibility of CYP1A enzymes is well preserved in compensated cirrhosis, whereas it is markedly reduced when liver dysfunction becomes severe. Induction appears to be impaired at the transcriptional level, due to the reduced expression of AhR, which controls the transcription of CYP1A genes.

Differential inducing effect of benzo[a]pyrene on gene expression and enzyme activity of cytochromes P450 1A1 and 1A2 in Sprague-Dawley and Wistar rats.

The objective of this study was to compare RT-PCR, Western blot and determination of enzyme activity in the assessment of the induction of cytochromes P450 (CYPs) 1A1 and 1A2 by benzo[a]pyrene (BaP) in Sprague-Dawley and Wistar rats. Inhibition studies and kinetic analyses confirmed literature data indicating that methoxyresorufin is a specific CYP1A2 substrate in both uninduced and BaP-treated rats, whereas ethoxyresorufin is a specific CYP1A1 substrate only in BaP-treated rats. BaP treatment increased mRNA and protein expressions of both CYP1A enzymes to a greater extent in Wistar than Sprague-Dawley rats. It consistently caused a higher increase in mRNA and protein expression of the aryl hydrocarbon receptor in the former rats. By contrast, CYP1A2 enzyme activity was much more markedly increased in Sprague-Dawley than Wistar rats and CYP1A1 activity was induced to similar levels. A BaP-induced increase in the turnover number of CYP1A enzymes in Sprague-Dawley rats, relative to Wistar rats, may provide a plausible explanation for the differential effect of BaP on gene expression and enzyme activity. These results have methodological implications, since they show that RT-PCR and Western blot may not provide a quantitative measure of induction of CYP1A activity, which is the actual measure of the change in CYP1A-mediated metabolism.

Inhibition of cytochrome P450 2C8-mediated drug metabolism by the flavonoid diosmetin.

The aim of this study was to assess the effects of diosmetin and hesperetin, two flavonoids present in various medicinal products, on CYP2C8 activity of human liver microsomes using paclitaxel oxidation to 6α-hydroxy-paclitaxel as a probe reaction. Diosmetin and hesperetin inhibited 6α-hydroxy-paclitaxel production in a concentration-dependent manner, diosmetin being about 16-fold more potent than hesperetin (mean IC(50) values 4.25 ± 0.02 and 68.5 ± 3.3 µM for diosmetin and hesperetin, respectively). Due to the low inhibitory potency of hesperetin, we characterized the mechanism of diosmetin-induced inhibition only. This flavonoid proved to be a reversible, dead-end, full inhibitor of CYP2C8, its mean inhibition constant (K(i)) being 3.13 ± 0.11 µM. Kinetic analysis showed that diosmetin caused mixed-type inhibition, since it significantly decreased the V(max) (maximum velocity) and increased the K(m) value (substrate concentration yielding 50% of V(max)) of the reaction. The results of kinetic analyses were consistent with those of molecular docking simulation, which showed that the putative binding site of diosmetin coincided with the CYP2C8 substrate binding site. The demonstration that diosmetin inhibits CYP2C8 at concentrations similar to those observed after in vivo administration (in the low micromolar range) is of potential clinical relevance, since it may cause pharmacokinetic interactions with co-administered drugs metabolized by this CYP.

Flavonoids diosmetin and hesperetin are potent inhibitors of cytochrome P450 2C9-mediated drug metabolism in vitro.

The aim of this study was to examine in vitro, by means of kinetic analysis and molecular docking simulations, the effects of the flavone diosmetin and its flavanone analog hesperetin on CYP (cytochrome P450) 2C9-mediated drug metabolism. To this purpose, the conversion of diclofenac to 4'-hydroxydiclofenac by human liver microsomes was used as a model assay for assessing the CYP2C9 inhibitory activity of these two flavonoids. Kinetic analyses showed that diosmetin and hesperetin were reversible, dead-end inhibitors of 4'-hydroxydiclofenac formation; their mean K(i) (inhibitor dissociation constant) values were 1.71 ± 0.58 and 21.50 ± 3.62 µM, respectively. Diosmetin behaved as a competitive inhibitor, since it increased markedly the K(m) (substrate concentration yielding 50% of V(max)) of the reaction without affecting the V(max) (maximum velocity of reaction). Hesperetin modified markedly K(m) and to a lesser extent also modified V(max), thus acting as a mixed competitive-noncompetitive inhibitor. The results of molecular docking simulations were consistent with those of kinetic analysis, since they showed that the putative binding sites of both diosmetin and hesperetin coincided with the CYP2C9 substrate binding site. The demonstration that diosmetin and hesperetin inhibit CYP2C9-mediated diclofenac metabolism at low micromolar concentrations is of potential clinical relevance because CYP2C9 is responsible for the biotransformation of various therapeutically important drugs that have narrow therapeutic indexes.

Glutathione transferases as targets for cancer therapy.

Besides catalyzing the inactivation of various electrophile-producing anticancer agents via conjugation to the tripeptide glutathione, some cytosolic proteins belonging to the glutathione transferase (formerly glutatione-S-transferase; GST) superfamily are emerging as negative modulators of stress/drug-induced cell apoptosis through the interaction with specific signaling kinases. In addition, several data link the overexpression of some GSTs, in particular GSTP1-1, to both natural and acquired resistance to various structurally unrelated anticancer drugs. Tumor overexpression of these proteins has provided a rationale for the search of GST inhibitors and GST-activated cytotoxic prodrugs. In the present review we discuss the current structural and pharmacological knowledge of both types of GST-targeting compounds.

Synthesis and preliminary in vitro biological evaluation of 4-[(4-hydroxyphenyl)sulfanyl]but-3-en-2-one, a 4-mercaptophenol derivative designed as a novel bifunctional antimelanoma agent.

We report the synthesis and preliminary in vitro biological evaluations of 4-[(4-hydroxyphenyl)sulfanyl]but-3-en-2-one, a compound designed as a potential bifunctional antimelanoma agent, bearing both a tyrosinase-activatable phenolic moiety and a GSH-reactive alpha,beta-unsaturated carbonyl group. Both the E (1) and Z (2) isomers of the synthesized compound proved to be very good substrates of mushroom tyrosinase, reacted quickly with GSH at physiological pH, and showed a significant cytotoxic activity against B16F1 murine melanoma cells.

Flavonoids diosmetin and luteolin inhibit midazolam metabolism by human liver microsomes and recombinant CYP 3A4 and CYP3A5 enzymes.

We evaluated the effects of increasing concentrations of the flavonoids salvigenin, diosmetin and luteolin on the in vitro metabolism of midazolam (MDZ), a probe substrate for cytochrome P450 (CYP) 3A enzymes, which is converted into 1'-hydroxy-midazolam (1'-OH-MDZ) and 4-hydroxy-midazolam (4-OH-MDZ) by human liver microsomes. Salvigenin had only a modest effect on MDZ metabolism, whereas diosmetin and luteolin inhibited in a concentration-dependent manner the formation of both 1'-OH-MDZ and 4-OH-MDZ, with apparent K(i) values in the 30-50mumol range. Both diosmetin and luteolin decreased 1'-OH-MDZ formation by human recombinant CYP3A4, but not CYP3A5, whereas they decreased 4-OH-MDZ formation by both recombinant enzymes. To assess whether any relationship exists between the physico-chemical characteristics of flavones and their effects on MDZ metabolism, we tested the effects of three other flavones (flavone, tangeretin, chrysin) on MDZ metabolism by human liver microsomes. Whereas flavones possessing more than two hydroxyl groups (luteolin, diosmetin) inhibited MDZ biotransformation, flavones lacking hydroxyl groups in their A and B rings (flavone, tangeretin) stimulated MDZ metabolism. We also found close relationships between the maximum stimulatory or inhibitory effects of flavones on 1'-OH-MDZ and 4-OH-MDZ formation rates and their log of octanol/water partition coefficients (logP) or their total number of hydroxyl groups. The results of the study may be of clinical relevance since they suggest that luteolin and diosmetin may cause pharmacokinetic interactions with co-administered drugs metabolized via CYP3A.

In vitro hepatic conversion of the anticancer agent nemorubicin to its active metabolite PNU-159682 in mice, rats and dogs: a comparison with human liver microsomes.

We recently demonstrated that nemorubicin (MMDX), an investigational antitumor drug, is converted to an active metabolite, PNU-159682, by human liver cytochrome P450 (CYP) 3A4. The objectives of this study were: (1) to investigate MMDX metabolism by liver microsomes from laboratory animals (mice, rats, and dogs of both sexes) to ascertain whether PNU-159682 is also produced in these species, and to identify the CYP form(s) responsible for its formation; (2) to compare the animal metabolism of MMDX with that by human liver microsomes (HLMs), in order to determine which animal species is closest to human beings; (3) to explore whether differences in PNU-159682 formation are responsible for previously reported species- and sex-related differences in MMDX host toxicity. The animal metabolism of MMDX proved to be qualitatively similar to that observed with HLMs since, in all tested species, MMDX was mainly converted to PNU-159682 by a single CYP3A form. However, there were marked quantitative inter- and intra-species differences in kinetic parameters. The mouse and the male rat exhibited V(max) and intrinsic metabolic clearance (CL(int)) values closest to those of human beings, suggesting that these species are the most suitable animal models to investigate MMDX biotransformation. A close inverse correlation was found between MMDX CL(int) and previously reported values of MMDX LD(50) for animals of the species, sex and strain tested here, indicating that differences in the in vivo toxicity of MMDX are most probably due to sex- and species-related differences in the extent of PNU-159682 formation.

Fluorescent, internally quenched, peptides for exploring the pH-dependent substrate specificity of cathepsin B.

Cathepsin B is a cysteine protease that in tumor tissues is localized in both acidic lysosomes and extracellular spaces. It can catalyze the cleavage of peptide bonds by two mechanisms: endoproteolytic attack with a pH optimum around 7.4, and attack from the C-terminus with a pH optimum at 4.5-5.5. In this work, seven fluorescent, internally quenched, decapeptides have been synthesized using the prototypical cathepsin B selective substrate Z-Phe-Arg-AMC as a lead, and used to identify the structural factors determining the susceptibility of peptides to hydrolysis at acidic and neutral pH values. Each peptide differs from the others in one amino acid (residue 6) and contains a highly fluorescent Nma group linked to the alpha-amino function of the N-terminal Orn residue and a Dnp group linked to the side chain of the Lys(8) residue acting as a quencher. Proteolytic cleavage was monitored by measuring the increase of fluorescence at 440 nm upon excitation at 340 nm, and the cleavage sites were determined by HPLC followed by ESI-MS analysis. Peptides containing Ala or Phe at position 6 are good substrates for the enzyme at both pH 5.0 and 7.4. By contrast, those containing Glu, Asp, Lys or Val are not cleaved at all by cathepsin B at pH 7.4, and are poorly hydrolyzed at pH 5.0. These findings provide new information for the rational design of cathepsin B-activated peptide-containing anticancer drugs.

In vitro evidence that carteolol is a nonconventional partial agonist of guinea pig cardiac beta1-adrenoceptors: a comparison with xamoterol.

The present study was designed to verify our previous hypothesis that carteolol, a beta1/beta2-adrenoceptor-blocking agent, is a nonconventional partial agonist of cardiac beta1-adrenoceptors. To this purpose, we characterized the effects of carteolol in guinea pig myocardial preparations and measured the affinities of carteolol for high- and low-affinity sites of beta1-adrenoceptors labeled by CGP12177 [(-)4-(3-t-butylamino-2-hydroxypropoxy)-2-benzimidazol-2-one]. All experiments were performed in comparison with xamoterol, a cardioselective beta1-adrenoceptor partial agonist. Both drugs caused cAMP-dependent positive inotropic and chronotropic effects, but carteolol was less effective and less potent than xamoterol, and its cardiac actions were not affected by conventional concentrations of the beta-blocker propranolol. Both carteolol and xamoterol antagonized the cardiac effects of isoprenaline, but although the antagonistic concentrations of xamoterol were almost equal to those producing cardiostimulation, the antagonistic concentrations of carteolol were 3 log units lower than those causing cardiostimulant effects. Both carteolol and xamoterol competed with (-)[3H]CGP12177 for a high-affinity site of beta1-adrenoceptors, but carteolol showed a higher affinity than xamoterol. Moreover, carteolol, unlike xamoterol, bound also to a low-affinity site of the receptors. The binding affinity constants of the drugs for the high-affinity site correlated well with the respective blocking potencies against isoprenaline, whereas the affinity constant of carteolol for the low-affinity site was well related to its agonist potency. In conclusion, our findings demonstrate that carteolol, unlike xamoterol, is a nonconventional partial agonist, which causes agonistic effects through interaction with the low-affinity propranolol-resistant site of beta1-adrenoceptors and antagonistic actions through the high-affinity site of the same receptors.

Role of antioxidant defences in the species-specific response of isolated atria to menadione.

In previous works we demonstrated that 2-methyl-1,4-naphthoquinone (menadione) causes a marked increase in the force of contraction of guinea pig and rat isolated atria. This inotropic effect was significantly higher in the guinea pig than in the rat and was strictly related to the amount of superoxide anion (O(2)(*-)), generated as a consequence of cardiac menadione metabolism through mitochondrial NADH-ubiquinone oxidoreductase. The present study was designed to further elucidate the basis of these quantitatively different positive inotropic responses. To this purpose, we measured O(2)(*-) and hydrogen peroxide (H(2)O(2)) produced by mitochondria isolated from guinea pig and rat hearts in the presence of 20 microM menadione. Moreover, we evaluated the menadione detoxification activity (DT-diaphorase) and the antioxidant defences of guinea pig and rat hearts, namely their GSH/GSSG content, Cu/Zn- and Mn-dependent superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (Gpx) activities. Our results indicate that DT-diaphorase activity and glutathione levels were similar in both animal species. By contrast, guinea pig mitochondria produced greater amounts of O(2)(*-) and H(2)O(2) than those of rat heart. This is probably due to both the higher Mn-SOD activity (2.93 +/- 0.02 vs. 1.95 +/- 0.06 units/mg protein; P < 0.05) and to the lower Gpx activity (10.09 +/- 0.30 vs. 32.67 +/- 1.02 units/mg protein; P < 0.001) of guinea pig mitochondria. A lower CAT activity was also observed in guinea pig mitochondria (2.40 +/- 0.80 vs. 6.13 +/- 0.20 units/mg protein; P < 0.01). Taken together, these data provide a rational explanation for the greater susceptibility of guinea pig heart to the toxic effect of menadione: because of the greater amount of O(2)(*-) generated by the quinone and the higher mitochondrial Mn-SOD activity, guinea pig heart is exposed to more elevated concentrations of H(2)O(2) that is less efficiently detoxified, because of lower Gpx and CAT levels of mitochondria.

Oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian Botryllus schlosseri.

When genetically incompatible colonies of the compound ascidian Botryllus schlosseri contact each other, a rejection reaction occurs, characterised by the appearance of cytotoxic foci along the touching borders. In the course of this reaction, morula cells, a common haemocyte-type in ascidians, release their vacuolar content, mainly phenoloxidase and its polyphenol substrata, upon the recognition of soluble factors diffusing from the alien colony through the partially fused tunic. In a previous paper, we demonstrated the relationship between phenoloxidase and cytotoxicity. Here, we investigated the effects of superoxide dismutase, catalase and sorbitol (scavengers of superoxide anions, peroxides and hydroxyl radicals, respectively) on the cytotoxicity observed in haemocyte cultures incubated with heterologous blood plasma. Although the above compounds have no effects on morula cell degranulation and phenoloxidase activity, they suppress cell death, suggesting that oxidative stress plays a key role in in vitro cytotoxicity. In addition, sorbitol reduces the extent of the cytotoxicity occurring in the rejection reaction between incompatible colonies, which stresses the important role of hydroxyl radicals in this process. The observation of a decrease in total and non-protein thiols in haemocytes previously incubated with heterologous blood plasma fits the hypothesis of oxidative stress as the main cause of phenoloxidase-related cytotoxicity.