Low concentrations of UD-CG 212 enhance myocyte contractility by an increase in calcium responsiveness in the presence of inorganic phosphate

Recent data show that UD-CG 212 in nanomolar concentrations increases myofibrillar Ca⁺⁺ responsiveness of chemically skinned cardiac preparations in the presence of elevated inorganic phosphate. We studied the effects of UD-CG 212 on cell shortening of intact myocytes and in addition measured the intracellular calcium transients with the aid of INDO-1 fluorescence in the presence of 5 mM inorganic phosphate.The validity of our experimental system was first tested with the calcium channel opener Bay k 8644. Bay k 8644 at 10^–8 M did not significantly influence myocyte shortening ( + 13.9 ± 4.6%, n = 9) but at 10^–7 M and 10^–6 M significantly increased contraction by 40.1 +- 13.6%and52.5 ± 17.0% respectively. Bay k 8644 at 10^–8 M increased the INDO-1 fluorescence ratio by 17.3 ± 4.7% (P < 0.01; n = 9), and at 10^–7 M by 21.5 + 4.3% (P < 0.01; n = 9), whereas 10^–6 M Bay k 8644 had no significant effect on peak INDO-1 ratio. However, 10^–7and 10^–6 M Bay k 8644 accelerated and broadened the calcium transients.Cell shortening of guinea pig ventricular myocytes electrically stimulated at 1 Hz was significantly increased by UD-CG 212 (10^–9-10^–7 M) and isoprena line(3 × 10^–8 M). An increase of 37.0 ± 14.0% (P < 0.05; n = 9) was observed at 10^–9 M UD-CG 212, 90.5±18.2% (P<0.05; n=9) at 10^–8 M UD-CG 212, 164.0 ± 34.9% (P < 0.05; n = 9) at 10^–7 M UD-CG 212, and 258.2 ± 67.4% (P < 0.05; n = 9) at 3 × 10-8 M isoprenaline. Peak INDO-1 fluorescence ratios were not significantly (P > 0.05) influenced after addition of 10^–9 M and 10^–8 M UD-CG 212, but significantly increased by 19.4 ± 4.9%(P < 0.05; n = 9) at 10^–7 MUD-CG 212 and by 81.1 ± 11.1% (P < 0.05; n = 9) at 3 x 10^–8 M isoprenaline.In conclusion, UD-CG 212 (10^–9 - 10^–7 M) Concentration-dependently increased myocyte shortening in the presence of 5 mM inorganic phosphate. Low concentrations of 10^–9 and 10^–8 M UD-CG 212 increased myocyte contractility without altering the peak INDO-1 fluorescence ratio whereas 10^–7 M UD-CG 212 and 3 × 10^–8 M isoprenaline increased cell shortening as well as peak INDO-1 fluorescence ratio. These data suggest that low concentrations of UD-CG 212 increase myocyte contractility by enhancing myofibrillar calcium responsiveness whereas higher concentrations elevate intracellular calcium probably via increased intracellular CAMP brought about by phosphodiesterase inhibition.     

Mechanism underlying histamine-induced intracellular Ca movement in PC3 human prostate cancer cells

The effect of histamine on intracellular free Ca ²⁺levels ([Ca ²⁺] _i) in PC3 human prostate cancer cells and the underlying mechanism were evaluated using fura-2 as a Ca ²⁺dye. Histamine at concentrations between 0.1 and 50 μM increased [Ca ²⁺] _iin a concentration-dependent manner with an EC ₅₀value of 1 μM. The [Ca ²⁺] _iresponse comprised an initial rise and a slow decay, which returned to baseline within 3 min. Extracellular Ca ²⁺removal inhibited 50% of the [Ca ²⁺] _isignal. In the absence of extracellular Ca ²⁺, after cells were treated with 1 μM thapsigargin (an endoplasmic reticulum Ca ²⁺pump inhibitor), 10 μM histamine did not increase [Ca ²⁺] _i. After pretreatment with 10 μM histamine in a Ca ²⁺-free medium for several minutes, addition of 3 mM Ca ²⁺induced [Ca ²⁺] _iincreases. Histamine (10 μM)-induced intracellular Ca ²⁺release was abolished by inhibiting phospholipase C with 2 μM 1-(6-((17 β-3- methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 10 μM pyrilamine but was not altered by 50 μM cimetidine. Collectively, the present study shows that histamine induced [Ca ²⁺] _itransients in PC3 human prostate cancer cells by stimulating H1 histamine receptors leading to Ca ²⁺release from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, and by inducing Ca ²⁺entry.     

Sulfation of resveratrol in human liver: Evidence of a major role for the sulfotransferases SULT1A1 and SULT1E1

Sulfation of resveratrol, a polyphenolic compound present in grapes and wine with anticancer and cardioprotective activities, was studied in human liver cytosol. In the presence of 3′-phosphoadenosine-5′-phosphosulfate, three metabolites (M1–3) whose structures were identified by mass spectrometry and NMR as trans-resveratrol-3-O-sulfate, trans-resveratrol-4′-O-sulfate, and trans-resveratrol-3-O-4′-O-disulfate, respectively. The kinetics of M1 formation in human liver cytosol exhibited an pattern of substrate inhibition with a K_i of 21.3?±?8.73?µM and a V_max/K_m of 1.63?±?0.41?µL?min^?1mg^?1 protein. Formation of M2 and M3 showed sigmoidal kinetics with about 56-fold higher V_max/K_m values for M3 than for M2 (2.23?±?0.14 and 0.04?±?0.01?µL?min^?1?mg^?1). Incubation in the presence of human recombinant sulfotransferases (SULTs) demonstrated that M1 is almost exclusively catalysed by SULT1A1 and only to a minor extent by SULT 1A2, 1A3 and 1E1, whereas M2 is selectively formed by SULT1A2. M3 is mainly catalysed by SULT1A2 and 1A3. In conclusion, the results elucidate the enzymatic pathways of resveratrol in human liver, which must be considered in humans following oral uptake of dietary resveratrol.     

Changes in antioxidant activity,total phenolic and abscisic acid constituents in the aquatic plants <Emphasis Type="Italic">Myriophyllum spicatum</Emphasis> L. and <Emphasis Type="Italic">Myriophyllum triphyllum</Emphasis> Orchard exposed to cadmium

Changes in antioxidant activity, total phenolic and abscisic acid (ABA) constituents of Myriophyllum spicatum L. and Myriophyllum triphyllum Orchard, cadmium (Cd) aqueous macrophytes, were investigated exposed to 0, 2, 4, 6, 8, 16 mg l^-1 Cd concentrations. M. triphyllum exhibited strong antioxidant activity but not M. spicatum before and after exposure. Free radical scavenging activity of M. triphyllum was significantly affected from the Cd concentrations and a significant increase was observed at 6 mgl^-1 Cd concentration. Total phenolic constituent and ABA concentration of M. triphyllum is higher than that of M. spicatum with or without heavy metal exposure (P < 0.05). While total phenolic constituents of both species were not significantly affected from Cd concentrations except for 6 mgl^-1 Cd concentration ABA contents did. ABA content of M. triphyllum increased from 1.81 ± 0.10 μg g^-1 (control) to 5.13 ± 0.15 μg g^-1 at 16 mg l^-1 Cd concentration and increase was from 0.59 ± 0.08 μg g^-1 (control) to 2.05 ± 0.10 μg g^-1 for M. spicatum at the same Cd concentration. Both species accumulated ABA indicating submerge plants can also accumulate ABA and its concentration increase with increasing Cd concentration. Such studies as this one may be important for evaluation of the metabolic variations of toxic metal tolerant macrophytes that grown in polluted aqueous ecosystem.     

Biological Evaluation of a Secondary Metabolite, 9-O-methylfusarubin, from Fusarium oxysporum

9-O-methylfusarubin was isolated from a novel source, Fusarion oxysporum, and evaluated for its phytotoxic and chlorotic inducing properties in plants as well as its potential as an antineoplastic agent. Etiolated wheat coleptiles were inhibited 100 and 40%, respectively, at 10^-3 and 10^-4 M. Bean plants were necrotic and chlorotic, with leaf malformations, following 10^-2 M treatments. Corn plants were chlorotic following 10^-3 and 10^-4 M treatments. Tobacco plants exhibited subtle interveinal chlorosis with 10^-2 M applications. All agrochemical effects were substantial, but transient. The anticancer properties were evaluated using ras transformed liver epithelial cells. Doses ranging from 12.5-50 μM provided a dose-dependent reduction in the number of viable cells when compared to controls. At the 50 μM dose, the number of viable cells was reduced by 65% suggesting potential antineoplastic effects of 9-O-methylfusarubin.     

Cardiovascular effects elicited by milonine, a new 8,14-dihydromorphinandienone alkaloid

Abstract: The mechanisms underlying the cardiovascular responses evoked by milonine (i.v.), an alkaloid, were investigated in rats. In normotensive rats, milonine injections produced hypotension and tachycardia, which were attenuated after N^w‐nitro‐l ‐arginine methyl esther (l ‐NAME; 20 mg/kg, i.v.). In phenylephrine (10 μM), pre‐contracted mesenteric artery rings, milonine (10^?10 M to 3 × 10^?4 M) caused a concentration‐dependent relaxation (EC₅₀ = 1.1 × 10^?6 M, E_max = 100 ± 0.0%) and this effect was rightward shifted after either removal of the vascular endothelium (EC₅₀ = 1.6 × 10^?5, p < 0.001), or after l ‐NAME 100 μM (EC₅₀ = 6.2 × 10^?5, p < 0.001), hydroxocobalamin 30 μM (EC₅₀ = 1.1 × 10^?4, p < 0.001) or ODQ 10 μM (EC₅₀ = 1.9 × 10^?4p < 0.001). In addition, in rabbit aortic endothelial cells, milonine increased NO₃^? levels. The relaxant effect induced by milonine was attenuated in the presence of KCl (20 mM), a modulator efflux K⁺ (EC₅₀ = 1.2 × 10^?5, p < 0.001), or different potassium channel blockers such as glibenclamide (10 μM) (EC₅₀ = 6.3 × 10^?5, p < 0.001), TEA (1 mM) (EC₅₀ = 2.3 × 10^?5 M, n = 6) or Charybdotoxin (0.2 μM) plus apamin (0.2 μM) (EC₅₀ = 3.9 × 10^?4 M, n = 7). In addition, pre‐contraction with high extracellular potassium concentration prevented milonine‐induced vasorelaxation (EC₅₀ = 1.0 × 10^?4, p < 0.001). Milonine also reduced CaCl₂‐induced contraction in Ca²⁺‐free solution containing KCl (60 mM). In conclusion, using combined functional and biochemical approaches, we demonstrated that the hypotensive and vasorelaxant effects produced by milonine are, at least in part, mediated by the endothelium, likely via nitric oxide release, activation of nitric oxide‐cGMP pathway and opening of K⁺ channels.     

Pharmacokinetic changes of M1, M2, M3 and M4 after intravenous administration of a new anthracycline,DA-125, to rats pretreated with phenobarbital, 3-methylcholanthrene,chloramphenicol, or SKF-525A

The pharmacokinetics of M1–M4, the metabolites of a new anthracycline antineoplastic agent, DA-125, were compared after intravenous (IV) administration of DA-125, 15 mg kg⁻¹, to rats pretreated with enzyme inducers, such as phenobarbital (PBT, n = 14) and 3-methylcholanthrene (MCT, n = 15), or enzyme inhibitors, such as SKF-525A (SKT, n = 11) and chloramphenicol (CMT, n = 15), and to their control rats (n = 15 for PBC, CMC or SKC, and n = 11 for MCC). After IV administration of DA-125, the plasma concentrations of both M1 and M2 declined slowly from 1 to 2 h onwards to 8 h in all groups of rats due to the continuous formation of M2 from M1. The AUC_{0–8 h} of M1 (47.1 versus 7.85 μg min mL⁻¹) and M2 (20.7 versus 44.3 μg min mL⁻¹) decreased significantly in the PBT group compared to those in the PBC group. However, the corresponding value of only M1 (74.6 versus 89.9 μg min mL⁻¹) decreased significantly in the MCT group. The above data indicate that metabolism of M1 is increased by pretreatment with both PB and 3-MC, and that of M2 with PB, but not with 3-MC. The AUC_{0–8 h} of both M1 (126 versus 78.5 μg min mL⁻¹) and M2 (69.2 versus 44.3 μg min mL⁻¹) increased significantly in the SKT group compared to the SKC group. However, the corresponding values were not significantly different between CMC and CMT groups. The above data indicate that the metabolism of both M1 and M2 is inhibited by pretreatment with SKF-525A, but not with CM. © 1998 John Wiley & Sons, Ltd.     

In Vitro Effect of Anti-Inflammatory Agents on Phagocytosis and Bacterial Killing by Human Neutrophilic Leukocytes

The phagocytosis of killed Staphylococcus epidermidis by neutrophilic leukocytes was inhibited in vitro by indomethacin in a concentration of 3 × 10^-4M, and by hydrocortisone, phenylbutazone, and paracetamol in a concentration of 10^-3M. Phagocytosis was slightly stimulated by 10^-7M phenylbutazone. Acetylsalicylic acid, mefenamic acid, and phenacetin had no effect. The bactericidal activity of leukocytes against live Staphylococcus epidermidis was reduced by 10^-4M phenacetin, 5 × 10^-4M mefenamic acid, and 10^-3M phenylbutazone and indomethacin, whereas concentrations of 10^-5M and above of paracetamol enhanced bacterial killing by leukocytes. Hydrocortisone and acetylsalicylic acid were ineffective. It is probable that concentrations of anti-inflammatory agents sufficient to affect phagocytosis and bacterial killing by neutrophils are as a rule not attained in the organism during treatment, although inhibition may occur locally, for example in inflamed tissue, as a consequence of a low pH, which potentiates the effect of acid anti-inflammatory drugs. This inhibition may be a factor in the mechanism of action of anti-inflammatory compounds.     

Transport properties of 3′-azido-3′-deoxythymidine and 2′,3′-dideoxyinosine in the rat choroid plexus

Transport properties of 3′-azido-3′-deoxythymidine (AZT) and 2′, 3′-dideoxyinosine (DDI) were characterized in the isolated rat choroid plexus. AZT and DDI competitively inhibited the active transport of [³H]benzylpenicillin, a prototypic organic anion, with K_i values of 85·4±13·1 and 155±22 μM, respectively. Accumulation of [³H]DDI was against an electrochemical potential via a saturable process (K_m=29·7±4·9 μM, V_max=13·5±2·4 pmol min⁻¹/μL tissue) that was inhibited by metabolic inhibitors (carbonylcyanide p -trifluoromethoxyphenylhydrazone, 10 μM, and rotenone, 30 μM) and sulphydryl reagents (p -chloromercuribenzoic acid, 100 μM, and p -chloromercuribenzenesulphonic acid, 100 μM), but did not require an inwardly directed Na⁺ gradient. Accumulation of [³H]DDI was inhibited by benzylpenicillin and AZT in a dose-dependent manner, with IC₅₀ values of 91·6±28·9 and 294±84 μM, respectively. In contrast, no significant accumulation of [³H]AZT was observed. These results suggest that DDI is transported, at least in part, by the transport system for organic anions located on the rat choroid plexus, whereas AZT is recognized, but not transported by this system. © 1997 John Wiley & Sons, Ltd.     

The lysosome among targets of metformin: new anti-inflammatory uses for an old drug?

Background: Rheumatoid arthritis and type-2 diabetes exhibit progressive co-morbidity. Chloroquine (CQ) reportedly improves both. CQ inhibits lysosomal function in cultured cells at supra-therapeutic concentration; however, this is doubted as target mechanism. Some anti-diabetic biguanides are metal-interactive lysosomal inhibitors; and all bind Zn²⁺.

Objectives: i) To bioassay the potency of CQ using ³H-leucine release from perfused myocardial tissue. ii) To determine whether metformin (MET) is CQ-mimetic, and interactive with Zn²⁺.

Results: Therapeutic CQ concentration (0.1 – 0.5 μM) clearly does cause lysosomal inhibition although delayed and submaximal. MET alone (10 μM) caused sub-maximal inhibition. Supra-physiological extracellular Zn²⁺ (5 – 50 μM) alone increased tissue Zn²⁺ content, and inhibited lysosomal proteolysis. Physiological equivalent Zn²⁺ (approximately 1 μM) had no effect. MET (≤ 25 μM) and Zn²⁺ (≤ 1 μM) exhibited astounding 10 – 100 fold anti-lysosomal synergy. Cathepsin B was 50% inhibited by 1 μM Zn²⁺, and is reportedly inhibited by gold agents.

Interpretation: MET somehow increases the natural inhibitory action of action of Zn²⁺ against cysteinyl proteases. TNF-alpha activates lysosomal function; and CatB is among post-receptor players. MET might decrease antigen processing in specialized cells, and lysosomal hyper-catabolism in other cells.

Conclusions: Trials of MET for new use as an anti-inflammatory agent are suggested. Guanidylguanidine is a practical pharmacophore for synthesis of future anti-lysosomal agents.     

Cytotoxicity of antineoplastic agents on rabbit corneal epithelium in vitro

Abstract

We studied the cytotoxic effects of antineoplastic agents in vitro including cytosine arabinoside (Ara-C), methotrexate, melphalan, 5-fluorouracil (5-FU), and triethylenethiophosphoramide (thiotepa), using rabbit corneal epithelial cell primary tissue cultures. Twenty-four hour [³H]thymidine incorporation was measured 6 hr after the addition of drug or vehicle. A dose-response effect was noted for all agents. Significant (p <0.05) inhibition of thymidine incorporation was reached with Ara-C (10^?7M), methotrexate (10^?3M), melphalan (10-⁶M), 5-FU (10^?4M), and thiotepa (10^?4M). Visual inspection of the cultures was a less sensitive indicator of drug-induced cytotoxicity; except for melphalan, drug concentrations much greater than those that inhibited thymidine incorporation did not alter cell morphology. At a concentration of 10^?4M, 2′-deoxycytidine, a competitive inhibitor of Ara-C, protected corneal epithelial cells exposed to Ara-C concentrations up to 10^?5M. When used alone, concentrations of deoxycytidine greater than 10^?4M were associated with significant impairment of thymidine uptake by cells. This in vitro model may be useful in quantitatively evaluating the epithelial cytotoxicity of other current and future antineoplastic agents, as a means for predicting and possibly moderating corneal toxicity of these agents clinically.     

Effect ofAlpha-2 adrenergic agonist onBeta adrenoceptor-mediated control of blood glucose in the fasted mouse

Dose-dependent increases in blood glucose were produced by epinephrine and clonidine in fasted male mice. Isoproterenol was ineffective in increasing blood glucose at lower doses (10⁻⁸ M/kg−10⁻⁷ M/kg); with higher dose (10⁻⁶ M/kg) the glucose level was increased. The hyperglycemia induced by epinephrine was inhibited by yohimbine, prazosin and propranolol, indicating that the hyperglycemic effect of epinephrine is mediated byalpha-1,alpha-2 andbeta adrenoceptor. When clonidine (10⁻⁶ M/kg) was administered simultaneously with isoproterenol (10⁻⁶ M/kg), an enhenced hyperglycemic effect was observed. The increment produced by clonidine plus isoproterenol was higher than that by clonidine alone. These results suggest that stimulation ofalpha-2 adrenoceptor may be responsible for the exertion of the hyperglycemic effect bybeta agonists in fasted mice.     

The effect of amperozide on uptake and release of [3H]-dopamine in vitro from perfused rat striatal and limbic brain areas

Amperozide, a putatively antipsychotic drug, was studied for its effects on uptake and release of [³H]-dopamine in rat brain in vitro. Amperozide inhibited uptake of [³H]-dopamine in striatal chopped tissue in vitro with an IC₅₀ of 18 μM. It also increased basal release of [³H]-dopamine from perfused rat striatal and limbic tissue in vitro at concentrations above 5 μM. Release of [³H]-dopamine from perfused rat striatal and limbic tissue stimulated with 5 μM amphetamine, was inhibited by 1 μM amperozide to 46%. No significant difference was found for the effect of amperozide on in vitro release of [³H]-dopamine from corpus striatum compared to tissue from limbic brain regions; neither on basal release nor on amphetamine-stimulated release of dopamine.     

Influence of calcium on the effects of okadaic acid and its interaction with caffeine and theophylline in rat myometrium

The effects of okadaic acid (OA), a monocarboxylic acid produced by marine dinoflagellates belonging to the genera Dinophysis and Prorocentrum, and their interactions with theophylline and caffeine were studied on the rat-isolated uterus in a calcium-containing medium and a calcium-free medium in the presence of 10^–3 M EGTA.Okadaic acid (5 × 10^–6 to 5 × 10^–5 M) induced a concentration-dependent contraction of the rat-isolated uterus corresponding, with 5 × 10^–5M, to 142.3±6.1% (n = 7) of the contraction induced by oxytocin 10^–6 M. The time to peak tension was inversely proportional to the maximum effect produced. The contraction was not sustained and was followed by a concentration-dependent decrease in tone. In a Ca²⁺-free medium containing 10^–3 M EGTA the contractile effects of OA were significantly inhibited or reduced. A 30 min pretreatment with theophylline (3 × 10^–3 M) or caffeine (2 × 10^–2 M) significantly reduced, in a Ca²⁺-containing medium, the maximum contractile effect of OA 10^–5 and/or 2 × 10^–5 M and shortened the relative time to peak tension. In a Ca²⁺-free medium containing 10^–3 M EGTA, only the second effect was observed. With a 1 min pretreatment and in a Ca²⁺-containing medium, theophylline 3 × 10^–3 M and caffeine 10^–2 M did not modify the maximum effect of OA 10^–5 M but shortened the time to peak tension. The same concentrations of the xanthines potentiated the E_max of OA 5 × 10^–6 M in the Ca²⁺-containing medium or in a Ca²⁺-free medium containing 10^–3 M EGTA. Okadaic acid 10^–6 M used as 30 min pretreatment versus OA 10^–5 M and 2 × 10^–5 M behaved like caffeine or theophylline.These results suggest that the OA-induced contraction of the rat uterine smooth muscle is partly effected by transmembrane calcium movements which can be inhibited in an O-Ca²⁺–10^–3 M EGTA solution or by theophylline or caffeine. This contraction also involves mobilization of Ca²⁺ from an intracellular pool which is also xanthine-sensitive. The latter effect seems to be important in inducing the contractile effect. This study does not exclude the possibility of other mechanisms being involved in the contraction induced by OA. Correspondence to: M. L. Candenas at the above address in Burjassot-València     

Radioligand binding characteristics of the chicken cardiac muscarinic receptor

Summary The muscarinic receptor present in chicken cardiac membranes was characterised using a ligand binding approach and compared to the M1, M2 and M3 receptors that can be identified in ligand binding studies at present. [³H]N-methylscopolamine and [³H]pirenzepine appeared to label the same population of muscarinic receptors in chicken cardiac membranes since the density of sites labeled by the two radioligands was similar. Furthermore, affinity estimates of 8 muscarinic receptor antagonists for chicken cardiac muscarinic receptors were the same irrespective of whether [³H]N-methylscopolamine or [³H]pirenzepine was used as the radioligand. The chicken cardiac muscarinic receptor displayed high affinity for pirenzepine (pK _i = 7.9) and so did not appear to represent an M2 receptor. Despite the high affinity of chicken cardiac muscarinic receptors for pirenzepine, affinity estimates for dicyclomine (pK _i = 8.0), CPPS (pK _i = 8.4) and 4DAMP (pK _i = 8.6) in chicken heart were not consistent with the presence of M1 receptors. The chicken cardiac muscarinic receptor also displayed significant differences to the M3 receptor since it displayed high affinity for AF-DX 116 (pK _i = 7.1) and methoctramine (pK _i = 8.4). Finally, chicken heart muscarinic receptors displayed high affinity for gallamine (pK _i = 7.0) and pirenzepine suggesting that the receptor was different to the M4 muscarinic receptor of the NG108-15 cell line. These findings suggest that chicken heart expresses a novel muscarinic receptor subtype distinct from the M1, M2, M3, and M4 subtypes already described. Send offprint requests to A. Michel.     

Identification of the human cytochrome P450s responsible for the in vitro metabolism of a leukotriene B4 receptor antagonist,CP-195,543

1.?The major human cytochrome P450 (CYP) form(s) responsible for the metabolism of CP-195,543, a potent leukotriene B4 antagonist, were investigated.

2.?Incubation of CP-195,543 with human liver microsomes resulted in the formation of three major metabolites, M1–3. M1 and M2 were diastereoisomers and formed by oxidation on the benzylic position. M3 was formed by aromatic oxidation of the benzyl group attached to the 3-position of the benzopyran ring.

3.?The results from experiments with recombinant CYPs, correlation studies and inhibition studies with form-selective inhibitors and a CYP3A antibody strongly suggest that the CYP3A4 plays a major role in the metabolism of CP-195,543. Recombinant CYP3A5 did not metabolize CP-195,543.

4.?The apparent K_m and V_max for the formation of M1–3 in human liver microsomes were determined as 36?μM and 4.1?pmol?min^?1?pmol^?1 P450, 44?μM and 10?pmol?min^?1?pmol^?1 P450, and 34?μM and 2.0?pmol?min^?1?pmol^?1 P450, respectively. The average in vitro intrinsic clearance for M2 was the highest both in human liver microsomes and recombinant CYP3A4 compared with M1 and M3. Intrinsic clearance for M2 in human liver microsomes and recombinant CYP3A4 was 0.231 and 0.736 ml?min^?1?pmol^?1 P450, respectively. The intrinsic clearances for M1 and M3 in human liver microsomes and CYP3A4 were 0.114 and 0.060 and 0.197 and 0.088 ml?min^?1?pmol^?1 P450, respectively. This suggests that benzylic oxidation is the predominant phase I metabolic pathway of CP-195,543 in man.     

Human mass balance,metabolite profile and identification of metabolic enzymes of [14C]ASP015K,a novel oral janus kinase inhibitor

Abstract

1.?The human mass balance of ¹⁴C-labelled ASP015K ([¹⁴C]ASP015K), an orally bioavailable Janus kinase (JAK) inhibitor, was characterized in six healthy male subjects after a single oral dose of [¹⁴C]ASP015K (100?mg, 3.7?MBq) in solution. [¹⁴C]ASP015K was rapidly absorbed with t_max of 1.6 and 1.8?h for ASP015K and total radioactivity in plasma, respectively. Mean recovery in urine and feces amounted to 36.8% and 56.6% of the administered dose, respectively. The main components of radioactivity in plasma and urine were ASP015K and M2 (5′-O-sulfo ASP015K). In feces, ASP015K and M4 (7-N-methyl ASP015K) were the main components.

2.?In vitro study of ASP015K metabolism showed that the major isozyme contributing to the formation of M2 was human sulfotransferase (SULT) 2A1 and of M4 was nicotinamide N-methyltransferase (NNMT).

3.?The in vitro intrinsic clearance (CL_{int_in?vitro}) of M4 formation from ASP015K in human liver cytosol (HLC) was 11-fold higher than that of M2. The competitive inhibitory effect of nicotinamide on M4 formation in the human liver was considered the reason for high CL_{int_in vitro} of M4 formation, while each metabolic pathway made a near equal contribution to the in vivo elimination of ASP015K. ASP015K was cleared by multiple mechanisms.     

Parabens enable suspension growth of MCF‐10A immortalized,non‐transformed human breast epithelial cells

Parabens (alkyl esters of p‐hydroxybenzoic acid) are used extensively as preservatives in consumer products, and intact esters have been measured in several human tissues. Concerns of a potential link between parabens and breast cancer have been raised, but mechanistic studies have centred on their oestrogenic activity and little attention has been paid to any carcinogenic properties. In the present study, we report that parabens can induce anchorage‐independent growth of MCF‐10A immortalized but non‐transformed human breast epithelial cells, a property closely related to transformation and a predictor of tumour growth in vivo. In semi‐solid methocel suspension culture, MCF‐10A cells produced very few colonies and only of a small size but the addition of 5 × 10^‐4 M methylparaben, 10^–5 M n‐propylparaben or 10^–5 M n‐butylparaben resulted in a greater number of colonies per dish (P < 0.05 in each case) and an increased average colony size (P < 0.001 in each case). Dose‐responses showed that concentrations as low as 10^–6 M methylparaben, 10^–7 M n‐propylparaben and 10^–7 M n‐butylparaben could increase colony numbers (P = 0.016, P = 0.010, P = 0.008, respectively): comparison with a recent measurement of paraben concentrations in human breast tissue samples from 40 mastectomies (Barr et al., 2012) showed that 22/40 of the patients had at least one of the parabens at the site of the primary tumour at or above these concentrations. To our knowledge, this is the first study to report that parabens can induce a transformed phenotype in human breast epithelial cells in vitro, and further investigation is now justified into a potential link between parabens and breast carcinogenesis. Copyright © 2012 John Wiley & Sons, Ltd.     

New N1-Hetarylmethylene-Substituted Amidrazones with Potential Antimycobacterial Activity

N¹-Hetarylmethylene (e.g. pyridyl, 2-benzoxazolyl, 2-benzofuryl) substituted picolinic acid amidrazones and pyrazine-2-carbox-amidrazones were investigated. The compounds were accessible by reaction of the unsubstituted amidrazones with aldehydes. Pyrazine-2-carbox-N¹-(2-benzoxazolylmethylene)amidrazones were obtained by a new multistep reaction. The investigated amidrazones were tested for their antibacterial activity against four strains of mycobacteria (M. tuberculosis, M. avium, M. intracellulare, M. lufu) and were shown to have rather low activity.     

Effect of doxepin on uptake and efflux of serotonin in human blood patelets in vitro

It is shown that the tricyclic antidepressant drug doxepin (comprising 15% of thecis- and 85% of thetrans-isomer) is a moderately potent competitive inhibitor of serotonin uptake in human blood platelets in vitro, with an inhibitory constantK _i of about 2×10^-7 M. The inhibitory effect was 6 times stronger in an artificial, protein-free medium than in diluted plasma, corresponding to about 85% protein binding. The efflux of serotonin from platelets preloaded with¹⁴C-serotonin was not affected by doxepin in concentrations up to 10^-6 M, but increased rapidly at concentrations above 10^-4 M.